A New Emerging Picoscale Biotechnology

In: Protein Analysis Techniques
(C)
Editors: Rakesh Sharma, Bharati D Shrinivas 2009 Innovations And Solutions, Inc.
___________________________________________________________________________
Lecture 3
SERUM PROTEOMICS FOR CANCER:

FROM BASIC RESEARCH TO CLINICAL APPLICATION
Rakesh Sharma, Bharati D Shrinivas
ABSTRACT
Proteomic analysis based on the separation and identification of proteins/peptides is
a po werful t ool for di scovering new b iomarkers. T wo-dimensional d ifferential gel
electrophoresis ( 2D-DIGE), t he P roteinChip a rray®, C linProt®, a nd liq uid
chromatography ar e k nown as s eparation t echnologies. Although t he d etection a nd
identification of proteins/peptides could be performed using mass spectrometry (MS), the
types o f M S used ar e d iverse an d t he ch aracteristics d epend o n t he ionization method
(e.g., m atrix-assisted la ser d esorption/ionization [ MALDI], s urface-enhanced l aser
desorption/ionization [ SELDI], a nd e lectrospray i onization [ ESI]) an d m ass an alyzer
(e.g., t ime-of-flight [ TOF], qua drupole [ Q], and i on t rap [ IT]). The di verse pr oteomic
systems t hat co uple s eparation t echnologies with MS h ave b een s uccessfully ap plied t o
search for cancer biomarkers in biological fluid such as serum. U ntil no w, many cancer
biomarkers h ave be en r eported t o be i dentified f rom serum a nd pl asma. I n or der t o
identify diagnostic biomarkers o f h epatocellular car cinoma ( HCC) an d ad ult T -cell
leukemia (ATL), which are both human viral malignancies, we have been analyzing sera
from patients using t he P roteinChip a rray®-SELDI s ystem a nd M ALDI-TOF-TOF-MS.
In t his r eview, we p resent a n o verview o f p roteomic t echnologies used t o s earch for
biomarkers a nd cancer b iomarkers discovered b y t hese technologies, i ncluding o ur d ata
on HCC and ATL. Cancer diagnosis using these proteomic biomarkers is also described.
2 Rakesh Sharma, Bharati D Shrinivas
INTRODUCTION
Cancer r emains t he m ajor de vastating di sease t hroughout t he w orld. C urrently, i t i s
estimated th at c ancer k ills o ver 6 mill ion pe ople pe r y ear w orldwide, w ith ov er 10 m illion
new c ases di agnosed e very year. D espite a dvances i n di agnostic i maging t echnologies,
surgical m anagement, a nd t herapeutic m odalities, t he l ong-term s urvival i s poor i n m ost
cancers. F or ex ample, t he f ive-year survival r ate is onl y 14% i n lung cancer and 4% in
pancreatic can cer [ 1, 2]. T he m ajority o f can cers ar e d etected i n t heir ad vanced s tages an d
some h ave d istant metastases, w hich ar e t hought t o b e t he cau ses o f t he c urrent t reatment
ineffectiveness.
It is widely accepted that early diagnosis and intervention are the best way to cure cancer
patients [3, 4]. Cancer biomarkers provide diagnostic, prognostic, and therapeutic information
about a particular cancer and show their ever-increasing importance in the early diagnosis of
cancer [5, 6]. Over the past several decades, enormous efforts have been made to screen and
characterize u seful can cer b iomarkers. S everal b iomarkers ap proved cl inically ar e available
for e arly di agnosis a nd/or the s uccessful m onitoring of t reatment a nd r elapses a nd ha ve
contributed s ignificantly t o reduce m ortality r ates an d i ncrease o verall s urvival for can cers
such a s prostate c ancer [5, 7]; however, unfortunately, most biomarkers a re not satisfactory
because o f t heir l imited s pecificity a nd/or s ensitivity [8–10]. T herefore, t here i s a n ur gent
need to discover better potential biomarkers for cancer diagnosis in clinical practice.
Global an alyses h ave o pened n ew av enues f or can cer b iomarker d iscovery [11–13].
Genomics, t ranscriptomics, pr oteomics, a nd m etobolomics a re s tudies of a ll ge nes,
transcripts, pr oteins, a nd metabolites, r espectively. From v arious s pecimens, such a s c ells,
tissues, a nd body f luids, bi omarkers de tected di fferentially be tween c ancer pa tients a nd
healthy in dividuals ha ve be en i nvestigated. In pa rticular, s erum/plasma i s a less-invasive
specimen that is suited to cancer diagnosis. Because serum/plasma has a high protein content
(60–80 m g/ml), a nalyzing all included proteins us ing pr oteomic a nalysis i s e fficient for
diagnostic biomarker discovery.
Proteomic analysis is widely used to discover cancer biomarkers and is based on protein
separation a nd i dentification t echnologies [ 14–17]. T o s earch f or di agnostic bi omarkers,
proteins/peptides i n sp ecimen a re fi rst se parated w ith s eparation t echnologies. T hese
expression pr ofiles a re t hen c ompared be tween c ancer a nd c ontrol gr oups. N ext,
proteins/peptides di fferentially e xpressed unde r di sease c onditions c an be i dentified us ing
mass spectrometry (MS). Using proteomic analysis of serum/plasma, many cancer biomarkers
have been reported to be identified [15, 17]. Using proteomic technologies, we have also
analyzed sera from patients with hepatocellular carcinoma (HCC) and adult T-cell leukemia
(ATL), w hich a re both h uman v iral m alignancies [ 18, 19] . I n t his r eview, w e p resent a n
overview of t he pr oteomic t echnologies us ed t o s earch f or bi omarkers a nd t he c ancer
biomarkers discovered b y these technologies, including our da ta on HCC a nd ATL. C ancer
diagnosis using these proteomic biomarkers is also described.
Proteomic Analysis in Biomarker Discovery for Cancer Diagnosis 3
T able 1. C har acter istics of global analyses.
1. PROTEOMIC ANALYSIS OF SERUM/PLASMA IS EFFECTIVE

TO SEARCH FOR CANCER DIAGNOSTIC MARKERS
There has been increasing interest in global analyses for the diagnosis of cancer [11–13].
Diagnostic biomarkers are currently being investigated throughout the world using genomic,
transcriptomic, pr oteomic, and m etabolomic a nalyses. I n ge neral, a bi omarker w hose
expression i s i ncreased o r decreased s pecifically u nder d isease c onditions i s r equired t o
develop a new diagnostic method. Although specimen matrices such as serum/plasma, urine,
and bi opsy t issues a re us ually a nalyzed, t he m ethod needs to be ch osen ap propriately
depending on the developmental purposes, cancer types, and specimen.
1.1. Characteristics of Global Analyses and Diagnostic Availability of

Biomarkers
Table 1 shows the characteristics of global analyses. Genomic analysis is based on DNA
sequencing of genes. By examining the whole genome sequence, we can obtain information
about a ltered ge netic a nd e pigenetic e vents. S ingle nuc leotide pol ymorphisms ( SNPs) a re
known a s ge netic events and thought t o be associated w ith disease s usceptibility and
individual responses to drug treatment [20]. While many SNPs are silent with no direct effect
on proteins as gene products, some affect the coding or regulatory (usually promoter) regions
of a gene and change the function and expression of translated proteins. There is a report that
SNPs o f th e liv er in testine-cadherin g ene ( CDH17) t rigger a berrant s plicing and a re
associated with the developmental risk of HCC [21]. As epigenetic events, DNA methylation
has b een s uggested t o b e as sociated w ith car cinogenesis [ 22]. Characteristically, D NA
methylation doe s not c hange ge netic in formation; h owever, it a lters th e r eadability of D NA
and results in gene inactivation. In particular, h ypermethylation of CpG islands in promoter
sequences is associated with the silencing of tumor suppressor genes and tumor-related genes
by s ubsequent down-regulated e xpression of mRNA t ranscripts. S uppressors of c ytokine
signaling 1 a nd 3 ( SOCS-1 and SOCS-3), w hich pl ay important r oles i n c ell gr owth a nd
immune reaction, have been reported to be inactivated by hypermethylation in several cancers
[23]. From these f indings, i t i s s uggested t hat S NPs a nd D NA m ethylation c ould be us ed

clinically as g enetic an d ep igenetic b iomarkers f or can cer d iagnosis, r espectively.
Considering clinical applications, SNP and methylation arrays, by which it becomes easy to
detect can cer-specific c hanges us ing e xtensive pr obe s ets, m ight be us eful. In a ddition, t he
translated proteins whose expression, amino acid sequences, and function are altered by SNPs
and methylation may also be available for diagnosis as protein biomarkers.
Transcriptomic analysis is based on DNA microarray and is used to investigate extensive
gene ex pression at t he m RNA l evel [24–25]. G enerally, mRNAs ex tracted f rom cells an d
tissues a re r everse-transcribed t o cDNAs and t hen hy bridized w ith c omplementary
oligonucleotide DNA pr obes. The expression levels of each mRNA can be m easured by
detecting hybridized cDNA. Because DNA probes for several tens of thousands of transcripts
are fixed on the basal plate over a couple of centimeters, the expressions of all genes can be
visualized s imultaneously i n j ust one experiment. I ndeed, us ing Genechip a rrays®
(Affymetrix, Santa Clara, CA), the expression of about 50,000 transcripts can be examined.
As part of the investigations into the ATL-developmental mechanism, Morishita’s group, in
collaboration with us, applied DNA microarray to biomarker search [26]. The comparison of
ATL cells derived from A TL patients with normal T -cells from healthy individuals showed
that tumor suppressor in lung cancer 1 (TSLC1), which is a cell adhesion molecule, is over-
expressed in ATL cells. While TSLC1 is a gene that associates with tissue invasion, its gene
product (protein) may be also useful for ATL diagnosis. Cancer-specific genes found by DNA
microarray analysis and these products could be used as diagnostic biomarkers [27–29].
Proteomic a nalysis ba sed o n t he s eparation a nd i dentification of pr oteins/peptides i s
widely used to discover biomarkers, investigate cancer development, and to clarify the action
mechanism of dr ugs [ 14–17]. T o s earch f or di agnostic bi omarkers, pr oteins/peptides i n a
specimen are first separated with diverse separation technologies and the expression profiles
are c ompared be tween di sease a nd c ontrol gr oups ( Chapter 2) . Next, pr oteins/peptides
differentially expressed i n d isease s tatus c an be i dentified us ing MS ( Chapters 3 and 4). In
clinical p ractice, n ovel d iagnostic m ethods ar e especially expected t o be developed us ing
identified biomarkers (Chapter 5).
Metabolomics is the global study of all small molecular metabolites existing in cells and
body fluids [30, 31]. Although metabolomic analysis consists of separation and identification
processes a s i n pr oteomics, t he t arget of a nalysis i s s mall m olecular c ompounds but n ot
proteins/peptides [32]. For metabolomic separation, gas chromatography (GC) [33], LC [34],
and capillary electrophoresis (CE) [35] are mainly used. Compounds are identified with MS
[33–35] and nuc lear m agnetic r esonance s pectroscopy ( NMR) [ 36, 37] , w hich a re coupled
together with separation technologies. Metabolomic analysis contributes to the understanding
of how m etabolites a nd t heir c oncentrations c hange u nder ph ysiological c onditions. A t t he
same ti me, a pplication o f th is a nalysis to th e b iomedical f ield may lead to th e d iscovery o f
novel drug candidates and diagnostic biomarkers [38, 39]. This analysis is most appropriate to
search for biomarkers from urine, owing to the paucity of misplaced materials such as blood
cells a nd pr oteins. R ecently, f rom more t han 2,000 m ass s pectra of ur ine metabolites, th e
expression of several peaks has been reported to be increased in renal cell carcinoma [40].
T able 2. Diagnostic availability of biomar ker s identified by global analyses.
1.2. Advantages of Proteomic Analysis in the Search for Diagnostic

Biomarkers
Genomic, t ranscriptomic, pr oteomic, metabolomic a nalyses pr ovide i nformation a bout

disease-specific genes (SNPs and methylation sites), mRNAs, proteins, and small molecular
metabolites, respectively. Considering that these biomarkers are actually used for diagnosis in
clinical p ractice, d eveloping m easurement s ystems f or t hem i s n ecessary. T he i mportant
requirements f or di agnostic a vailability a re s implicity of t he de tection pr ocedure, hi gh
throughput, specificity of biomarker detection, and good reproducibility.
Table 2 shows the diagnostic availability of biomarkers identified by global analyses. At
present, protein/peptide biomarkers appear to best meet these requirements. In particular, the
use of specific antibodies makes it possible to develop measurement systems such as enzyme-
linked immunosorbent assay (ELISA), which is used widely in clinical practice (Chapter 5).
In contrast to proteomic biomarkers, genomic and transcriptomic biomarkers are difficult
to make available for widely-used diagnostic systems at present. Genomic biomarkers such as
SNPs a nd D NA m ethylation a re d etected by t roublesome pr ocedures, i ncluding D NA
extraction from specimens and the amplification of target genes by polymerase chain reaction
(PCR). A lthough its performance ha s be en gr owing qu ickly r ecently, t he t hroughput i s s till
low for diagnostic a pplication. R ecently, a r apid S NP d etection s ystem na med s mart
amplification process version 2 (SMAP 2) has reportedly been developed [41]; however, it is
not c ommon yet in c linical p ractice. S imilarly, t ranscriptomic b iomarkers a re a lso p oorly
utilized f or di agnosis. A lthough m RNA i s de tected by r everse t ranscriptase ( RT)-PCR o r
Northern blotting, the method is tricky owing to mRNA flux. Collectively, it will take some
time b efore t he s pecific d etection o f g enomic an d t ranscriptomic b iomarkers b ecomes
common a s a c linical l aboratory t est; how ever, i f di fferential e xpression a nd S NPs c an be
detected at the protein level, ELISA using specific antibodies will become a widely used
diagnostic system.
Metabolomic b iomarkers ar e s mall molecular m etabolites. T hey ar e ex pected t o b e
applied in the diagnostic system; however, metabolomic analysis is still under development,
in c ontrast t o o ther gl obal a nalyses. A lthough t he i dentification o f s mall molecular

compounds is fundamental to the analysis, the database of mass and NMR spectra of
metabolites i s not y et c omplete. I n a ddition, r eference c ompounds f or confirming
identification are also lacking. Also, MS and NMR need extensive skills for their operation.
Further, specific detection methods must be established with respect to each target compound;
therefore, the development of a widely used diagnostic system using metabolic biomarkers is
difficult. R ecently, a ntibodies a gainst s mall molecular compounds ha ve be en r eported [ 42],
thereby leading to the development of methods, although the specificity of antibodies may be
low owing to few antigen determinants in the chemical structures of compounds.
As described above, measurement systems using protein biomarker appear to be effective
for t he de velopment of di agnostic m ethods. C onsequently, i t i s r easonable t hat pr oteomic
analysis using MS is focused on search for diagnostic biomarkers.
1.3. Specimen
Early d iagnosis is very i mportant t o r educe c ancer m ortality. P rotein bi omarkers i n

cancer di agnosis c an be obtained m ainly from s erum/plasma, ur ine, a nd pa thologic t issues.
Serum/plasma is a l ess-invasive specimen and is suitable for the early diagnosis. Because it
constantly perfuses tissues, a certain abnormality in the living body is believed to be reflected
in the expression and processing of contained proteins; for example, increased serum levels of
prostate-specific antigen (PSA) and cancer antigen (CA) 125 are used to detect cancer in the
prostate a nd ov ary, r espectively [43, 44] . Urine i s n on-invasive a nd us eful f or bi omarker
search i n ad dition t o s erum/plasma. Wh ile u rine b iomarkers ar e ef ficient, es pecially f or
urinary d iseases s uch as b ladder ca ncer [ 45], t hese co ncentrations i n ur ine un dergo m any
changes depending on diet, intake of water and drugs, and diseases. This is the difficulty in
urine a nalysis. P athologic t issues a re f requently us ed in t he de finitive di agnosis of s olid
cancer s uch as HCC and kidney car cinoma; h owever, t he pa thologic di agnosis i s hi ghly
invasive a nd uns uited t o e arly di agnosis. From t hese c haracteristics of s pecimens, i t i s
suggested t hat p roteomic a nalysis o f s erum/plasma i s ef fective in s earching f or can cer
diagnostic biomarkers.
High protein content (i.e., 60–80 mg/ml) in serum/plasma is attracting increasing interest
in proteomic analysis. The expectation is that the characterization of thousands of individual
proteins/peptides in serum/plasma will e nable the discovery of a number of reliable disease
biomarkers; how ever, t here is a di fficulty in t he pr oteomic a nalysis of s erum/plasma. O ver
99% pr otein content i n s erum/plasma i s dom inated b y 30 pr oteins, s uch a s a lbumin,
immunoglobulins, transferrin, ha ptoglobulin, complement C3, and l ipoproteins [46, 47]
(Table 3). Serum/plasma proteins are present across an extraordinary dynamic concentration
range that is likely to span more than 10 orders of magnitude. This dynamic range, namely the
existence of hi ghly a bundant pr oteins, makes t he de tection o f l ow a bundant pr oteins ( the
remaining 1%) extremely challenging. There are two remedies for this difficulty. The first is
the removal of highly a bundant proteins by a ffinity c olumns using a ntibodies a gainst them.
The s econd i s an alysis ai med at p eptides b ut not pr oteins. R egardless of t his difficulty,
considering t hat b iomarkers d iscovered f rom s erum/plasma ar e d irectly a vailable f or l ess-
invasive diagnosis, serum/plasma is an attractive specimen as the source of diagnostic
biomarkers.
T able 3. H igh abundant pr oteins in plasma der ived fr om healthy per sons
a
Accession No. from Swiss-Prot knowledge base.
2. SEPARATION TECHNOLOGIES IN PROTEOMIC ANALYSIS

Proteomic a nalysis ba sed on t he s eparation a nd i dentification of pr oteins/peptides is a
powerful t ool f or di scovering ne w bi omarkers. T o s earch f or di agnostic bi omarkers f rom
serum/plasma, d etecting p roteins/peptides e xpressed d ifferentially in d isease is e ssential.
Two-dimensional differential gel electrophoresis (2D-DIGE), ProteinChip array®, ClinProt®,
and LC are typical separation technologies for biomarker discovery (Figure 1).
2.1. 2D-DIGE
The gol d s tandard for protein separation i n p roteomic a nalysis i s 2 D-DIGE ( Figure 1A)
[48, 49] . First-dimensional s eparation is ach ieved b y i soelectric f ocusing, w hich s eparates
proteins on t he basis of their charge. Namely, protein samples are applied to immobilized pH
gradient (IPG) s trips. T he IPG s trip is typically polyacrylamide ge l in which the pH gradient
(e.g., pH3–10) is immobilized. When the focusing voltage is applied to strips, the movement of
a p rotein in a p H g radient i s d etermined s olely b y i ts p osition r elative t o its isoelectric p oint
(Figure 1A a). I f the pos ition of t he pr otein i n t he gr adient i s a t a pH t hat i s gr eater than i ts
isoelectric point (pI), its net charge is negative, due to the net effect of dissociated carboxylate
anions (negative charge) a nd unc harged a mines in the pr otein; therefore, the protein migrates
towards the positive electrode. If the position of the protein in the gradient is at a pH that is less
than i ts p I, i ts ne t c harge i s pos itive, due t o t he ne t e ffect of unc harged c arboxylic acid a nd
protonated amines (positive charge). The protein then migrates towards the negative electrode.
Any migration continues until the protein migrates to the position in the pH gradient that has no
net charge. On this principle, the protein is focused at its pI on the IPG strip.
Isoelectric f ocusing i s c oupled w ith s econd-dimensional s eparation, w hich e xploits
sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins
in accordance with molecular weight (Figure 1Ab). Under optimal conditions, the expression
pattern of s everal t housands of i ndividual pr oteins c an be vi sualized s imultaneously on a
single 2D gel. The resulting gel shows the proteins separated according to pI across the x-axis
of the 2D display and according to molecular weight across the y-axis.
(a) First-dimensional separation; Isoelectric focusing (b) Second-dimensional separation; SDS-PAGE
pH3 IPG strip pH10 Isoelectrically focused proteins

↓↓↓↓ ↓↓ ↓↓↓ ↓↓↓ ↓
O O − IPG strip
-
=
+ High
Focusing Time
C OH C O
＋
→
H →
NH2 NH2 ↑ ••••
• ● ••● • ●●
Molecular
→
↑ SDS-PAGE
Weight
●•
••• •
Protein ● ● ●
SDS-polyacrylamide gel
pI • • ●•
↑ ● • ●
O − • • • ●
↓ • ●
=
•
+ C O
H
NH2
＋ - Low
•
pH3 pH10
(A) 2D-DIGE: Protein sample is separated by isoelectric focusing (a) as first-dimensional separation,
followed by SDS-PAGE (b) as second-dimensional separation. Protein spots in separation gels are
visualized by CBB-, silver-, or fluorescent-staining.
Functional group Hydrophobic, cationic, anionic, Biomarker peak

Proteins/peptides metal ion presenting or hydrophilic
ProteinChip ↓
Peak Intensity
Patients
Patients 1→ ◆ ▼ ▽
◆
◎ △ ○
◇
◉ ▼ △
□
Peak detection
2→
△ ▼ ◆ △
○ with MS
● ◎ □
◆ → ▽
□ ▲ → →
3→
○ ▲ △
□ Incubation Wash
↑
○◇
●
◉ ◎ ○ ◎○ ○◇ ●
◉ ◎ ○ ◎○ mass
4→
Differential analysis
1→
Healthy Healthy individuals ↑
◆
2→
individuals ▼ ▽
◎ ◆
△
◇ △ ▼
◉ ▼ △
□ ◆ △
Peak detection
Biomarker peak
3→ ● ◎ □ ■ with MS
→ ▽
→ →
Peak Intensity
■
□
◆ ▲ △
Incubation
□ ▲
Wash ↓
4→ ● ■◎
◉ ◎ ■ ◇ ● ■◎
◉◎ ■ ◇
Sample
Just after sample addition Adsorption of proteins/peptides Removal of non-adsorbed
addition
having affinity with functional group proteins/peptides mass
(B) ProteinChip array®: Biological fluids, such as serum/plasma and urine, are treated with
ProteinChip arrays®. Proteins/peptides in fluids are adsorbed on spot surfaces modified with a
functional group (hydrophobic, cationic, anionic, metal ion presenting, or hydrophilic). After removing
non-adsorbed proteins/peptides by washing, peaks derived from those adsorbed on the surfaces are
detected with MS. For example, spectral comparison between diseased patients and healthy individuals
could lead to the discovery of biomarker peaks.
Biomarker peak
Hydrophobic, cationic, anionic,
Magnetic beads ↓
Peak Intensity
or metal ion presenting
Proteins/peptides
◎ △ ◎ △
◇ ◇
◎ □ □ ◎ ◎
● ◎ ● ◎ ◎ ◎ ◎
● ● ●
◎ ●●◇
■ ■ ● ■
▽ ◉ ◎ ▽ ◉ ◎
◉■ ◉◇ mass
↑Patients
■ ■
◇ ◆ ●
■ △ △
□ □
■ ◎ ◇ ◎
■ ◇ ◉
▼ ▼ ▽
□
▼
◆ →
▼
◇
▼
◉
▽
□
▼
◆ → → ■
◎■◎ ● → Differential analysis
◆ Incubation ◆ Wash Elution Peak detection ↑
△ △ ▼
◉ ◉ ◆ △ ■
△ ▼ ◎
■ ■
■
■■
with MS
◎ Healthy individuals
■ △ △
●
▲ ▲ ◇ ● ●
▲ △
□ ◆
● ▲ △ ◆ ◇ Harvest of adsorbed Biomarker peak
◇
Peak Intensity
◎ ● ● ◎ □ ■ ◎
■ △ proteins/peptides ↓
■ ■ ◎ △
◇
Adsorption of
Mix of sample (serum) proteins/peptides Removal of non-adsorbed
and magnetic beads having affinity with proteins/peptides
functional group
mass
(C) ClinProt®: Biological fluids are treated with ClinProt beads which have chromatographic surfaces
(hydrophobic, cationic, anionic, or metal ion presenting). Proteins/peptides in fluids are adsorbed on
bead surfaces. After washing, peaks derived from proteins/peptides adsorbed on bead surfaces are
detected with MS.
g p g
HPLC or nano LC
1st separation 2nd separation
Crude protein
extract
→ Peptide mixture → → MS
Strong cation
Reverse phase
Digestion with Injection exchanger Eluted peptides
proteolytic enzyme
(e.g., trypsin)
(D) Shotgun proteomics using LC: Crude protein extract containing intact proteins is digested with
proteolytic enzymes such as trypsin and then generated peptide mixture is subjected to HPLC or nano
LC. The expression and identification of peptides in the eluate are assessed with MS.
Figure 1. Protein separation technologies for biomarker discovery. :
Protein profiles can be compared using sophisticated software packages to detect protein
spots t hat ar e d ifferentially ex pressed b etween s amples. I n g eneral, p rotein s pots a re
visualized by Coomassie brilliant blue (CBB) or silver staining [49]; however, CBB staining
has low sensitivity and requires about 100 μg proteins per gel for spot detection, suggesting
that th is s taining is n ot s uited to p roteomic a nalysis of v aluable s pecimens. Although
detection by silver staining can be performed with a small amount of proteins (about 10 μg),
irregular staining is often seen, leading to low reproducibility and quantification, which can
be r esolved us ing f luorescent dy es [50, 51] . F luorescent 2D -DIGE ha s hi gh s ensitivity a nd
enables m ultiple pr otein e xtracts t o be s eparated on t he s ame 2D ge l b y l abeling proteins
using dy es w ith di fferent e xcitation a nd e mission w avelength pa irs. F urthermore, because
proteins a re fl uorescently l abeled be fore e lectrophoresis, i t i s not ne cessary t o c onsider t he
staining i rregularity of ge ls. R ecently, f luorescent 2D -DIGE ha s b ecome pop ular f or
proteomic analysis [51].
2.2. ProteinChip Array®
ProteinChip array® (Bio-Rad Laboratories, Inc., CA, USA) has been successfully applied
to di agnose v arious m alignancies, s uch a s ov arian c ancer a nd ga stric c ancer [ 52, 53]. As
shown i n F igure 1B , ProteinChip a rray® is a c onvenient s eparation to ol th at utilizes
chromatographic properties and its surfaces c an be modified with various functional groups
(hydrophobic, c ationic, a nionic, m etal i on pr esenting a nd h ydrophilic). In t his m ethod,
biological fluids, such as serum/plasma and urine, are directly applied to spot surfaces. After
a s eries of bi nding a nd w ashing s teps, pr oteins/peptides a nd a ny c ontaminants t hat do not
bind to the surface are removed. P rotein mixtures retained on the surface are then a nalyzed
with MS. In the binding step, the change of parameters (pH, salt concentration, and organic
solvent concentration) in buffer solution alters the affinity between proteins/peptides and the
surface, l eading t o t he de tection of a v ariety of pr oteins/peptides. A lso, pr etreatment of t he
specimen w ith pr otein de naturants ( e.g., urea) a nd de tergents ( e.g., CHAPS) a ffects the
variation. B ecause d ifferential an alysis can b e p erformed w ith a s mall am ount o f
serum/plasma ( a f ew microliters), P roteinChip array® is us eful i n bi omarker s earches us ing
valuable specimens; however, the limitations of a nalysis include high c ost a nd the fact that
MS used in analysis is exclusive (Chapter 3.2).
2.3. ClinProt®
ClinProt® (Bruker D altonics, Bremen, G ermany) is a p urification method ba sed on a ffinity

beads ( Figure 1C) [54, 55]. T his method uses chromatographic surfaces (hydrophobic, c ationic,
anionic, a nd metal i on pr esenting) on a n ou ter layer of m agnetic be ads. C ertain s ubsets of
proteins/peptides, which have affinity to the surface, can be selectively purified, allowing unbound
impurities to be removed by washing with buffers. Proteins/peptides bound to magnetic beads are
then eluted and directly analyzed by MS. The technical performance of affinity bead purification
is similar to that of ELISA, and it can be used to process many samples in parallel. In addition, the
cost of analysis is low and data can be obtained with diverse types of MS.
Inlet Ion source Mass analyzer Detector Control system Data system
Vacuum system
Figure 2. Block diagram of MS. Sample inlet, ion source, mass analyzer, detector, vacuum system,
control system, and data system are major components of MS. The mass analyzer, detector, and parts of
the ion source are maintained under vacuum. The control system monitors and controls all parts of the
instrument. Data are recorded by the data system.
2.4. Shotgun Proteomics Using LC
LC i s a s eparation t echnology us ed c ommonly in t he i solation of v arious biological

molecules, i ncluding pr oteins/peptides. The L C el uate i s d irectly an alyzed u sing M S i n
proteomic analysis [56, 57] (Figure 1D). Because intact proteins cannot be identified owing to
the measurement l imits of MS, crude protein extract is digested in advance with proteolytic
enzymes, such a s trypsin, with the a im of directly identifying the LC e luate. T he generated
peptides, de rived f rom a w ide v ariety of pr oteins i n e xtract, a re s ubjected t o L C a nd t hen
sequences of pe ptides i n t he el uate ar e continuously determined us ing M S. S imilarly, a
method that identifies digested peptides in complex mixtures using a combination of LC and
MS i s r eferred t o a s “ shotgun pr oteomics”. T he na me i s de rived f rom D NA s hotgun
sequencing, which is named for its analogy to the quasi-random firing pattern of a shotgun.
Considering that LC eluate is directly analyzed with MS, in which involatile buffering agents
and salts inhibit analysis, elution using a volatile buffer is necessary for peptide separation;
therefore, elution with a w ater-acetonitrile gradient using reversed phase chromatography is
mainly used to separate peptides prior to entering the MS. Furthermore, to enhance separation
capacity, multidimensional LC ( MDLC), w hich c ombines t wo or more fo rms o f L C i n
shotgun a nalysis, ha s thrived ov er t he pa st f ew years [58]. O ne of t he m ost w idely us ed
combinations for MDLC is strong-cation exchange and reversed phase. In addition to the type
of column, the flow rate is also important for separation. High performance LC (HPLC) and
nano LC are generally used for peptide separation. In particular, nano LC is efficient for
separating valuable specimens. In contrast to HPLC, its flow rate is extremely slow (typical
flow r ate: 20 nl /min) [ 49]. This s low rate enables s amples t o b e s eparated i n a co ndensed
state, indicating that analysis requires only a small amount of specimen.
3. MASS SPECTROMETRY
Proteins/peptides separated using diverse technologies can be identified using MS, which
is now the gold standard for identification. In this chapter, MS used in proteomic analysis is
described.
3.1. Fundamentals of MS
A bl ock di agram of a ba sic MS i s s hown i n Figure 2. T he s ample i nlet, i on s ource, m ass

analyzer, detector, vacuum s ystem, c ontrol s ystem, a nd d ata system a re t he major c omponents
[49]. Control system monitors and controls the other components. The mass analyzer, detector and
parts of the ion source are maintained under vacuum. MS equipment is mainly defined by details
of t he i on s ource a nd mass a nalyzer. A nalytes i ntroduced from the s ample i nlet a re ionized b y
various ionization methods and then ions are separated by a mass analyzer. Masses and quantities
of their ions are shown as the mass-to-charge ratio (m/z) and peak intensity, respectively.
3.2. Ionization Methods
Ionization methods for biological macromolecules, such as proteins/peptides, were developed

in the 1980s. Matrix-assisted laser/ionization (MALDI) is a soft ionization technique used in the
analysis of proteins/peptides, which are fragile when ionized b y conventional methods [59, 60].
As s hown i n Figure 3A , pr oteins/peptides a re d issolved i n a s olution of a U V-absorbing
compound, referred to as the “matrix”, and placed on sample stage. For peptide ionization, α-
cyano-4-hydroxycinnamic a cid ( CHCA) i s w idely used as the matrix. A lso, p rotein is ionized
using s innapic a cid ( SA) a nd 2,5 -dihydroxy acetophenone ( DHAP). A s t he s olvent d ries, t he
matrix compound c rystallizes and proteins/peptides m olecules are i ncluded i n m atrix c rystals.
Pulses of U V l aser l ight a re used t o vaporize t he matrix a nd t he i ncluded pr oteins/peptides a re
carried into t he ga s pha se. B ecause most matrixes a re a cidic c ompounds a nd d ilute acid ( e.g.,
trifluoroacetic acid; TFA) is added to the solvent, ionization occurs by protonation in the acidic
environment. W hile monomolecular p rotons a re no rmally a dded to a p rotein/peptide,
multicharged i ons a re oc casionally ge nerated. T hese ge nerated i ons a re t hen i ntroduced i nto a
mass a nalyzer. S urface-enhanced l aser desorption/ionization (SELDI) is a nother io nization
method that modifies MALDI and is a specialized method for ProteinChip array® (Chapter 2.2).
Along w ith M ALDI, t he ionization m ethod us ed w idely i n pr oteomic a nalysis i s
electrospray ionization (ESI) [61, 62]. Although the mechanism underlying ESI has not been
fully clarified, its well-regarded model is illustrated in Figure 3B. In ESI, an acidic solution
containing pr oteins/peptides i s s prayed di rectly i nto t he M S i nlet t hrough a s mall-diameter
needle. A high, positive voltage is applied to this needle and droplets of the solution are
sputtered f rom t he t ip. P rotons f rom a cidic c onditions gi ve t he dr oplets a pos itive c harge,
causing then to move from the needle towards the negatively charged instrument (relative to
the ne edle). D uring t his m ovement, the s ize of t he dr oplets i s r educed by e vaporation, a
process t hat c an be a ided by a f low of ni trogen ga s a nd he at. Protonated pr oteins/peptides
desorbed f rom dr oplets t o t he ga s pha se a re i ntroduced i nto t he m ass a nalyzer.
Characteristically, a significant portion of ions produced by ESI is multiply charged. Because
proteins/peptides di ssolved i n l iquid c an be a nalyzed di rectly, E SI i s c oupled t ogether w ith
LC and the eluate obtained by solvent gradient is analyzed in waves.
Laser light
Sample
stage
H＋
H＋
H＋
H＋
H＋
H＋ Mass
analyzer
H＋
H＋ H＋
H＋ H＋
H＋
H＋
Double-charged ions
Matrix ions and neutrals
Proteins/peptides ions (single charge)
Crystallized matrix
with included
proteins/peptides
(A) MALDI: The matrix crystals, including proteins/peptides, are vaporized by irradiating pulses of
UV laser light. Together, the included proteins/peptides are also carried into the gas phase. Ionization
occurs by protonation in the acidic environment and then generated ions are introduced into the mass
analyzer.
Charged droplets Desolvated ions
Capillary column
effluent ＋
●＋
＋＋●
＋●
＋＋＋＋＋
＋
＋＋●
＋● ＋
＋＋＋＋＋＋＋＋＋
＋
＋＋
＋
＋ ●＋
＋＋
＋＋
＋
＋＋＋＋
●＋
＋＋＋
＋
＋＋
＋●＋
＋＋＋＋
●＋ Mass
● ● ●●● ● ● ●
＋＋＋＋＋
→
LC→ ● ● ● ● ●
● ● ● ●● ●● ● ● ＋＋＋＋
＋
＋＋＋＋＋＋＋
＋
●＋
●＋
＋＋
＋●
＋●
analyzer
＋＋＋＋＋＋
＋
＋
＋＋＋
＋
＋＋＋＋＋＋＋
＋＋＋＋
＋
●＋＋●＋
＋
＋＋＋＋ ●＋
High voltage ＋＋
●＋
(+2 kV to +5 kV)
Heated desolvation region
(B) ESI: A: solution that contains proteins/peptides is sprayed directly into the inlet of MS through a
small-diameter needle. The solution is acidic and a positive voltage is applied to the needle. Droplets
from the needle are given a positive charge by the addition of protons and then move towards the
instrument. During this movement, the size of droplets is reduced by evaporation and then protonated
proteins/peptides desorbed from droplets are introduced into the mass analyzer. In ESI, a significant
portion of ions is multiply charged.
Figure 3. Ionization methods in proteomic analysis.

(a) Linear type (b) Reflector type

er se
r
L as La
Sample Sample
stage stage Reflectron
Ion flight path in field-free region Ion flight path in field-free region
→ →
↑ ↑ ↑ ↑ ↑
+25 kV +0 kV Detector +25 kV +0 kV
Acceleration Acceleration
region region
↑
Detector
(A) TOF mass analyzer: Ions are given a fixed amount of kinetic energy in the acceleration region and
then travel in a field-free region. Because their velocities are inversely proportional to their m/z, ions
with low m/z travel more rapidly than ions with high m/z. Using the time required for the ion to travel,
its m/z is calculated. In addition to the linear type (a), the reflector type (b) is used as a mass analyzer.
The use of a reflectron reduces accidental error in the velocity of traveling ions and increase of flight
distance, leading to high resolution analysis.
+
Metal rods (quadrupole)
+
+ - +
→Detector
+
+
-- +
↑
Ions
(B) Q mass analyzer: Ions pass into the electrostatic field created by electric potentials applied to four
parallel metal rods (quadrupole). Pre-selected ions make it through the inside diameter of the
quadrupole and strike the detector (circular ion). All other ions are deflected out of the quadrupole
(triangular ion).
Ring electrode
Ions Trapping Detector

region
Ring electrode
Endcap electrode Endcap electrode

(Ion entrance) (Ion exit)
(C) IT mass analyzer: All ions are initially trapped in the trapping region. Trapped ions are ejected out
of the region and into the detector by changing the electrostatic field. Because all ions injected into the
trap are sent to the detector, the detection sensitivity is extremely good.
Figure 4. Mass analyzers
3.3. Mass Analyzers
Mass analyzers separate ions according to their mass by applying electric and magnetic
fields. Although the unit for their mass is the mass-to-charge ratio (m/z), care should be taken
with the interchangeable use of “mass” and “m/z” because these values are not the same for
any ion that is multiply charged. For example, a peptide ion with a mass of 1,000 Da that is
doubly charged by the addition of two protons would be observed as an ion at 501 m /z. It is
critical to remember this distinction, particularly when ESI, by which multicharged ions are
mainly generated, is being used (Figure 3B). Time-of-flight (TOF), quadrupole (Q), and ion
trap (IT) mass analyzers are used in MS for protein/peptides analysis [49].
The operating principles of the TOF mass analyzer are simple. As shown in Figure 4Aa, ions
are first given a fixed amount of kinetic energy by acceleration in an electric field generated by a
high voltage, typically + 20 kV t o + 30 kV ( for pos itive i ons such as peptides). Following
acceleration, ions enter a field-free region where they travel at velocities based on a law. Namely,
their velocities are inversely proportional to their m/z; therefore, ions with low m/z travel more
rapidly than ions with high m/z. The time required for the ion to travel the length of the field-free
region is measured and used to calculate the m/z of the ion. To obtain a higher resolution, while
longer flight tubes or higher accelerating voltages are effective, an ion reflector, also referred to as
a “reflectron”, is built into high resolution MS. As shown in Figure 4Ab, the reflectron is an ion
mirror created by an electric field and reverses the flight path of the ion. In theory, ions with the
same mass travel in a flight tube with the same velocity; however, in fact, there is accidental error
in t he ve locity. F or example, ions w ith higher ve locity t han the t heoretical va lue penetrate the
flight tube further. In the reflectron, higher-velocity ions take longer to reverse the flight path. This
process reduces the error in the velocity. Also, another effect of a reflectron is the increased flight
distance, which also leads to better resolution.
The Q mass analyzer consists of four parallel metal rods (quadrupole) through which ions
pass (Figure 4B). When opposite and neighbor rods are subjected to electric potentials having
the s ame an d d ifferent p olarities, respectively, an electrostatic field of specific strength a nd
frequency is created inside the quadrupole. The path of ions is influenced by the fluctuating
electric field. Only ions of pre-selected m ass make it through the inside di ameter of the
quadrupole and strike the detector (circular ion in Figure 4B). All other ions are deflected out
of the quadrupole and never reach the detector (triangular ion in Figure 4B). Further, the Q
mass analyzer can scan pre-selected masses at rates on the order of 1000 m/z per sec so that
the acquisition of a mass spectrum between 200 m/z and 2000 m /z takes 1.8 s ec. This high-
speed scan is suitable for acquiring data on LC, from which analytes are continuously eluted.
The IT mass analyzer works on the same physical principle as the Q mass analyzer. The
term “ion trap” is derived from the fact that electrostatic fields are applied so that ions of all
m/z are initially trapped in the trapping region (Figure 4C). Trapped ions are ejected out of
the trapping region and into the detector by changing the electrostatic fields dependent on the
m/z o f io ns. A n im portant c haracteristic o f I T is th at t he detection sensitivity is e xtremely
good, be cause of the e fficient transmission a nd utilization of ions formed by a n ion source.
The overall dimensions of the trap are such that the distance from the trapping region to the
detector is only a few centimeters. In the course of the trapping and ejection process, all ions
injected into the trap can ultimately be sent to the detector; therefore, the compact size of ion
traps and the detection of all ions injected lead to good sensitivity.
3.4. Types of MS Used in Proteomic Analysis
As described above, although the types of ion source and mass analyzer are diverse, MS
is composed of their various combinations; for example, MALDI-TOF-MS, SELDI-TOF-MS,
ESI-Q-MS, a nd E SI-IT-MS a re us ed i n pr oteomic a nalysis. Furthermore, t o pe rform the
measurement w ith good m ass a ccuracy a nd hi gh r esolution, t andem M S, i n w hich multiple
mass an alyzers ar e co nnected i n s eries, i s used w idely at p resent. Tandem M S, s uch a s
MALDI-TOF-TOF-MS ( Figure 5 A), E SI-Q-TOF-MS ( Figure 5B ), a nd ESI-IT-TOF-MS
(Figure 5C), is indispensable for the identification of proteins/peptides.
4. IDENTIFICATION OF PROTEINS/PEPTIDES USING MS

The i dentification of pr oteins/peptides is an essential step in the f ield of p roteomics.
Examining protein expression patterns alone is, of course, important for diagnosis in various
diseases; however, without identifying the proteins/peptides associated with diseases, it is not
possible t o de lve i nto t heir f undamental c auses or t o d evelop c linical a pplications, s uch a s
ELISA, t hat r equire a ntibodies a gainst bi omarkers. T he i dentification of pr oteins/peptides
using MS is the key step in the development of clinical applications using proteomic
biomarkers.
4.1. Protein Identification by Peptide Mass Fingerprinting
Peptide m ass f ingerprinting ( PMF) i s a n a nalytical t echnique f or pr otein i dentification

and was de veloped in 1993 by several groups independently [63–67] ( Figure 6A ). In PMF,
after protein separation, the protein of interest is first digested with proteolytic enzymes such
as t rypsin. The m/z of generated pe ptides is accurately measured w ith MS. These act ual
measurement v alues ar e t hen in silico compared w ith theoretical v alues of know n pr oteins
registered in a database. A s da ta on know n proteins, registered proteins are t heoretically
digested into peptides with enzymes and the absolute masses of these peptides are calculated.
By comparing the actual measurement values of the interest protein with theoretical values of
the e normous num bers of p roteins r egistered i n t he da tabase s tatistically, the be st-matched
protein is searched. Databases of the National Center for Biotechnology Information (NCBI)
and S wiss-Prot a re po pular f or identification. NCBI i ncludes m ost of t he publ ic dom ain
sequence databases an d t he S wiss-Prot da tabase ha s a large a mount of a nnotation t ogether
with the sequences to allow for easier understanding of the functionality of the identified
proteins [ 68]. T he advantage of PMF i s t hat onl y t he m/z of ge nerated pe ptides h as t o b e
examined. De novo peptide s equencing, w hich i s t ime-consuming, i s not ne cessary. A
disadvantage is that the interest protein has to be registered in a database. Additionally, most
PMF a lgorithms a ssume t hat t he pe ptides c ome f rom a s ingle pr otein [ 69]; t herefore, t he
typical PMF samples are proteins isolated by 2D-DIGE and MDLC that have high separation
capacity.
4.2. Peptide Identification by MS/MS Ion Search
Mass analysis is essentially a separation of ions according to their m/z. Because tandem
MS h as m ultiple mass an alyzers t hat ar e co nnected i n s eries, t wo s tages o f m ass an alysis
(MS/MS a nalysis) c an be c arried out i n a s ingle e xperiment. A n i on w ith a s pecific m /z i s
selected i n t he f irst s tage of m ass a nalysis a nd s tructural i nformation a bout s elected i on i s
obtained in the second stage. In the c ase of pe ptide ions, the information is the a mino a cid
sequence of the peptide. At present, peptides with a molecular weight of less than ~3,000 can
be routinely identified by MS/MS ion search using tandem MS, such as MALDI-TOF-TOF-
MS, E SI-Q-TOF-MS, a nd E SI-IT-TOF-MS ( Figure 5) . M S/MS i on s earch i s a vailable t o
identify pe ptides de tected by di fferential a nalyses us ing P roteinChip a rray®, ClinProt®, a nd
shotgun proteomics, and to confirm the sequences of peptides used in protein identification by
PMF.
MS/MS i on s earch i s ba sed on t he f ragmentation r eaction of pe ptides a nd by da tabase
search [49, 70, 71] (Figure 6B). Namely, pe ptides ionized by ionization methods, including
MALDI and E SI, ha ve excess i nternal energy. After a peptide of interest (parent i on) i s
selected in the first mass analyzer, its peptide bonds are randomly cleaved by internal energy.
While t he f ragmentation r eaction o ccurs i n i ons w ith excess e nergy, most i ons f ormed b y
MALDI an d E SI h ave i nsufficient i nternal en ergy an d ar e s table t o d rive an y f ragment
reactions. Thus, what is required for MS/MS analysis o f peptides is a method to energize a
stable p arent io n a fter it h as b een m ass-selected a nd t o i nduce f ragmentation r eaction. T he
method used most often is collisionally induced dissociation (CID), in which a mass-selected
ion is transmitted into a high-pressure region of the tandem MS where it undergoes a number
of c ollisions w ith molecules of f iller ga s s uch a s a rgon. A s t he i on c ollides w ith t hese ga s
molecules, a portion of the kinetic energy of the ion is converted into internal energy to make
the i on uns table a nd drive t he f ragmentation r eaction. I n t he Q -TOF mass a nalyzer ( Figure
5B), a fter a n io n is s elected in th e f irst s tage ( Q m ass a nalyzer), it is f ragmented in th e
collision cell. Also, in the IT-TOF mass analyzer (Figure 5C), the trapping region of IT serves
as the cell. In contrast, in MALDI-TOF-TOF-MS (Figure 5A), CID is not often used for
fragmentation. T he s elected i on de cays i nto f ragments w ithout C ID a fter l eaving t he
acceleration region o f the ion s ource; therefore, this fragmentation reaction is referred to as
“post-source decay (PSD). In PSD, the reaction occurs mainly in the second stage of the mass
analyzer (TOF2 in Figure 5A). The fragment ions produced are m/z analyzed in the second
stage. B oth t he i ntact pe ptide m ass a nd f ragment i on m asses a re us ed f or pe ptide
identification through database searching (Figure 6B).
5. CANCER BIOMARKER AND DIAGNOSIS

Over t he p ast s everal d ecades, en ormous ef forts h ave b een m ade t o s creen and
characterize u seful can cer b iomarkers. A s cl inically i mportant b iomarkers, PSA,
carcinoembryonic a ntigen ( CEA), α-fetoprotein ( AFP), C A125, C A15-3, and C A19-9 ha ve
been identified [8–10]. Although they are commonly employed in clinical diagnosis, they are
not satisfactory because of their limited sensitivity and/or specificity; for example, CA125 is
widely us ed t o d etect ovarian can cer in post-menopausal women and i s t he b est cl inical
marker c urrently a vailable. Although CA125 l evels a re e levated in ~ 80% of pa tients w ith
advanced s tage o varian can cer, s erum levels are increased in o nly 5 0–60% o f p atients w ith
the ear ly stage [8, 72–74]. Early diagnosis of cancers, including ovarian cancer, remains
difficult. There is therefore an urgent need to discover better potential biomarkers for cancer
diagnosis in clinical practice.
MALDI source TOF1 TOF2

r
se Decision of the Fragmentation of
La
Sample precursor ion the mass-selected
stage Reflectron
takes place precursor ion occurs
↑ ↑
+25 kV +0 kV
Electrostatic gate
Acceleration for precursor ion
region selection
↑
Detector
B. ESI-Q-TOF-MS
ESI source Q mass analyzer TOF mass analyzer
(Reflector type) Reflectron
●
● ● • • • • • ••
LC→ ● → → Colision → →
●
• • • • ••
● •• •
cell
•
↑
Detector
C. ESI-IT-TOF-MS
IT mass analyzer
ESI source TOF mass analyzer

(Reflector type) Reflectron
●
● ● • • • • • •• •• • •
LC→ ●
● • • • • → → • • →→ →
• •• •
● •• •
• ••
•
Trapping
region
↑
Detector
Figure 5. Tandem MS Tandem MS, in which multiple mass analyzers are connected in series, is used
widely for analysis with good mass accuracy and high resolution. MALDI-TOF-TOF-MS (A), ESI-Q-
TOF-MS (B), ESI-IT-TOF-MS (C) are well-known as tandem MS for proteomic analysis.
Isolation of interest protein using

separation technologies
SDS-PAGE gel 2D-DIGE gel LC (e.g., HPLC, nano LC)
● • • •••●
● Separation column
• • •• ● •
● •
•• •
•••• • •
•
• •
•● • •
●
• ●• •
•
Interest proteins
→
→
Cutting
interest protein
Fraction including
Gel piece including only interest protein
only interest protein
Isolated protein
Digestion with
→
proteolytic enzymes
(e.g., trypsin)
Peptide derived
from isolated protein
→
The m/z measurement of

generated peptides using MS
Actual measurement values (m/z) of peptides

Peak Intensity
m/z
→
Database search
1. Proteins registered in database are in silico digested with
the same proteolytic enzyme.
2.
3. Actual measurement values of peptides derived from interest

protein are compared with theoretical values of all proteins
registered in database.
→
Best-matched protein
(Identification)
(A) PMF for protein identification. A protein isolated with SDS-PSGE, 2D-DIGE, and LC is digested
with proteomic enzymes such as trypsin. The m/z of generated peptides is measured using MS. Actual
measurement values (m/z) of the peptides are compared with theoretical values of those derived from
all proteins registered in the database to find the best-matched protein.
Figure 6. (Continued)
Biomarker peak
Peak Intensity
ProteinChip ClinProt ↓
→ ● ◎ ◎
Patients
◇
→ ◇
○ △ ▼
↑
→ m/z
◎
◉ Patients
→
→
◆
■
▲
▽
◎
◎
→ Differential analysis
↑
individuals
□ □▼
Healthy individuals
Healthy
→
Peak Intensity
■
■ △○ Biomarker peak
→ ◎ ◉
□
↓
→ ■ ●
m/z
PMF
Peak Intensity
→
Digestion
m/z
Selection of interest peptides

(amino acid sequence; A1-A2-A3------An-2-An-1-An)
e.g., Biomarker peaks detected using ProteinChip array and ClinProt
Peaks used in database search for protein identification by PMF
(peaks circled in broken lines)
＋
Fragmentation of selected
→
A1 A2 A3 A4 ------ An-3 An-2 An-1 An
peptide (parent ion) by CID
or PSD using tandem MS Parent ion
＋＋
Fragment ions A1 A2 A3 A4 ------ An-3 An-2 An-1 An
＋＋
A1 A2 A3 A4 ------ An-3 An-2 An-1 An
＋＋
A1 A2 A3 A4 ------ An-3 An-2 An-1 An
-----
-----
＋＋
A1 A2 A3 A4 ------ An-3 An-2 An-1 An
＋＋
A1 A2 A3 A4 ------ An-3 An-2 An-1 An
＋＋
A1 A2 A3 A4 ------ An-3 An-2 An-1 An
→
Detection of fragment ions
Actual measurement values (m/z) of parent ion and fragment ions
Fragment ions ←Parent ion

Peak Intensity
m/z
→
Database search
1. Peptides having the same m/z value as parent ion are searched from
database.
2. Candidate peptides are in silico fragmented.
3. The m/z values of fragment ions are calculated (theoretical values).
4. Actual measurement values of fragment ions are compared
with theoretical values.
→
Best-matched peptides
(Identification)
(B) MS/MS ion search for peptide identification. From spectra obtained by ProteinChip analysis,
ClinProt analysis, and PMF, the peptide of interest is selected (peaks circled in broken lines). The
selected peptide ion (parent ion) is fragmented by CID or PSD using tandem MS. After the m/z of the
parent ion and its fragment ions is measured, these actual measurement values are compared with
theoretical values from all peptides registered in the database to find the best-matched peptide.
Figure 6. Identification of proteins/peptides using MS
T able 4. C ancer biomar ker s identified by pr oteomic analysis of ser um/plasma.
5.1. Characteristics of Proteomic Cancer Biomarkers and Diagnostic

Availability
As described in Chapters 2–4, diverse types of protein separation technology and MS are
proficiently c ombined i n proteomic a nalysis. A lthough a v ariety of c ombined s ystems ha ve
been us ed i n a nalysis, t he 2D -DIGE–MS sy stem a nd P roteinChip a rray®–SELDI-TOF-MS

system are a t pr esent t he main s tandards t o s earch f or new di agnostic bi omarkers i n m any
cancers [14–17]. Many studies ha ve reported proteomic biomarkers in various c ancers [75–
105].
Decision tree
ProteinChip
First (CM10) m/z of peak → 7770 m/z
Threshhold (peak intensity) → 6.0646
Peak Intensity
analysis group 4467 m/z
→ ↓↓ 4470 m/z
1 7770 m/z ←Low High→
2→
↓
HCC 3890 m/z 3444 m/z
3→
(N=35) 3.1838 2.6786
↑
m/z
HCC
4→ • Increased in HCC Non- 4067 m/z
→ Differential analysis 4467, 4470, and 7770 m/z → 4435 m/z
1.0622 HCC
HCC
1.4707
1→ • Decreased in HCC
↑
Non-HCC 3444, 3890, and 4435 m/z
Non-HCC 2→ 4470 m/z Non- Non-
Peak Intensity
3444 m/z HCC

5.6677 HCC HCC
3→
(N=44) ↓3890 m/z
↓ 4435 m/z
4→ ↓ 7770 m/z
HCC
4.5484 Second analysis group
m/z (HCC, N=29; non-HCC, N=33)
Non- Sensitivity 83% (24/29)
HCC Specificity 76% (25/33)
HCC
Figure 7. Classification of HCC and non-HCC using decision tree. The first analysis group (HCC,
N=35; non-HCC, N=44) is subjected to ProteinChip array® (CM10) followed by obtaining spectra with
SELDI. Six peaks (3444, 3890, 4435, 4467, 4470, and 7770 m/z) were expressed differentially in the
HCC group compared to the non-HCC group. Using m/z and peak intensities of these peaks, the
decision tree was built up for HCC diagnosis. To determine the accuracy and validity of the algorithm
used, the decision tree was reevaluated using the second analysis group (HCC, N=29; non-HCC,
N=33). The decision tree correctly diagnosed 83% (24 of 29, sensitivity) of patients with HCC and 76%
(25/33, specificity) of patients without HCC.
Table 4 s hows can cer b iomarkers that h ave b een i dentified b y pr oteomic a nalysis of
serum/plasma. B iomarkers whose ex pression i s i ncreased an d/or d ecreased s pecifically i n a
particular can cer type h ave a g reat d eal o f p otential in the d iagnostic field; h owever, as the
expression of α-antitrypsin has been reported to be increased in colorectal cancer [78], HCC
[86], an d ovarian can cer [96], the expression of most biomarkers is increased and/or
decreased i n multiple t ypes of c ancer. W hile pr oteins/peptides t hat a re e xpressed
differentially in v arious c ancers co nstitute a l arge p ortion o f can cer b iomarkers, a f ew
biomarkers are possibly specific to a particular cancer.
Neutrophil-activating peptide 2 (NAP-2) and Ras-related protein Rab-7B are expected to
be s pecific bi omarkers of H CC a nd l ung c ancer, r espectively [ 87, 91]. NAP -2 is th e m ajor
form of C XCL7, w hich i s a m ember of t he c hemokine f amily involved i n r egulating
immunity a ngiogenesis, s tem-cell t rafficking, a nd m ediating or gan-specific m etastases o f
cancers [106]. CXCL7 is translated as a propeptide and then cleaved to several smaller forms,
including N AP-2. A lthough C XCL7 ha s be en r eported t o be a m arker of a dvanced
myelodysplastic s yndromes (MDS), hematologic s tem c ell m alignancies in e lderly patients
[107], no expression of NAP-2 is observed in tissues of other types of cancer. Furthermore,
serum levels of NAP-2 are correlated with the clinicopathological features and prognosis of
HCC [ 108]. Rab7B, w hich i s hi ghly e xpressed i n l ung c ancer, i s a m ember of the R as
oncogene f amily a nd a s mall R ab GTPase t hat r egulates v esicular t raffic f rom ear ly t o l ate
endosomal stages of the endocytic pathway. Rab7B is expressed in the heart, placenta, lung,
skeletal m uscle, a nd pe ripheral bl ood l eukocytes [ 109]; h owever, n o i ncrease o f i ts se rum

levels h as b een reported i n c ancers ot her t han l ung c ancer. Consequently, t hese f indings
suggest that N AP-2 an d R ab7B m ay b e s pecific b iomarkers o f H CC an d l ung can cer,
respectively, although many clinical studies must be performed using sera from patients with
a variety of diseases.
The expression of serum a myloid A (SAA) is increased in a v arious can cers (Table 4 ).
Although proteomic analysis of serum/plasma identified SAA in HCC [89], lung cancer [91],
nasopharyngeal carcinoma [94], ovarian cancer [99], and renal cancer [105], its increased
expression ha s a lso be en r eported i n ot her c ancers s uch a s ki dney, c olorectal, a nd pr ostate
cancers, as well as in leukemias and lymphomas [110]. In addition, SAA is an apolipoprotein
secreted in an acute phase of inflammation [111]; therefore, its increased expression may
represent the epiphenomenon of cancers. While SAA is unlikely to be of much clinical use for
diagnosing the onset of cancer, it may be important to monitor the stage of cancer progression
and relapse. In nasopharyngeal cancer, SAA has been reported to be useful for monitoring its
relapse [112].
Although most proteomic biomarkers a re not specific to a particular c ancer, a
combination of t hose w ith know n di agnostic m ethods ha s be en r eported t o i mprove t he
probability of cancer diagnosis [86, 101]. Using CA125 alone as the best clinical marker of
ovarian can cer, p atients w ith ear ly s tage can cer ar e d iagnosed w ith a r eceiver o perating
characteristic a rea und er t he c urve ( ROC AUC) of 0.6 13. In c ontrast, w hen f our proteomic
biomarkers (transthyretin, hemoglobin subunit β, apolipoprotein A1, a nd t ransferrin) a re
combined with CA125, the diagnosis is performed with a ROC AUC of 0.955 [101]. The four
biomarkers are highly abundant proteins and are not likely to be synthesized at the focal site.
Thus, these biomarkers, even if they are not released directly by the tumor, could be used in
combination w ith ot her markers, s uch a s CA125, t o i mprove t he di agnostic pr obability of
early s tage o varian can cer. I n an other instance, AFP is a biomarker for H CC diagnosis. B y
2D-DIGE of sera from HCC patients followed by protein identification using MALDI-TOF-
TOF-MS, heat-shock protein 27 (HSP27) was identified as an HCC biomarker [86] (Table 4).
HSP27 i s a st ress-inducible c ytosolic pr otein t hat i s u biquitously pr esent i n m any nor mal
tissues a nd ha s be en r eported t o be ov er-expressed i n many t ypes o f can cer, such as breast
cancer an d as trocytic b rain t umor [ 113, 11 4]. H SP27 is n ot a s pecific can cer b iomarker;
however, i t c ould be de tected i n H CC patients w ith n egative A FP [ 86]. T his su ggests t hat
HSP27 could be complementary to AFP in the diagnosis of HCC. Furthermore, combination
diagnosis of HSP27 and AFP improves the diagnostic probability of HCC patients with small
tumors (< 5 cm). By the same token, leucine-rich α2 glycoprotein (LRG) has been reported to
serve as a complementary biomarker of CA19.9, which is a tumor marker commonly used for
the diagnosis of pancreatic cancer [102].
5.2. Early Diagnosis of HCC Using ProteinChip Array®
In cancer d iagnosis, ear ly detection b efore t he o nset o f cl inical s ymptoms i s very

important. Early diagnosis can lead to curative treatment, significantly improving prognosis.
Diagnosing patients who present without symptoms, however, is difficult, because the tumor
is s till s mall a nd th ere a re f ew le sions. Using th e ProteinChip a rray®–SELDI-TOF-MS
system, early diagnostic methods have been reported in several cancers, including our data on
HCC [18, 115–117].
Approximately 170 million people worldwide are infected with hepatitis C virus (HCV),
which when persistent can progress to HCC. Interferon (IFN) or combined IFN and ribavirin
are cu rrently t he o nly ef fective t reatments f or ch ronic h epatitis C , l eading t o a d ecrease o f
HCC occurrence [118, 119]; however, some patients do not receive IFN treatment or fail to
recover f rom H CV e ven w ith I FN t reatment. In a ddition, a s ubset of i ndividuals r emains
unaware that they are infected with HCV. Namely, in these patients, HCC may present only
in the advanced stage. The prognosis of patients presenting with symptoms related to HCC is
extremely poor. O ne requirement is the e arly de tection of H CC be fore the onset of c linical
symptoms.
We have been studying the developmental mechanism of HCC [120–122]. As part of the
investigation, we sought to identify novel diagnostic markers of HCC using the ProteinChip
array®–SELDI-TOF-MS system and to develop a diagnostic system using these markers. The
decision t ree f or H CC di agnosis w as e stablished us ing s era f rom pa tients w ith a nd without
HCC (Figure 7) [18].
The f irst a nalysis gr oup ( HCC, N = 35; non -HCC, N= 44) w as s ubjected t o ProteinChip
array® (CM10) f ollowed b y obt aining s pectra w ith S ELDI. S ix pe aks ( 3444, 3890, 4435 ,
4467, 4470, and 7770 m/z) that were expressed differentially in the HCC group compared to
the non -HCC gr oup w ere u sed f or di agnosing HCC. The de cision t ree ge nerated u sing m /z
and pe ak i ntensities c orrectly c lassified 97% of H CC samples. Further, t o de termine t he
accuracy a nd v alidity of t he a lgorithm us ed, t he decision t ree w as r eevaluated using t he
second analysis group (HCC, N=29; non-HCC, N=33). The decision tree correctly diagnosed
83% ( 24 of 29, s ensitivity) of pa tients w ith H CC a nd 76% ( 25/33, s pecificity) of patients
without HCC. Importantly, the accuracy of decision-tree diagnosis for HCC was higher than
that of other known tumor markers such as AFP (sensitivity, 41%; specificity, 67%).
Cleavage Process of C3
Activation pathways S S S S
Pathogens C3
↑
YY Y
→ →
Classical C3a
● Antibody
● pathway
S S S S
Malignant cells
C3b
↑↑
Viral-infected cells ■
Bacteria
■
■
Non-self
material → Alternative
pathway → S S S S
C3f
iC3b
etc.
→
Mannose-
→
◇◇◇◇◇ C3dg
◇◇◇◇◇
Lectin
containing
◇◇◇◇◇ pathway
carbohydrate C3g C3d
S S S S
C3c
Figure 8. Activating mechanism of complement system. When pathogens (e.g., malignant cells, viral-
infected cells, and bacteria) exist in the living body, the system is activated through classical,
alternative, and lectin pathways, with the result that they are cleared by opsonization and/or lysis.
Classical, alternative, and lectin pathways are activated by an antibody bound to pathogens, certain non-
self materials, and mannose-containing carbohydrates, respectively. Whichever pathway is activated,
C3 is finally cleaved into C3a, C3b, iC3b, and C3f etc.
The most fundamental requirement for serum-based marker detection is the identification
of carcinoma in an early stage when treatment has the greatest impact on prognosis. Decision-
tree diagnosis was performed using serum samples taken from 7 patients 1 year before HCC
development. S ix of t he 7 (86%) pa tients w ho l ater de veloped H CC w ere di agnosed w ith
HCC using the de cision tree. O ne year be fore de velopment, H CC w as s till u ndetectable b y
the c urrent di agnostic methods. It i s s uggested t hat t his de cision t ree i s useful f or the e arly
diagnosis of HCC.
5.3. Proteomic Strategy for Discovering Specific Cancer Biomarkers
As s hown i n T able 4 , m any can cer b iomarkers h ave b een i dentified b y s erum/plasma
proteomic an alysis. Wh ile s pecific b iomarkers s uch as NAP-2 an d Rab7B ar e d iscovered
fairly infrequently [87, 91], most of these are unfortunately not specific to a particular cancer.
In addition, the proteomic biomarker is mostly highly abundant proteins (Table 3). Certainly,
non-specific a nd/or hi ghly expressed bi omarkers a re useful t o c omplement c onventional
diagnosis methods [86, 101] and for monitoring cancer relapse [112]; however, because they
are n ot n ecessarily r eleased d irectly f rom t umor t issues, t heir d iagnosis methods may h ave
little s cientific b asis. D iscovering s pecific can cer b iomarkers that ar e b ased o n s cientific
evidence are important for the development of novel diagnostic methods.
Proteomic a nalysis of l ow a bundant pr otein ( less t han 1% of s erum/plasma pr otein
content) i n s erum/plasma may l ead t o di scovering s pecific bi omarkers. A lthough s hotgun
proteomics us ing LC ( Figure 1D ) a nd t andem M S ( Figure 5B , C) ha s be en e stablished f or
serum/plasma a nalysis [123, 12 4], c ancer b iomarkers u seful fo r e arly d iagnosis are st ill
unknown. Proteomic approaches based on other perspectives might also be needed.
Many r esearchers have h ypothesized t hat t he b est can cer b iomarkers w ere l ikely t o b e
secreted p roteins [ 125]. I n a ddition, m any c lassical c ancer bi omarkers, s uch a s C EA a nd
CA15.3, are bound to the cell membrane, but their extracellular domains could be found,
through s hedding, i n t he c irculatory s ystem. O n t he ba sis of t his h ypothesis, R oeßler e t a l.
aimed t o i dentify n ovel s erum b iomarkers o f co lorectal can cer [ 126]. F irst, 16 m atched
colorectal cancer and adjacent normal tissue samples were subjected to 2D-DIGE followed by
protein i dentification us ing MS. F ive pr oteins w hose e xpression i s i ncreased m arkedly i n
colorectal cancer were identified as transforming growth factor-β-induced protein ig-h3 (βIG-
H3), ni cotinamide N -methyltransferase ( NNMT), nuc leoside di phosphate ki nase A ( nm23-
H1), p urine nuc leoside pho sphorylase ( PNPH), a nd m annose-6-phosphate receptor bi nding
protein 1 (M6P1). Validation using ELISA showed that elevated levels of NNMT were found
in sera from patients with c olorectal cancer. Furthermore, the accuracy of NNMT diagnosis
(ROC AUC, 0.8 4) w as hi gher t han t hat of t he e stablished t umor marker C EA ( ROC AUC,
0.78). S imilarly, proteomic an alysis o f t umor t issues o ccasionally l eads t o d iscovering
serum/plasma bi omarkers. Using t he s ame s trategy, ph osphoglycerate ki nase 1 ( PGK1) ha s
reportedly been identified as a serum biomarker of pancreatic ductal adenocarcinoma [127].
High abundant proteins are possibly suitable to search for specific cancer biomarkers. As
shown in Table 4, high abundant proteins expressed differentially in various cancers appear
capable of be ing c ategorized i n t hree gr oups: 1) c omponents of c omplement s ystem a nd
regulated pr oteins ( C3, C 4, and f actor B etc.), 2) a polipoproteins ( apolipoproteins A1, A 2,
and C 1 etc.), 3) iron-metabolic proteins (transferrin, haptoglobin, and hemopexin etc.).
Furthermore, as seen in C3a [75, 78, 85] and fibrinopetide A [81, 82, 90], which are fragment
peptides derived from complement C3 and fibrinogen, respectively, protein cleavage products
also s erve a s c ancer bi omarkers. C hanges i n t he e xpressions a nd/or pr ocessing pa tterns of
highly abundant proteins in cancer may be caused by specific molecules in pathogens. We are
focusing on the relationships between the complement system and ATL, a fatal leukemia
caused by infection with human T lymphotropic virus type-1 (HTLV-1) [19].
The c omplement s ystem is a major mediator o f innate immune defense [128–130]. T he
system c onsists o f a group of over 30 proteins, such as C 3, C4, and factor B. When p athogens
(e.g., m alignant cells, vi ral-infected c ells, a nd ba cteria) exist in the living bod y, t he s ystem is
activated, w ith t he r esult t hat t hey a re c leared b y opsonization a nd/or l ysis. T he c omplement
system h as three d istinct p athways (classical, a lternative, a nd lectin pa thways) ( Figure 8 ).
Classical, alternative, and lectin pathways are activated by an antibody bound to pathogens, certain
non-self m aterials, and m annose-containing carbohydrates e xpressed o n the p athogen s urface,
respectively. Whichever pathway is activated, C3 is finally cleaved into C3a, C3b, iC3b, and C3f
etc. The increase of C3 cleavage products in cancer patients is a reflection of this activation.
We i dentified C 3f as t he s erum b iomarker o f A TL by d ifferential a nalysis using t he
ProteinChip array®–SELDI-TOF-MS system followed by MS/MS ion search using MALDI-
TOF-TOF-MS [19]. Further, analysis of markers specific to the activation pathways showed
that the serum concentration of the marker of the lectin pathway was significantly higher in
ATL pa tients. T hese r esults s uggest t hat t he c omplement s ystem is a ctivated t hrough t he
lectin pathway in ATL. Furthermore, this implies the presence of a trigger that activates the
lectin pa thway. A s one possibility, i t i s c onsidered t hat a berrant gl ycoproteins a nd
glycolipids, including mannose-containing c arbohydrates, a re e xpressed on t he s urface of
ATL cells. C3f is not specific to ATL, because its expression is increased in various cancers
[19]. The trigger is, however, possibly suitable for diagnosis as an ATL-specific biomarker.
In a ddition t o i dentifying proteins/peptides b y pr oteomic a nalysis of s erum/plasma,
clarifying a molecule associated with the signal transduction pathway such as the complement
system may be necessary to discovery specific cancer biomarkers.
5.4. From Biomarker Discovery to Clinical Application
The most i mportant issue in de veloping diagnosis methods is reproducibility in c linical

practice as well as in the laboratory. In p articular, in the early d iagnosis o f can cer, because
tumors a re microscopic, i nsensible c hanges i n t he l iving body ha ve t o be c aptured. I n
addition, pr evious s tudies, including our da ta, i ndicated th at m easurement s ystems u sing
multiple bi omarkers a re e ffective i n e arly di agnosis [ 18, 11 5–117]; t herefore, m easurement
systems are required that reproducibly quantify multiple biomarkers simultaneously using just
one experiment.
The P roteinChip array®–SELDI-TOF-MS s ystem e nables th e d etection o f multiple
proteins/peptides i n j ust on e e xperiment. D iagnosis c an be pe rformed by t he decision t ree
using m /z a nd pe ak i ntensities w ithout t he i dentification of pr oteins/peptides. W hile m any
studies have reported diagnostic methods using decision trees, irreproducibility is a problem
[131]. Although our e xperience a lso s hows t his, t he i rreproducibility c ould be a ttributed t o
analytical s teps f rom th e time th e s erum is c ollected u ntil th e s pectrum is a cquired.
Unfortunately, no uni versal standard protocol exists for the collection, storage, and transport
of serum samples. Also, there is no universal quality control of the decision tree and spectra
obtained with MS. In particular, the spectral patterns acquired by SELDI-TOF-MS seem to be
affected by the aging degradation of the ionization laser. Whether this is true, the cause of the
irreproducibility is uncertain. If peaks useful for diagnosis are found, the better choice is to
identify proteins/peptides derived from these peaks followed by the development of other
measurement systems with good reproducibility and quantitation.
Sample addition
(Patient serum)
Secondary antibody Substrate
◆
◎ △
(enzyme linked) addition
◆ ● □
Detecting Y Y ★ Color developing
Capture ◆ antibody ◇ ◇ ☆
◎ Y Y Y
antibody
Antigen
○ ○ ○ ○
Y Y Y Y Y
Figure 9. Sandwich ELISA. After the plate is coated with a capture antibody, the sample is added and
any antigen present binds to capture the antibody. Next, the detecting antibody is added and binds to the
antigen, and then the enzyme-linked secondary antibody is added and binds to the detecting antibody.
Finally, the substrate is added and is converted by the enzyme to the detectable form.
ELISA is a biochemical technique used mainly in immunology to detect the presence of

an a ntibody or a n a ntigen i n a s ample [ 132]. E LISA ha s be en w idely us ed a s a di agnostic
system in clinical practice; for example, sandwich ELISA, which has goo d s ensitivity,
quantitation and reproducibility, is performed as follows (Figure 9): 1) the plate is coated with
a capture antibody; 2) sample is added and any antigen present binds to capture the antibody;
3) t he de tecting a ntibody i s a dded a nd bi nds t o t he a ntigen; 4) e nzyme-linked s econdary
antibody is added and binds to the detecting antibody; 5) substrate is added and is converted
by the enzyme to the detectable form. Early diagnosis of ovarian cancer has reportedly been
performed us ing E LISA f or pr oteomic biomarkers ( transthyretin, he moglobin subunit β,
apolipoprotein A 1, a nd t ransferrin) [101]. A lso, a s a hi gh-throughput and multiplex
measurement s ystem, an tibody ar ray h as r ecently b ecome o f i nterest [ 133]. T o apply
proteomic biomarkers in a clinical setting, the development of measurement systems, such as
ELISA and antibody array, may be a key step.
6. CANCER MALDI IMAGING TECHNIQUE AND DIAGNOSIS

CONCLUSION
Proteomic analysis based on protein/peptide separation and identification using MS is an
essential technology for biomarker discovery. Although most proteomic biomarkers may be
not specific for a particular cancer, they are certainly of help in the early diagnosis of several
cancers. There i s c onsiderable di fficulty i n di scovering s pecific bi omarkers; how ever, a
variety of strategies for discovering specific biomarkers may make it possible. We hope that
proteomic cancer biomarkers will be widely used in the future for early diagnosis in clinical
practice.
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Biochemical and Biophysical Research Communications 334 (2005) 1004–1013
BBRC
www.elsevier.com/locate/ybbrc
Demonstration of an in vivo generated sub-picomolar affinity

fully human monoclonal antibody to interleukin-8
Palaniswami Rathanaswami a,*, Shelly Roalstad b, Lorin Roskos c, Qiaojuan Jane Su c,
Steve Lackie b, John Babcook a
a
Abgenix Biopharma Inc., Burnaby, BC, Canada
b
Sapidyne Instruments Inc., Boise, ID, USA
c
Abgenix Inc., Fremont, CA, USA
Received 22 June 2005

Available online 13 July 2005
Abstract
The high specificity and affinity of monoclonal antibodies make them attractive as therapeutic agents. In general, the affinities of
antibodies reported to be high affinity are in the high picomolar to low nanomolar range and have been affinity matured in vitro. It
has been proposed that there is an in vivo affinity ceiling at 100 pM and that B cells producing antibodies with affinities for antigen
above the estimated ceiling would have no selective advantage in antigen-induced affinity maturation during normal immune
responses. Using a transgenic mouse producing fully human antibodies, we have routinely generated antibodies with sub-nanomolar
affinities, have frequently rescued antibodies with less than 10 pM affinity, and now describe the existence of an in vivo generated
anti-hIL-8 antibody with a sub-picomolar equilibrium dissociation constant. This confirms the prediction that antibodies with affin-
ities beyond the proposed affinity ceiling can be generated in vivo. We also describe the technical challenges of determining such high
affinities. To further understand the importance of affinity for therapy, we have constructed a mathematical model to predict the
relationship between the affinity of an antibody and its in vivo potency using IL-8 as a model antigen.
2005 Elsevier Inc. All rights reserved.
Keywords: Human antibodies; B cells; Affinity ceiling; High affinity; Sub-picomolar affinity; Affinity maturation; KinExA; SLAM; Interleukin-8 (IL-
8); XenoMouse
Ever since the development of hybridoma technology as human therapeutics was limited by their immunoge-
a quarter century ago, there has been a promise for nicity; patients treated with murine antibodies rapidly
monoclonal antibodies (mAbs) to be used as human mounted an immune response to these foreign proteins,
therapeutic agents. Eighteen therapeutic mAbs are a response known as a HAMA, for human anti-mouse
now on the market in the United States, with 16 of these antibody [2]. To avoid the HAMA response, we have
having been approved in the last decade, and over 100 developed XenoMouse strains that functionally recapit-
mAbs are currently in clinical development [1]. ulate the human antibody response, including a vast rep-
For the most part, the recent success of mAbs can be ertoire of high affinity, somatically hypermutated
attributed to advances in antibody generation technolo- human antibodies [3,4].
gies. The first products of hybridoma technology were In addition to immunogenicity, the kinetic properties
murine antibodies. The usefulness of these antibodies of the antibody often dictate their utility. A higher affin-
ity antibody will either be able to bind its ligand faster
* (determined by the association rate constant, kon),
Corresponding author. Fax: +1 604 676 8349.
E-mail address: swami.rathanaswami@abgenix.com (P. Rathana- remain bound longer (determined by the dissociation
swami). rate constant, koff) or possess both properties. Depend-
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2005.07.002
P. Rathanaswami et al. / Biochemical and Biophysical Research Communications 334 (2005) 1004–1013 1005
ing on the tissue distribution, expressed forms (mem- cent dye based on indodicarbocyanine) affinity-purified goat anti-
brane-bound or soluble), concentration, and biological human IgG (Fcc fragment-specific), goat anti-human IgG (H + L-
specific) and mouse anti-human IgG [F(ab 0 )2 fragment-specific] Abs
activity of a target, there can be a direct correlation be- (all three have minimal cross-reactivity to bovine, horse and mouse
tween the affinity and potency of an antibody [5]. The serum proteins) were purchased from Jackson ImmunoResearch
administration of an antibody of higher affinity and Laboratories (West Grove, PA). The recombinant human interleukin-8
potency may also translate into better in vivo efficacy (rhIL-8) (72 amino acid form) (CXCL8) (Cat. No. 200-08M) was
[6,7]. Antibodies of higher affinity may be able to be obtained from PeproTech, Canada (Ottawa, ON). Titermax gold
adjuvant was obtained from Sigma (Oakville, ON).
used at lower doses to achieve the desired clinical effects. Generation of fully human anti-human interleukin-8 monoclonal
Lower dosing may allow for more convenient routes of antibodies. XenoMouse strains were produced as described previously
administration and decreased injection volumes, which [4]. Cohorts of XenoMouse mice were immunized with rhIL-8. The
would translate into lower cost of goods. initial BIP (base of the tail by s.c. injection and i.p.) immunization was
Antibodies reported to be of high affinity are general- with 50 lg of rhIL-8, mixed 1:1 v/v with complete FreundÕs adjuvant
(CFA), per mouse. Subsequent boosts were made with 50 lg of rhIL-8,
ly in the nanomolar range [8,9] and occasionally in the mixed 1:1 v/v with incomplete FreundÕs adjuvant (IFA), per mouse.
sub-nanomolar range [10]. Extensive in vitro modifica- The animals were immunized on days 0, 14, 28, and 42. Then
tions have been employed to increase the affinity of immunizations were continued for some mice with 50 lg of rhIL-8 in
the antibodies to the picomolar range [7,11–13]. The Titermax gold adjuvant i.p. (on days 146, 160, and 181) and then with
use of XenoMouse mice, in conjunction with well-estab- 10 lg of rhIL-8 in PBS i.p. on day 205. A final boost was done with
10 lg of rhIL-8 in PBS i.p. on day 226 (for two mice) and on day 234
lished antibody generation procedures, reproducibly re- (for two mice). Spleen and lymph nodes from XenoMouse animals
sults in high affinity fully human mAbs suitable for were harvested 4 days after the last boost. The application of SLAM
repeated administration to humans [14,15]. technology [24,25] resulted in the rescue of multiple high affinity, fully
There are a number of technologies available to mea- human anti-hIL-8 mAbs from the hyper-immunized XenoMouse ani-
sure the affinity of an antigen–antibody interaction in mals. Briefly, B cells from the animals were harvested and cultured in
plates [25]. Wells were screened by ELISA to identify B cells producing
the nanomolar range [16–20]. However, to measure the IL-8-specific antibodies. 1063 wells were identified as being positive for
affinity of an interaction in the low picomolar range, a IL-8 binding. The single B cells secreting anti-IL-8 antibodies were
number of parameters may need to be modified [21]. It identified using an antigen-specific hemolytic plaque assay, picked by
was recently shown that for two of these technologies, micromanipulation, the heavy and light chain variable region cDNAs
Biacore and KinExA, similar kinetic rate constants and were amplified by RT-PCR and molecularly cloned into expression
vectors as previously described [25]. The expressed recombinant anti-
affinities could be measured for a number of antibodies bodies were purified using protein A–Sepharose affinity chromatog-
over a range of high affinity interactions [15]. Here, we de- raphy and analyzed by non-reducing SDS–PAGE to assess purity and
scribe the affinity characterization of very high affinity yield. Concentration was also confirmed by UV analysis at A280 and
(low picomolar to sub-picomolar) fully human antibodies ELISA.
generated from XenoMouse animals and the modifica- Preparation of antigen coated beads. Fifty micrograms of rhIL-8
was coupled to CNBr-activated Sepharose 4B. Alternatively, 25 lg/ml
tions required to measure these high affinities with preci- or 100 lg/ml of rhIL-8 was coupled to NHS-activated Sepharose. The
sion using KinExA. Experiments were performed in two remaining active groups on the beads were blocked as recommended
separate laboratories: Abgenix Biopharma, Burnaby, by the manufacturer. The beads were then finally blocked with 10 mg/
Canada and Sapidyne Instruments, Boise, USA. ml BSA in 1 M Tris and stored in the blocking solution.
Although it has been hypothesized that there could be KinExA equilibrium assays. The experiments with KinExA were
performed using an automated flow immunoassay system, KinExA
an intrinsic affinity ceiling (100 pM) for the selection of 3000 [26], in which rhIL-8-coupled beads served as the solid phase.
antibodies generated in vivo [22,23], this report supports Briefly, a constant amount of antibody between 0.67 and 5000 pM
the existence of higher affinity antibodies derived from binding site concentration (two binding sites for an IgG) was incubated
in vivo somatic hypermutation and affinity maturation, with titrating concentrations of rhIL-8 antigen starting at 100 nM in
with affinities in the sub-picomolar range. This implies PBS with 0.1% BSA (sample buffer) to reduce non-specific binding.
Antigen–antibody complexes were incubated at RT for 36–144 h to
that beyond the selection-driven affinity ceiling, higher allow equilibrium to be reached. The mixture was drawn through the
affinities can still occur by chance and are not selected rhIL-8-coupled beads to accumulate unbound antibody. The captured
against. Alternatively, the assumptions underlying the anti-hIL-8 mAb is directly proportional to the remaining free binding
limits of selection-driven affinity may need to be sites [26], and was detected using solutions containing Cy5-conjugated
re-visited. anti-human secondary antibody in sample buffer. The concentrations,
volumes, and flow rates of the secondary antibody solutions were
varied to optimize the signal to noise ratio in each experiment. The
schematics of the experiment and the measurement of free mAb have
Materials and methods been discussed previously [20,26]. The bound signals were converted
into relative values as a proportion of control in the absence of rhIL-8.
Instrumentation and materials. Solution based kinetic exclusion Three replicates of each sample (in some experiments two replicates)
assays were performed using the KinExA 3000 instrument (Sapidyne were measured for all equilibrium experiments. The equilibrium dis-
Instruments, Boise, ID). NHS-activated Sepharose 4 fast flow, CNBr- sociation constant (Kd) was obtained from non-linear regression
activated Sepharose 4B and protein A–Sepharose were obtained from analysis of the data using a one-site homogeneous binding model
Amersham Pharmacia (Amersham Biosciences, Piscataway, NJ). BSA contained within the software [20,27]. The software calculates the Kd
was obtained from Sigma (St. Louis, MO). Cy5-conjugated (fluores- and determines the 95% confidence interval by fitting the data points to
1006 P. Rathanaswami et al. / Biochemical and Biophysical Research Communications 334 (2005) 1004–1013
a theoretical Kd curve. The 95% confidence interval is given as Kd low The zero-order production rate of IL-8, S0, was varied to achieve
and Kd high. different initial conditions for baseline levels of unbound IL-8. In the
KinExA kinetic assays. For measuring the association rate con- simulations, parameter values for CLab, CLag, and V were fixed at
stant using KinExA, the same rhIL-8-coupled beads were used as the 0.0025, 0.493, and 0.064 L/kg, respectively. The clearance estimates
probe and the ‘‘Kinetics, Direct’’ method was used. The ‘‘Kinetics, were based on unpublished data, and the volume of distribution as-
Direct’’ experiments are identical to KinExA equilibrium assays with sumed limited distribution of mAb and IL-8 into extra vascular space.
respect to bead column height, antibody capture, antibody concen- Parameter sensitivity analysis indicated that changes in assumptions
tration, and antibody detection. However, the mAb was mixed with regarding clearance and distribution of mAb and IL8 did not influence
an amount of antigen that bound approximately 80% of the mAb in the estimate of optimum mAb affinity; however, the dose required to
the equilibrium experiments and the free antibody present in the achieve 90% suppression of unbound IL-8 was influenced by these
sample was probed repeatedly, pre-equilibrium. Since the binding assumptions. The association rate constant, kon, was fixed and changes
signals are proportional to the concentration of free antibody in the in affinity were assumed to be related to changes in the dissociation
solution, the signals decrease over time until the solution has equil- rate constant, koff. For any given affinity, changes in the values of koff
ibrated. The volumes and flow rates of the antigen–mAb mixtures and kon did not affect the simulated steady-state concentration of un-
and the Cy5-labeled secondary antibody were varied depending upon bound IL-8.
the mAb tested. Data were analyzed utilizing the KinExA analysis
software that comes with the KinExA 3000 instrument. This software
graphically represents the decrease in binding signals over time, and
fits the collected data points to an exact solution of the kinetic dif-
Results
ferential equations for binding. From this curve, an optimal solution
for the kon is determined. The koff is indirectly calculated from Affinity measurements of Anti-hIL-8 mAbs by KinExA
solutions for the kon and Kd.
Theoretical effect of affinity on potency. A mathematical model The affinity measurements of anti-hIL-8 mAbs from
was developed to simulate the effect of affinity on the dose of mAb
required to suppress the serum IL-8 levels in vivo by at least 90% at
XenoMouse determined by KinExA are shown in
steady-state following a multiple dose regimen. The model consisted Table 1. Generally, when the measured Kd of an anti-
of four differential equations describing the synthesis and elimination body was in the range of high picomolar or above, the
of endogenous IL-8, the dosing and elimination of mAb, and the 95% confidence interval of the predicted Kd was within
monovalent and divalent binding of mAb to the IL-8. The half-life of a narrow range. However, when the measured Kd was
the mAb was assumed to be 3 weeks, and dosing was conducted
every 3 weeks for 15 weeks. The simulation was conducted at 3, 30,
approaching low picomolar to sub-picomolar values,
300 pM, and 3 nM baseline levels of IL-8 to reflect concentrations the 95% confidence interval tended to be broader, indi-
that may be present in serum and at active sites of inflammation, cating the measured Kd was not as precise. For the
respectively [28–30]. Simulations were conducted using SAAM II v. low to sub-picomolar Kd measurements, the binding site
1.2 (SAAM Institute, Seattle, WA). Numerical integration was con- concentration of mAb used in the equilibrium mixture
ducted using the Rosenbrock method with an adjustable step size.
The time course in serum of unbound IL-8 (IL8u), unbound mAb
was higher than the Kd, causing the precision of these
(ABu), mAb monovalently bound to IL8 (ABmv), and mAb biva-
lently bound to IL8 (ABbv) was described by the following
equations:
dIL8u Table 1
¼ S 0 þ fluxunbindmv þ fluxunbindbv fluxbindmv
dt Kd measurements of anti-hIL-8 mAbs by KinExA using standard
CLag conditions
fluxbindbv IL8u;
V Anti-hIL-8 mAb Kd (pM) Kd range (pM) mAb conc. (pM)
dABu CLab 33 280 150–420 10
¼ fluxunbindmv fluxbindmv ABu; 142 400 190–680 25
dt V
203 190 64–340 25
dABmv 215 360 230–450 50
¼ fluxbindmv þ fluxunbindbv fluxunbindmv fluxbindbv
dt 469 870 640–1010 200
CLab 809 2.2 0.36–4.8 23
ABmv;
V 837 11 0.054–31 90
861 2.9 0.010–8.2 25
dABbv CLab 928 0.057 <0.010–1.8 20
¼ fluxbindbv fluxunbindbv ABbv; 1064 54 29–72 50
dt V
1080 630 160–980 25
where: 1093 200 76–300 100

ABuIL8u Serial dilutions of rhIL-8 were mixed with the respective binding site
fluxbindmv ¼ 2k on ;
V concentration of anti-hIL-8 mAbs and allowed to reach equilibrium
for 36 h. A proportionate amount of free mAb present in the equi-
ABmvIL8u librium mixture was measured by flowing 0.25–3 ml of the sample on
fluxbindbv ¼ k on ;
V rhIL-8 coupled to CNBr-activated Sepharose beads and detected using
1.0 ml of 1.7 lg/ml of Cy5-labeled goat anti-human IgG Fcc fragment-
fluxunbindmv ¼ k off ABmv; specific secondary antibody. Each sample was run in triplicate. The Kd
was calculated by the KinExA software and the 95% confidence
fluxunbindbv ¼ 2k off ABbv. interval is given as the Kd range.
measurements to be less accurate, thus warranting anti-hIL-8 mAb 928 was tested either flowing 0.5 ml at
KinExA signal optimization experiments. 0.25 ml/min or 3.0 ml at 1.5 ml/min for 120 s, it was deter-
mined that increasing the sample volume and flow rate
Signal optimization gave about 20% higher signal (data not shown).
The binding site concentration of the anti-hIL-8 mAb
In order to make an accurate Kd measurement by 928 was then lowered to 2 pM and a signal test was per-
KinExA, the active binding site concentration of the formed by flowing 30 ml at 1.5 ml/min for 1200 s. How-
antibody must be near or below the actual Kd. To mea- ever, when the sample flow volume and the rate were
sure antibodies with very high affinities, as described in raised, bubbles were forming in the bead pack, which
this paper, one needs to work at low picomolar binding affected the raw data traces. This problem was solved
site concentrations of antibody. At these low antibody by degassing the samples and label immediately before
concentrations the sensitivity of the assay needs to be running an experiment.
further optimized. To increase the detection sensitivity
of free antibody, bead coating concentrations and sam- Time to reach equilibrium and sample viability
pling parameters were optimized and different labels
were evaluated. The antibody binding site concentration was subse-
quently lowered to 1 pM, to measure the Kd of low to
Optimization of bead coating and labeled secondary sub-picomolar mAbs. At this very low binding site con-
antibody centration of antibody, the samples require a longer time
to reach equilibrium. Using the Kd curve obtained at a
Immobilization of certain antigens to beads may af- low mAb concentration and the measured kon, it was
fect their ability to bind antibodies, due to steric hin- estimated in the kinetic theory curve software from
drance by passive coating or destruction of epitopes by Sapidyne Instruments that the minimum time needed
side chain reactive coupling. To test whether the signal to reach equilibrium would be 6 days (data not shown).
in the bead pack could be increased, NHS-activated Otherwise, one could experimentally determine whether
Sepharose beads were coupled with rhIL-8 and the bead the antigen–antibody mixture reached equilibrium by
saturation was tested, as previously described [26]. It measuring the free mAb left in solution, which should
was determined that there was no signal gain above a remain constant during triplicate measurements, if
coupling concentration of 100 lg/ml of antigen. At equilibrium has been achieved. The viability of 1 pM
25 lg/ml of antigen, approximately 70% of the maxi- anti-hIL-8 mAb 928 was then checked at 0, 3, and 6
mum signal was observed. Subsequently, the NHS– day incubation times and found that the antibody
Sepharose beads were coupled with 100 lg/ml of activity had not decreased during this period at room
rhIL-8 for the low antibody binding site concentration temperature. Subsequently, all equilibrium reactions
(Kd controlled) experiments. The Kd measurements with were allowed to take place for 6 days.
high binding site concentration of mAb and on-rate
experiments were performed with 25 lg/ml of rhIL-8 Equilibrium and on-rate measurements of mAbs having
to conserve antigen. low picomolar affinity
Different Cy5-labeled anti-human secondary antibod-
ies were checked to identify one that would give the The Kd measurements were then repeated, for the
highest signal-to-noise ratio. When a 100 pM solution mAbs (809, 837, 861, and 928) which had Kds below
of anti-hIL-8 mAb was tested with various secondary la- 20 pM and broad 95% confidence intervals (Table 1),
bels (goat anti-human IgG (H + L)-specific, Fcc frag- with optimized parameters for bead coupling, sample
ment-specific or mouse anti-human IgG F(ab 0 )2 volume, flow rate, and secondary antibody-label. In
fragment-specific), the highest signal to noise ratio was KinExA, the percent of free mAb left in solution is plot-
generated using the Fcc-specific label (9.9 times higher ted against each concentration of antigen, generating a
than background). This Fcc fragment-specific goat sigmoidal curve, as shown in Fig 1. A theoretical curve
anti-human secondary antibody was used for subse- is fit to the data and 95% confidence intervals are calcu-
quent equilibrium and on-rate measurements. lated by the KinExA software. It is known that when the
binding site concentration of mAb is very close to, or be-
Optimization of sample volume and flow rate low the Kd value, the shape of the curve is constant and
is referred to as a Kd controlled curve (Fig. 1, curve 1).
Since the initial equilibrium analysis of a few anti-hIL- However, for mAb binding site concentrations above
8 mAbs showed low picomolar to sub-picomolar Kds with the Kd, the curve shifts towards the right and becomes
broader 95% confidence intervals (Table 1; mAb 809, 837, steeper (Fig. 1, curve 2). These steeper curves are re-
861, 928), the sample volume and flow rate were then opti- ferred to as mAb concentration controlled curves, and
mized to obtain a higher signal. When a 100 pM of will contain little information on the Kd. One rationale
120 Table 2
Kd measurements of the highest affinity anti-hIL-8 mAbs by KinExA
100
using optimized conditions
80 Anti-hIL-8 mAb Kd (pM) Kd range (pM) mAb conc. (pM)
% Free mAb
60 809 3.3 1.9–5.2 4.6, 27

Curve 1 Curve 2 837 16 9.3–25 18, 120
40 861 3.0 2.0–4.2 1.3, 13
928 0.61 0.38–0.94 0.68, 2.0, 14
20
Serial dilutions of rhIL-8 were mixed with the respective anti-hIL-8
0 mAbs in the binding site concentrations shown above and allowed to
1.00E-15 1.00E-12 1.00E-09
reach equilibrium for 36 h for the high binding site concentration of
Concentration of Antigen (M)
each mAb, or for 144 h for the low binding site concentration of each
mAb. A proportionate amount of free mAb present in the equilibrium
Fig. 1. Kd measurement of mAb 928 by KinExA. Twofold dilutions
mixture was measured in KinExA under optimized conditions. For
(50 pM to 24 fM) of rhIL-8 were mixed with 1 pM binding site
example, for the mixtures containing the low binding site concentra-
concentration of anti-hIL-8 mAb 928 and allowed to reach equilibrium
tion of mAb, 18–36 ml of the sample was flowed through rhIL-8
for 144 h. A proportionate amount of free mAb present in the
coupled to NHS-activated Sepharose beads and detected using 1–2 ml
equilibrium mixture was measured by flowing 22.5 ml of the sample at
of 2.0 lg/ml of Cy5-labeled goat anti-human IgG Fcc fragment-spe-
0.25 ml/min on rhIL-8 coupled to NHS-activated Sepharose beads.
cific secondary antibody. The Kd was calculated using KinExA soft-
The bead bound mAb was detected by flowing 2.0 ml of 2.0 lg/ml of
ware by n-curve analysis and the 95% confidence interval is given as the
Cy5-labeled goat anti-human IgG Fcc fragment-specific secondary
Kd range. Six experiments were performed for mAb 928 and two were
antibody at 0.25 ml/min. Each sample was run in duplicate. The Kd
performed for mAbs 809, 837, and 861.
calculated by the KinExA software by single curve analysis for a
representative experiment (curve 1) was 870 fM and the 95% confi-
dence interval calculated as the Kd high and Kd low was 1.3 pM and
500 fM, respectively. A representative mAb controlled experiment determined to be 610 fM (Kd high = 940 fM and Kd
(curve 2) was performed using 20 pM binding site concentration of low = 380 fM), which is very close to the Kd determined
anti-hIL-8 mAb 928, mixed with two fold dilutions (200 pM–97 fM) of
rhIL-8 and allowed to reach equilibrium for 48 h. A proportionate
by low mAb binding site concentration experiments ana-
amount of free mAb present in the equilibrium mixture was measured lyzed in single curves (590 ± 220 fM). The Kd distribu-
by flowing 5 ml of the sample at 0.5 ml/min on rhIL-8 coupled to tions of individual measurements of mAb 928 are
NHS-activated Sepharose beads. The bead bound mAb was detected given in Fig. 2.
by flowing 1.0 ml of 2.0 lg/ml of Cy5-labeled goat anti-human IgG The on-rate measurement by KinExA is not limited
Fcc fragment-specific secondary antibody at 0.25 ml/min. Each sample
was run in triplicate. The Kd was calculated to be 790 fM by using
by the mAb binding site concentration used in the exper-
n-curve analysis, by the KinExA software and the 95% confidence iment. Therefore, the measurements of kon for the very
interval was calculated to be between 1.1 pM (Kd high) and 570 fM (Kd high affinity antibodies were straightforward and did
low). Analysis of curve 2 also determined the active binding site
concentration of antibody present in each reaction to be equal to 67%.
Kd Distribution for mAb 928

for performing a mAb concentration controlled experi-
ment is that the KinExA software will be able to calcu- 1600
late precisely the active binding site concentration of 1400
mAb present in solution during the equilibrium 1200
reaction. 1000
Kd (fM)
Additionally, one can further increase the accuracy of 800

very high affinity measurements by performing one or 600
more experiments using a mAb binding site concentra- 400

tion near or slightly higher than its Kd value and at least 200
one or more experiments with a 10- to 100-fold higher 0
binding site concentration of mAb than the first experi- 0 1 2 3 4 5
Experiment no.
6 7 8 9
ment [15]. All these experiments can then be analyzed

simultaneously with the KinExA software by using the Fig. 2. Distribution of Kd measurements for the sub-picomolar affinity
anti-hIL-8 mAb 928 by KinExA. Two fold dilutions of rhIL-8 (50 pM–
‘‘n-curve analysis’’ selection. This ‘‘n-curve analysis’’ 24 fM) were mixed with anti-hIL-8 mAb 928 at an active binding site
selection allows one to obtain a precise Kd value (Table concentration of either 2 pM (Experiment No.1) or 670 fM (Experi-
2) by fitting all of the given curves to a single Kd value ments 2–4). Experiments 5–8 represent n-curve analysis, done either
simultaneously. We performed a number of experiments with 2 pM and 13.5 pM mAb (Experiment No. 5) or with 670 fM and
for n-curve analysis of mAb 928. In these experiments, 13.5 pM mAb (Experiments 6–8). The diamonds represent the optimal
Kd determined by KinExA data analysis for each experiment. The
we used mAb 928 either near the Kd concentration or error bars represent the 95% confidence intervals from KinExA data
at a concentration of about 15- to 20-fold higher than analysis. The error bars are not centered on the optimal Kd value
the Kd. By n-curve analysis, the Kd of mAb 928 was because of the non-linear fit of the theoretical curve to the equilibrium.
Table 3 are not predicted to yield a further improvement in

On-rate measurements for the highest affinity anti-hIL-8 mAbs in potency. For example, the mean concentration of IL-8
KinExA
in exudates from psoriatic plaques was reported to be
Anti-hIL-8 mAb kon (M1 s1) kon range (M1 s1) koff (s1) 250 pM [30]. Improvement of mAb affinity to approxi-
809 4.8E+6 4.5E+6–5.5E+6 1.6E5 mately 25 pM is predicted to decrease the dose required
837 1.1E+6 1.1E+6–1.2E+6 1.9E5 to suppress IL-8 concentrations in the psoriatic plaque;
861 4.4E+6 4.0E+6–5.0E+6 1.3E5
928 6.0E+6 5.6E+6–6.4E+6 3.7E6
a further increase in affinity is not predicted to improve
potency.
The binding site concentration of mAb used for on-rate experiments
was the same as those used in equilibrium binding experiments. An
amount of IL8 that bound 80% of the mAb in equilibrium binding Mutational analysis
experiments was mixed with the mAb. The amount of free mAb left in
the mixture was measured in KinExA by flowing 1 ml of the mixture The role of somatic mutation in optimizing hIL-8
repeatedly through rhIL-8 coupled to NHS-activated Sepharose beads binding affinity was analyzed for mAbs 928, 809, 215,
and detected using 1 ml of 2.0 lg/ml of Cy5-labeled goat anti-human
and 1064. These mAbs were derived from the same
IgG Fcc fragment-specific secondary antibody. The kon was measured
using the KinExA software by the kinetic direct method and the 95% germline sequence and thus the backbones should be
confidence intervals are given as kon range. The koff for the mAb was essentially identical. Table 4 lists the mutations located
calculated from the measured kon and Kd of the respective mAb. within the complementary determining regions (CDRs)
and framework regions (FRs) of each of these antibod-
ies in comparison with the germline residues. The resi-
not require further optimizations. The measured kon and dues are numbered according to the scheme of Kabat
the calculated koff are given in Table 3. et al. [31]. Mutational analysis indicates that these four
mAbs were derived from an ancestral clone and a
Simulated effect of affinity on potency ValH50 fi Asp mutation occurred within mAbs 215,
809, and 928. The light chain of the mAb 215 indepen-
The theoretical dose required to suppress IL-8 in vivo dently diverged from the precursor ValH50 fi Asp clone
by at least 90% at steady-state is shown in Fig. 3 as a and generated a SerL27A fi Asn mutation resulting in a
function of affinity for four baseline levels of endoge- high affinity Kd of 362 pM. Then, mAbs 809 and 928 di-
nous IL-8 in serum. Improvements in mAb affinity are verged from the ancestral ValH50 fi Asp clone with the
predicted to provide proportional improvements in addition of a HisH35 fi Leu mutation. Antibody 809
potency until the Kd falls to 1/10th the endogenous independently further diverged at TyrH79 fi Phe in
IL-8 level. When the antigen level is at least 10 times FR3 finally resulting in a higher affinity Kd of
greater than the Kd, the dose of mAb required to sup- 2.23 pM. The heavy chain of mAb 928 independently
press IL-8 by 90% is determined by antigen level and continued to diverge in FR3 at TyrH90 fi Phe. The light
not by affinity; thus, further improvements in affinity chain of the mAb 928 further mutated in the CDRs from
100
Table 4
Somatic mutations in the CDR and FR regions of mAb 928, 809, 215,
10
and 1064
Kabat Germline mAb mAb mAb mAb
Dose (mg/kg/3weeks)
number 928 809 215 1064

1
CDR regions
H50 Val Asp Asp Asp Leu
0.1 H35 His Leu Leu
L92 Gly Asp
3 pM Baseline IL-8 L32 Tyr Phe
0.01 30 pM Baseline IL-8 L27A Ser Asn
300 pM Baseline IL-8
3 nM Baseline IL-8
L91 Tyr Asp
H96 Arg His
0.001
H31 Ser Thr
0.1 1 10 100 1000
L52 Ser Tyr
Affinity (pM) L53 Ser Arg
Fig. 3. Theoretical effect of mAb affinity on potency. The dose (mg/
FR regions
kg/3 weeks) of mAb required to suppress IL-8 levels in vivo by at least
L39 Lys Arg
90% at steady-state was simulated as a function of mAb affinity (Kd) at
L72 Thr Ile
four baseline levels of endogenous IL-8 (3 pM, circles; 30 pM,
H79 Tyr Phe
triangles; 300 pM, squares; 3 nM, diamonds). When the Kd of the
H90 Tyr Phe
mAb is less than 1/10th the baseline IL-8 levels, further improvements
in affinity will not provide any improvements in potency. Residues are numbered according to the scheme of Kabat et al. [31].
GlyL92 fi Asp and TyrL32 fi Phe, as well as in FR3 The challenge in KinExA for sub-picomolar affinity
ThrL72 fi Ile, resulting in a final product with a superior measurements is to precisely measure the proportionate
affinity in the sub-picomolar range (Kd = 610 fM). How- amount of very low concentrations of free mAb left in
ever, mAb 1064 appears to have evolved independently solution after equilibrium is reached. To achieve this,
from a germline ancestral clone and resulted in muta- we modified a number of parameters. As shown for
tions in the CDRs at ValH50 fi Leu, SerH31 fi Thr, anti-hIL-8 mAb 928, by coupling the NHS-activated Se-
ArgH96 fi His, SerL52 fi Tyr, SerL53 fi Arg, and pharose beads with a saturating concentration of IL-8,
TyrL91 fi Asp, along with a mutation in FR2 at Ly- increasing the sample flow volume, and using the opti-
sL39 fi Arg, which ultimately resulted in a high affinity mized Cy5-labeled detection antibody, we were able to
Kd of 54 pM. increase significantly the signal-to-noise ratio. These
modifications together allowed us to measure the pro-
portionate amount of free antibody left in solution to
Discussion a very low concentration range. In addition, we incubat-
ed the antibody–antigen mixture for a sufficient period
There are a number of reports describing monoclonal of time to allow the reactants to reach equilibrium. By
antibodies with sub-nanomolar affinities [10,11,15]. applying these modified parameters, we performed a
However, in vitro affinity maturation by phage, yeast number of Kd controlled experiments, which were also
or ribosome display is often required to increase the used in n-curve analysis along with mAb controlled
affinity of an antibody to the picomolar range experiments and measured the average Kd of mAb 928
[11,13,32–34]. Here, we describe the affinity character- to be in the sub-picomolar range. Using n-curve analy-
ization of fully human mAbs generated in vivo from sis, one may even be able to extend the system to deter-
XenoMouse that were directly rescued using the selected mine equilibrium dissociation constants in the low
lymphocyte antibody method (SLAM) [24] and were not femtomolar range.
subjected to further in vitro affinity maturation. To our knowledge there has only been one publica-
Rigorous affinity analyses of these antibodies were tion describing a sub-picomolar affinity antibody [34].
performed at two separate laboratories utilizing This murine-derived, in vitro mutated and yeast display
KinExA technology. affinity matured antibody targets the hapten fluorescein.
A number of technologies, such as surface plasmon However, we have not seen any reports describing a sub-
resonance (SPR) (Biacore) [16], resonant mirror (IAsys) picomolar affinity antibody targeting a protein antigen,
[19], solution based kinetic exclusion assay (KinExA) generated in vivo from any species. The reason for this
[20], isothermal titration calorimetry [18], and fluores- might be due to either the inherent difficulties in rescuing
cence-polarization [17] are used to measure equilibrium very high affinity mAbs by available antibody genera-
constants. Commonly used Biacore technology utilizes tion technologies or in measuring high affinity interac-
surface-based biophysical methods to measure the kon tions with existing technologies. We have shown here
and koff, from which one can calculate the Kd. Whereas, that if the technologies that measure the affinity, such
KinExA utilizes solution-based biophysical methods to as solution-based KinExA, are used properly, high affin-
directly measure the Kd and kon. The koff can then be cal- ity measurements between a protein antigen and an anti-
culated using the measured Kd and kon. Both Biacore body can be measured with precision. The very high
and KinExA have been successfully used to measure affinity determined for mAb 928 was confirmed to be
affinities in the picomolar range [15,21,26,35]. Recently, sub-picomolar by two independent laboratories using
it has been shown that for some antigen–antibody affin- KinExA.
ity measurements, KinExA and Biacore correlate closely Additionally, we have demonstrated for the first time
[15]. However, Biacore requires proper experimental de- that fully human mAbs that exhibit an equilibrium dis-
sign to avoid avidity, mass transport, steric hindrance, sociation constant in the sub-picomolar range can be
aggregation, and crowding effects, as reviewed extensive- generated in vivo. In addition, we are now regularly iso-
ly before [21,36]. Here we have used KinExA technolo- lating low picomolar (less than 10 pM) fully human anti-
gy, where both binding constituents are present in bodies from XenoMouse [15]. Using SLAM technology,
solution without being modified, to precisely measure we have also generated a number of low picomolar affin-
low picomolar affinities. Since the concentration of ity antibodies directly from human peripheral blood B
IL-8 used in our kinetic equilibrium measurements were cells targeting a variety of antigens from human patho-
100 nM or below and IL-8 exists as a monomer at this gens. We have also generated many low picomolar affin-
concentration [37,38], we were able to measure the true ity antibodies, as well as a sub-picomolar affinity mAb
affinity, rather than the avidity, of the anti-hIL-8 mAbs. from rabbit peripheral blood B cells, using SLAM tech-
We also describe modifications to standard equilibrium nology (unpublished results). These observations indi-
measurement techniques for KinExA that allowed the cate that the generation of exceptionally high affinity
precise measurement of sub-picomolar affinities. antibodies occurs across species in vivo.
It was proposed by Foote and Eisen [22] that, under line sequence could have existed in multiple preexisting
physiological conditions, there is a ceiling at 100 pM on conformational isomers [47,48] and that somatic muta-
antibody affinity maturation in vivo for any antigen. It tions in the antibodies resulted in the stabilization of
was proposed that above this threshold there would be the high affinity binding isomers.
no selective in vivo advantage for somatic mutation Antibodies with affinities in the picomolar or femtom-
and affinity maturation to happen. This argument was olar range may provide significant improvements in mAb
based on the limits on both on- and off-rate constants potency, depending on the biological characteristics of
of an antibody. The on-rate constant of an antibody is the antigen, particularly the in vivo concentration
controlled by the diffusion coefficients of the antigen (Fig. 3). By using SLAM, a more efficient antibody gener-
and antibody [39,40] and the maximum on-rate constant ation procedure, to screen a larger proportion of the avail-
proposed was approximately 105–106 M1 s1. The pro- able immune repertoire in hyperimmune animals, it was
posed limit on the off-rate constant is based on the res- possible to identify a number of exceptionally high affinity
idence time of an antigen complexed to an antibody on a antibodies including one of sub-picomolar affinity with-
B cell surface before it is taken up and processed by the out the need for time consuming in vitro manipulations.
B cell [23,41]. These studies led to the conclusion that B We have determined the affinities of a panel of fully
cells cannot differentiate between antibodies that have human anti-hIL-8 antibodies generated in vivo from
off-rate constants less than 104 s1. However, no study XenoMouse animals to be in the low picomolar range.
describes a disadvantage for in vivo affinity maturation Furthermore, one of these antibodies was found to be a
beyond these affinity ceilings. In fact, Foote and Eisen sub-picomolar affinity antibody. As far as we are aware,
[22] themselves suggested that affinities beyond the ceil- this is the first such description of an in vivo generated
ing could happen randomly. We have generated anti- sub-picomolar affinity antibody. This report not only
bodies with on-rates greater than 106 M1 s1 [15,42], confirms the prediction that there can be affinity matura-
with off-rates less than 104 s1 [15] and the present tion in vivo beyond the proposed affinity ceiling [22], but
study extends those observations. Others have also in fact provides precise affinity measurements of an anti-
reported on-rates higher than 106 M1 s1 for antibod- body that does so by over two orders of magnitude.
ies [43] and for Fab fragments [44]. Increased on-rates
beyond the proposed diffusion limit may be explained
by additional electrostatic forces [45]. For the associa- Acknowledgments
tion of barnase with barstar, a basal association rate
constant of 105 M1 s1 was increased to over The authors thank Mike Gallo, Ian Foltz, and Geoff
5 · 109 M1 s1 by electrostatic forces [45]. For off- Davis for critical reading of the manuscript. We thank
rates, it is possible that B cell receptor turn-over in vivo Karen Richmond for her technical assistance.
may be slower or more heterogeneous than that mea-
sured in vitro using cell lines [23]. In this case, B cells
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Available online at www.sciencedirect.com
Picomolar Affinity Fibronectin Domains Engineered

Utilizing Loop Length Diversity, Recursive Mutagenesis,
and Loop Shuffling
Benjamin J. Hackel 1 , Atul Kapila 2 and K. Dane Wittrup 1,3 ⁎
1
Department of Chemical The 10th type III domain of human fibronectin (Fn3) has been validated as an
Engineering, Massachusetts effective scaffold for molecular recognition. In the current work, it was
Institute of Technology, desired to improve the robustness of selection of stable, high-affinity Fn3
Cambridge, MA 02139, USA domains. A yeast surface display library of Fn3 was created in which three
2 solvent-exposed loops were diversified in terms of amino acid composition
Department of Biology, and loop length. The library was screened by fluorescence-activated cell
Massachusetts Institute of sorting to isolate binders to lysozyme. An affinity maturation scheme was
Technology, Cambridge, developed to rapidly and broadly diversify populations of clones by random
MA 02139, USA mutagenesis as well as homologous recombination-driven shuffling of mu-
3
Department of Biological tagenized loops. The novel library and affinity maturation scheme combined
Engineering, Massachusetts to yield stable, monomeric Fn3 domains with 3 pM affinity for lysozyme. A
Institute of Technology, secondary affinity maturation identified a stable 1.1 pM binder, the highest
Cambridge, MA 02139, USA affinity yet reported for an Fn3 domain. In addition to extension of the
affinity limit for this scaffold, the results demonstrate the ability to achieve
Received 11 December 2007; high-affinity binding while preserving stability and the monomeric state.
received in revised form This library design and affinity maturation scheme is highly efficient, uti-
5 June 2008; lizing an initial diversity of 2 × 107 clones and screening only 1 × 108 mutants
accepted 19 June 2008 (totaled over all affinity maturation libraries). Analysis of intermediate
Available online populations revealed that loop length diversity, loop shuffling, and recursive
24 June 2008 mutagenesis of diverse populations are all critical components.
© 2008 Published by Elsevier Ltd.
Keywords: fibronectin type III domain; Fn3; protein engineering; loop
Edited by F. Schmid shuffling; affinity maturation
Introduction the β strands they connect, which are structurally

analogous to antibody complementarity-determin-
The 10th type III domain of human fibronectin ing regions (CDRs). Modification of the amino acid
(Fn3) has been demonstrated as an effective scaffold sequence in these loops can impart novel binding
for molecular recognition.1–9 It is a small (10 kDa), capacity on the scaffold. Koide and colleagues have
stable (Tm = 84 °C)7 cysteine-free beta-sandwich pro- diversified the BC and FG loops and screened
tein that maintains its native fold in reducing envi- libraries by phage display to yield a 5 μM binder to
ronments and can be produced at up to 50 mg/L in ubiquitin4 and a 250 nM binder to Src SH3 domain2;
Escherichia coli.9 The protein fold resembles an immu- FG loop libraries yielded improved integrin binders8
noglobulin variable domain and contains three and novel estrogen receptor binders,3 the latter of
solvent-exposed loops, termed BC, DE, and FG, for which was identified by a yeast two-hybrid screen.
Phage display and yeast surface display were used to
screen a three-loop library with tyrosine/serine di-
*Corresponding author. Department of Chemical versity to isolate binders of 5, 7, and 30 nM to yeast
Engineering, Massachusetts Institute of Technology, small ubiquitin-like modifier, human small ubiqui-
Cambridge, MA 02139, USA. E-mail address: tin-like modifier 4, and maltose-binding protein,
wittrup@mit.edu. respectively.5 mRNA display was used to screen
Abbreviations used: CDR, complementarity-determining three-loop libraries to identify a 20 pM binder to
region; DSC, differential scanning calorimetry; FACS, tumor necrosis factor α9 and a 340 pM binder to
fluorescence-activated cell sorting; Fn3, 10th type III vascular endothelial growth factor receptor 2. 7
domain of human fibronectin; PBSA, phosphate-buffered Lipovsek et al. used yeast surface display to engineer
saline with bovine serum albumin. a 350 pM binder to lysozyme from a library with
0022-2836/$ - see front matter © 2008 Published by Elsevier Ltd.

Fibronectin Domains of 1 pM Affinity 1239
diversified BC and FG loops.6 Engineered binders Batori et al. demonstrated that the BC, DE, and FG
have been used as intracellular3 and extracellular loops can tolerate insertions of four glycine residues
inhibitors8 as well as labeling reagents in flow cyto- while maintaining a native fold.14 In addition, phy-
metry8 and Western blots2 and also have been immo- logenetic analysis reveals significant length diversity
bilized for affinity purification1 and for use in protein in each of the three loops across different fibronectin
arrays.9 type III domains in multiple species (Fig. 1). Thus, we
Although wild-type Fn3 is highly stable and mono- hypothesize that loop length diversity will be stably
meric, previously reported high-affinity Fn3 clones tolerated and improve the functional binding capabi-
are oligomeric or unstable. Two clones with 300 pM lities of the Fn3 scaffold and perhaps could improve
and 1 nM affinity for tumor necrosis factor α exhibited the stability–affinity relationship.
midpoints of proteolysis susceptibility at appro- Yet, loop length diversity increases theoretical
ximately 30 and 42 °C, respectively.9 A clone with sequence space, which is already immense, increas-
340 pM affinity for vascular endothelial growth factor ing the demand for efficient protein engineering
receptor 2 exhibited Tm values of 32–52 °C; a different methods in terms of both clonal selection and se-
clone was more stable but only 40–50% monomeric quence diversification. Yeast surface display enables
and was of moderate affinity (13 nM).7 Stability quantitative selections using fluorescence-activated
engineering yielded a stable, monomeric 2 nM binder. cell sorting (FACS) and correspondingly fine affinity
The ability to engineer Fn3 domains with high affinity discrimination.15 The eukaryotic protein processing
and stability represents a critical need in scaffold of yeast also improves the functional stringency of
development, since both properties are required for selections.16 One potential limitation of yeast surface
clinical and biotechnological applications. display is that cellular transformation efficiency
Synthesis errors during our previous library holds library size to 107–109 without excessive effort.
creation resulted in rare clones with non-wild-type In vitro display methods enable larger theoretical
loop lengths. 6 These loop length variants were library size that correlates with selection of improved
preferentially selected during binder isolation, and binders17,18; however, as has been shown for phage
binding titrations revealed their critical importance display, nominal library size does not necessarily
to binding. Moreover, sequence analysis reveals non- equate with functional diversity.16 The intrinsic muta-
wild-type lengths in tumor necrosis factor α binders9 genesis from the requisite PCR step in mRNA and
and carcinoembryonic antigen binders (B.J.H., un- ribosome display is a key contributor to the success of
published data). The rarity of these length variants in these display technologies.19 We hypothesized that an
the initial library resulted in very low coverage of increase in the frequency of mutagenesis during
theoretical length variant sequence space. These re- directed evolution will improve the efficiency of
sults suggest that more extensive sampling of length cellular display methods by increasing the breadth
variant sequences could improve the binding cap- of the sequence space search in the vicinity of many
abilities of the Fn3 scaffold. This hypothesis is lead clones, rather than only a select few.
supported by the fact that CDR length diversity is Another important engineering component is the
already an acknowledged component of antibody manner in which selected sequences are diversified
engineering,10,11 and it has been demonstrated that throughout directed evolution. Successful techni-
antibody affinity maturation is improved with the ques include DNA shuffling,20 CDR shuffling,21,22
inclusion of length diversity.12,13 Koide and collea- CDR walking,23 and error-prone PCR mutagenesis24
gues incorporated loop length diversity in a recent either toward the entire gene or toward the sus-
Fn3 library and successfully obtained binders with pected paratopes. In the current work, we combine
length variation.5 From a structural perspective, error-prone PCR and an analog to CDR shuffling to
Fig. 1. Loop length variability of

fibronectin type III domains. The
relative frequency of loop lengths of
the BC (white), DE (gray), and FG
(black) loops of sixteen type III
domains of fibronectin, relative to
the 10th type III domain (10Fn3) of
human fibronectin, are shown.
Values and error bars represent the
average and standard deviation for
five species: human, cow, rat, mouse,
and chicken.
1240 Fibronectin Domains of 1 pM Affinity
yield both mild and significant changes in sequence ployed. This mutagenesis targeted the entire gene to
space both directed at the expected paratope and also enable selection of beneficial framework muta-
throughout the Fn3 domain. tions. Both mutagenesis strategies were used in
Here we demonstrate that loop length diversity parallel at each mutagenesis step throughout affinity
and a novel affinity maturation scheme enable maturation (Supplementary Fig. 1). Analysis of
robust and efficient selection of stable, high-affinity selected clones after the fact indicates that both loop
binders to lysozyme. The binders are characterized shuffling and whole-gene error-prone PCR contrib-
in terms of binding, stability, and structure includ- uted to improved phenotypes.
ing detailed analysis of the molecular basis of The Fn3 library was screened using yeast surface
binding of a picomolar affinity clone. display and FACS. Plasmids from thousands of lead
clones in a partially screened yeast population were
collected by yeast zymoprep. Quantitative real-time
Results PCR revealed that full population diversity was
recovered (data not shown). Both error-prone PCR
of the full gene and shuffling of mutagenized loops
Fn3 library construction were successfully employed to diversify the popula-
tion of lead clones (Fig. 2b). Gene mutagenesis was
A library was created in which 8, 5, and 10 amino performed by error-prone PCR with nucleotide ana-
acids of the BC, DE, and FG loops, respectively, were logs.27 Independent error-prone PCRs were conduc-
diversified both in amino acid length (Fig. 1) and in ted for each of the three loops using primers that
composition (Fig. 2a). Amino acid composition was overlap either the adjacent loop primer or the plas-
randomized using NNB degenerate codons to yield mid vector. This overlap enabled shuffled gene re-
all 20 amino acids with reduced stop codon fre- construction via homologous recombination during
quency. Four different loop lengths were chosen for yeast transformation (Fig. 2c). The number of diver-
each loop based partially on the loop lengths ob- sified transformants during affinity maturation
served in fibronectin type III domains in multiple ranged from 2 to 20 million (mean, 8.1 million) for
species (Fig. 1). The library of Fn3 genes was incor- full gene mutagenesis and 0.1 to 6.5 million (mean,
porated into a yeast surface display system by homo- 2.5 million) for loop mutagenesis. Sequence analysis
logous recombination with a vector incorporating an exhibited mutagenesis that matched the desired 1 to
N-terminal Aga2 protein for display on the yeast 5 amino acid mutations per gene (data not shown),
surface and a C-terminal c-myc epitope for detection which was achieved with a single combination of
of full-length Fn3.26 Library transformation yielded nucleotide analog concentration and number of PCR
6.5 × 107 yeast transformants. Sixteen (62%) of 26 cycles determined by mathematical modeling.
clones sequenced matched library design. Nine
(35%) contained frameshifts and 1 (4%) was annealed High-affinity binder engineering
improperly or underwent unintentional homologous
recombination in yeast. NNB diversification of the The efficacy of the new library and affinity matu-
loops yields stop codons in approximately 44% of ration scheme was tested by engineering binders to
clones. Thus, 34% [16/26 × (1 − 0.44)] of transformed lysozyme, a model protein that we have previously
cells should display full-length Fn3. This percentage used as an Fn3 target with different library and
was verified by flow cytometry analysis (data not maturation approaches.6
shown). The library contains approximately 2.3 × 107 The naive library was sorted three times by FACS
(6.5 × 107 × 0.34) full-length Fn3 clones. using multivalent biotinylated lysozyme preloaded
on streptavidin–fluorophore conjugates. The resul-
Population mutagenesis design tant population was diversified by both gene and
loop mutagenesis. Transformed mutants as well as
A negative side effect of loop length diversity is a the original clones were sorted twice by FACS, yield-
further increase in the vastness of possible protein ing enrichment of evident lysozyme-binding clones
sequence space to 1034 possible amino acid sequences (Fig. 3). Four additional rounds of isolation and
for the three loops. Thus, the 2.3 × 107 clones in the maturation, each consisting of two to three FACS
original library grossly undersample sequence space, selections followed by mutagenesis (Supplementary
so an extensive diversification method was desired to Fig. 1), were conducted using monovalent lysozyme,
broadly search sequence space during affinity ranging from 1 μM to 20 pM lysozyme. Labeling at
maturation. Error-prone PCR directed solely at the low picomolar concentrations while maintaining
solvent-exposed loops was used to focus diversity on stoichiometric excess of target to Fn3 would require
the likely paratope. Additionally, to make substantial impractically large volumes; thus, kinetic competi-
changes in sequence, a loop shuffling approach was tion sorting was used for the final three rounds of
developed. Fn3 genes were constructed with con- isolation and maturation. The final two sublibraries
served wild-type framework sequence and randomly were sorted an additional three times and the collect-
shuffled mutated loops from the pool of selected ed populations were sequenced to identify the high-
clones. Yet, as effective clones are selected, such subs- est affinity clones.
tantial sequence modification is not desired. Thus, Three dominant clones were identified by sequence
error-prone PCR without shuffling was also em- analysis (Table 1). Affinity titrations indicate that all
Fibronectin Domains of 1 pM Affinity
Fig. 2. Library design and affinity maturation scheme. (a) The naive library is randomized in the BC, DE, and FG loops (structure schematic derived from Main et al.25) at the
indicated positions to four possible loop lengths each. (b) Error-prone PCR is used to introduce random mutations either into the entire Fn3 gene or into the three loops (separate
PCRs). Arrowed lines indicate PCR primers; matching shades correspond to primers in a single PCR reaction. The framework (fw) and loop regions of the gene are indicated.
Zigzag lines represent nucleotide mutations. Smaller unlabeled images are used to emphasize that PCR templates and products are part of a repertoire based on many gene
variants. (c) Two yeast sublibraries are created by transformation via electroporation. During transformation, the full plasmid is created by homologous recombination of the
linearized vector with either the single mutated gene insert or the three mutated loop inserts.
1241
Fig. 3. Binder isolation and affinity maturation. Yeast libraries displaying Fn3 were labeled with mouse anti-c-myc
antibody followed by goat anti-mouse fluorophore as well as biotinylated lysozyme and streptavidin–fluorophore and
analyzed by flow cytometry. (a) Yeast population during second round of isolation and maturation labeled with 50 nM
multivalent lysozyme preloaded in a 3:1 stoichiometry on streptavidin–R-phycoerythrin. (b) Yeast population during
sixth round of isolation and maturation labeled with 0.5 nM monovalent lysozyme followed by streptavidin–
AlexaFluor488. (c) Yeast population during eighth round of isolation and maturation labeled with 2 nM monovalent
lysozyme for 15 min followed by 35 h of dissociation and labeling with streptavidin–R-phycoerythrin. Polygonal regions
represent sort gates for cell selection.
three clones have comparable equilibrium dissocia- produced in bacterial culture and purified for
tion constants (Kd) for binding to biotinylated biophysical characterization. Analytical size-exclu-
lysozyme: 2.6 ± 0.6 pM (clone L7.5.1), 2.9 ± 1.4 pM sion chromatography indicated that all three clones
(L8.5.2), and 2.8 ± 0.5 pM (L8.5.7) (Fig. 4a and Table 1). are predominantly monomeric with only L8.5.2
The high-affinity binding was validated using pur- present in a significant oligomeric state (80% mono-
ified Fn3 domains in an equilibrium competition meric; Table 1 and Supplementary Fig. 2a). It should
titration, which indicated an affinity of 6.9 ± 0.3 pM for be noted that L8.5.2 contains two cysteine residues
L7.5.1 (Fig. 4b). Dissociation rates for the three clones and intermolecular disulfide bonding contributes to
range from (2.5–5.4) × 10− 6 s− 1, which correspond to oligomerization as indicated by nonreducing SDS-
dissociation half times of 36–78 h (Fig. 4c). The PAGE (data not shown). The thermal stabilities of the
association rate of L7.5.1 was measured experimen- proteins were analyzed by differential scanning calo-
tally as (2.0± 0.5) × 106 M− 1 s− 1 (Fig. 4d). The cal- rimetry (DSC) and circular dichroism of purified
culated value of koff/kon is 1.2 ± 0.3 pM, which is protein as well as thermal denaturation of protein
reasonably consistent with the experimentally mea- displayed on the yeast surface (Table 1 and Supple-
sured Kd of 2.6 ± 0.6 pM. In addition to high affinity, mentary Fig. 3). L7.5.1 is the most stable clone with
the clones exhibit target specificity, as they do not midpoints of thermal denaturation ranging from 55.7
show appreciable binding to an array of other mole- to 58.8 °C for the three methods. It should be noted
cules (Fig. 5). that denaturation of L7.5.1 is not reversible because
Since previously reported high-affinity clones were of aggregation at high temperatures. Both other
unstable or oligomeric, it was desired to examine the clones are also stable with Tm values over 50 °C. In
behavior of the current Fn3 domains. The clones were addition, far-UV circular dichroism analysis reveals
Table 1. Characterization of wild-type, L7.5.1, L8.5.2, and L8.5.7
Amino acid sequence Tm (°C)

Kd koff Monomer T1/2
−5 −1
Clone BC loop DE loop FG loop Framework (pM) (10 s ) (%) DSC CD (°C)
WT DAPAVTR GSKST GRGDSPASSK N107 n/d 99 85.7 84.2 n/d
L7.5.1 RGYPWAT GVTN RVGRTFDTPG P15S, R33G, T35I, 2.6 ± 0.6 0.25 ± 0.02 99 58.1 58.8 ± 1.6 55.7 ± 0.1
P44L, V50M
L8.5.2 RGCPWAI GVTN RVGRMLCAPG R33G, I34V, N42S, 2.9 ± 1.4 0.32 ± 0.01 80 n/d 52.5 ± 0.2 50.6 ± 0.5
P44L, V45A, V50M,
K63E, K98R
L8.5.7 RDRPWAI GVTN RLSIVPYA D3G, L18I, R33G, 2.8 ± 0.5 0.54 ± 0.06 93 54.5 n/d 53.0 ± 0.4
N42S, P44L, V50M,
Y73H, N91T, S100P
Kd, equilibrium dissociation constant at 25 °C; koff, dissociation rate constant at 25 °C; Monomer, percent of purified protein present as
monomer in analytical size-exclusion chromatography; Tm, midpoint of thermal denaturation curve as determined by DSC or CD; T1/2,
midpoint of thermal denaturation curve as determined by yeast surface display residual activity assay.
Fig. 4. Determination of binding parameters for high-affinity clones. L7.5.1, L8.5.2, or L8.5.7 was displayed on the
yeast surface and assayed for binding to biotinylated lysozyme at 25 °C. Different symbols indicate replicate experiments.
Continuous lines indicate theoretical values. (a) Fraction of displayed L7.5.1 binding to biotinylated lysozyme at
equilibrium. Only L7.5.1 is shown for simplicity. (b) Relative binding of 20 pM biotinylated lysozyme to displayed L7.5.1
at equilibrium in the presence of indicated amount of soluble L7.5.1 competitor. ∞ indicates samples without biotinylated
lysozyme. (c) Fraction of displayed L7.5.1 (squares), L8.5.2 (diamonds), or L8.5.7 (triangles) binding to biotinylated
lysozyme after dissociation at 25 °C for the indicated time. (d) Fraction of displayed L7.5.1 binding to biotinylated
lysozyme after association at 25 °C; biotinylated lysozyme was present at 49 pM (crosses), 96 M (triangles), 185 pM
(diamonds), or 345 pM (squares). Results are presented from a single experiment, which is representative of triplicate
experiments performed at different sets of concentrations.
no significant differences in secondary structure Analysis of intermediate populations

between wild-type Fn3 and L7.5.1, indicating that
despite loop mutation and length variation the struc- Intermediate populations were investigated to elu-
tural integrity of the domain is maintained (Supple- cidate the progress of affinity maturation. Each sub-
mentary Fig. 2b). library (i.e., library of mutagenized clones created
Fig. 5. Binding specificity. Wild-

type Fn3 (wt), L7.5.1, L8.5.2, or L8.5.7
was displayed on the yeast surface,
washed, and incubated with 100 pM
biotinylated lysozyme (black) or
100 nM nontarget molecules: bioti-
nylated carcinoembryonic antigen
(bCEA, dark gray), biotinylated epi-
dermal growth factor receptor
domain IV (bEGFR, light gray), bio-
tinylated huntingtin peptide (bHtt,
white), or streptavidin–R-phycoery-
thrin (strep, striped). Binding was
detected with streptavidin–R-phy-
coerythrin secondary labeling and
flow cytometry. The data are pre-
sented as the mean fluorescence
units from binding (PE signal) normalized by the amount of Fn3 on the yeast surface (AlexaFluor488 signal). Values and
error bars represent mean ± standard deviation of duplicate measurements. Asterisk (*) indicates a value less than 0.005.
Table 2. Highest affinity Fn3 domains in each sublibrary
Amino acid sequence

Kd koff
Round BC loop DE loop FG loop Framework (pM) (10−5 s−1) t1/2 (h)
0 RDCPWAT WTPVCF SSQRGCM none ≫100,000 n/d n/d
1 SLDNQAN GQSD RCEPSRNSAV none N100,000 n/d n/d
2 same clone as round 1
3 SLDNQAN GVTN RVGRMLDTPG P44S, V50M 7600 ± 1100 460 0.04
4 SLDNQAK GATN RCKPFRNSAV P44S, V50M, T97I 330 ± 50 n/d n/d
5 RDCPWAI GVTN RVGRMSCTSG V1A, S1P, T14A, R33G, 16 ± 6 4.5 ± 0.3 4.2 ± 0.3
P44L, V50M
6 RGCPWAI GVTN RVGRMLCTPG P15S, R33G, N42S, 6.6 ± 1.3 0.72 ± 0.07 27 ± 3
P44L, V50M, K63E
7 RGYPWAT GVTN RVGRTFDTPG P15S, R33G, T35I, P44L, V50M 2.6 ± 0.6 0.25 ± 0.02 78 ± 1
8a RGCPWAI GVTN RVGRMLCAPG R33G, I34V, N42S, P44L, 2.9 ± 1.4 0.32 ± 0.01 60 ± 1
V45A, V50M, K63E
RDRPWAI GVTN RLSIVPYA D3G, L18I, R33G, N42S, 2.8 ± 0.5 0.54 ± 0.06 36 ± 4
P44L, V50M, Y73H, N91T
Round, number of mutagenesis cycles (round 0 indicates naive library); Kd, equilibrium dissociation constant at 25 °C; Koff, dissociation
rate constant at 25 °C; t1/2, time for 50% dissociation of the Fn3–lysozyme complex at 25 °C; n/d, not determined.
a
Two clones with similar affinities were identified from round 8.
during affinity maturation) was sorted by FACS The impact of loop shuffling is evident in multiple
without mutagenesis to identify the highest affinity cases. The BC loop from the round 1 clone is recom-
clone at each stage of affinity maturation. The high- bined with new DE and FG loops in round 3 to yield
est affinity clone was identified as the dominant an 8 nM binder. A mutated version of the FG loop
clone from sequencing several clones from the exten- present in round 1 is then recombined with mutated
sively sorted sublibraries. Sequences, equilibrium BC and DE loops from round 3 to achieve picomolar
dissociation constants and dissociation rate con- binding. The highest affinity clones in rounds 5–8
stants were determined (Table 2). The highest affinity result from apparent shuffling of the BC loop ob-
binder in the original library exhibits apparent mid- served in round 0 and the DE and FG loops observed
micromolar affinity. However, affinity maturation in round 3 as well as multiple framework mutations.
progressively improves binding performance to The appearance of point mutations is also apparent,
yield nanomolar and then picomolar binders (Fig. both within the loops and in the framework region.
6). It is noteworthy that affinity maturation exhibits a The impact of these framework mutations was inves-
relatively consistent correlation to the number of tigated in more detail in the context of L7.5.1.
clones analyzed throughout the course of affinity
maturation until the final round of directed evolu- L7.5.1 analysis
tion. A steady increase characteristic of a consistently
progressing affinity maturation scheme is observed L7.5.1 was selected for more thorough analysis be-
rather than one single exceptional increase in affinity, cause it has the fewest framework mutations and is
indicative of a fortuitous mutation or recombination. the most stable of the high-affinity clones. The engi-
Fig. 6. Affinity maturation pro-

gress. The highest affinity clone in
each affinity maturation sublibrary
was identified by FACS. The equili-
brium dissociation constant of the
first three clones could not be deter-
mined accurately by yeast surface
display titration because of the rela-
tively low affinity. Thus, an estimated
range of possible affinities consistent
with equilibrium labeling at high
nanomolar concentrations is indi-
cated. The equilibrium dissociation
constant of the latter six clones was
determined by titration and is repre-
sented as the mean±one standard
deviation of replicate measurements.
The number of total clones analyzed
is the cumulative total of the number
of full-length clones in the naive
library and total yeast transformants
in all affinity maturation sublibraries.
neered elements of the clone were analyzed for their Table 3. Affinity and stability of framework mutation
impact on affinity and stability. Each randomized reversions of L7.5.1
loop and framework mutation was independently
AA WT L7.5.1 Kd (pM) T1/2 (°C) Location
restored to wild-type sequence and both affinity and
stability were measured using yeast surface display. L7.5.1-parent clone 2.6 ± 0.6 55.7 ± 0.1
15 Pro Ser 3.4 ± 0.1 59.5 ± 0.7 AB loop
Reversion of the FG loop had the strongest effect on 33 Arg Gly 74 ± 23 56.2 ± 0.6 C strand
binding, as wild-type restoration decreased affinity to 35 Thr lle 5.8 ± 1.6 54.5 ± 0.5 C strand
the micromolar level; conversely, DE loop reversion 44 Pro Leu 14.3 ± 4.0 50.3 ± 1.2 CD loop
had a nearly negligible effect on binding, and the BC 50 Val Met 25 ± 16 53.7 ± 1.8 D strand
loop reversion had an intermediate effect with a 4700- The five framework mutations of L7.5.1 were individually re-
fold reduction (Fig. 7a). Both BC and DE loop rever- verted to wild-type amino acids. Binding affinity and thermal
sion significantly destabilize the domain (Fig. 7b). stability were determined by yeast surface display. AA, amino
Although these loop sequences confer high thermal acid position; WT, wild-type side chain; L7.5.1 clone, 7.5.1 side
chain; Kd, equilibrium dissociation constant at 25 °C; T1/2,
stability in the wild-type context, modification of midpoint of thermal denaturation as determined by yeast surface
adjacent loops apparently adjusts the intramolecular display.
contacts. Thus, during multiloop diversification,
selected sequences must provide not only proper
intermolecular contacts for high-affinity binding but conservative mutations and were beneficial to both
also proper intramolecular contacts for structural affinity and stability (Table 3). Conversely, R33G
integrity and stability. The stability of the FG loop replaces a large, positively charged side chain with a
reversion could not be accurately determined by this single hydrogen; this mutation provides 28-fold
method because of its weak binding. stronger binding without a substantial effect on sta-
Three of the five framework mutations in the se- bility. Perhaps the arginine side chain was not accom-
lected clone (T35I, P44L, and V50M) are relatively modated sterically and/or electrostatically at the
Fn3–lysozyme interface or the glycine enables a bene-
ficial backbone conformation. The P15S mutation had
only a very minor affinity improvement but substan-
tially destabilized the domain. The lack of impact on
binding is reasonable given its distance from the
expected paratope, and the substantial destabilization
is not surprising given the removal of the backbone-
constraining proline in the turn between the C and D
strands. Selection of this mutation was likely coin-
cident with another beneficial mutation, as it does not
impart significant benefit; it should be noted that
while the most beneficial mutations, R33G, P44L, and
V50M, are consistently observed throughout matura-
tion (Table 2), P15S and T35I are rare. Overall, these
data suggest the paratope is focused on the BC and FG
loops as well as potential key contacts on the C and D
strands of the β sheet.
Focused mutagenesis
The conservation of the DE loop sequence through-

out much of the affinity maturation (Table 2) despite
its relatively low impact on affinity relative to wild
type prompted further study of this loop. The func-
tionally tolerable diversity and the potential for
improved binding were explored through re-rando-
Fig. 7. L7.5.1 loop analysis. Each engineered loop of mization of the loop sequence. Within the context of
L7.5.1 was independently restored to wild-type sequence L7.5.1 S15P, the loop was randomized using NNB
and the resultant clones were displayed on the yeast surface. nucleotide diversity and the same four loop length
Filled and empty symbols indicate replicate experiments. (a) options as the original library. Labeling the library
Binding to biotinylated lysozyme at 25 °C was quantified by with 30 pM lysozyme, which yields 90% binding to
flow cytometry. Clone and equilibrium dissociation con- the parent clone L7.5.1 S15P, yields many clones that
stant: L7.5.1 (squares), 2.6± 0.6 pM; L7.5.1wtBC (diamonds), provide effective binding, indicating that the loop
12.4 ± 0.7 nM; L7.5.1wtDE (triangles), 3.7 ± 1.4 pM;
can tolerate significant diversity and maintain bind-
L7.5.1wtFG (crosses), 1.6± 0.2 μM. (b) Yeast displaying Fn3
were incubated at the indicated temperature for 30 min ing. Specifically, 7% of clones exhibit binding at 75%
followed by a flow cytometric assay for biotinylated of maximum, which is characteristic of a 10 pM Kd,
lysozyme binding. Clone and midpoint of thermal dena- or within threefold of the parent clone (Fig. 8). Yet,
turation: L7.5.1 (squares), 55.7± 0.1 °C; L7.5.1wtBC (dia- 30% of the full-length clones bind at less than 1% of
monds), 37.4 ± 1.5 °C; L7.5.1wtDE (triangles), 44.5± 0.1 °C. maximum, indicating that many loop sequences can
conserved amino acids were identified as the

positions of L7.5.1-homologous clones at which se-
quence diversity was observed in any sequence du-
ring affinity maturation or sublibrary analysis. The
nonconserved amino acids were randomized in two
modes: either to all 20 amino acids using NNB co-
dons or to amino acids observed during sequence
analysis using tailored codons at each position. The
library was sorted for binding to biotinylated lyso-
zyme using FACS. Sequence analysis after four
selections yielded a single clone, Cons0.4.1. The
affinity of Cons0.4.1 was measured as 1.1 ± 0.6 pM by
yeast surface display titration and 0.33 ± 0.15 pM by
equilibrium competition with purified Fn3 domain.
Fig. 8. L7.5.1 S15P DE loop randomization library The midpoint of thermal denaturation was mea-
binding analysis. The DE loop of L7.5.1 S15P was ran- sured as 52.5 ± 2.1 °C by circular dichroism analysis
domized using NNB codon diversity with four loop and 58.8 ± 3.4 °C by yeast surface display thermal
lengths. The yeast surface display library was labeled with resistance assay. Far-UV circular dichroism indicates
30 pM biotinylated lysozyme and mouse anti-c-myc that Cons0.4.1 maintains a secondary structure simi-
antibody followed by streptavidin–R-phycoerythrin and lar to that of the wild-type Fn3 domain (Supplemen-
goat anti-mouse antibody conjugated to AlexaFluor488.
tary Fig. 2b). Two of the eight codon changes were
Percentages indicate the relative number of c-myc positive
clones with bLys binding: c-myc display signals with 75% not possible with a single nucleotide mutation
of maximum (7%), 1–75% of maximum (63%), or b1% of making this clone unlikely to be reached by error-
maximum (30%). prone PCR. Thus, while error-prone PCR provides a
highly effective means of diversification, an im-
provement was observed through more thorough
searching of a focused region of sequence space once
greatly hinder binding either through direct interac- a consistent binding motif was identified.
tion with the binding partner or through structural
modification of the Fn3 paratope. In addition, the
library was sorted for binding to biotinylated lyso- Discussion
zyme using FACS. Sequence analysis after four selec-
tions identified a single clone, DE0.4.1, with GDLSHR In this work we explore the impact of multiple
replacing GVTN in the DE loop. Binding and stability components of engineering a high-affinity binding
analyses indicate a 3.1× improvement in binding to site in the Fn3 scaffold. The combination of loop
1.1 pM at the expense of an 8.5 °C decrease in the T1/2 length diversity and recursive mutagenesis includ-
(Table 4). The maintenance of glycine at position 52 ing mutated loop shuffling enabled selection of the
from wild type to L7.5.1 to DE0.4.1 is noteworthy for highest affinity Fn3 domains yet reported despite a
possible future library design especially considering relatively modest initial library size of 2.3 × 107 full-
the adjacent amino acid is proline; the proline–glycine length Fn3 clones. The results extend the affinity
pair provides an effective turn motif to begin the DE attainable by this single domain scaffold, further
loop. validating its use as a molecular recognition scaffold.
Lack of improvement from round 7 to round 8 in In addition, insights were gained for both Fn3 design
conjunction with sequence similarities during affi- and engineering and protein engineering in general.
nity maturation prompted investigation into a com- Analysis of the highest affinity binders in each
plementary method of affinity maturation. Since sublibrary (Table 2) demonstrates deviation from
only a fraction of protein sequence space is accessible wild-type loop lengths in all three loops implicating
through single nucleotide mutations, a library was the importance of this diversity element in Fn3
constructed in which the DNA encoding noncon- library design. Interestingly, the observed BC loops
served amino acids in the BC and FG loops was are all one amino acid shorter than wild type, which
randomized by degenerate oligonucleotides. Non- is observed both in other fibronectin type III domains
Table 4. Affinity and stability of clones from focused mutagenesis
Amino acid sequence

Kd T1/2
Clone BC loop DE loop FG loop Framework (pM) (°C)
L7.5.1 S15P RGYPWAT GVTN RVGRTFDTPG R33G, T35I, PAAL, V50M 3.4 ± 0.1 59.5 ± 0.7
Cons 0.4.1 REDPWAK GVTN RVGWASYTLG R33G, T35I, P44L, V50M 1.1 ± 0.6 59 ± 3
DE 0.4.1 RGYPWAT GDLSHR RVGRTFDTPG R33G, T35I, P44L, V50M 1.1 ± 0.5 51 ± 3
Kd, equilibrium dissociation at 25 °C; T1/2, midpoint of thermal denaturation curve as determined by yeast surface display residual
activity assay.
(Fig. 1) and in a previously engineered binder.6 DE with a substantially different FG loop, but conserved
loops are observed with either one less or one more DE and BC loops.
amino acid than wild type. Clone DE0.4.1 and the It is noteworthy that the focused mutagenesis
highest affinity clone from the naive library (Table 2) results indicate the potential for mild improvement
justify inclusion of a six-amino acid DE loop despite of affinity maturation through targeted randomiza-
its lack of existence in native fibronectin type III tion at multiple residues identified as variable through
domains. FG loops of wild-type length as well as sequence analysis. It is not yet clear if the success of
both two- and three-amino acid reductions were this avenue of affinity maturation resulted from the
observed. The observed length diversity, as well as relatively high number of mutations from the parent
the success of length diversity in a restricted diver- clone (eight amino acid changes in the two loops) or
sity Fn3 library published during preparation of this the ability to reach amino acids that would require
manuscript,5 support the inclusion of length diver- more than one nucleotide mutation. Regardless, the
sity in all future Fn3 engineering. Moreover, the fact that a secondary, semirational approach was only
combination of phylogenetic analysis and sequence able to yield 3.1× enhancement in affinity is indicative
analysis of engineered binders should continue to of a relatively effective search of sequence space by the
elucidate the relative preference of each length to initial affinity maturation.
further improve library design. Diversification of the DE loop is an important
Loop length diversity increases sequence space to aspect of Fn3 engineering. Because it is the shortest
1034 possible amino acid sequences for the three and least flexible wild-type loop, it is unclear if the
loops, although only 2.3 × 107 clones are present in binding potential gained by diversification offsets the
the yeast library. The two modes of diversity intro- possible structural destabilization. Thus, analysis of
duced during recursive mutagenesis effectively the contribution of the DE loop to binding and sta-
search this large sequence space despite the sparse bility are valuable to clone maturation and future Fn3
sampling. Sequence and binding analyses indicate library design. The originally selected DE loop,
that each of the elements of the mutagenesis method GVTN, stabilizes clone L7.5.1 relative to wild type
were beneficial. The loop mutagenesis path of the but does not significantly aid binding. Although loop
diversification was effective, as both loop shuffling reversion analysis suggests that the binding paratope
and loop-focused point mutations are evident (Table is dominated by the FG and BC loops, the DE loop can
2). In addition, the gene mutagenesis path was be engineered for improved binding (DE0.4.1) albeit
advantageous, as an effective loop combination was at the expense of stability. Since selections were
maintained in rounds 5–8 and beneficial framework explicit for affinity, the lack of binding performance
mutations were introduced throughout (Tables 2 and from the L7.5.1 DE loop likely resulted from a lack of
3). The importance of diversifying many clones du- DE loop diversity after a few rounds of directed evo-
ring each round of maturation is evident, since lution (Table 2). Thus, a future improvement on loop
homologs of the eventual DE and FG loops were not shuffling will be to incorporate a small percentage of
present in 30 sequences from the enriched popula- naive loops with the engineered loops. A broader
tions from the naive library. As a result of these uncertainty regarding the DE loop is the extent to
combined components, affinity maturation rapidly which it should be diversified in future Fn3 libraries.
and efficiently progressed to yield the clones with Previous binders have been engineered with wild-
3 pM affinity without rational intervention. The affi- type DE loops with 350 pM affinity for lysozyme,6
nity maturation procedure is straightforward and 30 nM for maltose-binding protein,5 250 nM for Src
simple; plasmid recovery, mutagenesis, amplification, SH3 domain,2 and approximately 5 μM for ubiquitin.4
and yeast transformation can be achieved in 1–2 days. The DE reversion of L7.5.1 represents a 3.7 pM binder
The increased frequency of mutagenesis is obviously with a wild-type DE loop. Collectively, it is clear that
applicable to any protein engineering method and is binders can be engineered without DE loop diversity.
strongly recommended to improve both the speed of Conversely, engineered DE loops in the L7.5.1 context
binder isolation and the overall efficacy. Shuffling can can either improve the affinity to 1.1 pM or stabilize
be implemented in any analogous protein scaffold the molecule by an 11 °C increase in T1/2, the latter of
provided the regions of interest are relatively prox- which is not surprising given its extensive contact
imal at the DNA level to enable overlap for homolo- with the BC loop residues as well as the shortened BC
gous recombination. Yeast surface display provides loop length. Moreover, engineered vascular endothe-
an effective system for shuffling because DNA frag- lial growth factor receptor 2 binders require their
ments can be recombined with high fidelity during engineered DE loop for binding, although it destabi-
cellular transformation simplifying the method. lizes the molecule by 2.0 kcal/mol or ∼30 °C decrease
The sequence diversity of the highest-affinity in Tm.7 Consequently, as expected, DE loop impact is
clones is striking. L8.5.2 has two cysteines at loca- context dependent. One possible general approach
tions consistent with formation of an interloop disul- would be to introduce mild DE diversity in the ori-
fide bond, as was found previously from a different ginal library to allow for binding constraint removal
Fn3 library screened by yeast display.6 However, and structural complementation of selected BC loops
unlike in that case, the disulfide is dispensable for as well as the possibility of beneficial binding contacts.
high-affinity binding, since L7.5.1 has highly related After binder selection, affinity or stability maturation
loop sequences but lacks both cysteines. A third could be employed with more diverse DE loop
clone with similar affinity (L8.5.7, 2.8 pM) was found shuffling in the context of effective BC and FG loops.
Unlike several examples of previous high-affinity were combined and thermally cycled at identical conditions.
Fn3 binder engineering, the selected high-affinity Two microliters of this product was combined with 0.4 μM
clones are relatively stable and monomeric. L7.5.1 is a primer (a1–b4n amplified with p1; c5nmix–d8n amplified
2.6 pM binder and N99% monomeric with a midpoint with p8) in a new 100-μL reaction and thermally cycled
under identical conditions to amplify the appropriate
of thermal denaturation of 56–59 °C. Moreover,
strand. The products were combined and thermally cycled
reversion of serine to proline at position 15 improves at identical conditions. The final products were concentrated
the T1/2 to 60 °C. In addition, Cons0.4.1 is a 1.1 pM with PelletPaint (Novagen). The plasmid acceptor vector
binder with Tm of 53 °C, and L8.5.7 is a 2.8 pM binder, pCTf1f4 (Ref. 6) was digested with NcoI, NdeI, and SmaI
is 93% monomeric, and has a Tm of 53–55 °C. (New England Biolabs, Ipswich, MA). Multiple aliquots of
Although stability was not an explicit element of ∼10 μg of Fn3 gene and 3 μg plasmid vector were combined
selection, the eukaryotic secretion machinery of yeast with 50–100 μL of electrocompetent EBY100 and electro-
provides some level of quality control against mis- porated at 0.54 kVand 25 μF. Homologous recombination of
folded proteins.28,29 In general, Fn3 clones of higher the linearized vector and degenerate insert yielded intact
stability are displayed at high densities on the yeast plasmid. Cells were grown in YPD (1% yeast extract, 2%
peptone, 2% glucose) for 1 h at 30 °C, 250 rpm. The number
cell surface (data not shown); thus, unstable clones are
of total transformants was 6.5× 107 cells as determined by
slightly selected against based on the two-color sort serial dilutions plated on SD-CAA plates (0.1 M sodium
regions. It remains to be seen if stable clones will phosphate, pH 6.0, 182 g/L sorbitol, 6.7 g/L yeast nitrogen
generally result from selections by yeast surface base, 5 g/L casamino acids, 20 g/L glucose). The library was
display or if this was a fortuitous result; regardless, propagated by selective growth in SD-CAA, pH 5.3 (0.07 M
yeast surface display provides a means for stability sodium citrate, pH 5.3, 6.7 g/L yeast nitrogen base, 5 g/L
engineering either during or after binder selection. casamino acids, 20 g/L glucose, 0.1 g/L kanamycin,
Overall, the method can be further improved 100 kU/L penicillin, and 0.1 g/L streptomycin) at 30 °C,
through refinement of naive loop length distribution, 250 rpm.
an increase in magnitude of the initial library,
inclusion of naive loops during loop shuffling, and Fluorescence-activated cell sorting
perhaps more expansive mutation than attainable by
error-prone PCR with nucleotide analogs. Never- Yeast were grown in SD-CAA, pH 5.3, at 30 °C, 250 rpm
to logarithmic phase, pelleted, and resuspended to 1 × 107
theless, the combination of yeast surface display, loop
cells/mL in SG-CAA, pH 6.0 (0.1M sodium phosphate, pH
length diversity, and dual-mode affinity maturation 6.0, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids,
rapidly yielded stable, high-affinity binders. The 19 g/L dextrose, 1 g/L glucose, 0.1 g/L kanamycin,
method should be valuable toward development of 100 kU/L penicillin, and 0.1 g/L streptomycin) to induce
Fn3 binders to additional targets as well as transfer- protein expression. Induced cells were grown at 30 °C,
able to engineering of other proteins for any screen- 250 rpm for 12–24h.
able functionality. Round 0 (three FACS selections) and round 1 (two FACS
selections) were conducted with multivalent lysozyme
prepared by incubating streptavidin–fluorophore (R-phy-
Materials and Methods coerythrin, AlexaFluor488, or AlexaFluor633; Invitrogen,
Carlsbad, CA) with biotinylated lysozyme (Sigma, St.
Louis, MO) in a 1:3 ratio in phosphate-buffered saline with
Fn3 library construction bovine serum albumin (PBSA). Yeast were pelleted,
washed in 1 mL PBSA (0.01 M sodium phosphate, pH
Oligonucleotides were purchased from MWG Biotech 7.4, 0.137 M sodium chloride, 1 g/L bovine serum
(High Point, NC) and Integrated DNATechnologies (Coral- albumin), resuspended in PBSA with 10–40 mg/L mouse
ville, IA) (see Supplementary Data online for sequences). anti-c-myc antibody (clone 9E10, Covance), and incubated
The Fn3 library was constructed to produce wild-type on ice. Cells were washed with 1 mL PBSA and resus-
sequence in the framework regions and to randomize the pended in PBSA with multivalent lysozyme (0.5 μM for the
BC, DE, and FG loops of Fn3. The DNA encoding for amino first four sorts and 50 nM for the fifth sort) and goat anti-
acids 23–30 (DAPAVTVR) was replaced by (NNB)x where mouse antibody conjugated to R-phycoerythrin, Alexa-
x = 6, 7, 8, or 9 to yield a loop length that is −2, −1, 0, or +1 Fluor488, or AlexaFluor633.
amino acids relative to wild type. Similarly, the DNA for Intermediate FACS selections were conducted with
amino acids 52–56 (GSKST) was replaced by (NNB)y where near-equilibrium labeling with monovalent lysozyme.
y = 4, 5, 6, or 7, and the DNA for amino acids 77–86 Three, two, two, and three selections were performed in
(GRGDSPASSK) was replaced by (NNB)z where z = 5, 6, 8, rounds 2–5, respectively. Yeast were pelleted, washed in
or 10. 1 mL PBSA, resuspended in PBSA with biotinylated
The library was constructed by sequential annealing and lysozyme (ranging from 1 μM to 20 pM) and mouse anti-c-
extension of eight overlapping oligonucleotides.6 The myc antibody, and incubated on ice. Cells were then
following components were combined in a 50-μL reaction: washed with 1 mL PBSA and resuspended in PBSA with
two oligonucleotides (0.2 μM a2, b3nmix, c6t, or d7nmix + streptavidin–fluorophore and fluorophore-conjugated
0.4 μM a1, b4n, c5nmix, or d8n, respectively), 1× polymerase goat anti-mouse antibody.
buffer, 0.2 mM deoxynucleotide triphosphate (dNTP), 1 mM FACS selections of very high affinity populations were
MgSO4, 1 U KOD Hot Start DNA Polymerase (Novagen, conducted with kinetic competition. Two, three, and two
Madison, WI), 1 M betaine, and 3% dimethyl sulfoxide. The selections were performed in rounds 6–8. Yeast were
mixture was denatured at 95 °C for 2 min followed by 10 washed and incubated briefly with 1–2 nM biotinylated
cycles of 94 °C for 30 s, 58 °C for 30 s, and 68° for 1 min and a lysozyme. Yeast were then washed and resuspended with
final extension of 68 °C for 10 min. Forty microliters of the PBSA with 140 nM unbiotinylated lysozyme (to prevent
products (a1+a2, b3nmix+b4n, c5nmix+c6t, or d7nmix+d8n) further association of labeled target) and incubated at
room temperature for 2 h to 7 days to enable dissociation of DNA sequencing

biotinylated lysozyme. Cells were washed in PBSA,
resuspended in PBSA with mouse anti-c-myc antibody, Plasmid DNA was isolated using the Zymoprep kit II,
and incubated on ice. Cells were washed and labeled with cleaned using the Qiagen PCR Purification kit, and trans-
secondary reagents as in equilibrium labeling. formed into DH5α (Invitrogen) or XL1-Blue E. coli (Stra-
In all cases, labeled cells were washed with 1 mL PBSA, tagene, La Jolla, CA). Individual clones were grown,
resuspended in 0.5–2.0 mL PBSA and analyzed by flow miniprepped, and sequenced using BigDye chemistry on
cytometry using either a MoFlo (Cytomation) or Aria an Applied Biosystems 3730.
(Becton Dickinson) cytometer. c-myc+ cells with the top
0.2–3% of lysozyme binding/c-myc display ratio were Measurement of Kd, kon, and koff
selected. Collected cells were grown in SD-CAA, pH 5.3, at
30 °C, 250 rpm and either induced in SG-CAA, pH 6.0, for The equilibrium dissociation constant for a clone was
further selection or used for plasmid recovery. determined essentially as described.26 Briefly, yeast contain-
ing the plasmid for an Fn3 clone was grown and induced as
Fn3 mutagenesis for FACS selection. Cells were washed in 1 mL PBSA and
resuspended in PBSA containing biotinylated lysozyme in
Plasmid DNA from 1 × 108 cells was isolated using two concentrations generally spanning 4 orders of magnitude
columns of Zymoprep kit II (Zymo Research, Orange, CA) surrounding the equilibrium dissociation constant. The
according to the manufacturer's instructions except for numbers of cells and sample volumes were selected to
additional centrifugation of neutralized precipitate. The ensure excess lysozyme relative to Fn3. For clones of low
zymoprep elution was cleaned using the Qiagen PCR picomolar affinity, this criterion necessitates very low cell
Purification kit (Qiagen, Valencia, CA), and eluted in 40 μL density, which makes cell collection by centrifugation
of elution buffer. Error-prone PCR of the entire Fn3 gene procedurally difficult. To obviate this difficulty, uninduced
was performed in a 50-μL reaction containing 1× Taq buffer, cells are added to the sample to enable effective cell pelleting
2 mM MgCl2, 0.5 μM each of primers W5 and W3, 0.2 mM during centrifugation with no effect on lysozyme binding of
(each) dNTPs, 5 μL of zymoprepped DNA template, 2 mM the Fn3-displaying induced cells. Cells were incubated at
8-oxo-deoxyguanosine triphosphate (TriLink, San Diego, 25 °C for sufficient time to ensure that the approach to
CA), 2 mM 2'-deoxy-p-nucleoside-5'-triphosphate (Tri- equilibrium was at least 98% complete. Cells were then
Link), and 2.5 U of Taq DNA polymerase (Invitrogen). pelleted, washed with 1 mL PBSA, and incubated in PBSA
In parallel, error-prone PCR of the loop regions was with 10 mg/L streptavidin–R-phycoerythrin for 10–30 min.
performed via three separate 50 μL reactions with Cells were washed and resuspended with PBSA and
20 mM 8-oxo-deoxyguanosine triphosphate and 20 mM analyzed with an Epics XL flow cytometer. The minimum
2'-deoxy-p-nucleoside-5'-triphosphate and primers BC5 and maximum fluorescence and the Kd value were
and BC3 for the BC loop, DE5 and DE3 for the DE loop, determined by minimizing the sum of squared errors.
and FG5 and FG3 for the FG loop. The reaction mixtures For determination of the dissociation constant, koff,
were denatured at 94 °C for 3 min, cycled 15 times at 94 °C clonal cell cultures were grown, induced, and washed as
for 45 s, 60 °C for 30 s, and 72 °C for 90 s, and finally above. Cells were incubated in PBSA with a saturating
extended at 72 °C for 10 min. Multiple preliminary concentration of biotinylated lysozyme at 25 °C. At various
mutagenesis reactions of the wild-type plasmid were times, an aliquot of cells was washed with PBSA with
conducted at different nucleotide analog concentrations. excess unbiotinylated lysozyme, resuspended in PBSA
Sequence analysis and comparison to a theoretical with excess unbiotinylated lysozyme, and incubated at
framework30 indicated the aforementioned conditions 25 °C. Simultaneously, all samples of differing dissociation
yield one to five amino acid mutations per gene. The PCR times were washed with PBSA and incubated in 10 mg/L
products were purified by agarose gel electrophoresis and streptavidin–R-phycoerythrin for 10–30 min. Cells were
each amplified in four 100-μL PCR reactions containing 1× washed and resuspended with PBSA and analyzed with an
Taq buffer, 2 mM MgCl2, 1 μM of each primer, 0.2 mM (each) Epics XL flow cytometer. The minimum and maximum
dNTPs, 4 μL of error-prone PCR product (of 40 μL from gel fluorescence and the koff value were determined by
extraction), and 2.5 U of Taq DNA polymerase. The reactions minimizing the sum of squared errors.
were thermally cycled at the same conditions except that 35 For determination of the association constants, kon,
cycles were used. Reaction products were concentrated with clonal cell cultures were grown, induced, and washed as
PelletPaint (Novagen) and resuspended in 1 μL of water. above. At various times, an aliquot of cells was resus-
Plasmid pCT-Fn3 was digested with PstI, BtgI, and pended in biotinylated lysozyme and incubated at 25 °C.
BamHI to create linearized vector pCT-Fn3-Gene with the Simultaneously, all samples of differing association times
entire Fn3 gene removed. Plasmid pCT-Fn3 was digested were washed with PBSA with excess unbiotinylated
with BclI, BtgI, and PasI to create linearized vector pCT- lysozyme and incubated in PBSA with 10 mg/L strepta-
Fn3-Loop with the wild-type gene removed from the BC vidin–R-phycoerythrin for 10–30 min. Cells were washed
loop through the FG loop. and resuspended with PBSA and analyzed with an Epics
Electrocompetent EBY100 (100 μL) was combined with XL flow cytometer. The maximum fluorescence and kon
0.5–2.0 μg of pCT-Fn3-Gene and the gene-based PCR were determined by minimizing the sum of squared errors
product and electroporated at 0.54 kV and 25 μF in a 2 mM assuming a 1:1 binding model. The experimentally
electroporation cuvette. Similarly, 100 μL of electrocompe- determined value of koff was used to determine the
tent EBY100 was combined with 0.5–2.0 μg of pCT-Fn3- effective association rate, kon[lysozyme] + koff.
Loop and the three loop-based PCR products and electro- The equilibrium dissociation constant was also deter-
porated. Homologous recombination of the linearized mined for the soluble forms of L7.5.1 and Cons0.4.1 by
vector and mutagenized insert(s) yielded intact plasmid. equilibrium competition titration. Varying concentration of
Cells were grown in YPD for 1 h at 30 °C, 250 rpm. The purified Fn3 domains were incubated with 20 pM biotiny-
medium was switched to SD-CAA to enable selective lated lysozyme in 50 mL of PBSA. Yeast displaying L7.5.1
propagation of successful transformants via growth at were added and incubated for 7 days to near equilibrium.
30 °C, 250 rpm, for 24–48 h. Cells were then pelleted, washed with 1 mL PBSA, and
incubated in PBSA with 10 mg/L streptavidin–R-phycoer- two PCR products created with a gene-terminal primer
ythrin for 15 min. Cells were washed and resuspended with and a primer that annealed adjacent to the loop of interest
PBSA and analyzed with an Epics XL flow cytometer. A but was extended to include wild-type sequence. Specifi-
two-state binding model was assumed and the minimum cally, one PCR reaction contained a 5′ gene terminal primer
and maximum fluorescence and equilibrium dissociation and a primer that annealed to the 25 nucleotides imme-
constant were determined by minimizing the sum of diately 5′ of the loop of interest but included a nonanneal-
squared errors. ing ‘tail’ encoding for the wild-type loop sequence. In
parallel, PCR was performed with a 3′ gene terminal
primer and a primer annealing to the 25 nucleotides
Fn3 production and biophysical characterization immediately 3′ of the loop and including a nonannealing
tail. The first PCR product encodes from the start of the
Fn3 clones were produced as previously described.6 gene to the loop of interest and the second PCR product
Briefly, BL21(DE3)pLysS E. coli (Invitrogen) containing the encodes from the loop of interest to the end of the gene.
pET-24b-based Fn3 plasmid were grown in Luria–Bertani These two products are annealed and extended to yield the
medium with 50 μg/mL kanamycin and 34 μg/mL full Fn3 gene containing the wild-type sequence in the loop
chloramphenicol at 37 °C, 250 rpm, to an A600 of 0.1–0.2 of interest. Framework reversions were introduced by
and induced with 0.5 mM IPTG for 18–24 h at 30 °C, standard site-directed mutagenesis using the QuikChange
250 rpm. Cells were lysed by sonication and the insoluble Mutagenesis Kit (Stratagene) according to the manufac-
fraction was removed by centrifugation at 19,000g for turer's instructions. Clone construction was verified by
40 min. His6-tagged Fn3 was purified from the soluble DNA sequencing.
fraction with TALON Superflow Metal Affinity Resin
(Clontech), dialyzed against PBS, and concentrated to
0.5 mL with an Amicon Ultra centrifugal filter (Millipore). Focused library construction
The oligomeric state was analyzed by size-exclusion
chromatography on a Superdex 75 HR10/300 column The DE randomization library was created in a manner
(Amersham Pharmacia Biotech, Piscataway, NJ). Mono- similar to that of the L7.5.1 loop reversion clones. One PCR
mer was isolated for biophysical analysis. PBS standards or amplified the L7.5.1 S15P gene fragment 5′ of the DNA
monomeric protein in PBS was thermally denatured from encoding for the DE loop. A second PCR amplified the
25 to 95 °C at a rate of 1 °C/min in a differential scanning gene 3′ of the DNA encoding the DE loop using a primer
calorimeter (VP-DSC, MicroCal). Irreversible aggregation that included a degenerate (NNB) DE loop sequence and
of L7.5.1 occurs at high temperatures. The midpoint of 20 nucleotides of overlap with the other PCR product. The
thermal denaturation for this clone is identified as the PCR products were annealed, extended to produce the full
temperature of maximum heat capacity. Samples were gene, and amplified. This process was conducted inde-
dialyzed in 10 mM sodium phosphate buffer, pH 7.0, and pendently with four oligonucleotides encoding the four
diluted to 8–10 μM for far-UV circular dichroism analysis. different DE loop lengths. The gene fragments were
Ellipticity was measured from 250 to 190 nm on an Aviv electroporated into electrocompetent EBY100 along with
202 spectrometer (Aviv Biomedical, Lakewood, NJ) with a pCT-Fn3-Loop vector. The resulting library encoded for
quartz cuvette with a 1-mm path length (New Era, Vine- L7.5.1 S15P with a fully random DE loop of length 4, 5, 6, or
land, NJ). Thermal denaturation was conducted by mea- 7 amino acids.
suring ellipticity at 216 nm from 25 to 95 °C and calculating A library randomizing the unconserved residues of clones
Tm from a standard two-state unfolding curve. similar to L7.5.1 was constructed by PCR of L7.5.1 S15P. The
Thermal stabilities were also determined using a yeast 5′ primer contained 17 nucleotides 5′ of the BC loop, 21
surface display thermal denaturation assay derived from nucleotides to encode the BC loop, and 22 nucleotides to
Orr et al.31 Fn3 was displayed on the yeast surface as for anneal 3′ of the BC loop. The 3′ primer contained 19
measurement of kinetic and equilibrium binding con- nucleotides 3′ of the FG loop, 30 nucleotides to encode for
stants. Cells were washed and resuspended with PBSA, the FG loop, and 10 nucleotides 5′ of the FG loop (note that
incubated at 20–85 °C for 30 min, and incubated on ice for the nucleotides encoding the first three conserved amino
5 min. Biotinylated lysozyme was added at a saturating acids of the FG loop also enable annealing during PCR). The
concentration (e.g., 20 nM for L7.5.1) and mouse anti-c-myc PCR products were amplified with extended primers to
antibody was added at 40 mg/L and incubated on ice for increase the length of the conserved sequence flanking the
20 min. Cells were washed and incubated in PBSA with loops to improve homologous recombination. The gene
10 mg/L streptavidin–R-phycoerythrin and 25 mg/L fragments were electroporated into electrocompetent
AlexaFluor488 conjugated goat anti-mouse antibody. EBY100 along with pCT-Fn3-Loop vector. Two versions of
Cells were washed and resuspended in PBSA and ana- the BC and FG loops were included. One oligonucleotide
lyzed on an Epics XL flow cytometer. The minimum and completely randomized the unconserved residues using
maximum fluorescence (Fmin and Fmax, respectively), the NNB degeneracy (BC, RXXPWAX; FG, RVGRXXXXXG).
T1/2, and the enthalpy of unfolding at T1/2 (ΔHm) were The other oligonucleotide restricted diversity to amino acids
determined by minimizing the sum of squared errors observed during affinity maturation [BC, R(D/G)(C/H/R/
between experimental data and theoretical values accord- Y)PWA(I/T); FG, RVG(R/W)(A/M/T/V)(F/L/P/S)(C/D/
ing to a two-state unfolding equation: G/Y)(A/T)(L/P/S)(G/S)].
1
F Fmin DHm 1 1
¼ ffolded ¼ 1 þ exp
Fmax Fmin R T1=2 T
Acknowledgements
L7.5.1 reversion clone construction
The research was funded by CA96504, CA101830,
Reversion of engineered loops of L7.5.1 to wild-type a National Defense Science and Engineering Grad-
sequence was accomplished by annealing and extending uate Fellowship and a National Science Foundation
Graduate Fellowship. Assistance from the MIT ranta, P. (1999). Expanding the conformational diver-
Flow Cytometry Core Facility is greatly appre- sity by random insertions to CDRH2 results in
ciated. The Biophysical Instrumentation Facility for improved anti-estradiol antibodies. J. Mol. Biol. 291,
the Study of Complex Macromolecular Systems 589–602.
13. Lee, C. V., Liang, W. C., Dennis, M. S., Eigenbrot, C.,
(NSF-0070319 and NIH GM68762) is gratefully
Sidhu, S. S. & Fuh, G. (2004). High-affinity human
acknowledged. antibodies from phage-displayed synthetic fab lib-
raries with a single framework scaffold. J. Mol. Biol.
340, 1073–1093.
Supplementary Data 14. Batori, V., Koide, A. & Koide, S. (2002). Exploring the
potential of the monobody scaffold: effects of loop
Supplementary data associated with this article elongation on the stability of a fibronectin type III
can be found, in the online version, at doi:10.1016/ domain. Protein Eng. 15, 1015–1020.
j.jmb.2008.06.051 15. VanAntwerp, J. J. & Wittrup, K. D. (2000). Fine affinity
discrimination by yeast surface display and flow
cytometry. Biotechnol. Prog. 16, 31–37.
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Picomolar sensitivity MRI and photoacoustic imaging
of cobalt nanoparticles
Louis-S. Boucharda,1, M. Sabieh Anwarb,1, Gang L. Liuc,2, Byron Hannc, Z. Harry Xied, Joe W. Grayc, Xueding Wange,1,
Alexander Pinesf,g,1, and Fanqing Frank Chenc,h,i,1
aDepartment of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; bSchool of Science and Engineering, Lahore University of
Management Sciences, Opposite Sector U, D.H.A. Lahore 54792, Pakistan; cComprehensive Cancer Center, University of California, San Francisco, CA 94143;
dMinispec Division, Bruker Optics, Inc., 2700 North Crescent Ridge Drive, The Woodlands, TX 77381; eDepartment of Radiology, University of Michigan,
Ann Arbor, MI 48109-0553; fCollege of Chemistry, University of California, Berkeley, CA 94720; gMaterials Sciences Division and hLife Sciences Division,
Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and iZhejiang California Nanosystems Institute, Zhejiang University, Hangzhou 310029,
People’s Republic of China
Contributed by Alexander Pines, December 22, 2008 (sent for review November 6, 2008)
Multimodality imaging based on complementary detection principles spatial resolution of PAT is not limited by optical diffusion, but
has broad clinical applications and promises to improve the accuracy instead by the bandwidth of the acoustic detectors. It has been
of medical diagnosis. This means that a tracer particle advantageously shown that PAT can depict subsurface tissue structures and func-
incorporates multiple functionalities into a single delivery vehicle. In tional changes noninvasively with resolution up to 100 ␮m (14, 15).
the present work, we explore a unique combination of MRI and Like other optical modalities, PAT is highly sensitive in mapping
photoacoustic tomography (PAT) to detect picomolar concentrations and quantifying the dynamic distribution of optical contrast agents
of nanoparticles. The nanoconstruct consists of ferromagnetic (Co) such as metallic nanocolloids and organic dyes (16–19, 27).
particles coated with gold (Au) for biocompatibility and a unique
MEDICAL SCIENCES
In the present work, we have fabricated a composite-material
shape that enables optical absorption over a broad range of frequen- nanoparticle, which we call ‘‘nanowonton.’’ The nanowonton has a
cies. The end result is a dual-modality probe useful for the detection Co core and an Au thin-film coating and is a construct similar to the
of trace amounts of nanoparticles in biological tissues, in which MRI Chinese eatable called the wonton (see Fig. 1 and Fig. S2). The
provides volume detection, whereas PAT performs edge detection. nanowontons have been characterized by scanning and transmis-
sion electron microscopies, absorption spectroscopy, and NMR
dual-modality imaging 兩 ferromagnetic nanoparticle 兩 relaxometry (Fig. 2). The nanowonton is shown to exhibit a
molecular imaging 兩 MRI contrast 兩 photoacoustic tomography combination of ferromagnetic and optical responses (Fig. 2), mak-
ing it amenable to dual-modality MRI and PAT studies. NMR T2
CHEMISTRY
relaxivity measurements reveal a per-particle relaxivity of 1 ⫻ 107
W e have synthesized nanoparticles for dual-modality (1–7)
MRI and photoacoustic tomography (PAT). The incorpo-
ration of MRI and PAT into a single probe offers the unique
s⫺1mM⫺1 (Table 1).
Previously, the oxidation-induced instability and toxicity of Co
possibility of combining the complementary strategies of contrast- nanoparticles have prohibited their wide use as MRI contrast
based volume imaging and edge detection. Our nanoconstruct agents, but in the present case, the Au coating circumvents this
consists of zero-valence ferromagnetic cobalt (Co) particles (8) with issue. Furthermore, the shape and thickness of the Au capping layer
a gold (Au) coating for biocompatibility and a unique shape are designed so that the center of its optical absorption range
rendering increased optical absorption over a broad range of matches the near infrared laser excitation wavelength used in PAT
frequencies (9–19). This research theme follows the rapid devel- imaging (700 nm) optimizing the photothermal response. We have
opments in nanotechnology, diagnostic radiology, and targeted also reported the geometry-dependent optical absorption for sim-
molecular imaging (20), whereby nanoparticulate contrast agents, ilarly shaped nanostructures such as nanocrescents (28). The
with the desirable properties of high chemical specificity, biocom- nanowonton design provides wavelength tunability for PAT (Fig.
patibility, and a reasonable half-life, are administered within a S3) and can be further improved through control of the fabrication
specific region of interest. In nanoparticle-based imaging studies, procedure.
higher particle concentrations lead to better signal-to-noise con- The PAT imaging contrast is demonstrated in Fig. 3A, for an
trasts, but this also poses a tradeoff with the toxicity. Therefore, one porcine gel containing several inclusions of different nanowonton
of the most important parameters when developing particle-based concentrations. The inclusion with a nanowonton concentration of
contrast is the safest and lowest nanoparticle concentration that 13 pM can hardly be recognized from the background, leading us
offers sufficient contrast sensitivity. to conclude that this PAT system has a detection sensitivity of the
In MRI, magnetic materials such as gadolinium chelates and order of 25 pM. Spin echo MRI images of Co nanowonton agarose
magnetic nanoparticles are often used (21–23) to enhance image gel phantoms A and B are shown in Fig. 4 A and B. Spin echo images
contrast. The magnetic nanoparticles are passivated by biocompat- show that higher concentrations lead to shorter T2 values for the
ible coatings such as dextrin, citrate, polystyrene/divinylbenzene, water protons. The smallest detectable concentration is 2.5 pM and
and elemental gold. These coatings also detoxify the particles,
resulting in enhanced lifetimes in vivo. Typical examples of mag-
netic nanoparticulate core-shell configurations include magnetite– Author contributions: L.-S.B., M.S.A., G.L.L., X.W., A.P., and F.F.C. designed research; L.-S.B.,
dextrin, magnetite–silica (24) and iron–gold (25). M.S.A., G.L.L., B.H., Z.H.X., and X.W. performed research; A.P. contributed new reagents/
analytic tools; L.-S.B., M.S.A., G.L.L., and X.W. analyzed data; and L.-S.B., M.S.A., X.W., and
Laser-based PAT (9–19) is a hybrid imaging modality [see F.F.C. wrote the paper.
supporting information (SI) Fig. S1]. It uses a pulsed laser source
The authors declare no conflict of interest.
to illuminate a biological sample. Light absorption by the tissue
1Towhom correspondence may be addressed. E-mail: bouchard@chem.ucla.edu,
results in a transient temperature rise on the order of 10 mK. The xdwang@umich.edu, sabieh@1ums.edu.pk, pines@berkeley.edu, or f㛭chen@lbl.gov.
rapid thermoelastic expansion excites ultrasonic waves that are 2Present address: Department of Electrical and Computer Engineering, University of Illinois at
measured by using broadband ultrasonic transducers conformally Urbana-Champaign, 3104 Micro and Nanotechnology Laboratory, 208 North Wright Street,
arranged around the sample. Finally, a modified back-projection MC-249, Urbana, IL 61801.
reconstruction algorithm (26) is used to construct a map of the This article contains supporting information online at www.pnas.org/cgi/content/full/
distribution of the optical energy deposition within the sample. The 0813019106/DCSupplemental.
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0813019106 PNAS 兩 March 17, 2009 兩 vol. 106 兩 no. 11 兩 4085– 4089
PBS shows no such contrast enhancement. In Fig. S4, PAT images
acquired before and after a rat tail injection show the type of
contrast enhancement which can be expected from a local injection
of 100 pM contrast agent. Because of high-frequency ultrasound
detection, the PAT modality is generally better suited at delineating
edges at the location of the contrast. These nanowonton particles
advantageously combine the strengths of both MRI and PAT
modalities into a single delivery vehicle.
This dual-modality PAT/MRI contrast agent demonstrates, so
far, the most sensitive detection experiment of magnetic nanopar-
ticles with particle concentrations in the picomolar and tens of
picomolars range. The particles may even be used for stand-alone
MRI or PAT. For example, in the MRI studies, the T2 contrast is
clearly visible to concentrations as low as 2.5 pM in phantoms and
50 pM in tissues. These detection thresholds are 7 orders of
magnitude better than those demonstrated by Lu et al. (23) for
monocrystalline iron oxide particles. Our T2 relaxivity (see Table 1)
per-particle concentration is 5 orders of magnitude better than the
cited work. The particle relaxivity and T2-weighted MRI detection
threshold are also better than those demonstrated by Cho et al. (25)
for 18 nm-diameter Fe/Au nanoparticles. This degree of sensitivity
is, to our knowledge, unprecedented and compares with sensitivities
Fig. 1. Fabrication procedure of the nanowontons including 6 steps: (1) Etching
polysilicon nanopillars on the surface of single crystalline silicon wafer (for simpler
approaching those of radioactive labels. This improved perfor-
presentation, we omitted from the illustration the preparatory step of depositing mance is in large part contributed by our choice of a ferromagnetic
5 nm of chromium before going to step 2, see Methods); (2) deposition of a 10-nm material, cobalt, which has a saturation magnetization 3.42 times
gold thin film; (3) deposition of 10-nm cobalt thin film; (4) deposition of 10-nm larger than magnetite, leading to a per-particle relaxivity that is
gold thin film; (5) etching polysilicon nanopillars in KOH batch solution; and (6) nearly 12 times larger. Because T2-weighted MRI depends expo-
complete removal of polysilicon and chromium by KOH etching and separation of nentially on the relaxation rate, this leads to a substantial difference
nanowontons. in contrast observed in our experiments. We refer the reader to the
SI section for a more extensive discussion of relaxivity effects,
including a comparison with results from other agents reported in
the contrast with respect to 5 pM is also clearly visible in Fig. 4 the literature.
B and D. The highly stable, thin (10 nm) film (Au) coating provides
A T2-weighted spin echo image from a slice through the mouse’s biocompatibility, as demonstrated by experimental results (Fig. S5).
leg muscles is shown in Fig. 5. The injection of PBS-buffered Co Furthermore, the Au thin film deposition process can be well
nanoparticles at 50 pM concentration results in a substantial drop controlled to allow tunable absorption spectra, allowing PAT at
in the MR signal in this region whereas the control injection with different optical wavelengths (see Fig. S3). It has been demon-
Fig. 2. Characterization of the cobalt nanowontons.

(A) Scanning electron microscopy image of
nanowontons. (B) Transmission electron microscopy im-
age of 3 nanowontons in various diameters; notice that
the lighter regions in the nanoparticle are relatively hol-
low, and are responsible for photothermal tuning prop-
erties of the nanowonton (38). (C) Particle diameter dis-
tribution of 150 nanowontons. Because of the
inhomogeneous polysilicon nanopillar diameter, the size
of the nanowontons varies from 30 to 90 nm, and the
average diameter is 60 nm. (D) Absorption spectrum of
nanowonton, medium peak wavelength is ⬇700 nm. (E)
Spin–spin relaxation time T2 measured at 20 MHz proton
frequency and 37 °C. T2 begins to change when the con-
centration exceeds 20 pM. Note that although 20 MHz is
much less than 300 MHz (used for the MRI), this gives a
lower bound on relaxivity and shows that the contrast
works even at low fields, such as those from portable
NMR devices.
4086 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0813019106 Bouchard et al.

Table 1. T2 relaxivity per particle concentration is calculated Methods
to be 1 ⴛ 107 sⴚ1mMⴚ1 For additional information on materials and methods used, see SI Text.
Sample no. Conc, pM T2, ms Conc, mM 1/T2, s⫺1
Fabrication of the Nanowontons. The schematic diagram of the fabrication
1 1,000 123 0.000001 8.1301 procedure is illustrated in Fig. 1. First, a batch-fabricated vertical silicon nanopillar
2 800 124 0.0000008 8.0645 array was fabricated on the surface of a 4-inch diameter silicon wafer. The
3 600 163 0.0000006 6.1350 coverage of the nanopillar structure was ⬎90% of the total wafer surface area.
4 500 204 0.0000005 4.9020 On the top of each silicon nanopillar, there was a spherical silicon oxide nano-
structure. Four metallic layers of 5-nm chromium, 10-nm gold, 10-nm cobalt, and
5 400 239 0.0000004 4.1841
10-nm gold were sequentially deposited on the wafer surface. However, after
6 300 333 0.0000003 3.0030
deposition, the sidewalls of all of the nanopillars remained exposed. The silicon
7 200 451 0.0000002 2.2173 wafer was therefore immersed in a 10% KOH bath solution at 80 °C, etching away
8 100 828 0.0000001 1.2077 the nanopillars from the unprotected sidewalls in 10 min, The multilayer metallic
9 50 1384 0.00000005 0.7225 nanostructure on the top of the nanopillars was lifted off and suspended in the
10 25 1788 0.000000025 0.5593 KOH bath solution. Because silicon oxide and chromium were also etched away
11 10 2250 0.00000001 0.4444 by KOH, only the gold– cobalt– gold sandwich nanostructures, the nanowontons,
12 5 2258 0.000000005 0.4429 remained in solution. These were finally separated by centrifugation. The SEM,
13 2 2268 0.000000002 0.4409 TEM, and size distribution measurements are shown in Fig. 2 A–C. After fabrica-
tion, these samples were chemically analyzed by inductively coupled plasma mass
14 1 2270 0.000000001 0.4405
spectrometry (ICP-MS), measuring the total amount of Co or Au ions. By assuming
The T2 reaches saturation point above 800 pM, so the 1,000-pM data point bulk parameters of the materials and the size of the nanoparticles, we deduced
was excluded from the fit to obtain linearity for calculating 1/T2. the mass of Co and Au per nanoparticle, and calculated the nanoparticle
concentration.
strated that a variety of gold nanocolloids are already entering in PAT System. The PAT system is schematically shown in Fig. S1. An OPO (Vibrant
MEDICAL SCIENCES
B; Opotek) pumped by an Nd/YAG laser (Brilliant B; Bigsky) was used to provide
vivo clinical trials (19, 29–33). Among them, Au nanorods present
laser pulses with a repetition rate of 10 Hz and a pulse width of 5 ns. In this study,
particularly good optical absorption in the near-infrared region, the wavelength of the laser light was tuned to 700 nm, which was in the
tunable by changing the aspect ratio. It has already been demon- near-infrared region and enabled good penetration in biological tissues. The
strated that gold nanorod contrast agents can be imaged with PAT, laser beam, after being expanded and homogenized, illuminated the imaged
both ex and in vivo (34, 35). Our study has shown that the sensitivity sample with an input energy density of ⬇10 mJ/cm2, well below the American
of PAT in imaging the nanowonton is equivalent to that for gold National Standards Institute safety limit of 22 mJ/cm2 at the applied wavelength.
The laser light penetrated into the sample and generated photoacoustic signals
nanorods. In fact, the MRI contrast is also expected to be strongly
that were scanned by an ultrasonic transducer (XMS-310; Panametrics) with a
dependent on the shape of the nanoconstruct. It is envisaged that center frequency at 10 MHz and a receiving bandwidth of 100%. To realize 2D
CHEMISTRY
nanorods or needle-shaped structures can elicit greater contrast cross-sectional imaging, the sample was rotated axially in the xy plane while the
because of larger shape-induced susceptibility gradients. The transducer and the laser beam were kept static. To couple the signals, the sample
present nanowonton shape is, to first order, a compromise between and the transducer were immersed in water. After a preamplifier (PR5072;
optical and magnetic responses. However, further work on shape Panametrics), the detected signals were digitized by an oscilloscope (TDS 540B;
Tektronics) and then collected by a computer. The current PAT system exhibits
optimization will be required to conclusively comment on this.
spatial resolution of 200 ␮m in the xy plane, which has been verified by measuring
Furthermore, the Au sandwich structure also allows additional the line spread function (LSF) (40).
tuning of absorbed wavelengths (28, 36, 37). This can further
improve the sensitivity of the PAT technique. Last, the Au coatings Photoacoustic Imaging on Phantoms. To demonstrate the PAT imaging contrast,
are especially attractive because of the possibility of conjugating the we constructed a phantom made of 5% porcine gel in which 4 inclusions with
particles with specific molecules such as antibodies, specific ligands, different concentrations of nanowontons are embedded (Fig. 3). Cylindrical-
thiol functional groups and therapeutic drugs, opening up prospects shaped phantoms with a 20-mm diameter were made from porcine gel. Spher-
ical-shaped droplets with a size of 2.8 mm were made with the same gel and
for targeted molecular imaging (20). An additional imaging mo- contained different concentrations of the contrast agent. These droplets were
dality built into our nanoconstruct is the optical thermal conversion embedded 1 cm deep in the phantoms. For example, the phantom shown in Fig.
capability making these structures highly suited for photothermal 3 contains 4 such embedded droplets, where the nanowonton concentrations
therapy (32, 38–41). were 100, 50, 25, and 13 pM. The corresponding PAT image is shown in Fig. 3A,
Fig. 3. Photoacoustic imaging of nanowonton phan-

tom gels. (A) PAT image of 4 absorbing objects contain-
ing nanowonton contrast agent embedded in a gel
phantom (5% agarose). The concentrations of
nanowontons were 100, 50, 25, and 13 pM, respectively,
for objects A, B, C, and D. (B) Intensity profiles extracted
from the image along 4 lines (horizontal and vertical
dashed lines indicated on the image) going through the
absorbing centers are plotted to highlight the visibility of
nanowonton inclusions in the reconstructed image. With
a CNR close to 1, the object D, where the nanowonton
concentration is 13 pM, can hardly be recognized from
the background, showing that the current PAT system
has detection sensitivity on the order of 25 pM.
Bouchard et al. PNAS 兩 March 17, 2009 兩 vol. 106 兩 no. 11 兩 4087
Fig. 4. MRI of nanowonton gel phantoms. The nanowonton gels are arranged along the perimeter of a circle. (A and B) Spin-echo images for the phantoms A and
B, respectively, with an echo time (TE) of 50 ms and recycle time (TR) of 1 s. The higher-concentration samples appear darker in the images, with doped water used as
a control and exhibiting the strongest T2-weighted intensity. The concentrations of the gels are given in the figure along with the T2 values that are deduced from a
7-point curve-fitting procedure. The field of view for this image is 3 ⫻ 3 cm, the number of points is 256 ⫻ 128, and the slice thickness is 1 mm. (C and D) Intensity profiles
for the images in A and B. The relative intensities along a circular contour drawn through the middle of the gels are plotted as a function of the gel azimuthal angle
from the x axis. The nanoparticles contrast remains detectable down to 2.5 pM. The images for phantom A and B are plotted to different (normalized) scales.
the locations of the objects being marked with dashed circles. In Fig. 3A, we have
also quantified the contrast-to-noise ratio (CNR) ⫽ (So ⫺ Sb)/␴, where So is the
average intensity within the object, Sb is the average intensity within the back-
ground defined by the mean of all of the pixels in the image beyond the big
dashed circle (i.e., the area out of the gel phantom), and ␴ is the standard
deviation. The computed CNRs for the objects A, B, C, and D are 10.6, 5.7, 2.1, and
1.0. It is clear that the objects A, B, and C have been imaged with sufficient optical
absorption contrast.
MRI of the Phantoms. For the phantom MRI studies, 6 holes were drilled into a (⬇3
cm diameter) Teflon cylinder, azimuthally distributed around the center. The
phantom is shown in Fig. 4. Each hole was 5 mm in diameter and ⬇1 cm deep. Two
similar pieces (A and B) were machined. Nanocolloidal solutions of the
nanowonton in (5%) agarose gel were prepared in concentrations of 500, 375,
250, 125, 50, 12.5, 5, and 2.5 pM. The agarose gel was heated until it became
transparent, and the nanoparticles were subsequently transferred to the hot
agarose, preparing the desired concentrations. Precise volumes of the nanocol-
loids were then slowly transferred to the cylindrical recesses and allowed to cool
in ambient conditions, ensuring that no air bubbles were formed during the
cooling. The solutions were intermittently pried to ensure that the distribution of
the nanowontons would be kept as uniform as possible. The tops and bottoms of
the phantoms were sealed with polystyrene to prevent leakage during phantom
handling. The phantom A contained the concentrations 500, 375, 250, 125, and
50 pM, whereas B contained 125, 12.5, 5, and 2.5 pM. The latter had 1 cylinder
empty, and each of the phantoms was also loaded with 0.6 mM MnCl2 doped
water to act as the control reference.
The MRI was performed in a 300-MHz NMR spectrometer (Varian Inova)
equipped with triple-axis magnetic field gradients. The phantoms were imaged
Fig. 5. Transverse (axial) MRI image in mouse leg muscle injected with Co by using a spin-echo pulse sequence, with slice selection along the z axis, phase
nanoparticles in PBS solution. The position of the blue arrows indicates the sites encoding along the y axis, and readout along the x axis. We used an echo time (TE)
of injection for the Co nanoparticles (upper right corner) and the PBS control of 50 ms and recycle time (TR) of 1 s. The field of view was 3 ⫻ 3 cm, the number
(lower left corner). Two water-carrying test tubes are also visible in the scans for of points was 256 ⫻ 128, and the slice thickness along the z direction was 1 mm.
purposes of MRI slice alignment (red arrows). MRI parameters were: TE ⫽ 50 ms; T2 measurements were also performed by repeating the spin echo sequence with
TR ⫽ 1 s; field of view is 2.6 cm ⫻ 2.6 cm, and slice thickness is 0.5 mm. varying TEs; the values used were 10, 30, 40, 50, 100, 150, and 200 ms.
4088 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0813019106 Bouchard et al.

MRI on Mouse Muscle. For the animal studies, the mouse was i.p. anesthetized ACKNOWLEDGMENTS. This work was supported by the Prostate Cancer Foun-
with 400 ␮L of Avertin. After 5 min, 50 ␮L of 60-nm-sized gold-coated Co dation and University of California, San Francisco (UCSF), Prostate Cancer
nanoparticles at 50 pM in a solution of PBS were intramuscularly injected into Specialized Program of Research Excellence (SPORE) ; Camille and Henry
the leg. As a control, PBS solution without the nanoparticles was also injected Dreyfus Foundation (L.-S.B.); National Natural Science Foundation of China
in the diametrically opposite position to the site of injection of the cobalt Grant NSFC-30828010, the Zhejiang California Nanosystem Institute, Depart-
ment of Defense Breast Cancer Research Program Grants BC045345 and
nanowonton. After an additional 10 min, the mouse was killed and placed into
BC061995, and University of California San Francisco Prostate Cancer SPORE
the vertical bore of the Varian 300-MHz NMR spectrometer. The mouse tail award (National Institutes of Health Grant P50 CA89520) (F.F.C.); Director,
was then imaged by using a T2-weighted spin-echo sequence. Multiple trans- Office of Science, Office of Basic Energy Sciences, Materials Sciences Division,
verse slices were imaged, the slice thickness (along the z direction) being 0.5 of the U.S. Department of Energy Contract DE-AC03-76SF00098; National
mm, the TR was 1 s, the number of points was 256 ⫻ 128, and the field of view Institutes of Health Grant R01 AR055179; and Department of Defense Prostate
was 2.6 ⫻ 2.6 cm. Cancer Research Program Grant W81X WH-07-1-0231.
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Current Nanoscience, 2005, 1, 47-64 47
Nanosystems in Drug Targeting: Opportunities and Challenges
Jaspreet K. Vasir1, Maram K. Reddy1 and Vinod D. Labhasetwar1,2,*
1
Department of Pharmaceutical Sciences, College of Pharmacy, 986025 Nebraska Medical Center, Omaha, NE 68198-
6025, USA, 2Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE
68198, USA
Abstract: The long cherished goal of targeting drugs to specific sites in the body, where the pharmacological action is
desired and sparing other tissues has been actively pursued all these years. The concept of ‘magic bullets’ given by
Ehrlich has now seen a metamorphosis to ‘magic wands’, in the form of targeted drug delivery systems. The magic, all
due to the specific targeting ligands which guide the drug carriers to the molecular targets be it on cell surface or nuclear
membranes. Nanosystems including the nano-sized (<1000 nm) drug carrier systems, such as polymeric nanoparticles,
liposomes, micelles and polymer-drug conjugates are the vanguards of this ever-evolving field. Targeting drugs to specific
sites, and maintaining pharmacologically relevant drug levels at the site for a period required for desired therapeutic action
is what makes the nanosystems – the burgeoning magic wands. Substantial challenges still exist in terms of biological
barriers. Nevertheless, the approaches like directly reaching the target using catheters, or using exogenous guiding
mechanisms (magnetic fields and ultrasound), and exploiting the accessible targets on vascular endothelium are emerging
as new and promising trends. It is conceivable, that despite all the formidable challenges, interplay of different disciplines
ranging from engineering to biology will make the dream of drug targeting come true!
Keywords: Nanoparticles, nanosystems, targeting, ligands, drug and gene therapy.
1. INTRODUCTION emerging targeting technologies such as transcriptional

targeting, physical targeting, targeting of vascular and
In the past few decades, rapid advances in cell and
intracellular targets.
molecular biology have allowed us to develop a better
understanding of the pathophysiology of various diseases.
2. BARRIERS TO DRUG TARGETING
This has cast a floodlight on the possible cellular and
molecular targets which can be exploited not only for drug Targeting of drugs offers enormous advantages but is
discovery but also for imaging and therapy. Further, equally challenging. A better understanding of the
integration of high throughput techniques and combinatorial physiological barriers which a drug needs to overcome
chemistry with biology has brought an explosive growth in should enable the pharmaceutical scientists to develop
the number of new drug molecules (in discovery pipeline) successful design of targeted drug delivery systems. Main
aimed to act on the identified targets. Therapeutic hurdles to drug targeting include physiological barriers,
armamentarium has thus seen an exponential growth and biochemical challenges to identify and validate the molecular
now ranges from low molecular weight drugs to targets and the pharmaceutical challenges to devise
macromolecules like proteins and plasmid DNA. However, appropriate techniques of conjugating targeting ligands to the
molecular complexity associated with drugs and nanosystems. The challenge in drug targeting is not only the
inaccessibility of most physiological targets presents the targeting of drug to a specific site but also retaining it for the
Holy Grail of drug delivery - to deliver these specific drugs desired duration to elicit pharmacological action. For a
to their site of action at therapeutically relevant levels. Thus, nanosystem administered intravenously, the first and
drug targeting has evolved as the most desirable but elusive foremost barrier is that of the vascular endothelium and the
goal in drug delivery science. It can potentially increase basement membrane [1]. Also, plasma proteins have the
efficacy and reduce toxicity of new and pre-existing drugs by ability to affect the biodistribution of drug carrier systems
altering their pharmacokinetics and biodistribution and introduced in the blood stream. The in vivo biodistribution
restricting the action of drugs to the treated tissue. The and opsonization of nanosystems in blood circulation is
purpose of this review is to present an overview of the governed by their size and surface characteristics. For the
barriers to drug targeting, various approaches designed to nanosystem to remain in blood circulation for a long time,
overcome these barriers and to focus on the development of the major problem is to avoid its opsonization and
nanosystems as potential carriers for drugs to enable site subsequent uptake by the phagocytic cells. The passage of
specific targeting (Fig. 1). Special mention is made for drug molecules and drug delivery systems across the
endothelium is sensitive to the molecular weight and size of
the system, respectively. The tight endothelial cells in brain
constitute the blood brain barrier (BBB), which restricts the
*Address correspondence to this author at the 986025 Nebraska Medical entry of most drugs and delivery systems. However vascular
Center, Omaha, NE 68198-6025, USA; Tel: (402) 559-9021; Fax: (402)
559-9543; E-mail: vlabhase@unmc.edu endothelium is not uniform throughout. The altered
1573-4137/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

48 Current Nanoscience, 2005, Vol. 1, No. 1 Labhasetwar et al.
Fig. (1). Schematic describing barriers to drug targeting and the role of nanosystems in overcoming these barriers.
endothelia in tumors allow an enhanced permeability to the physiological barriers, and for drug therapy, deliver the
macromolecules and the particulate drug delivery systems. pharmacological agent to its site of action at therapeutically
Another barrier is that of the extracellular matrix, which relevant drug levels for a time sufficient to allow therapeutic
should be crossed to access the target cells in a tissue. If the action. Conjugation of targeting ligands to drugs or drug
whole tissue constitutes a target then the uniform distribution carrier nanosystems is the most popular way of directing
of drug throughout the tissue is another problem [1]. them to their target sites. To this end, various techniques
Targeting nanosystems to specific receptors or antigens on have been devised, including covalent and non-covalent
the cell surface provides the driving force for diffusion of the conjugation [4]. The emphasis is that the ligand must be
system to the specific cells. attached stably and accessibly to the drug carrier, so that the
For drugs whose targets are located in the ligand is presented in its right orientation for binding to the
cytoplasm/nucleus of a cell, further barrier needs to be target receptors. For example, the monoclonal antibodies
crossed to allow internalization of nanosystems into specific must bind to the drug/its carrier with their F c part, so that
cells [2]. The barriers not end here, a number of endocytic their antigen binding site (F ab) is free to interact with the
pathways have been described for the cellular entry of antigenic targets on cells [5]. The coupling reactions must
nanosystems. Drugs/nanosystems need to diffuse through the not affect the biological activity of ligand and should not
viscous cytosol to access the particular cytoplasmic targets adversely affect the structure of drug delivery nanosystems.
where site of action is located. Nuclear membrane poses Further, such coupling reactions must be optimized so that
another formidable barrier for drugs such as oligo- binding of ligands takes place in a homogeneous manner on
nucleotides, plasmid DNA and other low molecular weight the surface of the drug carrier nanosystems.
drugs whose site of action is located in the nucleus of a cell.
3. APPROACHES TO DRUG TARGETING
Although a number of cellular and molecular targets are
emerging, the real problem lies with the poor accessibility of An ideal targeted drug delivery approach would not only
drugs/ nanosystems to the target tissue. The presence of such increase therapeutic efficacy of drugs but also decrease the
barriers leads to a poor in vitro/in vivo correlation when the toxicity associated with drug to allow lower doses of the
targeted delivery systems are tested in receptor bearing cells drug to be used in therapy. A vast array of methods, which
in vitro and fail in vivo [3]. Thus, to harness the potential of can further be classified into two key approaches – active
new targets in imaging and therapy, one would need to and passive have been explored for targeting drugs by means
develop targeted systems which can successfully overcome of designing innovative nanosystems (Table 1). Some of the
Nanosystems in Drug Targeting: Opportunities and Challenges Current Nanoscience, 2005, Vol. 1, No. 1 49
Table 1. Drug Targeting Approaches
PASSIVE TARGETING ACTIVE TARGETING
1. Pathophysiological factors 1. Biochemical targets

• Inflammation/infection • Organs
• EPR effect • Cellular
2. Physicochemical factors • Organelles
• Size • Intracellular
• Molecular weight 2. Physical/External stimuli
3. Anatomical opportunities • Ultrasound
• Catheterization • Magnetic field
• Direct injection 3. Pretargeting/Sandwich targeting
4. Chemical approaches 4. Promoter/Transcriptional targeting
• Prodrugs
• Chemical delivery systems
methods like catheterization, direct injection, prodrugs or blood vessels, the vascular endothelium of angiogenic blood
chemical delivery systems have inherent capability to deliver vessels has large gaps (600-800 nm) in between the adjacent
drugs or their appropriate modifications to the specific site of endothelial cells [8]. This increased vascular permeability
action. However, others require design of a suitable carrier coupled with the impaired lymphatic drainage in tumors
system to be able to deliver the drug to its target site. allows an enhanced permeability and retention (EPR) effect
Following is a description of such approaches to drug of the nanosystems in the tumor [11, 12]. For passive tumor
targeting. accumulation using the EPR effect, the targeted system
should have long blood circulation time, should not lose drug
3.1. Passive Targeting Approaches activity while in circulation and the drug should stay with the
carrier until accumulation of drug at the target is attained.
Passive targeting refers to the accumulation of drug or Other factors which may influence tumor accumulation by
drug-carrier system at a particular site due to physico- the EPR effect include the degree of tumor
chemical or pharmacological factors [1]. Drug or drug carrier
vascularization/angiogenesis and the size of the delivery
nanosystems can be passively targeted making use of the system. Liposomes, polymeric nanoparticles, micellar
pathophysiological and anatomical opportunities. systems as well as polymeric-drug conjugates have been
successfully used to target drugs to tumor tissues in a passive
3.1.1. Pathophysiological Opportunities
manner. The EPR effect has also been explored to target
The physiology of diseased tissues may be altered in a drugs to the injured artery following balloon angioplasty to
variety of pathological conditions, and can be exploited for inhibit restenosis. Since the angioplasty disrupts the
passively targeting drugs. Release of various chemotactic endothelium of the target artery, it is more permeable to
factors from the infected/inflamed tissues results in vascular nanosystems than the normal artery [13].
remodeling to enable leukocyte extravasation and hence also
increases permeability for particulate drug carriers [6, 7]. 3.1.2. Anatomical Opportunities
Such pathophysiological opportunities include increased Drugs may be introduced into “discrete anatomical
vascular permeability in various inflammatory conditions
compartments” for example: lungs, knee joints, respiratory
which allows extravasation of the nanosystems and their tract, and eye by means of minimally invasive procedures,
selective localization in the inflamed tissue [8]. It was shown using catheters or direct injections at local sites. These
that polymeric nanoparticles administered per orally could be methods of site-specific localized drug delivery prevent
selectively targeted to the inflamed colonic mucosa in
unwanted systemic exposure of the drug and thus avoid
inflammatory bowel disease [9]. Similarly, the blood brain adverse effects of drugs in the non target tissues. This not
barrier can also be crossed to access the target sites for brain only minimizes the dose but also the cost of therapy. The
delivery in some inflammatory conditions [10]. above approach of overcoming biological barriers offers an
Solid tumors present much more favorable conditions for excellent method of localizing the cytotoxic action of
preferential accumulation of macromolecular drugs and antineoplastic agents to tumors and delivering drugs to
colloidal sized drug delivery systems like polymeric-drug inaccessible organs like brain.
conjugates, liposomes etc. Rapidly growing or metastasizing We have been studying the catheter based local delivery
tumors recruit new blood vessels through the process of
of poly-D,L-lactide- co-glycolide (PLGA) nanoparticles for
angiogenesis to meet the nutritional demands of increasing inhibiting restenosis. In restenosis or the reobstruction of an
number of cells. Unlike the tight endothelium of normal artery following an interventional procedure, the pathology
Fig. (2). Angiogram demonstrating infusion of nanoparticles in the target artery following angioplasty using a cardiac infusion catheter in a
porcine coronary model of restenosis. LAD: Left Anterior Descending coronary artery.
Fig. (3). Fluorescent PLGA nanoparticles were injected stereotactically into the brain through the striatum of the rat and the animals were
allowed to survive for 3 days post injection. Fig 3A is the digital camera view of a frozen section of the brain showing injection site and Fig
3B is the fluorescent low-magnification image of the injection site. Outlined area in Fig 3A represents the site of injection. Nanoparticles
were found to be spreading from the injection site to surrounding parenchyma. Scale bar in Fig 3A is 1 mm and in Fig 3B 100 µm.
is limited to the localized area of endothelial injury following and fluid-phase endocytosis, the transport of therapeutic
angioplasty procedures. We have demonstrated nanoparticle agents via systemic administration to the brain is limited [16,
localization in arterial tissue after injecting a suspension at 17]. To overcome these challenges, a promising approach
the site of injured artery using a cardiac infusion catheter could be the direct injection of therapeutic agent or a
(Fig. 2). Nanoparticles, due to their colloidal size, can nanosystem to the brain tissue using microinjection
penetrate the arterial wall through the disrupted epithelium techniques. This approach could be more effective in
and are immobilized in the arterial wall [14]. This allows targeted delivery to a certain area of the brain, however
sustained levels of the pharmacological agent at the localized would not be as effective if the whole brain is the target for
site. Using the above strategy, we have demonstrated 35% drug therapy. This is because of poor diffusion of therapeutic
inhibition of restenosis in a rat carotid model with a single agents through the brain tissue. To improve diffusion of the
localized intraluminal delivery of dexamethasone-loaded injected drug, some of the strategies include conjugating the
nanoparticles. Dexamethasone levels were sustained for therapeutic proteins with non-toxic neuronal binding domain
more than one week in the infused section of artery and not of tetanus toxin (tetanus toxin fragment C-TTC). Proteins
in the adjacent carotid artery; presenting nanoparticles as linked to TTC not only show a better retention but also a
localized, sustained drug delivery approach for restenosis. superior distribution in the extracellular space on direct
Blood brain barrier (BBB), which is formed by the tight injection into brain [18]. In our studies, we are exploring the
junctions within the capillary endothelium of the brain, strategy of directly injecting nanoparticles into the brain
constitutes a formidable barrier to the CNS delivery of (Fig. 3). The drug encapsulated into nanoparticles will be
protected and also will be released slowly, thus can be
therapeutic agents [15]. Although selective transport
mechanisms are present in the BBB such as diffusion, effective in providing targeted and sustained drug delivery.
carrier-mediated transport, receptor-mediated, adsorptive, In a preliminary study, we have demonstrated the feasibility
of the above approach and the injected nanoparticles are seen targeting employs specific modification of a drug/drug-
to distribute through the brain parenchyma. It would be carrier nanosystems with “active” agents having selective
interesting to determine in the future studies, how effective affinity for recognizing and interacting with a specific cell,
are these nanoparticles in drug distribution in the brain and tissue or organ in the body [23]. Direct coupling of drugs to
sustaining the drug retention and effect. targeting ligand, restricts the coupling capacity to a few drug
molecules. In contrast, coupling of drug carrier nanosystems
3.1.3. Physicochemical Factors to ligands allows import of thousands of drug molecules by
means of one receptor targeted ligand.
The clearance kinetics and in vivo biodistribution of
nanosystems depend on the physicochemical factors like Drug targeting to specific cells has been explored
size, surface charge and surface hydrophobicity and can be utilizing the presence of various receptors, antigens/proteins
manipulated to enable passive targeting [19]. A major part on the plasma membrane of cells and also by virtue of the
(~90%) of the nanosystems injected intravenously generally lipid components of the cell membranes. The receptors and
is lost to the reticulo-endothelial system (RES), mainly fixed surface bound antigens may be expressed uniquely in
macrophages in the liver and spleen after opsonization by diseased cells only or may exhibit differentially higher
proteins present in the blood stream [20]. Particles < 100 nm expression in diseased cells as compared to the normal cells.
can pass through the fenestrations in the liver endothelium Active agents, such as ligands for the receptors and
and the sieve plates of sinusoids to localize in the spleen and antibodies to the surface proteins have been used extensively
bone marrow. This natural tendency of nanosystems to to target specific cells. The following section gives a brief
localize in the RES presents an excellent opportunity for overview of the targets and the ligands which can be used as
passive targeting of drugs to the macrophages present in the active targeting moieties to target nanosystems to specific
liver and the spleen. It has been utilized for targeting cells in the body.
antibiotics and antiviral agents for intracellular infections.
Nanoparticles are promising drug carrier systems for 3.2.1. Targets
antiparasitic drugs used in the treatment of leishmaniasis (A) Receptors
caused by an intracellular parasite- Leishmania donovani.
The binding of anti-leishmanial drugs to various kinds of The presence of receptors on cell membranes potentiates
nanoparticles was found to increase their therapeutic index active targeting by not only allowing specific interaction of
(by 5 times) due to passive targeting of the reticulo- drug carrier system with cells but also facilitating its uptake
endothelial system [21]. Acute lethal toxicity studies, via receptor mediated endocytosis [24].
performed in healthy mice after intravenous injection of free
primaquine and primaquine-loaded poly-lactic acid (PLA) (a) Folic Acid Receptor
nanoparticles, reported a two folds increase in the lethal dose
(LD50) with the nanoparticle formulation. Rapid clearance of Folic acid - a vitamin essential for de novo nucleotide
the drug from the blood stream due to the RES uptake synthesis is taken up by cells via receptor mediated
restricted the drug accumulation to liver and spleen only and endocytosis using membrane associated folate receptors.
thus was responsible for reducing the lethal toxicity of drug. Folate receptors are expressed only on certain epithelial cells
in humans, but are differentially over-expressed in cells of
Polymeric nanoparticles have also demonstrated cancers with epithelial origin. It has been primarily used for
significant promise for anti-HIV therapy by delivering the tumor specific drug delivery in many cancers including
anti-HIV drugs with poor selectivity and short plasma half- breast, ovary, brain and lung malignancies [25].
lives into the phagocytic cells. Polyacrylcyanoacrylate
(PACA) nanoparticles loaded with Saquinavir were found to (b) LDL Receptors
be effective in HIV- infected human macrophage culture
[22]. Large sized liposomes especially multi-lamellar Low density lipoprotein (LDL) receptors are a family of
vesicles (MLV) are cleared from the blood circulation by the nine endocytic receptors that transport cholesterol rich
RES system and have been explored to replace genetically lipoproteins (LDL) into cells via receptor mediated
deficient enzymes in these cells and to target antimicrobial endocytosis. Many drugs and some lipid-based systems such
agents to intracellular pathogens. as liposomes and cholesterol rich emulsions have been
proposed to interact with these receptors [26]. Anionic
Although the RES uptake of the colloidal sized liposomes [27] and apolipoproteinE (apoE) enriched
nanosystems holds promise for passive targeting of many liposomes [28] were found to mimic LDL and provided site
drugs; it becomes a major hurdle for drugs whose site of specific delivery of antitumor agents to cancer cells via the
action is located in tissues other than the RES. A variety of LDL receptors. B16 melanoma cell line showing higher
methods have been explored to overcome the rapid expression of LDL receptors was used to assess the specific
opsonization and the subsequent RES uptake of nanosystems binding affinity of apoE labeled liposomes in vitro.
and to make them long circulating for effective targeting of Moreover, increased uptake of liposomes was observed in
drugs to tissues other than the RES. liver and adrenals- organs showing relatively higher
expression of LDL receptors in normal rats pretreated with
3.2. Active Targeting Approaches 17-α-ethinyl estradiol (used to selectively upregulate LDL
Passive targeting approaches are limited in their scope receptors on liver and adrenals) (Fig. 4). This approach,
and thus, tremendous effort has been directed towards the however, is not very specific as LDL receptors are expressed
development of active approaches for drug targeting. Active on cells of almost all organs.
Fig. (4). Organ distribution of apoE-enriched liposomes in control and 17 α-EE-pretreated rats. [ 3H]CO-labeled liposomes (1.0 mg of
phospholipid) were injected after previous incubation (30 min at 37°) with 100 mg of apoE into anesthetized propylene glycol treated rats ( )
versus 17 α-EE-treated rats ( ). After 120 min of circulation, the total ( left) and specific (i.e., per gram wet weight) ( right) organ distri-
butions were determined. Recoveries of 3H-radioactivity in the rats exceeded 95%. Values are corrected for serum radioactivity and represent
mean ± standard error from three experiments. Reprinted with permission from ref [28].
(c) Peptide Receptors targeting, would be the one expressed exclusively by tumor
cells and similarly by all cells present in the tumor.
A large number of peptide receptors are expressed in
large quantities in certain tumor cells. Receptors for peptides Prostate specific antigen, differentially expressed on
such as somatostatin analogs, vasoactive intestinal peptide, prostate cancer cells, and erbB2- a growth factor that is over
gastrin related peptides, cholecystokinin, leutanising hor- expressed in 20-30% human breast adenocarcinomas, are
mone releasing hormone have been localized on tumor cells examples of tumor specific antigens. erbB2, expressed on the
[29]. Peptides/peptide analogs can be conjugated to a drug tumor cell surface is readily accessible, has low expression
carrier system to allow tumor specific targeting of cytotoxic on normal cells and shows a homogeneous distribution
agents, following interaction with peptide receptors. within the tumor. It has been used for immunotherapy with
liposomal doxorubicin formulation coupled to anti-erbB2-
(B) Lipid Components of Cell Membranes antibody [31]. A significant increase in the in vivo efficacy
was observed with anti-erbB2 immunoliposomes as
Lipid components of cellular membranes are emerging as compared to the unmodified liposomes and saline control in
novel targets for antineoplastic drugs [30]. Interaction of a human erbB2-overexpressing breast cancer model (Fig. 5).
synthetic phospholipid analogs with cellular membranes Immunotherapy of a non-uniformly distributed tumor
changes the lipid composition, membrane permeability and antigen in a tumor mass, may result in emergence of resistant
fluidity, thereby influencing signal transduction mechanisms cells- which do not express the specific antigens. Tumor
and inducing apoptotic cell death. Two such phospholipid specific antigens are the emerging and promising targets for
analogs- Edelfosine and Miltefosine can selectively kill successful and exquisitely specific tumor therapy. But,
malignant cells and thus offer promising approaches in clinical use of such targets requires appropriate validation of
cancer chemotherapy. targets in human tumor tissues to ensure that these antigens
are specific to tumor cells and are not shed into the
(C) Surface Antigens/Proteins circulation.
Expression of different proteins on the surface of cells
constitutes the biochemical writing on the cells, which can 3.2.2. Targeting Ligands
be deciphered using monoclonal antibodies against these Active targeting of drugs can be realized by the use of
proteins. The diseased cells may express either new proteins active agents or ligands which interact with some degree of
or may exhibit differential (under/over) expression of the exclusivity with the specific targets/receptors identified on
proteins found on normal cells. The advent of Proteomics particular cell types. Targeting ligands employed for actively
technology has brought a revolution in the field of targeted drug delivery include simple molecules like sugars,
identification and validation of tumor specific antigens. An folic acid, peptides and specifically engineered antibodies.
ideal tumor specific antigen, which can be used for drug
porcine brain. High affinity of WGA for the brain endothelia

allows drug targeting by binding to cell surface and
subsequent internalization without disrupting the barrier
properties of brain endothelia [36, 37].
(D) Modified Albumins

Cationized albumin (CBSA) coupled to sterically
stabilized liposomes has been demonstrated as a promising
targeting ligand for brain specific targeting [38]. CBSA
coupled liposomes showed better interaction with brain
endothelial cells and higher intracellular accumulation as
compared to bovine serum albumin (BSA). It was concluded
that cationized albumin is taken up into the brain endothelia
via a caveolae mediated endocytic pathway. Sugar modified
albumins have been proposed as suitable carriers for
targeting drugs selectively to various cells in liver [39].
(E) Peptides
Peptides like vascular endothelial growth factor (VEGF),
vasoactive intestinal peptide (VIP), leutanising hormone
releasing hormone (LHRH), somatostatin etc. are used for
targeting drugs to their specific receptors which are
Fig. (5). Efficacy of anti-erbB2 immunoliposomes in a human expressed abundantly in different disease states. Protein
erbB2- overexpressing breast cancer model. Anti-erbB2 delivery to the brain has been achieved successfully
immunoliposome-Doxil containing the F5 scFv (triangles) following conjugation of protein to TAT peptide [40]. In
administered on days 21, 28, and 35 (arrows, same dose as above)
another approach, non-toxic binding fragment of tetanus
are compared to control (PBS) treatment (open circles). Data
represent mean tumor volumes; mm 3
± S.E. Reprinted with toxin has been used to improve CNS delivery of proteins by
permission from ref [31], Copyright (2002), with permission from systemic administration [41].
Elsevier.
(F) Antibodies
(A) Folic Acid The discovery of hybridoma technology has heralded an
Significant over expression of folate receptors on a extensive use of antibodies as targeting moieties, against
variety of tumor cells allows targeting using folic acid as a specific antigens/proteins expressed on the surface of cells.
targeting moiety. Folate has been conjugated to radiolabelled Intact antibodies have been used as highly specific targeting
dendrimers and to liposomes to effect preferential uptake and agents with a high affinity towards their targets. These can
accumulation of therapeutic agents in tumor cells [32, 33]. be conjugated either directly to the drugs (immuno-
Induction of folate receptors with retinoid compounds therapeutics) or to the nano-sized delivery systems (e.g.,
followed by folate conjugated liposomes has been used immunoliposomes, immunomicelles, etc.) to effect drug
successfully for therapy of acute myelogenous leukemia. targeting. However, the use of mouse monoclonal antibodies
This treatment modality was also found to bypass the P- (mAbs) in humans presents some problems. Their in vivo use
glycoprotein (Pgp) mediated drug efflux mechanisms [34]. in humans is restricted for repeated administration, due to the
development of immune response against murine antibody
(B) Sugars domains, producing human anti-mouse antibody (HAMA).
Presence of endogenous lectins on the epithelial cells of Moreover, the success of complete mAbs in solid tumors is
different gut regions can be used for drug targeting by using limited by their high molecular weight and poor penetration
sugars as the targeting moieties. Galactose shows greater power which results in non-uniform distribution in the solid
interaction in proximal regions of gut while fucose binds to tumors. Solutions to these problems lie in the recombinant
the distal positions. Asialoglycoprotein receptor expressed technology which has revolutionized the field of ‘engineered
on the surface of hepatocytes is another example of antibodies’ for human use (Fig. 6). Newer strategies to
endogenous lectin, useful for drug targeting by galactose overcome these problems include fusion of mouse variable
bearing drug carrier system [35]. regions to human constant regions (chimeric antibodies),
removal of T cell epitopes (de-immunization) and grafting
(C) Lectins mouse antigen binding regions onto human acceptor
antibody frameworks (humanization of antibodies) [42].
Different cell types, particularly diseased cells express These three strategies to generate humanized mAbs can
different glycans on their surfaces in comparison to their avoid the problems due to host anti- antibody reactions.
normal counterparts. Thus, selection of suitable lectins Further engineering of antibodies to generate smaller but
allows targeting of drugs to specific cells [35]. An effective antibody fragments has allowed a successful use of
outstanding example of such an interaction is that of wheat the immuno-therapies for solid tumors. Novel antibody
germ agglutinin (WGA) with primary endothelial cells of formats include the monovalent F ab fragments, single chain
Fig. (6). Schematic representation of an intact Ig together with Fab and Fv fragments and single V (colored ovals; dots represent antigen-
binding sites) and C domains (uncolored). Engineered recombinant antibodies are shown as scFv monomers, dimers (diabodies), trimers
(triabodies) and tetramers (tetrabodies), with linkers represented by a black line. Minibodies are shown as two scFv modules joined by two C
domains. Also shown are Fab dimers (conjugates by adhesive polypeptide or protein domains) and Fab trimers (chemically conjugated).
Colors denote different specificities for the bispecific scFv dimers (diabodies) and Fab dimers and trimers. Reprinted with permission from
ref [42].
Fv fragments (scFv) and the bispecific antibodies. A F ab formulation, availability of polymers and newer charac-
fragment takes one fourth of the time taken by an intact terization tools have tremendously expanded the research
immunoglobin molecule to move a distance of 1mm into a interests in such nanosystems. For the purposes of this
solid tumor [43]. Thus smaller antibody fragments exhibit review, the term “nanosystems” shall include all the nano-
better pharmacokinetics for tissue penetration with full sized (<1000 nm) drug carriers systems, such as polymeric
binding specificity but show poor retention time at the target nanoparticles, liposomes, micelles and polymer-drug
site. F ab fragment of anti HER-2 monoclonal antibody conjugates. Some of the newer nanosystems that are being
conjugated to liposomes was shown to bind specifically to developed include nanocages, nanogels, nanofibers, nano-
breast cancer cell line SK-BR-3 which overexpressed HER-2 shells, nanorods, nanocontainers, etc. As new systems and
[44]. A scFv fragment against the human transferrin receptor molecular complexes are being developed, the existing drug
was used to target cationic lipoplexes to breast tumor cells delivery systems are undergoing transformation to meet the
for p53 gene therapy upon systemic administration. These needs of effective drug targeting. For example, liposomes
immunolipoplexes demonstrated increased tumor cell from simple lipid vesicles are modified to stealth liposomes
binding, improved gene delivery and transfection efficiency to more complex systems with specific ligands and peptides
in vitro and in vivo [45]. to achieve cellular and tissue targeted drug therapy. Also, the
integrated approaches to drug delivery by scientists from
4. NANOSYSTEMS different disciplines have created new opportunities for
applications of nanosystems in medicine and drug therapy.
Better appreciation of biology, advances in polymer
Submicron size range of nanosystems offers excellent
chemistry and their interaction with nanotechnology has
opportunities to breach the physiological barriers and access
brought a renaissance in the field of drug delivery, whereby
different tissues followed by an efficient cellular uptake and
nano-sized drug delivery systems are emerging as a panacea
intracellular internalization. This is not only being explored
for the global quest of drug targeting. A plethora of highly
for the purposes of drug delivery but widely accepted and
optimized methods for the preparation, versatility of employed as imaging tools. Moreover, these nanosystems
have the potential to effectively carry and target drugs by near to transition temperature and can be induced to deliver
virtue of their colloidal size range and ability to be drugs to target tissues, for example, tumors on external
coupled/conjugated with different targeting ligands. application of heat. Relatively inaccessible inflammation
sites (e.g. brain) have been accessed by the use of negatively
4.1. Liposomes charged and arginine-glycine-aspartic acid (RGD) sequence
coated magnetic liposomes, which mediate delivery of drugs
Liposomes have been extensively investigated as a through monocytes/neutrophils [50].
potential drug delivery system due to the enormous diversity
of structure and compositions that can be achieved [5]. They “Stealth” or long-circulating liposomes designed to evade
are capable of targeting drugs by passive as well as active detection by the RES, have found significant applications
means. Unmodified liposomes undergo rapid clearance from pertinent to ligand mediated targeted drug delivery [51]. This
blood stream due to sequestration by macrophages of the can be attained by producing a hydrophilic surface by
RES. This property and the flexibility of altering size of grafting polyethylene glycol (PEG) chains on the liposome
liposomes have allowed its use in passive targeting of a surface or by inclusion of phosphatidylinositol and
number of drugs. Liposomes can be used to deliver gangliosides in the formulation. These sterically stabilized
immunomodulators, cytotoxic and anti-microbial agents to liposomes act as circulating drug reservoirs and enable
the macrophages by means of passive targeting. Activating targeting of drugs to non-RES target sites [52]. Ligand
factors such as cytokines have been delivered to mediated targeting has been avidly explored with such long
macrophages to render them tumoricidal. Such activated circulating liposomes but still no products are available on
macrophages selectively kill tumor cells and this approach market. The concept of antibody targeted liposomes or
has been proposed for treatment of metastasis after surgical immunoliposomes has been existing for quite a few decades
removal of primary tumors. The EPR effect in tumors now. Antibodies can be conjugated to the PEG chains on
proposed by Maeda has been successfully employed to PEG stabilized liposomes to achieve specific tissue targeting.
selectively deliver cytotoxic agents to tumor cells by means Accumulation of liposomes at the target site doesn’t
of appropriately sized liposomes. The rapid uptake and essentially guarantee therapeutic efficacy of the encapsulated
accumulation of liposomes in tumor cells has been proposed drug. Cellular uptake of the liposomes/ encapsulated drug
to be a reason for reduced plasma levels of the anthracycline can occur either by endocytotic pathway or by fusion of
compounds and thus reduced chances of myocardial toxicity. liposomal surfaces with the cell membrane. Receptor
Two such formulations are currently commercially available- mediated endocytosis has been shown to be responsible for
Daunosome™ liposome (NeXstar, Inc.) incorporates internalization of liposomes coupled to an anti-transferrin
daunorubicin and Doxil™ (Sequus Pharmaceuticals) based receptor antibody at the BBB.
on doxorubicin. These formulations have an extended Liposomes have also been actively pursued as a potential
circulation time by virtue of their small size and sterically tool for targeted gene delivery to specific cells in the body.
stabilized surfaces [46]. Cationic liposomes can be complexed with polyanionic
Selective uptake of unmodified liposomes by the plasmid DNA to form highly compact nanostructures with a
phagocytic cells has also been exploited for intracellular net positive charge called the “lipoplexes”. This non-viral
delivery of antimicrobial agents, thereby increasing their gene delivery system was designed for gene targeting to liver
therapeutic index and decreasing toxicity substantially. This by conjugating modified galactolipids to the lipoplex.
has been used for intracellular pathogens such as Leishmania Labeling of lipoplexes with asialofetuin and protamine
donovani, systemic fungal infections, HIV virus and sulfate has documented improved in vivo efficiency due to
mycobacterial infections, particularly in immunocom- efficient targeting to nucleus by the nuclear localization
promised patients [5]. The natural tendency of liposomes to signal present in protamine sulfate [53]. The addition of
be cleared by the RES has widened the scope of their protamine sulfate to asialofetuin-lipoplexes increased the in
applications for imaging as well. Liposomes have been used vivo gene expression levels by 75 folds as compared to those
as carriers for radioisotopes and contrast agents to be used in by conventional lipoplexes (Fig. 7). This was attributed to
diagnostic imaging [47]. Liposomes carrying contrast agents the smaller size of the lipoplexes produced in combination
accumulate more in surrounding cells than in tumor cells due with protamine and the possible nuclear targeting by nuclear
to the low phagocytic activity of tumor cells. This can be localization signals present in protamine. Despite the
used for tumor imaging, wherein tumor cells are observed as significant progress made so far in the formulation
holes within the surrounding background. Localized site technology and the new methods of ligand conjugation, there
specific delivery of drugs is possible by introducing drug are still numerous lacunas to be filled before liposomes can
carrying liposomes by means of catheters. Selective be projected as the ultimate nanosystems for targeted drug
transfection of a plasmid DNA associated with liposomes delivery. Major obstacles include gene transfer to the nucleus
was observed in vivo after injecting into an isolated region of of a cell and obtaining a sustained long term expression of
artery by a catheter [48]. This approach is applicable for the genes.
diseases such as atherosclerosis or neointimal hyperplasia to
treat particular sections of the tissues. Liposomes can also be 4.2. Nanoparticles
localized to a particular target tissue by external application
of localized heat and use of temperature sensitive liposomes Nanoparticles are sub-micron sized polymeric colloidal
[49]. Phospholipids, with a transition temperature slightly particles with a therapeutic agent of interest encapsulated
higher than body temperature are used in this design. Such within their polymeric matrix or adsorbed or conjugated onto
formulations show increased permeability at temperatures the surface. Polymeric nanoparticles constitute a versatile
Fig. (7). Histologic evaluation of GFP gene expression in hepatocytes after tail vein injection of PBS and naked DNA (a,b), plain (c,d) and
protamine-AF-lipoplexes (e,f). Cells under phase contrast microscopy (a,c,e) and under fluorescence microscopy (b,d,f) at an original
magnification of X 200. A representative overview is shown. Reprinted with permission from ref [53].
drug delivery system, which can potentially overcome Like all colloidal drug carrier systems, nanoparticles are
physiological barriers, and guide the drugs to specific cells rapidly opsonized and cleared by the macrophages of RES.
or intracellular compartments; either by passive or ligand Thus, intravenously injected nanoparticles are mostly (90%)
mediated targeting approaches. It also allows controlling the taken up by the liver and the spleen within minutes following
release pattern of drug and sustaining drug levels for a long drug administration [20]. This characteristic biodistribution
time by appropriately selecting the polymeric carriers. is of special benefit for targeting drugs to macrophages and
has been employed for chemotherapy of the RES localized line. IC 50 values for paclitaxel were 5 folds lower with
tumors. Anticancer drug loaded nanoparticles are mainly transferrin conjugated nanoparticles than that with solution
concentrated in Kupffer cells in liver on intravenous or unconjugated nanoparticles. A complete regression of
injection. These cells act as reservoirs and allow prolonged tumor and a significantly higher survival rate of animals was
diffusion of anticancer drug into the neighboring tumor cells. observed with a single-dose intra-tumoral injection of
This approach of passive targeting has been useful for the transferrin conjugated nanoparticles in a murine model of
treatment of hepatic metastasis [54]. prostate cancer as compared to that with unconjugated
nanoparticles or drug dissolved in Cremophor ® EL (Fig. 8).
The RES uptake of nanoparticles depends on the particle
The mechanism of greater efficacy of transferrin conjugated
size, surface charge and surface hydrophobicity. Particles
nanoparticles appears to be due to the greater cellular uptake
<100 nm, with hydrophilic surfaces undergo relatively less
of drug. Thus, nanoparticles are emerging as a promising
opsonization and clearance by RES uptake. It prolongs the
tool for the intracellular delivery of practically insoluble
circulation time of nanoparticles in the blood and allows
drugs (such as taxol) and sensitive drugs (such as proteins
targeting of nanoparticles to tissues other than the RES. Such
and oligonucleotides or DNA) [62]. Nanoparticles not only
long circulating nanoparticles have been designed as
act as an effective carrier for such drugs protecting them
‘biomimetic nanoparticles’ or ‘sterically stabilized nano-
from undesirable physiological conditions but also allow a
particles’ [55-57]. The former approach involves preventing
selective and controlled release of drugs at their target sites.
the recognition of surface of nanoparticles by the RES by
coating the nanoparticles with biomimetic substances such as They are of specific importance in cancer chemotherapy,
sialic acid, albumin or inactivated complement moieties. where cellular uptake of drugs is limited due to efflux of
This approach has not been successful due to rapid drug by P-glycoprotein and drug resistance is a common
desorption of the coatings in vivo. Steric stabilization of problem. Anticancer drugs encapsulated in nanoparticles
nanoparticles has been achieved by making the surface of cannot be recognized by these efflux mechanisms and thus
particles more hydrophilic so as to prevent opsonization in circumvent multidrug resistance [63].
the blood stream. This is done either by adsorbing The versatility of formulation, colloidal size, bio-
hydrophilic surfactants on nanoparticle surface or using compatibility and sustained release properties of nano-
block/branched copolymers [58]. Polyethylene oxide (PEO)
particles have already been accepted with growing interest
or poly ethylene glycol (PEG) is the most successful non- for a wide range of applications. However, the recent
ionic hydrophilic polymer used for this purpose [58, 59]. strategy gaining widespread interest and attention is that of
Overcoating of nanoparticles with polysorbates is known “functionalized nanoparticles”. Methods are being devised to
to result in greater transport of nanoparticles across the BBB, tailor make the surface characteristics of nanoparticles to
allowing brain targeting following an intravenous injection achieve specific ligand mediated targeting of therapeutic and
[60]. Polysorbates on the surface of nanoparticles act as imaging agents. Optimization of these techniques, coupled
anchors for apolipoprotein E (present in plasma), which with the considerable progress made in the field of
adsorbs on the nanoparticles. The particles then mimic LDL nanoparticle characterization and a better understanding of
particles and interact with LDL receptors on endothelial cells their in vivo behavior has raised hopes for the successful
in the brain capillaries leading to their cellular uptake. development of commercial products based on targeted
Kreuter’s group has enlisted a number of possible nanoparticles for use in therapy and imaging.
mechanisms for nanoparticle mediated drug transport across
the BBB. These include endocytotic/transcytotic pathway 4.3. Polymeric Micelles
with a possibility of inhibition of P-glycoprotein efflux
Amphiphilic copolymers self-assemble in an aqueous
mechanisms [60]. Lymphatic targeting of drugs is possible
milieu to form spherical colloidal nanoparticles called
by a sub-cutaneous injection of nanoparticles. This can be
polymeric micelles [64]. Polymeric micelles have been
used as a tool for chemotherapy against lymphatic tumors or
proposed as novel carrier systems in drug targeting due to
metastasis [23].
their increased loading capacities, stability in physiological
Nanoparticles offer numerous opportunities for targeting conditions and the possibility of engineering the core-shell
by surface modifications which would allow specific bio- architecture for different applications [65, 66]. The size of
chemical interactions with the proteins/receptors expressed polymeric micelles (usually <100 nm) and their hydrophilic
on target cells. We have recently demonstrated increased surface is of crucial importance in overcoming the major
efficacy of paclitaxel loaded nanoparticles on conjugation obstacle of drug targeting- the RES uptake. This unique
with transferrin in a murine model of prostate cancer [61]. disposition characteristic can be used for effective tumor
Transferrin receptors are over expressed in most cancer cells targeting by means of the EPR effect [67]. Functionalization
by 2-10 folds than in normal cells. Conjugation of transferrin of polymeric micelles enables ligand-targeted systems to be
ligand to nanosystems or the antibodies against transferrin developed for cell-specific targeting. A pre-requisite for the
receptor has been explored for targeting drugs to tumor cells. development of such actively targeted micelles is an
We have demonstrated that the transferrin conjugation to effective synthetic method for end-functionalization of the
nanoparticles enhances the therapeutic efficacy of the amphiphilic polymers and conjugation of ligands to the ends
encapsulated paclitaxel as compared to that with of the hydrophilic segments. A variety of PEG-PLA block
unconjugated nanoparticles. The cellular uptake of copolymers having amino/saccharide end functional groups
conjugated nanoparticles was about 3 times more than that of have been prepared. Such micelles with peptide/sugar
unconjugated nanoparticles in a prostate cancer (PC3) cell moieties on the surface can be used to target specific peptide
Fig. (8). Antitumor activity of Tx-NPs-Tf in a murine prostate tumor model. PC3 cells (2 × 10 6 cells) were implanted s.c. in athymic nude
mice. Tumor nodules were allowed to grow to diameter of about 50 mm 3 prior to receiving different formulations as a single-dose treatment.
Tx-NPs-Tf (l, 24 mg/kg; ¨, 12 mg/kg), Tx-NPs ( Χ, 24 mg/kg), Tx-Cremophor ® EL formulation ( s, 24 mg/kg), ( u) control NPs and ( n)
Cremophor® EL formulation. Data are means + s.e.m., n=6. * p <0.005 Tx-NPs-Tf versus Tx-NPs and Tx-Cremophor ® EL groups. Reprinted
with permission from ref [61], Copyright (2004) Wiley-Liss, Inc., A Wiley Company.
receptors on cell surface for cell specific targeting of drugs found to passively target tumors in a rodent model by EPR
[68]. effect, without showing any signs of immunogenicity or
polymer related toxicity [73]. Ligand directed polymer-drug
Stability of micelles is another critical factor for effective
conjugate can also be prepared to target specific receptors on
drug delivery in vivo. The micelles must dissociate to release
cellular surfaces. The essential features of such nanosystems
the entrapped drug at the target site. Polyion complexes
include a polymeric carrier, a bio-responsive linker and a
(PIC) micelles were prepared with polyethylene glycol and
specific ligand. HPMA copolymers have been extensively
poly lysine (PEG-PLys) and oligo-DNA and the cores were
used as the carrier in such systems. Polymer-drug linkers
reversibly cross linked by oxidation of thiol groups [69].
must be selected such that the conjugate remains intact
This allowed specific intracellular release of oligo-DNA
during transport to the target site, but allows drug release at
because the disulfide cross-links could be cleaved only in the
appropriate rate on reaching its target. The tetrapeptidyl
presence of strong reducing environment. Immunomicelles
linker, Gly-Phe-Leu-Gly, which is cleaved by endolysosomal
prepared by conjugating antitumor antibodies to polymeric
micelles were shown to recognize and bind to various cancer proteases is the most popular linker, which is stable in
cells in vitro [70]. Taxol- a water insoluble anticancer drug circulation but cleaves subsequent to its endocytic uptake by
cells. pH-sensitive cis-aconityl, hydrazone and acetal
loaded into these immunomicelles showed higher
linkages are alternatives for polymer-drug conjugation [74].
accumulation in experimental tumors in mice than non-
targeted micelles. The only clinically tested polymer-drug conjugate
bearing a targeting ligand is the galactosamine directed
4.4. Polymer-Drug Conjugates HPMA-tetrapeptidyl-doxorubicin [75]. The system is
designed for treatment of liver cancer by targeting the
The concept of polymer-drug conjugation was developed asialoglycoprotein receptor on hepatocytes. Doxorubicin
as a means of improving cell specificity of low molecular
concentration was found to be 12-50 folds higher (in
weight anticancer drugs, to effect targeting to tumor cells
hepatoma tissue) than could be achieved by free drug.
[71]. Conjugation of a polymeric carrier to low molecular
weight drugs brings a drastic change in the pharmacokinetic
5. EMERGING TRENDS
disposition of drug at both the whole body and the cellular
levels [72]. These conjugates allow passive targeting of 5.1. Vascular Targeting
tumors via EPR effect and their long circulation time in
blood stream. N-(2-hydroxy propyl) methacrylamide The blood and lymphatic vessels of individual tissues in
(HPMA) copolymer conjugated doxorubicin system was normal and pathological conditions are biochemically
Fig. (9). Athymic WEHI mice were subcutaneously injected with M21-L cells (5 X10 6), and tumors were allowed to grow to ~ 100 mm 3.
Mice were then injected i.v. with 450 nmoles of NP electrostatically coupled to 25 µg of plasmid expressing firefly luciferase. One group also
received a coinjection of 20-fold molar excess of the soluble αvβ3-targeting ligand. After 24 hours, mice were killed, tissues were surgically
removed, and luciferase activity was quantified. ( Inset) Luciferase expression as a function of DNA dose injected. Each bar represents the
mean ± SD of five replicates. Reprinted with permission from ref [79]. Copyright [2002] American Association for the Advancement of
Science.
distinct from each other. The vasculature, especially the normal and neoplastic tissues [80]. Using integrated
endothelial cells express molecules that are specific to a analytical techniques such as subtractive proteomics, bio-
particular tissue and can be utilized for targeting drugs. informatics and molecular imaging, they identified new
Vascular targeting of drugs is thus highly gaining importance molecular targets on endothelial cells of lung and lung tumor
due to these biochemical differences and the easy tissues. They validated that aminopeptidase-P and annexin-
accessibility by all drugs absorbed or injected in the blood A1 are distinctly present on the endothelial cells of lungs and
stream [76]. Thus, recently the focus has shifted to identify lung tumors, respectively. These novel accessible tissue
and validate such biochemical markers in the normal and specific targets can be used for molecular imaging and
pathological tissues, especially in tumor tissues. Tumor targeted delivery of drugs/genes in vivo. Specific molecular
vasculature is distinct from normal vasculature due to the markers have also been identified in inflammatory lesions
process of neovascularization which is accompanied by such as arthritic synovium and atherosclerotic plaques [81].
expression of many new proteins or over expression of many
5.2. Intracellular Targeting
normal proteins on the cell surfaces. Molecular markers of
angiogenesis include additional peptidases/proteases and Intracellular targeting is desirable for drugs whose site of
integrins. Cell adhesion receptors, integrins are also over action is located in the intracellular compartments or which
expressed in tumor vasculatures [77, 78]. One such molecule undergo extensive efflux from the cell by the efflux
is the integrin- αvβ3 which is preferentially expressed in transporters such as multidrug resistance proteins (MRP) and
angiogenic endothelium of tumors and is a potential target P-glycoproteins (Pgp) [82]. Drugs such as glucocorticoids
for drugs. αvβ3 has been used as a targeting ligand coupled (e.g., dexamathasone) have cytoplasmic receptors, while
with lipid based cationic nanoparticles to affect gene most anticancer drugs (DNA intercalators) have their site of
delivery targeted to neo vascularization in tumor bearing action located in the nucleus. Macromolecular drugs such as
mice [79]. A group of tumor bearing mice was injected with proteins and plasmid DNA usually have their site of action in
non-targeted nanoparticles, other one with targeted nano- the cytoplasm and nucleus, respectively. The ultimate goal of
particles and third one received targeted nanoparticles co- gene delivery can be fulfilled only if the transgene is able to
injected with 20-fold molar excess of the soluble αvβ3 localize and integrate with the nuclear or mitochondrial
targeting ligand. The specificity of gene delivery using this DNA. Further, steps need to be taken to ensure the protection
approach was evaluated by quantifying the gene expression of proteins/plasmid DNA from the nucleases or the
levels in various tissues in tumor bearing mice (Fig. 9). endolysosomal enzymes while delivering them into the
Tumor specific gene expression was completely blocked cytoplasm or targeting to nucleus. A number of drug carrier
when mice were co-injected with excess of the targeting systems (lipid based) have been explored for specific
ligand. intracellular delivery of proteins and DNA. Although such
Recently Oh and others have described a novel approach systems result in relatively higher intracellular delivery
for identifying and validating distinct molecular signatures in efficiency, they are incapable of maintaining therapeutic
added. Nanoparticles could be detected inside the cytoplasm

of cells even 14 days after nanoparticle incubation with cells
(Fig. 10). Thus, it was hypothesized that nanoparticles
escape endo-lysosomes and then release dexamethasone in a
sustained manner, while present in the cytoplasm.
Intracellular drug levels were maintained for up to 8 days of
study and therapeutic efficiency was shown in terms of anti-
proliferative activity. Dexamethasone loaded nanoparticles
showed significantly higher and more sustained inhibition of
cell proliferation as compared to drug solution.
In another study, we have demonstrated sustained
intracellular localization of DNA with nanoparticles as
compared to transient localization of DNA in cells
transfected with naked DNA alone. Breast cancer cells
(MDA-MB-435S) transfected with wt-p53 plasmid DNA
loaded nanoparticles showed significantly higher and more
sustained (7 days) intracellular DNA levels as opposed to
transfection with naked DNA and DNA-Lipofectamine™
complex [85]. This study has significant implications in
Fig. (10). Inhibition of VSMC proliferation with dexamethasone in cancer gene therapy, where sustained gene expression is of
solution and encapsulated in nanoparticle formulations. Cell growth importance for greater therapeutic benefit. Thus, biode-
was followed by an MTS assay, where absorbance is proportional gradable nanoparticles can not only be used for efficient
to the number of viable cells. Data are means ± the standard cytoplasmic delivery of low molecular weight drugs but also
deviation (n= 6). Reprinted with permission from ref [84], for macromolecules. Further, ligands can be chemically
Copyright (2004) American Chemical Society. coupled to nanoparticles surface to mediate targeting of
nanoparticles to mitochondria or nucleus of the cell. Various
drug concentrations for long times [83]. Thus the focus is cell penetrating peptides including synthetic peptides, protein
now-a-days shifted to the development of drug delivery transduction domains (PTD), and membrane-translocating
systems which not only target the drug to its site of action sequences (MTS) have been described for the intracellular
but also maintain the drug concentrations at therapeutically delivery of drugs especially macromolecules [88]. In general,
relevant levels for a sustained period of time. We are these peptides are made up of 30 amino acids, are positively
investigating biodegradable nanoparticles formulated from charged and have the ability to translocate the cell membrane
poly-(D,L-lactide-co-glycolide) for cytoplasmic delivery of and deliver drugs into the cytoplasm or the nucleus. HIV
drugs and plasmid DNA [84, 85]. protein derived trans-activating regulatory protein (HIV-
TAT) and penetratin are the most extensively investigated
We have earlier demonstrated that nanoparticles are
peptides for intracellular delivery of drugs. Efficient gene
internalized into vascular smooth muscle cells (VSMCs) and
transfection has been demonstrated by means of TAT-
vascular endothelial cells by means of endocytosis, partly
conjugated liposomes both in vitro and in vivo [89]. The
through fluid phase pinocytosis and partly through clathrin
conjugation of TAT on the surface of liposomes was found
coated pits [86]. Following cellular uptake, nanoparticles are
to influence their intracellular uptake. The TAT conjugated
transported to primary endosomes and to sorting endosomes.
liposomes take a non-endocytic route for cellular entry and
A fraction of nanoparticles can be recycled back to the
the uptake was observed to be energy independent. However,
exterior of cell by means of exocytosis, while the rest
the mechanism of cell penetration of such peptides is yet to
reaches secondary endosomes. Secondary endosomes fuse
be elucidated completely and thus the issue of cell specificity
with lysosomes to form endo-lysosomes and the efficiency
by means of these peptides is still unresolved [88].
of cytoplasmic delivery is governed by the ease and rapidity
of escape of nanoparticles from endo-lysosome. We For drugs with nucleus as the site of action, the nuclear
proposed and confirmed that the surface charge reversal of membrane offers another challenge. Nuclear uptake of
nanoparticles in the acidic pH of endo-lysosomes was plasmid DNA is the rate limiting step in efficient transfection
responsible for the endo-lysosomal escape of nanoparticles and successful gene therapy [90]. Molecules smaller than 40-
[87]. Thus, surface modification of nanoparticles can be 45kDa and less than 100 nm have been shown to cross the
attempted in order to affect targeting of drugs either to endo- nuclear membrane by passive transport, while other
lysosomes or to cytoplasm. A rapid escape of nanoparticles molecules interact with cytosolic factors to cross the nuclear
from endo-lysosomes would result in efficient delivery of membrane through the nuclear pore complexes [91]. Certain
nanoparticles into the cytoplasm. The therapeutic efficacy of peptide sequences, better known as nuclear localization
drugs with cytoplasmic targets depends on the intracellular signals (NLS) have been reported to specifically interact
drug levels and their maintenance for sustained period of with the cytoplasmic factors which can then target molecules
time. We have reported efficient cytoplasmic delivery of to the nucleus. NLS are peptides with no general consensus
dexamethasone and sustained intracellular drug levels by sequence, however mostly composed of basic amino acids.
using biodegradable nanoparticles [84]. VSMCs were treated NLS present in the SV-40 large T antigen was the first and
with dexamethasone in solution or entrapped in nanoparticles the most extensively studied classical NLS [92]. Various
for 3 days, following which no further dose of drug was strategies have been devised to target plasmid DNA to the
nucleus of a cell, including conjugation of NLS peptide to providing effective drug targeting by using an external
the end of a linear DNA molecule and non covalent stimulus. Ultrasound could not only trigger the drug release
modification of a plasmid DNA with NLS among others at the target site but also enhanced the cellular uptake of
[91]. Non-viral vectors for gene delivery (liposomes, drug. Although the precise mechanism of ultrasound affected
nanoparticles) can be surface modified and conjugated to the drug targeting is not completely understood, some of the
identified NLS sequences, to effect nuclear targeting. In proposed mechanisms include, ultrasonic triggered extra-
pioneering work, gold nanoparticles coupled with SV-40 vasation of polymeric micelles, ultrasonic activated release
large T antigen on their surface were shown to efficiently of drug from micelles and ultrasonic enhanced uptake into
translocate into the nucleus, upon microinjection into the the cells [99]. A decrease in tumor volume was demonstrated
cells [93]. A recent study proposed enzymatically digested by focusing ultrasound on the tumors following an
®
low molecular weight protamine as a non-toxic and efficient intravenous injection of Doxorubicin loaded Pluronic
gene carrier. Plasmid DNA complexed with low molecular micelles.
weight protamine was found to efficiently translocate into
the cell and then transferred to the nucleus of cell owing to 5.4.2. Magnetic Field
the structural similarity of protamine with HIV-TAT peptide
Magnetic nanoparticles offer exciting new opportunities
[94]. Another evolving concept is that of utilization of the
towards developing an effective drug targeting delivery
viral proteins for nuclear targeting of molecules or the design
system. It is possible to produce, characterize and
of synthetic viruses. However, two or more targeting
specifically tailor the functional properties of magnetic
peptides may be used to target the nanosystems to specific
nanoparticles for their clinical applications [100]. A potential
cells and then translocate the drug to the nucleus. One such
benefit of using magnetic nanoparticles is the use of
approach using a combination of cell penetrating and nuclear
localized magnetic field gradients to attract the particles to a
targeting peptides has been explored with gold nanoparticles
chosen site, and the possibility to hold them there until the
[95].
therapy is complete and then to remove them. Magnetic
nanoparticles loaded with a therapeutic agent may be
5.3. Promoter/Transcriptional Targeting injected intravenously, and then the blood circulation could
It’s a novel approach towards targeted gene therapy transport these particles to the region of interest under the
which involves the use of specific tissue, tumor, or induced influence of magnetic field gradient. Alternatively,
promoters that can limit gene expression to target cells which depending upon the pathological condition (e.g., solid
express a specific transcription factor [96]. This specifically tumors), one could inject these particles directly into the
restricts the transgene expression in the target tissue. artery leading to the desired site and then use magnetic field
Moreover, it allows the flexibility to regulate the duration gradient to localize them into the general area where the
and level of expression exogenously by using promoters that treatment is desired [101]. An effective targeting of magnetic
are preferentially activated under certain conditions. An ideal nanoparticles via systemic administration would require a
tumor specific promoter is the one highly active in tumor targeting magnetic force that would offset the force due to
cells with little or no activity in normal cells. Promoter for linear blood-low rates of ~0.05 cm/s in capillaries to 10 cm/s
telomerase gene can be genuinely classified as tumor in arteries and 50 cm/s in the aorta [102]. To overcome the
specific and is being used to drive transgene expression in a above difficulty, magnetic nanoparticles with higher
variety of cancer cells [97]. Other examples include α- magnetic moments or magnets that can provide higher
fetoprotein gene which is transcriptionally silent in adult magnetic field gradient are being developed.
liver but expressed in fetal and hepatocellular carcinoma
cells [98]. Furthermore, many aspects of tumor 5.5. Pretargeting or Sandwich Targeting
microenvironment (like specific promoters for endothelial The basic objective of pretargeting is to decouple the
cells and hypoxia) and many exogenous factors (radiation, targeting ligand and the active pharmacological moiety. It
heat and drugs) can also be used to induce promoters to involves pretargeting of specific cells with a ligand followed
allow transgene expression specifically in tumor cells [96]. by treatment with a drug linked to another molecule having
This new approach is extensively pursued for cancer gene high affinity for the ligand used for pretargeting. Avidin-
therapy to target damaging events to tumor cells, while biotin is one of the biological pairs employed for
sparing normal tissues. pretargeting by Hoya et al. [103]. The proteins of target cells
(endothelial cells) were biotinylated by accessing the tissue
5.4. Physical Targeting using a catheter. The active drug or drug carrier system
bound to avidin was then injected intravenously. In vivo
In addition to the aforementioned active targeting experiments demonstrated that drugs bind to biotinylated
methods, targeting and release from a drug delivery system endothelial cells through avidin, without getting cleared by
can be achieved by applying external stimuli such as blood flow in the artery. This novel approach for targeted
ultrasound or magnetic fields. drug delivery has implications in targeting drugs for
intravascular diseases which need continuous presence of
5.4.1. Ultrasound drug at the target site for a long period of time, for example
angiogenesis in tumors. Pretargeting has been proposed to
Ultrasound focused on tumor tissue has been shown to
deliver targeted radio immunotherapy for treatment of
trigger the release of drugs from polymeric micelles only at
cancer; so that cell killing high doses of radiation can be
the tumor site. This is a new drug delivery modality
selectively delivered to tumor cells with reduced exposure to
normal healthy cells. Mab’s against tumor antigens bound to CONCLUDING REMARKS
streptavidin can be used to target the desired tissue.
Nanosystems offer opportunities to achieve drug
Radioisotopes conjugated with biotin bind only to the tumor
targeting with newly discovered disease-specific targets,
cells through the biotin-streptavidin interaction. This can
however challenges remain, primarily because of the
potentially reduce the dose limiting toxicities of directly
complexity of the body and the layers of barriers that these
radio labeled antibodies (such as bone marrow toxicity).
systems need to overcome to reach to the target. Effective
However, this strategy is in its infancy and needs further
targeting would require dual focus, a better understanding of
evaluation before it can be used in humans.
the target and a simultaneous development of the targeting
system. Some of the physical approaches such as by directly
5.6. Chemical Strategies
reaching to the accessible targets using a catheter system or
The objective of targeting drugs is not just an using guiding mechanisms such as a magnetic field gradient
afterthought but now-a-days it’s implicit in the drug could also be effective in drug targeting. As nanotechnology
development process. Chemical approaches towards drug for biomedical applications evolves, the caution is also
targeting include the well-known prodrug approach and the expressed about their safety and it should be investigated
emerging chemical delivery systems. Two prodrug thoroughly.
technologies for targeting cytotoxic compounds selectively
to tumor cells are undergoing evaluation at present. These ACKNOWLEDGEMENTS
include the antibody directed enzyme prodrug therapy We gratefully acknowledge the financial support from
(ADEPT) and the gene directed enzyme prodrug therapy the Nebraska Research Initiative and the National Institutes
(GDEPT). ADEPT involves antibody targeted against tumor of Health. JKV is supported by a pre-doctoral fellowship
antigen to deliver a non endogenous enzyme specifically to from the Department of Defense, US Army Medical
tumor cells. The prodrug is administered subsequently and Research and Materiel Command (DAMD17-02-1-0506).
gets converted into an active drug by the enzymatic action VL acknowledges the contribution of his laboratory
only in the desired cell population. GDEPT approach is members and collaborators for some of the data that are
similar to ADEPT however, here inspite of using enzyme, described in this review. We would also like to thank Ms.
the cells are transfected with the gene responsible for Elaine Payne and Ms. DeAnna Loibl for providing
expression of the non endogenous enzyme [104]. The gene is administrative support.
targeted to specific cells and expressed intracellularly so that
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Received: 13 September, 2004 Accepted: 29 September, 2004

Research Article
Tumor Imaging Using a Picomolar Affinity HER2 Binding

Affibody Molecule
1,3 1 1 1 1
Anna Orlova, Mikaela Magnusson, Tove L.J. Eriksson, Martin Nilsson, Barbro Larsson,
1 2 3
Ingmarie Höidén-Guthenberg, Charles Widström, Jörgen Carlsson,
1,3 4 1,3
Vladimir Tolmachev, Stefan Ståhl, and Fredrik Y. Nilsson
1
Affibody AB, Bromma; 2Department of Hospital Physics, Uppsala University Hospital; 3Department of Oncology, Radiology, and
Clinical Immunology, Rudbeck Laboratory, Uppsala University, Uppsala; and 4Department of Biotechnology,
AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden
Abstract molecules should be rapidly excreted, thus facilitating high-

contrast tumor imaging and reducing the time between injection
The detection of cell-bound proteins that are produced due to
and examination. Although antibodies can be selected for good
aberrant gene expression in malignant tumors can provide
specificity, they are too large to allow for rapid kinetics. Therefore,
important diagnostic information influencing patient man-
smaller antibody fragments have gained increasing interest and
agement. The use of small radiolabeled targeting proteins
there are a number of reports on the use of antibody fragments for
would enable high-contrast radionuclide imaging of cancers
in vivo imaging (3–5). However, further size reduction of these
expressing such antigens if adequate binding affinity and
affinity proteins (27-54 kDa) could potentially improve extravasa-
specificity could be provided. Here, we describe a HER2-
tion, increase tissue penetration, and speed up blood clearance (6),
specific 6 kDa Affibody molecule (hereinafter denoted Affi-
thereby increasing imaging contrast. Short peptides, on the other
body molecule) with 22 pmol/L affinity that can be used for
hand, are small and have very rapid kinetics. Natural peptide
the visualization of HER2 expression in tumors in vivo using
receptor ligand derivatives, for example, show great potential for
gamma camera. A library for affinity maturation was
radionuclide imaging (7, 8), but there is a limited repertoire of
constructed by re-randomization of relevant positions identi-
natural peptides to choose from. Unfortunately, peptides derived
fied after the alignment of first-generation variants of nano-
from screening using phage display libraries seldom have the high
molar affinity (50 nmol/L). One selected Affibody molecule,
affinity necessary to be considered a general class of targeting
ZHER2:342 showed a >2,200-fold increase in affinity achieved
molecules (9). Therefore, it would be attractive to develop new
through a single-library affinity maturation step. When
targeting agents, which are smaller than antibody fragments in
radioiodinated, the affinity-matured Affibody molecule
order to improve tumor penetration but have better binding
showed clear, high-contrast visualization of HER2-expressing
specificity and affinity than short peptides. Efforts towards
xenografts in mice as early as 6 hours post-injection. The
improved affinity proteins have recently been made based on
tumor uptake at 4 hours post-injection was improved 4-fold
different protein scaffolds (10–12), e.g., ‘‘trinectins’’ (13, 14),
(due to increased affinity) with 9% of the injected dose per
‘‘anticalins’’ (15), ‘‘ankyrin repeats’’ (16), engineered T cell receptors
gram of tissue in the tumor. Affibody molecules represent a
(17), and ‘‘Affibody molecules’’ (18).
new class of affinity molecules that can provide small sized,
Affibody molecules represent a new class of affinity proteins
high affinity cancer-specific ligands, which may be well suited
based on a 58–amino acid residue protein domain, derived from
for tumor imaging. (Cancer Res 2006; 66(8): 4339-48)
one of the IgG-binding domains of staphylococcal protein A. This
three-helix bundle domain has been used as a scaffold for the
Introduction construction of combinatorial phagemid libraries, from which
Molecular phenotyping of gene products that are aberrantly Affibody variants that target the desired molecules can be selected
expressed in tumors, thus allowing for diagnosis of tumors by using phage display technology (18, 19). The simple, robust
targeted visualization of cancer markers (e.g., HER2), holds promise structure of Affibody molecules in combination with their low
for a shift in clinical practice towards personalizing cancer molecular weight (6 kDa), make them suitable for a wide variety of
treatment. However, development has been hampered by the lack applications, for instance, as detection reagents (20) and to inhibit
of specific agents that bind such gene products, which in addition receptor interactions (21). In this work, an Affibody molecule
have properties making them suitable for use as medical imaging specific for HER2 is described.
agents. HER2 is a transmembrane protein belonging to the human
A targeting agent for medical imaging should have high affinity epidermal growth factor tyrosine kinase receptor family and is
for its target (1) and should be able to specifically target relevant overexpressed in several cancer types, but is present only to a small
disease-related structures in the body while avoiding normal tissue extent or not at all in normal adult tissue (22, 23). In breast and
(2). In the patient, it should quickly find its target whereas unbound ovarian cancers, HER2 is overexpressed in 23% to 30% of all cases
(24). In patients with breast cancer, the overexpression of HER2 is
associated with shorter time to disease progression and decreased
Note: A. Orlova and M. Magnusson contributed equally to this study.
overall survival, and it has been shown that the overexpression is
Requests for reprints: Fredrik Y. Nilsson, Affibody AB, Box 20137, SE-161 02 preserved in metastases (25, 26). Clinical practice guidelines of the
Bromma. Phone: 46-8-5988-3851; Fax: 46-8-5988-3801; E-mail: fredrik.nilsson@ American Society of Clinical Oncology recommend detection of
affibody.se.
I2006 American Association for Cancer Research. HER2 expression in all newly diagnosed or recurrent breast
doi:10.1158/0008-5472.CAN-05-3521 carcinomas in order to select patients who will benefit from
www.aacrjournals.org 4339 Cancer Res 2006; 66: (8). April 15, 2006
Cancer Research
treatment with Herceptin and anthracyclines (27). The use of nucleotides 238 to 2,109, was used as target proteins during selection. The
radionuclide molecular imaging of HER2 expression may help to protein was biotinylated as previously described (25) using EZ-Link Sulfo-
rapidly identify HER2 expression in tumor lesions, it may assist to NHS-LC-Biotin (Pierce, Rockford, IL) with a 30 times molar excess of
avoid false-negative results due to sampling errors and hetero- biotin.
The library was subjected to five rounds of selection in solution on
geneity of expression from biopsy probes, and can provide
biotinylated HER2-ECD using a 10-fold decreasing target concentration for
information on HER2 expression in cancers resistant to trastuzu- each round. To avoid nonspecific binders, all tubes used in this procedure
mab treatment. Together, these factors provide strong arguments were pretreated with PBST supplemented with 0.1% gelatin and the phage
for the development of radionuclide targeting strategies directed stock/s were preincubated with streptavidin beads (Dynabeads M-280
against HER2. Streptavidin; 112.06, Dynal A.S., Oslo, Norway) for rounds 1 and 2. The phage
A previously selected Affibody molecule, ZHER2:4 (28), was library was subjected to biopanning against HER2-ECD for 1 hour and
shown to bind selectively to the extracellular domain of HER2 45 minutes at room temperature under continuous rotation. To capture
with an affinity of 50 nmol/L. Literature data shows that an phage binding biotinylated target, SA beads were incubated with the phage-
increase of affinity beyond 10 8 mol/L improves the tumor target mixtures for 15 minutes. The beads were washed and the bound
uptake of targeting proteins (29, 30). To increase the affinity, we phages were subsequently eluted and propagated as previously described
(36, 37).
tried dimerization of ZHER2:4 (31, 32) and affinity maturation by
ELISA-based ranking of second generation binders. Single colonies
directed evolution, as reported here. For antibodies, secondary were inoculated in 1 mL TSB-YE medium supplemented with 100 Amol/L
libraries for affinity maturation have proven successful in isopropyl-L-thio-h-D-galactopyranoside and 100 Ag/mL ampicillin in deep
generating high-affinity scFv binders to HER2 (e.g., 13 pmol/L; well plates (Nunc, Roskilde, Denmark), and grown on a shaker overnight
ref. 33). However, to this date, it has not been shown whether at 37jC. Cells were pelleted by centrifugation at 3,000 g for 10 minutes.
single domain scaffold proteins, such as the 6 kDa Affibody The pellets were resuspended in 300 AL PBST and frozen for 30 minutes
molecule, could yield such high-affinity binders. Here, we at 80jC. The samples were thawed and centrifuged at 3,500 g for
describe a monovalent HER2-binding Affibody molecule with a 20 minutes. The supernatants (100 AL), containing ABD-tagged (38)
2,200-fold increased affinity obtained through an affinity Affibody molecules were loaded in microtiter wells, which had been
maturation process of the parental binder (28). The binder previously coated with 6 Ag/mL HSA in 15 mmol/L Na2CO3 and 35 mmol/L
NaHCO3 (pH 9.6) overnight at 4jC and blocked with 2% skimmed milk
was characterized in vitro and in vivo and compared with the
powder in PBST for 1 hour at room temperature. The plates were washed
nanomolar parental molecule. Tumor imaging efficiency was four times with PBST prior to the addition of 100 AL of 1 Ag/mL biotinylated
shown in mice using gamma camera. HER2 per well and incubated for 1.5 hours. After washing the wells four
times, 100 AL streptavidin-horseradish peroxidase (1:5,000; DAKO Cytoma-
tion, Denmark) per well were added and incubated for 1 hour. The wells
Materials and Methods were washed four times and 100 AL developing solution ImmunoPure TMB
Construction of a secondary phagemid library. The secondary library substrate kit (Pierce) was added to each well. After 30 minutes, 100 AL of the
was created by PCR amplification from a single 129-nucleotide template stop solution (2 mol/L H2SO4) was added to each well. The absorbance at
oligonucleotide with certain degenerated codons (5V-ctc gag gta gac aac aaa 450 nm was measured in an ELISA reader.
ttc aac aaa gaa nnk mrm mmm gcg tat tgg gag atc nnk nnk tta cct aac DNA sequencing and sequence clustering. DNA sequencing was done
tta aac nnk nnk caa nnk cgc gcc ttc atc cgc agt tta tat gat gac cca agc caa with ABI PRISM dGTP, BigDye Terminator v3.0 Ready Reaction Cycle
agc-3V), encoding helices 1 and 2 of the Z domain. The gene fragment Sequencing Kit (Applied Biosystems) according to the manufacturer’s
was amplified using the forward primer 5Vcccccccccctcgaggtaga caacaaatt- recommendations. The sequences were analyzed on an ABI PRISM 3100
caa-5V (Xho I site underlined) and the reverse primer 5Vcccccctg- Genetic Analyser.
ctagcaagttagcgctttggcttgggtcatc-3V (NheI site underlined), with 100 fmol The Affibody molecules were clustered using the so-called average-link
template oligonucleotide for each of 95 parallel reactions. The amplifica- hierarchical clustering method. Initially, each molecule’s sequence was
tions were done using AmpliTaq Gold polymerase (Applied Biosystems, defined as a separate subset. A complete clustering was obtained by finding
Foster City, CA) for 10 cycles (15 seconds at 96jC, 15 seconds at 60jC, and 1 the two subsets that were least dissimilar to each other and selected
minute at 72jC), pooled, purified using the QIAquick PCR purification kit subsets were merged to create a new subset, decreasing the number of
(Qiagen, Hilden, Germany), XhoI/NheI digested and ligated to XhoI/NheI- subsets by one. This procedure was repeated until all subsets were
phagemid vector pAffi128 encoding the third nonvariegated a-helix of the clustered. A subset was regarded as a cluster when it showed up as a node
Z domain. Electrocompetent Escherichia coli RRIDM15 (34) cells were relatively close to the vertical axis in a dendrogram, whereas the length
transformed with 60 aliquots of ligated material using 0.2-cm gap size of the branch to its parent was relatively large. Cluster validation was done
cuvettes in an ECM 630 set (BTX, Genetronics) at 2,500 V, 125 V and 50 AF. by applying the so-called Nearest-Exterior-Neighbor/Furthest-Interior-
Cells were grown in SOC medium [tryptone soy broth (TSB) + yeast extract Neighbor cluster validation method to the subset output from the clus-
(YE) supplemented with 1% glucose, 10 mmol/L MgCl2, 10 mmol/L MgSO4, tering method. First, a cluster quality index was computed for each subset.
10 mmol/L NaCl, and 2.5 mmol/L KCl] for 50 minutes and transferred to six A high value of this index indicates a compact and isolated subset. In a
Erlenmayer flasks, each containing 1 L of TSB + YE (30 g/L TSB and 5 g/L second step, a reference distribution of cluster quality indices was created,
YE; both from Merck, Darmstadt, Germany) supplemented with 2% glucose against which the cluster quality indices for the obtained subset were
and 25 Ag/mL carbencillin and grown overnight at 37jC. The cells were compared. This comparison also took subset size into account. The
centrifuged at 2,000 g, following resuspension in PBS/glycerol solution outcome of these comparisons was a number, the so-called significance
to a final approximate concentration of 20% glycerol, aliquoted and stored value, for each subset.
at 80jC. Protein expression and purification. Gene fragments encoding the
Phage selection procedures. Preparation of phage stocks from the selected second-generation Affibody molecules ZHER2:473, ZHER2:382, ZHER2:470,
affinity maturation library (a portion of Zlib2004HER2mat) and between ZHER2:487, ZHER2:489, ZHER2:477, ZHER2:492, ZHER2:475, ZHER2:342, and ZHER2:336, as
selections was done according to previously described procedures (18) well as the first-generation binder, ZHER2:4 (28), were subcloned from the
using the helper phage M13K07 (New England Biolabs, Beverly, MA). phagemid vector to a pET-derived expression vector pAY442 under the
Polyethylene glycol/NaCl precipitation yielded phage titers of f1013 control of the T7 promoter. The proteins were expressed in E. coli
colony-forming units/mL. A 100 kDa recombinant extracellular domain of BL21(DE3) cells and the His6-tagged proteins were IMAC purified under
HER2 (HER2-ECD) comprising 624 amino acids (35), corresponding to denatured conditions using QIAfilter 96 Plates Ni-NTA Superflow kit and
Cancer Res 2006; 66: (8). April 15, 2006 4340 www.aacrjournals.org
Affibody Tumor Imaging
QIAsoft 4.1, medium-scale protein/Ni-NTA Superflow 96 Denat program on used as a reference surface, and human IgG (Amersham Biosciences) was
a Biorobot 3000 (Qiagen). The protein samples refolded when dialyzed in immobilized (f5,000 resonance units) on a separate flow-cell surface on
PBS using Slide-A-Lyzer (Pierce) according to the manufacturer’s recom- CM5 sensor chips, to serve as negative controls. Binding analyses were done
mendations. The protein concentrations were calculated from the measured at 25jC, and PBS was used as the running buffer. In a first experiment,
absorption values at 280 nm. Extinction coefficients were theoretically 50 nmol/L of each Affibody molecule (diluted in PBS) was injected over all
calculated from the primary amino acid sequence. surfaces with a flow rate of 10 AL/min.
Biosensor analyses. A BIAcore 2000 instrument (Biacore AB, Uppsala, In a second experiment, the ZHER2:342 and ZHER2:477 Affibody variants
Sweden) was used for real-time biospecific interaction analysis between were subjected to a kinetic analysis, in which the proteins were injected
selected Affibody molecules and the target protein. HER2-ECD [diluted in over a HER2-ECD surface (approximate immobilization levels of 1,200,
10 mmol/L NaAc (pH 4.5)] was immobilized (f3,500 resonance units) on a 1,600, and 2,000 resonance units, respectively) at different concentrations
CM5 sensor chip (BR-1000-14, Biacore) according to the manufacturer’s (0-6 nmol/L, with 0.19 nmol/L as the lowest protein concentration diluted
instructions. Another flow-cell surface was activated and deactivated to be in PBS) with a flow rate of 50 AL/min. The dissociation equilibrium constant
Figure 1. A, the strategy for affinity

maturation. Amino acid sequence of the
wild-type Z domain aligned to deduced
amino acid sequences of different Affibody
variants selected against HER2-ECD (28).
The 13 randomized amino acid residues
(Q9, Q10, N11, F13, Y14, L17, H18, E24,
E25, R27, N28, Q32, and K35) are
presented. An amber stop codon is included
in the ZHER2:25 variant. Horizontal bars,
amino acid identities. Right, the number of
times each variant was found upon DNA
sequencing of 49 colonies. The design of
the constructed library aimed for affinity
maturation ZLibHER2:mat is presented in
single letter code, with positions selected for
variation in boldface (X = any amino acid).
Note, that a bias for arginine or lysine
residues is introduced at position 10, and a
bias for glutamine or threonine residues
at position 11. The three a-helices in the
wild-type Z domain are boxed. B, sequence
clustering. The Affibody molecules were
clustered using an average-link hierarchical
clustering method, and the result is
illustrated in a dendrogram. A subset was
regarded as a cluster when it showed up as a
node relatively close to the vertical axis in
the dendrogram, whereas the length of the
branch to its parent was relatively large.
Cluster validation was done by applying
a Nearest-Exterior-Neighbor/Furthest-
Interior-Neighbor cluster validation method
to the subset output from the clustering
method. Positions of the 10 second-
generation Affibody molecules selected for
further characterization. C, sequences of
selected second-generation Affibody
variants. Amino acid sequence of the
wild-type Z domain aligned to deduced
amino acid sequences of 10 different
second-generation Affibody variants
selected in the affinity maturation effort
against HER2-ECD. The amino acid
residues found in the variegated position in
boldface. Amino acid residues other than
R/K at position 10 and Q/T at position
11 could appear by alternative combinations
of the included nucleotides. Right, the
selection cycle from which the clone was
isolated/the selection principle applied for
this specific clone/the number of times each
variant was found on DNA sequencing of
160 randomly picked colonies from selection
cycles four and five.
Cancer Research
(K D), the association rate constant (k a), and the dissociation rate (Molecular Probes), 1 Ag/mL for 5 seconds, washed, dried for 1 hour, and
constant (k d) were calculated using BIAevaluation 3.2 software (Biacore), mounted with antifading reagent (Vector Laboratories, Inc.).
assuming a one-to-one Langmuir binding model taking mass transfer ZHER2:342 and anti-HER2 antibody were used to simultaneously stain
effects into account. A long dissociation phase was applied (60 minutes), HER2 on SKOV-3 cells. Slides with formaldehyde-fixed cells were stained
due to the slow off rate, to get fully reliable results. with ZHER2:342 (5 Ag/mL) Affibody molecule followed by goat anti-Affibody
In a third experiment, the difference in binding to HER2-ECD between IgG and rabbit anti-goat IgG Alexa Fluor 488 (Molecular Probes). Antibody
the monovalent ZHER2:4 (28) and the divalent (ZHER2:4)2 (31) first-generation staining was done on Affibody molecule–stained cells and the antibody was
Affibody variants and the affinity-matured second-generation ZHER2:342 detected with anti-human IgG Alexa Fluor 555 as described above. The
Affibody molecule, was evaluated by injection of 5 Amol/L of each protein slides were counterstained with 20 AL of 4V,6-diamidino-2-phenylindole
over the HER2-ECD surface, with a flow rate of 5 AL/min. (Molecular Probes), 1 Ag/mL for 5 seconds, washed, dried for 1 hour, and
Immunofluorescence. The human ovarian adenocarcinoma SKOV-3 mounted with antifading reagent (Vector Laboratories).
(HTB-77, ATCC), and the human neuroblastoma cell line, SH-SY5Y (CRL- Membrane fluorescence was analyzed in a DMLA microscope, equipped
2266, ATCC), were grown in 5% CO2 incubator at 37jC in 25 cm2 flasks. with a digital camera (Leica Microsystems AG). Digital images of green, red
SKOV-3 cells were cultured in McCoy’s 5a and SH-SY5Y in DMEM medium and blue fluorescence were acquired and saved as overlays using the IM1000
(Life Technologies, Invitrogen) supplemented with 2 mmol/L glutamine and software (Leica Microsystems).
10% fetal calf serum (Life Technologies, Invitrogen). Cells were trypsinated Immunohistochemical staining. SKOV-3 xenograft tumors (described
(Trypsin-EDTA, Life Technologies, Invitrogen) the day before analysis, below) were excised and snap-frozen in liquid nitrogen. Six-micron-thick
washed in medium, and resuspended to a concentration of 2.5 105 cells/ cryosections of the tumors (Ljung CM3000, Leica Microsystems) were fixed
mL. Approximately 5,000 to 10,000 cells were added per well of an eight- in 3% formaldehyde in PBS for 15 minutes and blocked in 1% FCS in PBS for
well, multi-well slide (Histolab, Sweden). The cells were incubated on multi- 1 hour before staining. The sections were either stained with ZHER2:342 at a
well slides overnight to obtain a flat morphology. The next day, medium was concentration of 5 Ag/mL, followed by polyclonal rabbit anti-Affibody IgG
gently removed from the slides and cells were stained for 1 hour with the (prepared in-house) and horseradish peroxidase-conjugated anti-rabbit IgG
ZHER2:342 Affibody molecule (5 Ag/mL) or 0.5 Ag/mL of the monoclonal (DAKO Cytomation) or with trastuzumab (0.5 Ag/mL) followed by
antibody trastuzumab (Herceptin, Apoteket AB, Sweden), with a volume of horseradish peroxidase–conjugated anti-human IgG (DAKO Cytomation).
20 AL/well. After staining, cells were fixed with 3% formaldehyde in PBS, one The staining was developed for 7 minutes with 3,3V-diaminobenzidine
drop per well for 15 minutes. The slides were rinsed in PBS and the ZHER2:342 chromogen substrate (DAKO Cytomation) and then washed for 2 to
Affibody molecule was detected with mouse anti-Affibody mouse serum 3 minutes. The slides were counterstained with Mayers HTX (Histolab) for
(prepared in-house) followed by 5 Ag/mL of goat anti-mouse IgG Alexa 20 seconds and then rinsed for 10 minutes before mounting with Mount-
Fluor 488 (Molecular Probes, Eugene, OR). Trastuzumab was detected with quick (Histolab). The staining was analyzed in a DMLA microscope,
5 Ag/mL of goat anti-human IgG Alexa Fluor 488 (Molecular Probes). The equipped with a digital camera (Leica Microsystems). Digital images were
slides were counterstained with 20 AL of 4V,6-diamidino-2-phenylindole acquired and saved using the IM1000 software (Leica Microsystems).
Figure 2. Biacore ranking of second-

generation Affibody variants and comparison
to first generation binders. A, sensorgrams
obtained after injection of the 10 different
second-generation Affibody molecules
over a sensor chip flow-cell surface
containing amine-coupled HER2-ECD.
Sensorgrams are: (a ) ZHER2:336, (b )
ZHER2:342, (c ) ZHER2:475, (d) ZHER2:477,
(e ) ZHER2:492, (f ) ZHER2:487, (g ) ZHER2:489, (h)
ZHER2:473, (i) ZHER2:382, and (j) ZHER2:470.
B, sensorgrams obtained after injection of
monovalent ZHER2:4 Affibody molecule,
compared with divalent (ZHER2:4)2 Affibody
molecule and second-generation ZHER2:342
Affibody molecule over a sensor chip
containing amine-coupled HER2-ECD.
Radioiodination. The radiolabeling with 125I of the ZHER2:342 Affibody

molecule was done using N-succinimidyl p-(trimethylstannyl)benzoate as a
precursor, as previously described (25). Labeling conditions were adjusted
to provide attachment for a single prosthetic group per protein molecule.
Radiochemical purity was controlled using ITCL SG eluted with 70%
acetone in water and was always better than 95%. The radiolabeled Affibody
molecules were analyzed using Biacore technology to verify that the labeling
procedure had not affected the binding affinity to HER2-ECD.
Biodistribution in tumor-bearing mice. Animal experiments were
approved by the local ethics committee for animal research. SKOV-3
xenografts were established on female BALB/c nu/nu mice (10-12 weeks old
on arrival), 4 to 8 weeks before the experiment by implanting f5 106
cells s.c. on the right hind leg. All mice were injected with f50 AL (0.5 Ag,
100 kBq) of the 125I-labeled ZHER2:4, (ZHER2:4)2, or ZHER2:342 Affibody
molecules into the tail vein. Groups of four mice were sacrificed after 1,
4, and 24 hours, and organs were dissected, weighted, and their radioactivity
content was measured in the automated gamma counter. Tissue uptake was
calculated as the percentage of injected radioactivity per gram of tissue
(% IA/g). To show that tumor uptake was target-mediated, a control group
of four animals was pretreated with 0.5 mg of cold, nonlabeled Affibody
molecules 45 minutes before the injection of 0.5 Ag of the radioiodinated
counterpart. Four hours later, these animals were sacrificed and tumor
radioactivity uptake was measured.
Gamma camera–based imaging. SKOV-3 xenografts were established
for f2 months, as described above. The tumors had an average volume of
f0.5 cm3 at the day of the experiment. In total, 60 Ag of ZHER2:342 was
labeled with 111 MBq 125I, resulting in a specific activity of 1.3 MBq/Ag.
Each mouse was injected with 90 AL (2.3 Ag, 2.9 MBq) 125I-ZHER2:342. After
6 or 24 hours, respectively, mice were euthanized with a lethal dose of
Ketalar/Rompun i.p. The mice were imaged using an e.CAM gamma
camera (Siemens, Erlangen, Germany) with a low-energy high-resolution
collimator.
Results
Affinity maturation of first-generation HER2 binders. We
designed and constructed the affinity maturation library based on a
primary set of HER2-binding Affibody molecules (28). These were
Figure 3. Fluorescence staining of SKOV-3 and SH-SY5Y cells with ZHER2:342
aligned with an allowed bias for the strongest binders, and we and trastuzumab. A, SKOV-3 cells stained for HER2 expression with ZHER2:342
found it reasonable to fix 5 (13, 14, 28, 32, and 35), and allow a bias Affibody molecules; B, SKOV-3 cells stained for HER2 expression with
for R and K in position 10, and Q and T in position 11, of the 13 trastuzumab; C, the HER2-negative cell line SH-SY5Y stained with ZHER2:342;
and D, the HER2-negative cell line SH-SY5Y stained with trastuzumab. E and F,
originally randomized amino acid residues. Thus, positions 9, 17, the same cells double-stained for ZHER2:342 (green ) and trastuzumab (red),
18, 24, 25, and 27 were targeted for randomization (Fig. 1A) using respectively. G, immunohistochemical staining of HER2 with ZHER2:342 Affibody
molecules was done on a section of SKOV-3 cell xenograft tumor. The
NNG/T degenerated codons. Due to the small size of the Affibody section was counterstained with hematoxylin (blue ). The transversing mouse
molecule, it was possible to use a single 129 nucleotide connective tissue remains negative. H, a consecutive section of SKOV-3 cell
oligonucleotide with degenerated codons, encoding helices 1 and xenograft tumor was stained with trastuzumab.
2 of the Z-domain, to create the secondary library. The
oligonucleotide was PCR-amplified and subsequently ligated into
a phagemid vector encoding the third a-helix of the Z-domain. The clustering of the sequenced clones using an average-link hierar-
resulting library consisted of f3 108 members, which should be chical clustering method developed in-house, the relationship
enough to sample a majority of all theoretical variants. Phage between selected clones was visualized (Fig. 1B). Based on the
stocks were prepared and selections done essentially as previously values in the ELISA screening and the clustering results, 10 clones
described (18, 28), using decreasing concentrations of target were selected for further characterization (Fig. 1C). The cycle from
protein and intensive washing to select for the strongest HER2- which the particular clone was isolated, and the frequency of
binding Affibody variants in the library. occurrence are indicated (Fig. 1C), as well as their position in the
Clones obtained after four and five rounds of selection were clustering analysis (Fig. 1B).
cultivated in 96-well plates, freeze-thawed to release periplasmic Biosensor screening, stability, and affinity determination.
content, and subjected to an ELISA screening procedure for HER2 To obtain an initial ranking of binding affinities, the 10 selected
binding activity (data not shown). When subjecting 160 randomly Affibody variants as well as the ZHER2:4 and dimeric (ZHER2:4)2 were
picked clones to this ELISA screening, using the first-generation expressed and analyzed for their HER2 binding using a Biacore
Affibody molecule ZHER2:4 as reference, a majority of the clones instrument. The different Affibody molecules were separately
gave significantly higher absorbance values than the reference injected over sensor chip flow-cell surfaces containing the
ZHER2:4, thus, indicating stronger HER2 binding (data not shown). immobilized target protein HER2-ECD and a control protein IgG,
The same 160 clones were subjected to DNA sequencing, and upon respectively. An overlay plot of the obtained sensorgrams suggested
Cancer Research
Table 1. Biodistribution of ZHER2:4, (ZHER2:4)2, and ZHER2:342
Organ 1 hour 4 hours 24 hours

(% IA/g)
ZHER2:4 (ZHER2:4)2 ZHER2:342 ZHER2:4 (ZHER2:4)2 ZHER2:342 ZHER2:4 (ZHER2:4)2 ZHER2:342
Blood 2.61 F 0.35 3.83 F 0.29 4.50 F 0.78 0.39 F 0.09 0.56 F 0.14 0.25 F 0.03 0.03 F 0.00 0.02 F 0.01 0.04 F 0.01
Heart 1.00 F 0.16 1.34 F 0.12 2.11 F 0.64 0.13 F 0.06 0.31 F 0.14 0.11 F 0.02 0.01 F 0.00 0.01 F 0.01 0.01 F 0.02
Lung 2.25 F 0.40 2.56 F 0.10 4.56 F 1.02 0.40 F 0.09 0.44 F 0.12 0.37 F 0.03 0.04 F 0.01 0.05 F 0.02 0.07 F 0.02
Liver 4.08 F 0.70 5.10 F 0.25 8.32 F 1.18 0.77 F 0.09 1.00 F 0.38 0.30 F 0.05 0.07 F 0.03 0.02 F 0.00 0.04 F 0.00
Spleen 1.81 F 0.40 1.41 F 0.10 2.68 F 0.37 0.58 F 0.17 0.19 F 0.04 0.12 F 0.02 0.06 F 0.03 0.01 F 0.01 0.03 F 0.01
Pancreas 0.93 F 0.10 2.43 F 0.08 2.48 F 0.48 0.16 F 0.06 0.26 F 0.05 0.13 F 0.03 0.01 F 0.00 0.02 F 0.01 0.01 F 0.00
Kidney 24.98 F 3.69 68.90 F 10.62 49.64 F 6.72 6.04 F 0.64 29.85 F 3.16 9.51 F 0.78 0.16 F 0.01 0.87 F 0.13 0.46 F 0.21
Stomach 1.18 F 0.10 2.26 F 0.29 2.21 F 0.46 0.59 F 0.30 0.76 F 0.21 0.25 F 0.04 0.02 F 0.00 0.06 F 0.08 0.03 F 0.01
Small intestine 2.35 F 0.70 ND F ND 2.65 F 1.01 0.41 F 0.13 ND F ND 0.61 F 0.27 0.01 F 0.01 ND F ND 0.01 F 0.01
Large intestine 0.90 F 0.15 ND F ND 1.93 F 0.67 0.12 F 0.06 ND F ND 0.66 F 0.50 0.00 F 0.00 ND F ND 0.01 F 0.01
Salivary glands 1.33 F 0.18 2.70 F 0.53 2.33 F 0.27 0.60 F 0.14 1.39 F 0.31 0.33 F 0.09 0.02 F 0.00 0.03 F 0.02 0.02 F 0.02
Skin 1.33 F 0.24 3.31 F 1.18 2.58 F 0.75 0.25 F 0.07 0.27 F 0.09 0.26 F 0.10 0.02 F 0.00 0.02 F 0.01 0.03 F 0.01
Muscle 0.36 F 0.06 0.84 F 0.12 0.66 F 0.14 0.05 F 0.02 0.07 F 0.03 0.06 F 0.03 0.00 F 0.00 ND F ND 0.00 F 0.00
Bone 0.54 F 0.08 0.56 F 0.11 1.03 F 0.24 0.06 F 0.05 0.08 F 0.04 0.11 F 0.02 0.00 F 0.00 0.05 F 0.04 0.03 F 0.04
Brain 0.07 F 0.01 ND F ND 0.12 F 0.02 0.00 F 0.00 ND F ND 0.04 F 0.02 0.00 F 0.00 ND F ND 0.00 F 0.00
Tumor 4.05 F 1.28 3.23 F 0.12 8.23 F 2.08 2.40 F 0.17 2.27 F 0.55 9.46 F 1.60 0.17 F 0.03 0.34 F 0.11 4.13 F 0.46
Ratios
Tumor/blood 1.6 0.8 1.8 6.2 4.1 37.8 5.7 17.0 103.3
Tumor/kidney 0.2 0.0 0.2 0.4 0.1 1.0 1.1 0.4 9.0
Tumor/liver 1.0 0.6 1.0 3.1 2.3 31.1 2.5 17.0 103.3
NOTE: Tumor and normal organ uptakes are expressed as the percentage of injected activity per gram F SD (n = 4).
that approximately half of the analyzed Affibody variants bound to HER2-ECD was determined to be f22 pmol/L for ZHER2:342 and
the target with significantly higher affinities than ZHER2:4 (Fig. 2A). f32 pmol/L for ZHER2:477. The association rate constant (k a) was
The slower off-rate kinetics of second-generation variants could calculated to be f4.8 106 M 1 s 1 and the dissociation rate
clearly be seen by visual inspection of the sensorgrams. As constant (k d) f1.1 10 4 s 1 for ZHER2:342, and for ZHER2:477, the
expected, no significant binding could be seen in the control k a was f4.3 106 M 1 s 1 and the k d was f1.4 10 4 s 1. None
protein IgG (data not shown). A group of four Affibody variants, of the Affibody molecules bound EGFR-ECD (HER1), a protein
ZHER2:336, ZHER2:342, ZHER2:475, and ZHER2:477, with good binding which has strong homology with HER2. Based on these data,
profiles were selected for further studies. ZHER2:342 was selected for further characterization.
Differences in solubility between the Affibody variants was Comparing first- and second-generation binders in vitro.
investigated. The threshold was set at no loss of protein at a The affinity-matured ZHER2:342 (K D f22 pmol/L for HER2) was
concentration of 4 mg/mL upon freezing and thawing, and with compared with a monovalent (K D f50 nmol/L for HER2) and a
no signs of aggregates in gel filtration (data not shown). ZHER2:336 divalent form (K D f3 nmol/L for HER2) of the first-generation
and ZHER:475 did not meet these criteria. The melting temperatures Affibody molecule (28, 31), ZHER2:4 using Biacore analysis (Fig. 2B).
(Tm’s) of the selected Affibody variants varied substantially: 73jC The monovalent and bivalent first-generation Affibody molecule
for ZHER2:336, 67jC for ZHER2:342, 46jC for ZHER2:475, and 49jC for had similar association rates (31), whereas the bivalent (ZHER2:4)2
ZHER2:477, respectively. The Affibody variants were also tested had a 13-fold slower dissociation rate. Interestingly, the affinity-
in vivo for tumor uptake and kidney retention at 4 hours with matured ZHER2:342 Affibody molecule did not only have a 90-fold
tumor to blood ratios for ZHER2:336, ZHER2:342, ZHER2:475, and slower dissociation rate than the parental ZHER2:4, but also a 27-fold
ZHER2:477 of 18 F 6, 40 F 3, 18 F 4, and 21 F 6, respectively, and faster association rate (Fig. 2B).
tumor to kidney ratios of 0.6 F 0.2, 1.0 F 0.1, 0.5 F 0.2, and 0.62 F Fluorescence and immunohistochemical analysis. The ability
0.06, respectively. Taking these different factors into account, the of the matured Affibody molecules to detect membrane-bound
ZHER2:342 followed by the ZHER2:477 variant seemed to be most HER2 was analyzed using fluorescence microscope analysis. The
promising for further studies. Affibody molecule ZHER2:342 molecule worked very well as a
The ZHER2:342 and ZHER2:477 Affibody molecules were subjected to detection reagent and gave a membrane staining on HER2-positive
a kinetic analysis in order to determine the kinetic binding SKOV-3 cells (Fig. 3A), but not on HER2-negative SH-SY5Y cells
constants. Prior to the kinetic analysis, the protein concentration (Fig. 3C). Approximately 50% of the SKOV-3 cells were strongly
was carefully determined by amino acid analysis, and gel filtration stained with ZHER2:342 with an almost identical staining pattern as
was done (data not shown) to confirm a monomeric state. The the monoclonal antibody trastuzumab, which was used as a
Affibody molecules were then injected over a HER2-ECD flow-cell positive control (Fig. 3B and D). Double staining with trastuzumab
surface or a control EGFR-ECD flow-cell surface at different and ZHER2:342 Affibody molecules showed that both reagents
concentrations, and the dissociation equilibrium constant (K D) for bind to the same cells and with identical staining morphology
(Fig. 3E and F). Importantly, in accordance with earlier studies (28), was indeed target mediated, one group of mice was pretreated
ZHER2:342 and trastuzumab could bind simultaneously to SKOV-3 with a 1,000-fold (0.5 mg) excess of unlabeled ZHER2:342 Affibody
cells without blocking each other’s binding sites (data not shown) molecule before injection of radiolabeled ZHER2:342 Affibody
confirming that they bind to different epitopes on HER2 (31). molecule. Tumor uptake at 4 hours post-injection was reduced
Furthermore, ZHER2:342 and trastuzumab were used for traditional from 9.5 F 2.3% to 1.5 F 1.5% ID/g (P < 0.01) by the pretreatment,
immunohistochemical staining of cryosections of SKOV-3 xeno- demonstrating that tumor uptake was target-specific. The same
graft tumor tissues. Both the Affibody molecule and trastuzumab confirmation of target specificity was obtained both for the
bound to SKOV-3 tumor cells leaving the surrounding and monomeric and dimeric form of first-generation of Affibody
transversing mouse connective tissue negative (Fig. 3G and H) molecules, where tumor uptake was significantly reduced by the
with no qualitative difference in the staining behavior between injection of a large excess of unlabeled forms of the same peptide
ZHER2:342 and trastuzumab. (P < 0.001 and P < 0.01, respectively). The only sites of nonspecific
In vivo biodistribution. To investigate whether improved accumulation were the kidneys, which could be expected for
affinity would result in improved tumor uptake, the affinity- proteins with a size significantly below renal filtration threshold.
matured ZHER2:342 Affibody molecule (K D f22 pmol/L) was However, the radioactivity was rapidly washed out from the
compared with the first-generation Affibody molecule in its kidneys.
monovalent (ZHER2:4, K D f50 nmol/L for HER2) and divalent It was apparent that the ratio of uptake in tumor compared
forms (K D f3 nmol/L). Mice carrying SKOV-3 xenografts of f3 with other organs was improved with increased affinity when the
mm in diameter were injected with 125I-labeled Affibody molecules. radioactivity concentration in tumor and blood was compared over
The radioactivity in excised tumors was measured at 1, 4, and 24 time for the different Affibody molecules (Fig. 4B; Table 1). In spite
hours post-injection. and presented as the percentage of injected of the increased affinity of the dimer, the difference in tumor-to-
dose per gram of tissue (% ID/g; Table 1). Very little difference was blood ratio between the monomeric and dimeric variants of the
observed between the two variants of the first-generation binder first-generation binder was modest. In contrast, a dramatic
whereas the uptake for ZHER2:342 compared with the parental improvement was found for the ZHER2:342 Affibody molecule,
ZHER2:4 monomer was improved by 2.03, 3.94, and 24.29 times at 1, showing an impressive tumor-to-blood ratio of 38:1 at 4 hours
4, and 24 hours, respectively, with a tumor uptake of f9% ID/g at and 100:1 at 24 hours post-injection. At 4 hours post-injection,
4 hours (Fig. 4A). To verify that the observed tumor-targeting effect tumor-to-organ ratios for most other organs were at least 2-fold
Figure 4. Evaluation of the Affibody molecules in vivo.

A, biodistribution of 125I-labeled Affibody molecules 4 hours
post-injection. Percentage of injected dose per gram of tissue is
plotted for ZHER2:4 (white ), and ZHER2:342 (gray ), monovalent Affibody
molecules (columns, mean; bars, F SE; n = 4). B, in a second
experiment, the relation in uptake between tumor and blood
was investigated at different time points relevant to imaging;
tumor (squares ) and blood (circles ) values for ZHER2:4 (dashed line ),
and ZHER2:342 (solid line ). Points, mean; bars, F SE (n = 4).
Cancer Research
better for ZHER2:342 in comparison with ZHER2:4. Twenty-four hours be an avenue for improving tumor specificity (39, 40)—and thus,
post-injection, that difference was >10-fold. Furthermore, at the contrast of a radionuclide image—over existing larger affinity
24 hours, the tumor to kidney ratio was improved to 9:1 for the proteins like antibodies and fragments thereof (41, 42). To obtain
ZHER2:342 as compared with the ZHER2:4, where the tumor to kidney small high affinity binders for HER2, a 6 kDa HER2-specific
ratio was 1:1. Affibody molecule was affinity maturated in vitro. The procedure
Gamma camera–based imaging of HER2 expression in used for affinity maturation resulted in an increased affinity by
SKOV-3 xenografts. To evaluate whether the matured ZHER2:342 three orders of magnitude in one single maturation step. The
could be used for in vivo imaging of HER2 expression, the matured Affibody molecule with highest affinity (K D of 22 pmol/L)
Affibody molecule was labeled with 125I and injected into mice was selected through various in vitro and in vivo characterizations
carrying SKOV-3 tumors f0.5 cm3 in size. After 6 or 24 hours, and showed improved tumor targeting in biodistribution studies
images of tumor-bearing mice were acquired using a gamma and gamma imaging.
camera. After 6 hours, the tumor could clearly be visualized with It is interesting to compare the in vivo data of the ZHER2:342
high contrast. At this time point, the only site with elevated Affibody molecule described in this study with published results for
radioactivity uptake besides the tumor were the kidneys. However, other HER2-targeting proteins. We have restricted ourselves to the
radioactivity accumulation in the tumor was appreciably higher analysis of targeting of HER2 using radioiodinated proteins.
than in the kidneys (Fig. 5B). The increase in contrast due to Moreover, all studies which were selected for comparison used
increase in affinity is striking when compared with the first- SKOV-3 ovarian carcinoma xenografts, the same as that used in this
generation (ZHER2:4)2 Affibody molecule, in which the kidney study. Together, this should minimize differences in agent-target
uptake is much more prominent than the tumor after 6 hours interaction, antigen density, and cellular processing. Comparisons
(Fig. 5A). After 24 hours, the nonspecific radioactivity accumu- in the literature show that the uptake of the ZHER2:342 Affibody
lation was further reduced whereas a high dose remained on the molecule at 4 hours post-injection (9.5 F 1.6%, ID/g) was similar to
tumor (Fig. 5C). the uptake in tumor of the C6.5 scFv diabody (10.1% ID/g; ref. 43),
and exceeded the uptake of the 741F8-2 sFv (2.9% ID/g; ref. 44), and
its homodimer (5.6% ID/g; ref. 45). However, better clearance of
Discussion ZHER2:342 provided tumor-to-blood ratios, which at this time point,
In this study, we describe in vivo tumor targeting by use of a new was 7.8 times better than for the C6.5 scFv diabody, 4.5 times better
class of small affinity proteins called Affibody molecules. We than for the 741F8-2 sFv, and 9.3 times better than for the 741F8-2
reasoned that the very high affinity for the HER2 tumor target, (sFvV)2. Advantages in contrast remained over time even at
combined with the very small size of the targeting molecule, may 24 hours.
Figure 5. Gamma camera–based imaging of xenografted mice, at 6 and 24 hours after injection of 125I-labeled Affibody molecules. In a set of experiments, the
first-generation Affibody molecule, ZHER2:4 (A) was compared with the affinity-matured Affibody molecule ZHER2:342 (B) at 6 hours post-injection. Intensity of color
corresponds to radioactive accumulation: low (blue ) and high (yellow ) accumulation. Kidney uptake is more prominent for the ZHER2:4 Affibody molecule (A).
The tumor uptake of ZHER2:342 was stable up to at least 24 hours post-injection (C ).
It has been suggested that bivalently binding antibody fragments is important to choose the optimal time point for the image, as
have superior tumor specificity compared with their monovalent there is an initial high uptake of activity in the kidneys due to
counterparts (46), and in a recent study, Wu and colleagues showed excretion. The activity is, however, rapidly decreasing, from almost
that even relatively large (f80 kDa) HER2-specific divalent 50% ID/g at 1 hour post-injection to <10% ID/g at 4 hours post-
minibodies could be used for tumor imaging in mice (47). However, injection. Six hours post-injection, the uptake in the tumor is much
a bivalent antibody fragment is, by necessity, larger than a more prominent than the kidneys, as can be seen in Fig. 5B.
monovalent one, and should thus have poorer tissue penetration If the molecule was to be tested in the clinic, obviously a
properties (39). It remains an open question if the targeting benefit single-photon emitter 123I (T 1/2 = 13.3 hours) or positron-emitter
124
of divalency could be overcome by increased affinity for a I (T 1/2 = 4.17 days) should be used instead of 125I. The
monovalent smaller fragment. In this study, the first-generation development of suitable chemistry to label the ZHER2:342 molecule
bivalent HER2 Affibody molecule (K D 3 nmol/L) did not show with generator-produced 99mTc (T 1/2 = 6 hours) or 68Ga (T 1/2 = 68
improved tumor-to-blood ratios compared with the monovalent minutes) could further facilitate future imaging studies.
molecule (K D 50 nmol/L), in spite of higher affinity. In contrast,
when improving the affinity, both the total tumor uptake and
tumor-to-blood contrast improved dramatically. These results may Acknowledgments
suggest that for very small proteins, keeping the size small is more Received 10/6/2005; revised 12/19/2005; accepted 2/13/2006.
important than gaining functional affinity by dimerization. Grant support: P25882-1 from the Swedish Agency for Innovation Systems
(VINNOVA) and by two research grants from the Swedish Cancer Society.
Taken together, these results suggest that the radioiodinated The costs of publication of this article were defrayed in part by the payment of page
ZHER2:342 Affibody molecule may be a suitable agent for in vivo charges. This article must therefore be hereby marked advertisement in accordance
imaging of HER2 expression, and a literature comparison suggests with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Per-Åke Nygren and Elin Gunneriusson for valuable advice on the affinity
that ZHER2:342 may provide better tumor contrast at early time maturation, Per-Olof Fjällström for computing the software for clustering of selected
points than HER2-specific antibodies and fragments thereof (48). It clones, and Lars Abrahmsén and Birger Jansson for comments on the manuscript.
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Targeted
cancer nanotherapy
by Gloria J. Kim and Shuming Nie*
Significant progress has been made in the Conventional cancer therapy and diagnostics involves
development of new agents against cancer and new the application of catheters, surgery, biopsy,
chemotherapy, and radiation. Most current anticancer
delivery technologies. Proteomics and genomics
agents do not greatly differentiate between
continue to uncover molecular signatures that are
cancerous and normal cells. This leads to systemic
unique to cancer. Yet, the major challenge remains in toxicity and adverse effects. Consequently, the
targeting and selectively killing cancer cells while systemic application of these drugs often causes
affecting as few healthy cells as possible. severe sideeffects in other tissues (e.g. bone marrow
Nanometer-sized particles have novel optical, suppression, cardiomyopathy, and neurotoxicity),
electronic, and structural properties that are not which greatly limits the maximal allowable dose of
available from either individual molecules or bulk the drug. In addition, rapid elimination and
widespread distribution into nontargeted organs and
solids. When linked with tumor-targeting moieties,
tissues requires the administration of a drug in large
such as tumor-specific ligands or monoclonal
quantities, which is uneconomical and is often
antibodies, these nanoparticles can be used to target complicated because of nonspecific toxicity.
cancer-specific receptors, tumor antigens Nanotechnology could offer a less invasive alternative,
(biomarkers), and tumor vasculatures with high enhancing the life expectancy and quality of life of the
affinity and precision. patient. The diameter of human cells spans 10-20 µm. The size
of cell organelles ranges from a few nanometers to a few
hundred nanometers. Nanoscale devices can readily interact
with biomolecules on the cell surface and within the cells in
a noninvasive manner, leaving the behavior and biochemical
properties of those molecules intact. In their ‘mesoscopic’
size range of 10-100 nm in diameter, nanoparticles have
more surface areas and functional groups that can be linked
to multiple optical, radioisotopic, or magnetic diagnostic and
therapeutic agents. When linked with tumor-targeting
Department of Biomedical Engineering, ligands such as monoclonal antibodies, these nanoparticles
Emory University and Georgia Institute of Technology,
101 Woodruff Circle Suite 2001, can be used to target tumor antigens (biomarkers), as well as
Atlanta, GA 30322, USA
*E-mail: snie@emory.edu
tumor vasculatures with high affinity and specificity. In this
28 August 2005 ISSN:1369 7021 © Elsevier Ltd 2005

RESEARCH REPORT
Glossary
Passive Targeting EPR Effect
Agonist A drug that triggers an action from a cell or
another drug.
Tumor Environment
Angiogenesis The process of vascularization of a
tissue involving the development of new capillary blood Direct Local Delivery
vessels. This is caused by the release of chemicals by

Active Targeting Lectin-carbohydrate Interaction
the tumor.
Antagonist A compound that inhibits the effect of a Ligand-receptor Interaction

hormone or drug.
Antibody-antigen Interaction
Apoptosis A form of programmed cell death induced
by external or internal signals that trigger the activity Scheme 1. Targeting strategies for nanoscale drug delivery systems.
of proteolytic caspases, whose actions dismantle the
article, we discuss different targeting strategies for nanoscale
cell and result in cell death.
drug delivery systems (see Scheme 1), and offer a perspective
Doxorubicin A type of anticancer drug called an on cancer nanotherapy.
‘anthracycline glycoside’. It works by impairing DNA
synthesis, and hence can target rapidly dividing cells. Passive targeting
Solid tumors have a diffusion-limited maximal size1,2 of
Epitope A part of a molecule that an antibody will
about 2 mm3 and will remain at this size until angiogenesis
recognize and bind to.
occurs, thus granting them access to the circulation3. Rapid
Integrin Superfamily of cell surface proteins that are vascularization to serve fast-growing cancerous tissues
involved in binding to extracellular matrix components inevitably leads to a leaky, defective architecture and
in some cases. impaired lymphatic drainage. This structure allows an
enhanced permeation and retention (EPR) effect4,5 (first
Liposome Lipid bilayers that form colloidal particles in
described by Matsumura et al.6), as a result of which
an aqueous medium.
nanoparticles accumulate at the tumor site. For such a
Matrix metalloproteinase A member of a group of passive targeting mechanism to work, the size and surface
enzymes that can break down proteins, such as properties of drug delivery nanoparticles must be controlled
collagen, normally found in the spaces between cells to avoid uptake by the reticuloendothelial system (RES)7. To
in tissues (i.e. extracellular matrix proteins). They maximize circulation times and targeting ability, the optimal
need Zn or Ca ions to work properly. Matrix size should be less than 100 nm in diameter and the surface
metalloproteinases are involved in wound healing, should be hydrophilic to circumvent clearance by
angiogenesis, and tumor cell metastasis. macrophages (large phagocytic cells of the RES). A
hydrophilic nanoparticle surface safeguards against plasma
Reticuloendothelial system A system composed of protein adsorption, and can be achieved through hydrophilic
monocytes and macrophages located in reticular polymer coating (e.g. by polyethylene glycol (PEG),
connective tissue. These cells are responsible for poloxamines, poloxamers, and polysaccharides) or the use of
engulfing (phagocytosis) and removing cellular debris, branched or block copolymers8,9. The covalent linkage of
old cells, pathogens, and foreign substances from the amphiphilic copolymers (polylactic acid, polycaprolactone,
bloodstream. and polycyanonacrylate) chemically coupled to PEG9-11 is
generally preferred, as it avoids aggregation and ligand
Xenobiotics Chemicals that are foreign to the body.
desorption when in contact with blood components7.
Xenograft Cells of one species transplanted to another. An alternative passive targeting strategy is to use the
unique tumor environment in a scheme called tumor-activated
August 2005 29
RESEARCH REPORT
Biocompatible polymer
Lectin-carbohydrate is one of the classic examples for
Anticancer
agent targeted drug delivery15. Lectins are proteins of
nonimmunological origin that are capable of recognizing and
binding to glycoproteins expressed on cell surfaces. Lectin
Cleavable linkage
responsive to tumor environment interactions with certain carbohydrates are very specific.
Carbohydrate moieties can be used to target drug delivery
systems to lectins (direct lectin targeting), and lectins can be
used as targeting moieties to target cell surface
carbohydrates (reverse lectin targeting). However, drug
delivery systems based on lectin-carbohydrate have been
nucleus
developed mainly to target whole organs16, which can harm
normal cells. Therefore, in most cases, the targeting moiety is
Fig. 1 Tumor-activated prodrug delivery and targeting. The anticancer agent is conjugated directed toward specific receptors or antigens expressed on
to a biocompatible polymer via an ester bond. The linkage is hydrolyzed by cancer-specific
enzymes, or by high or low pH, at the tumor site, at which time the nanoparticle releases the plasma membrane or elsewhere at the tumor site.
the drug.
Multiple drug resistance (MDR), which is a major challenge
prodrug therapy. The drug is conjugated to a tumor-specific in chemotherapy, often stems from the overexpression of the
molecule and remains inactive until it reaches the target12 plasma membrane P-glycoprotein (Pgp)17,18. In general, Pgp
(Fig. 1). Overexpression of the matrix metalloproteinase acts as an efflux pump to extrude positively charged
(MMP), MMP-2, in melanoma has been shown in a number of xenobiotics – including some anticancer drugs – out of the
preclinical as well as clinical investigations. Mansour et al.13 cell. Many tumor cells are resistant to doxorubicin, which is a
reported a water-soluble maleimide derivative of doxorubicin Pgp substrate. To overcome the resistance,
(DOX) incorporating an MMP-2-specific peptide sequence poly(cyanocarylate) nanoparticles have been developed19,20.
(Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln) that binds rapidly and Adsorption of the nanoparticles onto the plasma membrane
selectively to the cysteine-34 position of circulating albumin. and the subsequent release of doxorubicin leads to saturation
The albumin-doxorubicin conjugate is cleaved efficiently and of Pgp. Furthermore, the negatively charged degradation
specifically by MMP-2, releasing a doxorubicin tetrapeptide products of the polymer form an ion pair and neutralize the
(Ile-Ala-Gly-Gln-DOX) and subsequently doxorubicin. pH and positive charge of doxorubicin19, enhancing the diffusion of
redox potential have also been explored as drug-release the drug across the plasma membrane. Blagosklonny
triggers at the tumor site14. proposed an approach to selectively kill resistant cancer cells
Yet another passive targeting method is the direct local that is based on a temporary increase in the resistance of
delivery of anticancer agents to tumors. This approach has sensitive cells against certain drugs by specific protectors,
the obvious advantage of excluding the drug from the such as pharmacological inhibitors of apoptosis21. These
systemic circulation. However, administration can be highly protectors are pumped out by MDR cells, while increasing the
invasive, as it involves injections or surgical procedures. For resistance in sensitive cells that do not have active drug
some tumors that are difficult to access, such as lung cancers, efflux pumps. After applying a cytotoxic drug, sensitive cells
the technique is nearly impossible to use. are protected and survive the exposure, while unprotected
MDR counterparts are killed. By abolishing dose-limiting side-
Active targeting effects of chemotherapy, this strategy might provide a means
Active targeting is usually achieved by conjugating to the to selectively treat aggressive and resistant cancers. Tsuruo
nanoparticle a targeting component that provides preferential suggested that antibodies to P-glycoprotein overexpressed on
accumulation of nanoparticles in the tumor-bearing organ, in multidrug resistant (MDR) cells could make an attractive
the tumor itself, individual cancer cells, intracellular targeting moiety22.
organelles, or specific molecules in cancer cells. This approach The overexpression of receptors or antigens in many
is based on specific interactions such as lectin-carbohydrate, human cancers lends itself to efficient drug uptake via
ligand-receptor, and antibody-antigen. receptor-mediated endocytosis (cellular ingestion) – see Fig. 2.
30 August 2005
RESEARCH REPORT
Since glycoproteins cannot remove polymer-drug conjugates Cargo
that have entered the cells via endocytosis23,24, this active Ligand
targeting mechanism provides an alternative route for Cleavable Bond
overcoming MDR. Receptor
The cell surface receptor for folate is inaccessible from the 1. Internalization
circulation to healthy cells because of its location on the apical Cancer Cell Surface
H+
H+
membrane of polarized epithelia, but is overexpressed on the
6. Membrane Fusion
surface of various cancers, including ovary, brain, kidney, H+
breast, and lung malignancies. Surface plasmon resonance

2. Early Endosomes H+
studies reveal that folate-conjugated PEGylated cyanoacrylate H+
5. Recycling Endosomes
nanoparticles have a ten-fold higher affinity for the folate
H+
H+ H+
receptor than free folate11. Folate receptors are often H+
organized in clusters and bind preferably to the multivalent 3. Acidified Endosomes 4. Endosomal Release
forms of the ligand. Furthermore, confocal microscopy

Fig. 2 Nanoparticle drug delivery and targeting using receptor-mediated endocytosis.
demonstrated selective uptake and endocytosis of folate-
conjugated nanoparticles by tumor cells that bear folate with better success. For example, anti-ERBB2
receptors. Interest in exploiting folate receptor targeting in immunoliposomes loaded with doxorubicin show greater
cancer therapy and diagnosis has increased rapidly, as attested antitumor activity than the free drug or the drug loaded in
by many conjugated systems, including proteins, liposomes, nontargeted liposomes in several tumor xenograft
imaging agents, and neutron activation compounds9,11,25-31. models39,40. Moreover, the systemic toxicity of the
Tumor targeting by antibodies with engineered properties immunoliposome-targeted doxorubicin was much less than
(Table 1) is in its infancy, but holds much promise. The that of free doxorubicin. Bispecific antibodies, which are non-
monoclonal antibody (mAb) BR96 (anti-sialyl Lewis Y natural antibodies with two different epitopes, have been
antigen), conjugated with doxorubicin, has proved highly used most widely for the delivery of immune effector cells
efficacious in tumour xenograft studies32 but, unfortunately, and, to a lesser extent, for the delivery of radionuclides,
has shown little or no efficacy in Phase II trials for metastatic drugs, and toxins to tumors41,42.
breast cancer33 and advanced gastric adenocarcinoma34, Alternatively, tumor vasculatures can be targeted to allow
respectively. Moreover, dose-limiting gastrointestinal targeted delivery to a wide range of tumor types. A number of
toxicities are observed in the breast cancer trial, because the angiogenesis inhibitors are undergoing clinical trials (Table 2).
immunoconjugate binds to antigen-positive normal cells in Antiangiogenic therapy prevents neovascularization (the
gastric mucosa, small intestine, and pancreas33. proliferation of blood vessels in different tissues) by
Calicheamicins35-37 and maytansinoids38 are the most inhibiting proliferation, migration, and differentiation of
extensively evaluated of many small-molecule toxins that are endothelial cells43. Vascular endothelial growth factor (VEGF)
used for direct antibody arming, but indirect arming has met is expressed in many solid tumors. A potent angiogenesis-
Table 1 Antibodies in cancer therapeutics.
Mechanism Antibody target Trade name

Agonist activity CD40, CD137 Various
Antagonist activity CTLA4 MDX-010
Angiogenesis inhibition VEGF Avastin™
Antibody-dependent cell-mediated cytotoxicity CD20 Rituxan®, HuMax-CD20
HER-2/neu Herceptin®
EGF receptor HuMax-EGFr
Toxin-mediated killing CD33 Mylotarg®
Disruption signaling HER-2/neu Pertuzumab (2C4)
Complement-dependent cytotoxicity CD20 Rituxan®, HuMax-CD20
Blockage ligand binding EGF receptor Erbitux™
August 2005 31
RESEARCH REPORT
Table 2 Angiogenesis inhibitors undergoing clinical trial.
Mechanism Antiangiogenic drug Target Stage of clinical development

Monoclonal antibodies targeting VEGF-A Avastin™ VEGF-A Phase I, II, III
VEGF-Trap VEGF-A Phase I
Antibodies targeting VEGFR-2 IMC-ICII VEGFR-2 Phase I
Receptor tyrosine kinase inhibitors SU5416 VEGFR-2 Phase I, II, III
SU6668 VEGFR-2, bFGFR, PDGFR Phase I, II
ZD 6474 VEGFR-2, EGFR Phase I, II
Endothelial cell proliferation inhibitors ABT-510 Endothelial CD-36 Phase I, II
Angiostatin Various Phase I
Endostatin Various Phase I
TNP-470 Methionine aminopeptidase, Phase I
cyclin dependent kinase 2
Integrin activity inhibitors Vitaxin Integrin ανβ3 Phase I, II
Medi-522 Integrin αvβ3 Phase I
Vascular targeting agents ZD 6126 Endothelial tubulin Phase I
DMXAA Endothelial tubulin Phase I
stimulating protein, it also increases the permeability of nonionizable compounds, micronization, soft-gel technology,
tumor blood vessels. This leads to swelling of the tumor, cosolvents, surfactants, or complexing agents have been
ultimately hindering the ability of cancer cells to recruit used52,34. Since it is faster and more cost effective to
blood supply through angiogenesis. VEGF has been used in redesign the molecule than to develop a new one, a broadly
liposomes and polymeric nanospheres to deliver angiostatin based technology applicable to poorly water-soluble drugs
and endostatin44,45. The αvβ3 integrin is one of the most could have a tremendous impact.
specific biomarkers that can differentiate newly formed Paclitaxel (Taxol™) is a microtubule-stabilizing agent that
capillaries from their mature counterparts46. Although all promotes tubulin polymerization, disrupting cell division and
endothelial cells use integrin receptors to attach to the causing cell death54. It displays neoplastic activity against
extraluminal submatrix, one unique receptor (αvβ3 integrin) primary epithelial ovarian carcinoma, breast, colon, and lung
is found on the luminal surface of the endothelial cell only cancers. Because it is poorly soluble in aqueous solution, the
during angiogenesis47. High-affinity αvβ3 selective ligands, formulation that is currently available is Chremophor EL
Arg-Gly-Asp (RGD), have been identified by phage display (polyethoxylated castor oil) and ethanol55. In a new
studies. The cyclic form, which contains a conformationally formulation used in Abraxane (recently approved by the FDA
constrained RGD, has a higher binding affinity than the linear to treat metastatic breast cancer), paclitaxel was conjugated
form48. Doxorubicin-loaded PEG nanoparticles conjugated to
cyclic RGD49 and paclitaxel-cyclic RGD nanoparticles50 have Targeting ligand Self-assembled polymer
been reported recently. Anticancer

agent
Covalent bond Tumor specific
Perspective enzyme cleavable linkage
100 nm Receptor-mediated
Nanotechnology is beginning to change the scale and endocytosis
methods of drug delivery. For decades, researchers have been

developing new anticancer agents and new formulations for
delivering existing and new agents.
More than 40% of active substances identified through nucleus
combinatorial screening programs are poorly soluble in Solid tumor
water51. The conventional, and most current, formulations of Receptor nucleus

Release of the anticancer agent
such drugs are frequently plagued with problems such as poor
and inconsistent bioavailability. The most widely used Fig. 3 Self-assembled polymeric nanoparticles for both tumor targeting and therapeutic
functions. Inset: delivery of the nanoparticle drugs by receptor-mediated endocytosis and
method for enhancing solubility is to generate a salt. For controlled drug release inside the cytoplasm.
32 August 2005
RESEARCH REPORT
to albumin nanoparticles56. In addition to circumventing side- Self-assembled polymer
effects of the highly toxic Chremophor EL, which include Anticancer agent
Imaging agent
hypersensitivity reactions and toxicity to kidney cells and
nerve tissue (nephrotoxicity and neurotoxicity)55,57, the
system cleverly takes advantage of albumin receptors for Tumor specific
enzyme cleavable linkage
improved drug delivery to cancer cells.
Covalent bond
For specific targeting, the differences between cancerous
100 nm Targeting ligand 100 nm
cells and normal cells, which include uncontrolled
proliferation, insensitivity to negative growth regulation, and Fig. 4 Multifunctional nanoparticles for integrated cancer imaging and therapy. As
antigrowth signals, angiogenesis and metastasis can be nanoparticles are developed for clinical applications, an exciting opportunity is to monitor
drug delivery and treatment efficacy by using embedded imaging agents.
exploited. Thanks to recent advances in proteomics and
genomics, there is a growing body of knowledge of unique biomedical applications. Many of the principles used to target
cancer markers. They form the basis of complex interactions delivery of drugs to cancers may also be applied to target
between bioconjugated nanoparticles and cancer cells. Carrier imaging and diagnostic agents. The full in vivo potential of
design and targeting strategies may vary according to the cancer nanotechnology in targeted drug delivery and imaging
type, developmental stage, and location of cancer. There is can be realized by using strategically engineered
much synergy between imaging and nanotechnology in multifunctional nanoparticles (Figs. 3 and 4). NT
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August 2005 33
INSTITUTE OF PHYSICS PUBLISHING JOURNAL OF PHYSICS D: APPLIED PHYSICS
J. Phys. D: Appl. Phys. 36 (2003) R167–R181 PII: S0022-3727(03)40035-1
TOPICAL REVIEW
Applications of magnetic nanoparticles

in biomedicine
Q A Pankhurst1 , J Connolly2 , S K Jones3 and J Dobson4
1
Department of Physics and Astronomy, and London Centre for Nanotechnology,
University College London, Gower Street, London WC1E 6BT, UK
2
Department of Applied Physics, Curtin University of Technology, GPO Box U1987,
Perth 6845, Western Australia
3
SIRTeX Medical Limited, PO Box 760, North Ryde, New South Wales 2113, Australia
4
Department of Biomedical Engineering and Medical Physics, Centre for Science and
Technology in Medicine, Keele University, Stoke-on-Trent ST4 7QB, UK
E-mail: q.pankhurst@ucl.ac.uk
Received 4 April 2002

Published 18 June 2003
Online at stacks.iop.org/JPhysD/36/R167
Abstract
The physical principles underlying some current biomedical applications of
magnetic nanoparticles are reviewed. Starting from well-known basic
concepts, and drawing on examples from biology and biomedicine, the
relevant physics of magnetic materials and their responses to applied
magnetic fields are surveyed. The way these properties are controlled and
used is illustrated with reference to (i) magnetic separation of labelled cells
and other biological entities; (ii) therapeutic drug, gene and radionuclide
delivery; (iii) radio frequency methods for the catabolism of tumours via
hyperthermia; and (iv) contrast enhancement agents for magnetic resonance
imaging applications. Future prospects are also discussed.
1. Introduction body, such as a tumour. Third, the magnetic nanoparticles

can be made to resonantly respond to a time-varying magnetic
Magnetic nanoparticles offer some attractive possibilities in field, with advantageous results related to the transfer of energy
biomedicine. First, they have controllable sizes ranging from from the exciting field to the nanoparticle. For example, the
a few nanometres up to tens of nanometres, which places particle can be made to heat up, which leads to their use
them at dimensions that are smaller than or comparable to as hyperthermia agents, delivering toxic amounts of thermal
those of a cell (10–100 µm), a virus (20–450 nm), a protein energy to targeted bodies such as tumours; or as chemotherapy
(5–50 nm) or a gene (2 nm wide and 10–100 nm long). This and radiotherapy enhancement agents, where a moderate
means that they can ‘get close’ to a biological entity of interest. degree of tissue warming results in more effective malignant
Indeed, they can be coated with biological molecules to make cell destruction. These, and many other potential applications,
them interact with or bind to a biological entity, thereby are made available in biomedicine as a result of the special
providing a controllable means of ‘tagging’ or addressing it. physical properties of magnetic nanoparticles.
Second, the nanoparticles are magnetic, which means that In this paper we will address the underlying physics of
they obey Coulomb’s law, and can be manipulated by an the biomedical applications of magnetic nanoparticles. After
external magnetic field gradient. This ‘action at a distance’, reviewing some of the relevant basic concepts of magnetism,
combined with the intrinsic penetrability of magnetic fields including the classification of different magnetic materials
into human tissue, opens up many applications involving the and how a magnetic field can exert a force at a distance,
transport and/or immobilization of magnetic nanoparticles, or we will consider four particular applications: magnetic
of magnetically tagged biological entities. In this way they separation, drug delivery, hyperthermia treatments and
can be made to deliver a package, such as an anticancer drug, magnetic resonance imaging (MRI) contrast enhancement. We
or a cohort of radionuclide atoms, to a targeted region of the will conclude with an outlook on future prospects in this area,
0022-3727/03/130167+15$30.00 © 2003 IOP Publishing Ltd Printed in the UK R167

Topical Review
especially with regard to the challenges faced in bringing volume, where m is the magnetic moment on a volume V
current laboratory-tested technologies into mainstream use. of the material. All materials are magnetic to some extent,
with their response depending on their atomic structure and
2. Basic concepts temperature. They may be conveniently classified in terms of
their volumetric magnetic susceptibility, χ, where
2.1. M–H curves
M = χH, (2)
Figure 1 shows a schematic diagram of a blood vessel into
which some magnetic nanoparticles have been injected. The describes the magnetization induced in a material by H.
magnetic properties of both the injected particles and the In SI units χ is dimensionless and both M and H are
ambient biomolecules in the blood stream are illustrated by expressed in A m−1 . Most materials display little magnetism,
their different magnetic field response curves. To understand and even then only in the presence of an applied field;
these curves better, we need to be aware of some of the these are classified either as paramagnets, for which χ
fundamental concepts of magnetism, which will be recalled falls in the range 10−6 –10−1 , or diamagnets, with χ in the
briefly here. Further details can be found in one of the many range −10−6 to −10−3 . However, some materials exhibit
excellent textbooks on magnetism (e.g. [1, 2]). ordered magnetic states and are magnetic even without a field
If a magnetic material is placed in a magnetic field of applied; these are classified as ferromagnets, ferrimagnets
strength H, the individual atomic moments in the material and antiferromagnets, where the prefix refers to the nature
contribute to its overall response, the magnetic induction: of the coupling interaction between the electrons within the
material [2]. This coupling can give rise to large spontaneous
B = µ0 (H + M), (1) magnetizations; in ferromagnets M is typically 104 times larger
than would appear otherwise.
where µ0 is the permeability of free space, and the The susceptibility in ordered materials depends not just
magnetization M = m/V is the magnetic moment per unit on temperature, but also on H, which gives rise to the
characteristic sigmoidal shape of the M–H curve, with
M M
M approaching a saturation value at large values of H.
DM PM Furthermore, in ferromagnetic and ferrimagnetic materials
(x1000) (x100) one often sees hysteresis, which is an irreversibility in the
magnetization process that is related to the pinning of magnetic
H H domain walls at impurities or grain boundaries within the
material, as well as to intrinsic effects such as the magnetic
anisotropy of the crystalline lattice. This gives rise to open
M–H curves, called hysteresis loops. The shape of these
loops are determined in part by particle size: in large particles
(of the order micron size or more) there is a multi-domain
ground state which leads to a narrow hysteresis loop since it
takes relatively little field energy to make the domain walls
move; while in smaller particles there is a single domain
ground state which leads to a broad hysteresis loop. At even
smaller sizes (of the order of tens of nanometres or less) one
can see superparamagnetism, where the magnetic moment
of the particle as a whole is free to fluctuate in response to
thermal energy, while the individual atomic moments maintain
their ordered state relative to each other. This leads to the
anhysteretic, but still sigmoidal, M–H curve shown in figure 1.
M M
The underlying physics of superparamagnetism is founded
FM SPM on an activation law for the relaxation time τ of the net
magnetization of the particle [3, 4]:

H H
E
τ = τ0 exp , (3)
kB T
where E is the energy barrier to moment reversal, and

Figure 1. Magnetic responses associated with different classes of kB T is the thermal energy. For non-interacting particles
magnetic material, illustrated for a hypothetical situation in which the pre-exponential factor τ0 is of the order 10−10 –10−12 s
ferromagnetic particles of a range of sizes from nanometre up to and only weakly dependent on temperature [4]. The energy
micron scale are injected into a blood vessel. M–H curves are barrier has several origins, including both intrinsic and
shown for diamagnetic (DM) and paramagnetic (PM) biomaterials extrinsic effects such as the magnetocrystalline and shape
in the blood vessel, and for the ferromagnetic (FM) injected
particles, where the response can be either multi-domain (- - - - in anisotropies, respectively; but in the simplest of cases it
FM diagram), single-domain (—— in FM diagram) or has a uniaxial form and is given by E = KV , where
superparamagnetic (SPM), depending on the size of the particle. K is the anisotropy energy density and V is the particle
R168
Topical Review
volume. This direct proportionality between E and V is domain wall motion imposed by the intrinsic anisotropy and
the reason that superparamagnetism—the thermally activated microstructural impurities and grain boundaries in the material.
flipping of the net moment direction—is important for small This energy is delivered by the applied field, and can be
particles, since for them E is comparable to kB T at, say, characterized by the area enclosed by the hysteresis loop.
room temperature. However, it is important to recognize that This leads to the concept that if one applies a time-varying
observations of superparamagnetism are implicitly dependent magnetic field to a ferromagnetic or ferrimagnetic material,
not just on temperature, but also on the measurement time τm one can establish a situation in which there is a constant flow
of the experimental technique being used (see figure 2). If of energy into that material, which will perforce be transferred
τ τm the flipping is fast relative to the experimental time into thermal energy. This is the physical basis of hyperthermia
window and the particles appear to be paramagnetic (PM); treatments, which are discussed further in section 5. Note that
while if τ τm the flipping is slow and quasi-static properties a similar argument regarding energy transfer can be made for
are observed—the so-called ‘blocked’ state of the system. A SPM materials, where the energy is needed to coherently align
‘blocking temperature’ TB is defined as the mid-point between the particle moments to achieve the saturated state; this also is
these two states, where τ = τm . In typical experiments τm discussed in more detail in section 5.
can range from the slow to medium timescales of 102 s for DC
magnetization and 10−1 –10−5 s for AC susceptibility, through 2.2. Forces on magnetic nanoparticles
to the fast timescales of 10−7 –10−9 s for 57 Fe Mössbauer
To understand how a magnetic field may be used to manipulate
spectroscopy.
magnetic nanoparticles, we need to recall some elements of
All of the different magnetic responses discussed above
vector field theory. This is not always intuitive, and the reader
are illustrated in figure 1 for the case of ferromagnetic
is directed to recent reviews for further details [5–7]. It is also
or ferrimagnetic nanoparticles injected into a blood vessel.
important to recognize that a magnetic field gradient is required
Depending on the particle size, the injected material exhibits
to exert a force at a distance; a uniform field gives rise to a
either a multi-domain, single-domain or superparamagnetic
torque, but no translational action. We start from the definition
(SPM) M–H curve. The magnetic response of the blood
of the magnetic force acting on a point-like magnetic dipole m:
vessel itself includes both a PM response—for example, from
the iron-containing haemoglobin molecules, and a diamagnetic Fm = (m · ∇)B, (4)
(DM) response—for example, from those intra-vessel proteins
which can be geometrically interpreted as differentiation with
that comprise only carbon, hydrogen, nitrogen and oxygen
respect to the direction of m. For example, if m = (0, 0, mz )
atoms. It should be noted that the magnetic signal from the
then m · ∇ = mz (∂/∂z) and a force will be experienced on the
injected particles, whatever their size, far exceeds that from
dipole provided there is a field gradient in B in the z-direction.
the blood vessel itself. This heightened selectivity is one of the
In the case of a magnetic nanoparticle suspended in a weakly
advantageous features of biomedical applications of magnetic
DM medium such as water, the total moment on the particle
nanoparticles. can be written m = Vm M, where Vm is the volume of the
Returning to the hysteresis which gives rise to the open particle and M is its volumetric magnetization, which in turn
M–H curves seen for ferromagnets and antiferromagnets, is given by M = χH, where χ = χm − χw is the effective
it is clear that energy is needed to overcome the barrier to susceptibility of the particle relative to the water. For the case
of a dilute suspension of nanoparticles in pure water, we can
(a) (b) approximate the overall response of the particles plus water
system by B = µ0 H, so that equation (4) becomes:
Vm χ
Fm = (B · ∇)B. (5)
µ0
Furthermore, provided there are no time-varying electric fields
or currents in the medium, we can apply the Maxwell equation
∇ × B = 0 to the following mathematical identity:
∇(B · B) = 2B × (∇ × B) + 2(B · ∇)B = 2(B · ∇)B, (6)
to obtain a more intuitive form of equation (5):
2
B
Fm = Vm χ∇ or
2µ0 (7)
1
Figure 2. Illustration of the concept of superparamagnetism, where Fm = Vm χ∇ 2 B · H ,
the circles depict three magnetic nanoparticles, and the arrows
represent the net magnetization direction in those particles. in which the magnetic force is related to the differential of the
In case (a), at temperatures well below the magnetostatic field energy density, 21 B · H. Thus, if χ > 0
measurement-technique-dependent blocking temperature TB of the the magnetic force acts in the direction of steepest ascent of the
particles, or for relaxation times τ (the time between moment energy density scalar field. This explains why, for example,
reversals) much longer than the characteristic measurement time τm , when iron filings are brought near the pole of a permanent
the net moments are quasi-static. In case (b), at temperature well
above TB , or for τ much shorter than τm , the moment reversals are so bar magnet, they are attracted towards that pole. It is also the
rapid that in zero external field the time-averaged net moment on the basis for the biomedical applications of magnetic separation
particles is zero. and drug delivery, as will be discussed in sections 3 and 4.
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3. Magnetic separation smaller magnetic particle sizes can also be advantageous, for
example, in reducing the likelihood that the magnetic material
3.1. Cell labelling and magnetic separation will interfere with further tests on the separated cells [20].
In biomedicine it is often advantageous to separate out
specific biological entities from their native environment 3.2. Separator design
in order that concentrated samples may be prepared for
Magnetic separator design can be as simple as the application
subsequent analysis or other use. Magnetic separation using
and removal of a permanent magnet to the wall of a test tube
biocompatible nanoparticles is one way to achieve this. It
to cause aggregation, followed by removal of the supernatant
is a two-step process, involving (i) the tagging or labelling
(figure 3(a)). However, this method can be limited by slow
of the desired biological entity with magnetic material, and
accumulation rates [21]. It is often preferable to increase the
(ii) the separating out of these tagged entities via a fluid-based
separator efficiency by producing regions of high magnetic
magnetic separation device.
field gradient to capture the magnetic nanoparticles as they
Tagging is made possible through chemical modification
float or flow by in their carrier medium. A typical way
of the surface of the magnetic nanoparticles, usually by coating
to achieve this is to loosely pack a flow column with a
with biocompatible molecules such as dextran, polyvinyl
magnetizable matrix of wire (e.g. steel wool) or beads [22]
alcohol (PVA) and phosopholipids—all of which have been
and to pump the magnetically tagged fluid through the column
used on iron oxide nanoparticles [8–10]. As well as providing
while a field is applied (figure 3(b)). This method is faster
a link between the particle and the target site on a cell or
than in the first case, although problems can arise due to the
molecule, coating has the advantage of increasing the colloidal
settling and adsorption of magnetically tagged material on the
stability of the magnetic fluid. Specific binding sites on the
matrix. An alternative, rapid throughput method which does
surface of cells are targeted by antibodies or other biological
macromolecules such as hormones or folic acid [11–13]. As not involve any obstructions being placed in the column is the
antibodies specifically bind to their matching antigen this use of specifically designed field gradient systems, such as
provides a highly accurate way to label cells. For example, the quadrupolar arrangement shown in figure 4 which creates
magnetic particles coated with immunospecific agents have a magnetic gradient radially outwards from the centre of the
been successfully bound to red blood cells [8, 14], lung cancer flow column [23].
cells [15], bacteria [16], urological cancer cells [17] and Golgi As well as separating out the magnetically tagged material,
vesicles [18]. For larger entities such as the cells, both the spatially varying magnitude of the field gradient can
magnetic nanoparticles and larger particles can be used: for be used to achieve ‘fluid flow fractionation’ [24]. This
example, some applications use magnetic ‘microspheres’— is a process in which the fluid is split at the outlet into
micron sized agglomerations of sub-micron sized magnetic fractions containing tagged cells or proteins with differing
particles incorporated in a polymeric binder [19].
The magnetically labelled material is separated from its (a)
native solution by passing the fluid mixture through a region in
which there is a magnetic field gradient which can immobilize
the tagged material via the magnetic force of equation (7). This
force needs to overcome the hydrodynamic drag force acting
on the magnetic particle in the flowing solution,
Fd = 6πηRm v, (8)
where η is the viscosity of the medium surrounding the cell (b)

(e.g. water), Rm is the radius of the magnetic particle, and
v = vm − vw is the difference in velocities of the cell and the
water [6]. There is also buoyancy force that affects the motion,
but this is dependent on the difference between the density of
the cell and the water, and for most cases of interest in biology
and medicine can be neglected. Equating the hydrodynamic
drag and magnetic forces, and writing Vm = 43 πRm 3
, gives the
velocity of the particle relative to the carrier fluid as:
Rm
2
χ ξ
v = ∇(B 2 ) or v = ∇(B 2 ), (9)
9µ0 η µ0
Figure 3. The standard methods of magnetic separation: in (a) a
where ξ is the ‘magnetophoretic mobility’ of the particle—a magnet is attached to the container wall of a solution of magnetically
parameter that describes how manipulable a magnetic particle tagged (•) and unwanted (◦) biomaterials. The tagged particles are
is. For example, the magnetophoretic mobility of magnetic gathered by the magnet, and the unwanted supernatant solution is
microspheres can be much greater than that of nanoparticles, removed. In (b) a solution containing tagged and unwanted
biomaterials flows continuously through a region of strong magnetic
due to their larger size. This can be an advantage, for example, field gradient, often provided by packing the column with steel wool,
in cell separations, where the experimental timeframe for the which captures the tagged particles. Thereafter the tagged particles
separations is correspondingly shorter. On the other hand, are recovered by removing the field and flushing through with water.
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(a) (b) separation is used to increase the concentration of the material.

The mobility of the magnetic nanoparticles allows a shorter
reaction time and a greater volume of reagent to be used than
in standard immunoassays where the antibody is bound to a
plate. In a variation of this procedure, magnetic separation
has been used to localize labelled cells at known locations
for cell detection and counting via optical scanning [14]. The
cells are labelled both magnetically and fluorescently and move
through a magnetic field gradient towards a plate on which lines
of ferromagnetic material have been lithographically etched.
The cells align along these lines and the fluorescent tag is used
for optical detection of the cells.
Figure 4. A rapid throughput method of magnetic separation, in
which an annular column containing a flowing solution of
magnetically tagged (•) and unwanted (◦) biomaterials is placed 4. Drug delivery
within a set of magnets arranged in quadrature: (a) longitudinal
cross-section of the annular column; (b) transverse cross-section of 4.1. Motivation and physical principles
the four magnets with the resulting magnetic field lines. Under the
action of the magnetic field gradient the tagged particles move to the The major disadvantage of most chemotherapies is that
column walls, where they are held until the field is removed and they are relatively non-specific. The therapeutic drugs
they are recovered by flushing through with water. The central core are administered intravenously leading to general systemic
of the column is made of non-magnetic material to avoid distribution, resulting in deleterious side-effects as the drug
complications due to the near-zero field gradients there.
attacks normal, healthy cells in addition to the target tumour
cells. For example, the side effects of anti-inflammatory
magnetophoretic mobilities. In a variant of this, the fluid is drugs on patients who have chronic arthritis can lead to the
static while an applied magnetic field is moved up the container discontinuation of their use. However, if such treatments could
[25]. The particles move up the container in the resulting be localized, e.g. to the site of a joint, then the continued use of
field gradient at a velocity dependent on their magnetophoretic these very potent and effective agents could be made possible.
mobility. At the top of the container they enter a removable Recognition of this led researchers in the late 1970s
section and are held here by a permanent magnet. The bottom to propose the use of magnetic carriers to target specific
section of the container moves to the next section, a magnetic sites (generally cancerous tumours) within the body [34–36].
field with different strength to the first is applied and the process The objectives are two-fold: (i) to reduce the amount of
repeats. The result is a fractionation of the sample into aliquots systemic distribution of the cytotoxic drug, thus reducing
of differing magnetophoretic mobility. the associated side-effects; and (ii) to reduce the dosage
required by more efficient, localized targeting of the
3.3. Applications drug. In magnetically targeted therapy, a cytotoxic drug is
attached to a biocompatible magnetic nanoparticle carrier.
Magnetic separation has been successfully applied to many These drug/carrier complexes—usually in the form of a
aspects of biomedical and biological research. It has proven biocompatible ferrofluid—are injected into the patient via
to be a highly sensitive technique for the selection of rare the circulatory system. When the particles have entered the
tumour cells from blood, and is especially well suited to the bloodstream, external, high-gradient magnetic fields are used
separation of low numbers of target cells [26]. This has, for to concentrate the complex at a specific target site within the
example, led to the enhanced detection of malarial parasites body (figure 5). Once the drug/carrier is concentrated at the
in blood samples either by utilizing the magnetic properties of target, the drug can be released either via enzymatic activity or
the parasite [27] or through labelling the red blood cells with changes in physiological conditions such as pH, osmolality,
an immunospecific magnetic fluid [28]. It has been used as or temperature [37], and be taken up by the tumour cells.
a pre-processing technology for polymerase chain reactions, This system, in theory, has major advantages over the normal,
through which the DNA of a sample is amplified and identified non-targeted methods of cytotoxic drug therapy.
[29]. Cell counting techniques have also been developed. One The physical principles underlying magnetic targeting
method estimates the location and number of cells tagged by therapy are similar to those used in magnetic separation,
measuring the magnetic moment of the microsphere tags [30], and are derived from the magnetic force exerted on a SPM
while another uses a giant magnetoresistive sensor to measure nanoparticle by a magnetic field gradient, as in equation (7).
the location of microspheres attached to a surface layered with The effectiveness of the therapy is dependent on several
a bound analyte [31]. physical parameters, including the field strength, gradient
In another application, magnetic separation has been used and volumetric and magnetic properties of the particles.
in combination with optical sensing to perform ‘magnetic As the carriers (ferrofluids) are normally administered
enzyme linked immunosorbent assays’ [32, 33]. These assays intravenously or intra-arterially, hydrodynamic parameters
use fluorescent enzymes to optically determine the number such as blood flow rate, ferrofluid concentration, infusion
of cells labelled by the assay enzymes. Typically the target route and circulation time also will play a major role—as will
material must first be bound to a solid matrix. In a modification physiological parameters such as tissue depth to the target site
of this procedure the magnetic microspheres act as the surface (i.e. distance from the magnetic field source), reversibility and
for initial immobilization of the target material and magnetic strength of the drug/carrier binding, and tumour volume [38].
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Tissue
F
F
Magnetic
Nanoparticles Blood Vessel
F
F F
F
Tissue SiO
SiO2 2
Magnet F
F
Ferrite
Fe rr ite F
F
Core
Cor e
F
F F
F
Figure 5. A hypothetical magnetic drug delivery system shown in
cross-section: a magnet is placed outside the body in order that its
magnetic field gradient might capture magnetic carriers flowing in F
F
the circulatory system.
Figure 6. Schematic diagram of a functionalized magnetic
In general, larger particles (e.g. magnetic microspheres, ca nanoparticle showing a core/shell structure with a shell of silica,
SiO2 , and functional groups attached to the shell.
1 µm in diameter, comprising agglomerates of SPM particles)
are more effective at withstanding flow dynamics within the
circulatory system—particularly in larger veins and arteries. polymer or (ii) a porous biocompatible polymer in which
In most cases the magnetic field gradient is generated by magnetic nanoparticles are precipitated inside the pores [45].
a strong permanent magnet, such as Nd-Fe-B, fixed outside Recent developmental work on carriers has largely focused on
the body over the target site. Preliminary investigations new polymeric or inorganic coatings on magnetite/maghemite
of the hydrodynamics of drug targeting suggest that for nanoparticles [10, 46–52], although noble metal coatings such
most magnetite-based carriers, flux densities at the target as gold are also being considered [53]. Research also continues
site must be of the order of 0.2 T with field gradients of into alternative magnetic particles, such as iron, cobalt or nickel
approximately 8 T m−1 for femoral arteries and greater than [54–56] or yttrium aluminium iron garnet [57]. Cobalt/silica
100 T m−1 for cartoid arteries [39]. This suggests that targeting
carriers are currently being investigated for their use in eye
is likely to be most effective in regions of slower blood
surgery to repair detached retinas [58, 59].
flow, particularly if the target site is closer to the magnet
source. More recently, Richardson and others have developed
mathematical models to determine particle trajectories for 4.3. Targeting studies
a variety of field/particle configurations in two dimensions,
including consideration of their motion as they approach the Magnetic carriers were first used to target cytotoxic drugs
vessel wall [40]. This is significant since near the walls the (doxorubicin) to sarcoma tumours implanted in rat tails [60].
particle motion is no longer governed by Stoke’s law for the The initial results were encouraging, showing a total remission
drag force, equation (8), and the hydrodynamic parameters of the sarcomas compared to no remission in another group
are modified. Experimental work is underway to inform the of rats which were administered with ten times the dose
development of such models [41]. but without magnetic targeting. Since that study, success
in cytotoxic drug delivery and tumour remission has been
reported by several groups using animals models including
4.2. Magnetic drug carriers
swine [61, 62], rabbits [37] and rats [38, 63, 64]. Kubo
Since the first magnetic polymer carriers of the 1970s, a et al recently offered a variation on these techniques. They
variety of magnetic nanoparticle and microparticle carriers implanted permanent magnets at solid osteosarcoma sites in
have been developed to deliver drugs to specific target sites hamsters and delivered the cytotoxic compounds via magnetic
in vivo. The optimization of these carriers continues today. liposomes. They reported a four-fold increase in cytotoxic
Generally, the magnetic component of the particle is coated drug delivery to the osteosarcoma sites when compared with
by a biocompatible polymer such as PVA or dextran, although normal intravenous (non-magnetic) delivery [65], as well as a
recently inorganic coatings such as silica have been developed. significant increase in anti-tumour activity and the elimination
The coating acts to shield the magnetic particle from the of weight-loss as a side effect [66].
surrounding environment and can also be functionalized by This technique has also been employed to target cytotoxic
attaching carboxyl groups, biotin, avidin, carbodi-imide and drugs to brain tumours. Brain tumours are particularly
other molecules [42–44]. As shown in figure 6, these difficult targets due to the fact that the drug must cross the
molecules then act as attachment points for the coupling of blood–brain barrier. Pulfer and Gallo [63] demonstrated that
cytotoxic drugs or target antibodies to the carrier complex. particles as large as 1–2 µm could be concentrated at the site
The carriers typically have one of two structural of intracerebral rat glioma-2 (RG-2) tumours. Though the
configurations: (i) a magnetic particle core (usually magnetite, concentration of the particles in the tumour was low, it was
Fe3 O4 , or maghemite, γ -Fe2 O3 ) coated with a biocompatible significantly higher than non-magnetic particles. In a later
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study the group demonstrated that 10–20 nm magnetic particles external magnetic fields, the virus is in contact with the tissue
were even more effective at targeting these tumours in rats [64]. for a longer period of time, increasing the efficiency of gene
Electron microscopic analysis of brain tissue samples revealed transfection and expression [77, 78]. New magnetic carriers
the presence of magnetic carriers in the interstitial space in are being developed specifically for these applications [79]
tumours but in normal brain tissue, they were only found in and this is an area which shows great promise.
the vasculature. Mykhaylyk and others [67] recently had less
success using magnetite–dextran nanoparticles but were able 5. Hyperthermia
to target rat glial tumours by disrupting the blood–brain barrier
immediately prior to particle injection. 5.1. Catabolism of tumours by hyperthermia
Studies of magnetic targeting in humans were rare up to
now. A Phase I clinical trial conducted by Lübbe and others The possibility of treating cancer by artificially induced
[68–70] demonstrated that the infusion of ferrofluids was well hyperthermia has led to the development of many
tolerated in most of the 14 patients studied. In addition, the different devices designed to heat malignant cells while
authors reported that the ferrofluid was successfully directed sparing surrounding healthy tissue [80–82]. Experimental
to the advanced sarcomas without associated organ toxicity. investigations of the application of magnetic materials for
More recently, FeRx Inc. was granted fast-track status to hyperthermia date back to 1957 when Gilchrist et al [83]
proceed with multi-centre Phases I and II clinical trials of heated various tissue samples with 20–100 nm size particles
their magnetic targeting system for hepatocellular carcinomas of γ -Fe2 O3 exposed to a 1.2 MHz magnetic field. Since
(a type of liver tumour). This appears to be the most promising then there have been numerous publications describing a
clinical application at present. variety of schemes using different types of magnetic materials,
As promising as these results have been, there are several different field strengths and frequencies and different methods
problems associated with magnetically targeted drug delivery of encapsulation and delivery of the particles [84–102]. In
[38, 71]. These limitations include (i) the possibility of broad terms, the procedure involves dispersing magnetic
embolization of the blood vessels in the target region due to particles throughout the target tissue, and then applying an AC
accumulation of the magnetic carriers, (ii) difficulties in scaling magnetic field of sufficient strength and frequency to cause
up from animal models due to the larger distances between the the particles to heat. This heat conducts into the immediately
target site and the magnet, (iii) once the drug is released, it is no surrounding diseased tissue whereby, if the temperature can
longer attracted to the magnetic field, and (iv) toxic responses be maintained above the therapeutic threshold of 42˚C for
to the magnetic carriers. Recent pre-clinical and experimental 30 min or more, the cancer is destroyed. Whereas the
results indicate, however, that it is still possible to overcome majority of hyperthermia devices are restricted in their utility
these limitations and use magnetic targeting to improve drug because of unacceptable coincidental heating of healthy tissue,
retention and also address safety issues [68, 72]. magnetic particle hyperthermia is appealing because it offers
a way to ensure only the intended target tissue is heated (see
figure 7).
4.4. Radionuclide and gene delivery
One way to overcome the limitation caused by the release of the 5.2. Operational constraints
drug from the carrier is to use a system in which the therapeutic
agent remains coupled to the magnetic carrier throughout A number of studies have demonstrated the therapeutic efficacy
the duration of the treatment. Based on this idea, the of this form of treatment in animal models (see, e.g. the review
possibility of targeting radionuclides via magnetic carriers
has been investigated. The advantage these complexes have 46
over cytotoxic drug/magnetic carrier complexes is that the
effectiveness of the radionuclide does not require the tumour 44
Temperature (˚C)
cells to actually take up the agent. If the radionuclide is targeted 42

to a region near the tumour site and held there, the radiation will
40
affect the surrounding tumour tissue while it is still attached to
the magnetic carrier. This type of system was tested in 1995 38
using in vitro and mouse models. In both cases, targeting of 36
a magnetic carrier coupled to a β-emitter (Y-90) was effective
at concentrating radiation to the desired site. In the mouse 34
tumour model, there was a significant increase in radioactivity 32
at the tumour site compared to using the same complex without
30
a magnetic field: 73 ± 32% vs 6 ± 4% [73]. Since this study,
Häfeli and others [74–76] have demonstrated the effectiveness 0 10 20 30 4
40 50
of this technique in both animal and cell culture studies using Time (minutes)
both yttrium-90 and rhenium-188.
Investigations also have begun with a view towards using Figure 7. Animal trial data on hyperthermia treatments in rabbits,
showing preferential heating of a tumour using intra-vascularly
magnetic carriers for gene therapy. In this case, a viral vector infused ferromagnetic microspheres; ( ) tumour edge, () tumour
carrying the therapeutic gene is coated onto the magnetic centre, () normal liver 1–2 cm from tumour, (×) alternative lobe,
carrier’s surface. By holding the carrier at the target site via and (♦) core body temperature.
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by Moroz et al [103]). To date, however, there have been have been investigated. Particle sizes less than about 10 µm are
no reports of the successful application of this technology to normally considered small enough to enable effective delivery
the treatment of a human patient. The challenge lies in being to the site of the cancer, either via encapsulation in a larger
able to deliver an adequate quantity of the magnetic particles moiety or suspension in some sort of carrier fluid. Nanoscale
to generate enough heat in the target using AC magnetic particles can be coupled with antibodies to facilitate targeting
field conditions that are clinically acceptable. Most of the on an individual cell basis. Candidate materials are divided into
laboratory and animal model based studies reported so far are two main classes; ferromagnetic or ferrimagnetic (FM) single
characterized by the use of magnetic field conditions that could domain or multi-domain particles, or SPM particles. The heat
not be safely used with a human patient. In most instances, generating mechanisms associated with each class are quite
reducing the field strength or frequency to safer levels would different, each offering unique advantages and disadvantages,
almost certainly lead to such a reduction in the heat output as discussed later.
from the magnetic material as to render it useless in this
application. 5.3. Heating mechanisms
It is important therefore to understand the underlying
physical mechanisms by which heat is generated in small FM particles possess hysteretic properties when exposed to a
magnetic particles by alternating magnetic fields. Enough time varying magnetic field, which gives rise to magnetically
heat must be generated by the particles to sustain tissue induced heating. The amount of heat generated per unit
temperatures of at least 42˚C for 30 min or so. Calculating volume is given by the frequency multiplied by the area of
the heat deposition rate required to achieve this is complicated the hysteresis loop:
by the presence of blood flow and tissue perfusion, both of
which are dominant sources of tissue cooling and both of PFM = µ0 f H dM. (10)
which vary actively as tissue is heated. Several authors have
analysed the heat transfer problem whereby a defined volume This formula ignores other possible mechanisms for
of tissue is heated from within by evenly dispersed sources such magnetically induced heating such as eddy current heating
as microscopic magnetic particles [104–106]. The problem and ferromagnetic resonance, but these are generally irrelevant
posed by the cooling from discrete blood vessels is generally in the present context. The particles used for magnetic
avoided because of the mathematical complexity and lack of hyperthermia are much too small and the AC field frequencies
generality of the results. However, an often-used rule of thumb much too low for the generation of any substantial eddy
is that a heat deposition rate of 100 mW cm−3 of tissue will currents. Ferromagnetic resonance effects may become
suffice in most circumstances. relevant but only at frequencies far in excess of those generally
The frequency and strength of the externally applied considered appropriate for this type of hyperthermia.
AC magnetic field used to generate the heating is limited For FM particles well above the SPM size limit there
by deleterious physiological responses to high frequency is no implicit frequency dependence in the integral of
magnetic fields [107, 108]. These include stimulation of equation (10), so PFM can be readily determined from
peripheral and skeletal muscles, possible cardiac stimulation quasi-static measurements of the hysteresis loop using, for
and arrhythmia, and non-specific inductive heating of tissue. example, a VSM or SQUID magnetometer. As discussed
Generally, the useable range of frequencies and amplitudes is in section 2, the re-orientation and growth of spontaneously
considered to be f = 0.05–1.2 MHz and H = 0–15 kA m−1 . magnetized domains within a given FM particle depends on
Experimental data on exposure to much higher frequency fields both microstructural features such as vacancies, impurities
comes from groups such as Oleson et al [109] who developed a or grain boundaries, and intrinsic features such as the
hyperthermia system based on inductive heating of tissue, and magnetocrystalline anisotropy as well as the shape and size of
Atkinson et al [110] who developed a treatment system based the particle. In most cases it is not possible to predict a priori
on eddy current heating of implantable metal thermoseeds. what the hysteresis loop will look like.
Atkinson et al concluded that exposure to fields where the In principle, substantial hysteresis heating of the FM
product H · f does not exceed 4.85 × 108 A m−1 s−1 is safe particles could be obtained using strongly anisotropic magnets
and tolerable. such as Nd-Fe-B or Sm-Co; however, the constraints on the
The amount of magnetic material required to produce amplitude of H that can be used mean that fully saturated
the required temperatures depends to a large extent on the loops cannot be used. Minor (unsaturated) loops could be
method of administration. For example, direct injection used, and would give rise to heating, but only at much reduced
allows for substantially greater quantities of material to be levels. In fact, as is evident in equation (10), the maximum
localized in a tumour than do methods employing intravascular realizable PFM should involve a rectangular hysteresis loop.
administration or antibody targeting, although the latter two However, this could only be achieved with an ensemble of
may have other advantages. A reasonable assumption is that uniaxial particles perfectly aligned with H , a configuration
ca 5–10 mg of magnetic material concentrated in each cm3 that would be difficult if not impossible to achieve in vivo. For
of tumour tissue is appropriate for magnetic hyperthermia in a more realistic ensemble of randomly aligned FM particles
human patients. the most that can be hoped for is around 25% of this ideal
Regarding the choice of magnetic particle, the iron oxides maximum.
magnetite (Fe3 O4 ) and maghemite (γ -Fe2 O3 ) are the most However, over the last decade the field of magnetic particle
studied to date because of their generally appropriate magnetic hyperthermia has been revitalized by the advent of ‘magnetic
properties and biological compatibility, although many others fluid hyperthermia’, where the magnetic entities are SPM
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nanoparticles suspended in water or a hydrocarbon fluid to 6. MRI contrast enhancement

make a ‘magnetic fluid’ or ‘ferrofluid’ [98, 111, 112]. When a
ferrofluid is removed from a magnetic field its magnetization 6.1. Physical principles
relaxes back to zero due to the ambient thermal energy of
its environment. This relaxation can correspond either to MRI relies on the counterbalance between the exceedingly
the physical rotation of the particles themselves within the small magnetic moment on a proton, and the exceedingly
fluid, or rotation of the atomic magnetic moments within each large number of protons present in biological tissue, which
particle. Rotation of the particles is referred to as ‘Brownian leads to a measurable effect in the presence of large magnetic
rotation’ while rotation of the moment within each particle fields [122, 123]. Thus, even though the effect of a steady
is known as ‘Néel relaxation’. Each of these processes state field of B0 = 1 T on a collection of protons, such as
is characterized by a relaxation time: τB for the Brownian the hydrogen nuclei in a water molecule, is so small that it
process depends on the hydrodynamic properties of the fluid; is equivalent to only three of every million proton moments
while τN for the Néel process is determined by the magnetic m being aligned parallel to B0 , there are so many protons
anisotropy energy of the SPM particles relative to the thermal available—6.6×1019 in every mm3 of water—that the effective
energy. Both Brownian and Néel processes may be present in signal, 2 × 1014 proton moments per mm3 , is observable. As
a ferrofluid, whereas only τN is relevant in fixed SPM particles illustrated in figure 8, this signal can be captured by making use
where no physical rotation of the particle is possible. The
of resonant absorption: applying a time-varying magnetic field
relaxation times τB and τN depend differently on particle size;
in a plane perpendicular to B0 , tuned to the Larmor precession
losses due to Brownian rotation are generally maximized at
frequency ω0 = γ B0 of the protons. For 1 H protons the
a lower frequency than are those due to Néel relaxation for a
given size. gyromagnetic ratio γ = 2.67 × 108 rad s−1 T−1 , so that in a
The physical basis of the heating of SPM particles by AC field of B0 = 1 T the Larmor precession frequency corresponds
magnetic fields has been reviewed by Rosensweig [113]. It is to a radio frequency field with ω0 /2π = 42.57 MHz. In
based on the Debye model, which was originally developed to practice the radio frequency transverse field is applied in a
describe the dielectric dispersion in polar fluids [114], and the pulsed sequence, of duration sufficient to derive a coherent
recognition that the finite rate of change of M in a ferrofluid response from the net magnetic moment of the protons in the
means that it will lag behind H . For small field amplitudes, and MRI scanner. From the instant that the radio frequency pulse is
assuming minimal interactions between the constituent SPM turned off the relaxation of the coherent response is measured
particles, the response of the magnetization of a ferrofluid to an via induced currents in pick-up coils in the scanner. These
AC field can be described in terms of its complex susceptibility resonantly tuned detection coils enhance the signal by a quality
χ = χ + iχ , where both χ and χ are frequency dependent. factor of ca 50–100. As shown in figure 8, for B0 parallel to
The out-of-phase χ component results in heat generation
given by [113]:
(a) (b)
B0 B0
PSPM = µ0 πf χ H 2 , (11)
m
which can be interpreted physically as meaning that if M
lags H there is a positive conversion of magnetic energy
m
into internal energy. This simple theory compares favourably
with experimental results, for example, in predicting a square
dependence of PSPM on H [91], and the dependence of χ on (c)
the driving frequency [115–117]. mxy
amplitude
Measurements of the heat generation from magnetic

signal
particles are usually quoted in terms of the specific absorption

rate (SAR) in units of W g−1 . Multiplying the SAR by the tim
time
density of the particle yields PFM and PSPM , so the parameter
allows comparison of the efficacies of magnetic particles
covering all the size ranges [88, 111, 118–121]. It is clear (d) mz
from such comparisons that most real FM materials require
amplitude
applied field strengths of ca 100 kA m−1 or more before they

signal
approach a fully saturated loop, and therefore only minor

hysteresis loops can be utilized given the operational constraint
of ca 15 kA m−1 , giving rise to low SARs. In contrast, SPM time
materials are capable of generating impressive levels of heating
at lower fields. For example, the best of the ferrofluids reported Figure 8. Illustration of magnetic resonance for a large ensemble of
by Hergt et al [121] has a SAR of 45 W g−1 at 6.5 kA m−1 protons with net magnetic moment m in the presence of a external
and 300 kHz which extrapolates to 209 W g−1 for 14 kA m−1 , magnetic field B0 . In (a) the net moment precesses around B0 at the
compared to 75 W g−1 at 14 kA m−1 for the best FM magnetite characteristic Larmor frequency, ω0 . In (b) a second external field is
sample. While all of these samples would be adequate for applied, perpendicular to B0 , oscillating at ω0 . Despite being much
weaker than B0 , this has the effect of resonantly exciting the
magnetic particle hyperthermia, importantly, it seems clear that moment precession into the plane perpendicular to B0 . In (c) and (d)
ferrofluids and SPM particles are more likely to offer useful the oscillating field is removed at time zero, and the in-plane (c) and
heating using lower magnetic field strengths. longitudinal (d) moment amplitudes relax back to their initial values.
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the z-axis these relaxation signals are of the form: Iron oxide nanoparticles are the most commonly used
SPM contrast agents. Dextran coated iron oxides are
mz = m(1 − e−t/T1 ) (12) biocompatible and are excreted via the liver after the treatment.
They are selectively taken up by the reticuloendothelial system,
and
a network of cells lining blood vessels whose function is to
mx,y = m sin(ω0 t + φ)e−t/T2 , (13)
remove foreign substances from the bloodstream; MRI contrast
where T1 and T2 are the longitudinal (or spin–lattice) and relies on the differential uptake of different tissues [126]. There
transverse (or spin–spin) relaxation times, respectively, and is also a size effect: nanoparticles with diameters of ca 30 nm
φ is a phase constant. The longitudinal relaxation reflects a or more are rapidly collected by the liver and spleen, while
loss of energy, as heat, from the system to its surrounding particles with sizes of ca 10 nm or less are not so easily
‘lattice’, and is primarily a measure of the dipolar coupling recognized. The smaller particles therefore have a longer
of the proton moments to their surroundings. The relaxation half-life in the blood stream and are collected by reticulo-
in the xy-plane is relatively rapid, and is driven by the endothelial cells throughout the body, including those in the
loss of phase coherence in the precessing protons due to lymph nodes and bone marrow [127, 128]. Similarly, such
their magnetic interactions with each other and with other agents have been used to visualize the vascular system [129],
fluctuating moments in the tissue. Dephasing can also be and to image the central nervous system [130]. It is also notable
affected by local inhomogeneities in the applied longitudinal that tumour cells do not have the effective reticuloendothelial
field, leading to the replacement of T2 in equation (13) by the system of healthy cells, so that their relaxation times are not
shorter relaxation time, T2∗ : altered by the contrast agents. This has been used, for example,
to assist the identification of malignant lymph nodes [131],
1 1 B0
= +γ , (14) liver tumours [132] and brain tumours [133].
T2∗ T2 2 Iron oxide nanoparticles also lend themselves to
encapsulation into target-specific agents, such as a liposome
where B0 is the variation in the field brought about either
that is known to localize in the bone marrow [134]. Dendrimer
through distortions in the homogeneity of the applied field
coatings comprising a highly branched polymer structure that
itself, or by local variations in the magnetic susceptibility of
has a high rate of non-cell-specific binding and intracellular
the system [124, 125].
uptake have also been used to good effect [135], as has the use
6.2. MRI contrast enhancement studies of lipofection agents, normally used to carry DNA into the cell
nucleus, to enable intracellular incorporation of the magnetic
Both T1 and T2∗ can be shortened by the use of a magnetic
nanoparticles into stem cells [136]. Magnetic nanoparticles
contrast agent. The most common contrast agents currently
have also been utilized for the in vivo monitoring of gene
used are PM gadolinium ion complexes, although agents based
expression, a process in which cells are engineered to over-
on SPM nanoparticles are commercially available, such as
express a given gene. This process leads to the production of
‘Feridex I. V.’, an iron oxide contrast agent marketed by
increased numbers of certain cell wall receptors, which in turn
Advanced Magnetics Inc. for the organ-specific targeting
can be targeted using specially coated nanoparticles, thereby
of liver lesions. The SPM particles used are magnetically
allowing a differentiation between the expressing cells and
saturated in the normal range of magnetic field strengths used
their surroundings [137]. Another aspect of the targeting of
in MRI scanners, thereby establishing a substantial locally
cell receptors has been to selectively study cells that are in the
perturbing dipolar field which leads, via equation (14), to a
process of cell death [138].
marked shortening of T2∗ (see figure 9) along with a less marked
reduction of T1 [123].
7. Discussion and future prospects
In this paper we have reviewed some basic concepts regarding

(a) (b) the interactions between magnetic nanoparticles and a static
or time-varying external magnetic field, and shown how
these pertain to current biomedical applications of magnetic
nanoparticles. We have focused in particular on magnetic
separation, drug delivery, hyperthermia and MRI contrast
enhancement, although these are only four of the many
biomedical applications of magnetic nanoparticles that are
currently being explored. For example, research is being
conducted into magnetic twisting cytometry, a process in which
ferromagnetic microspheres are bound to specific receptors on
a cell wall. Changing the direction of an applied magnetic field
twists the microsphere by a measurable amount, which can then
be related to the mechanical properties of the cell membrane
time and cytoskeleton [139–143]. Magnetic nanoparticles are
Figure 9. Effect of magnetic particle internalization in cells on T2∗ also being tested for tissue engineering applications, for
relaxation times: (a) the protons in cells tagged by magnetic particles example, in the mechanical conditioning of cells growing in
have a shorter T2∗ relaxation time than those in (b) untagged cells. culture [144–146]. In such systems magnetic particles are
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Topical Review
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Journal of Nanobiotechnology BioMed Central
Review Open Access

Applications of nanoparticles in biology and medicine
OV Salata*
Address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
Email: OV Salata* - oleg.salata@path.ox.ac.uk
* Corresponding author
Published: 30 April 2004 Received: 23 December 2003

Accepted: 30 April 2004
Journal of Nanobiotechnology 2004, 2:3
This article is available from: http://www.jnanobiotechnology.com/content/2/1/3
© 2004 Salata; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for
any purpose, provided this notice is preserved along with the article's original URL.
nanotechnologynanomaterialsnanoparticlesquantum dotsnanotubesmedicinebiologyapplications
Abstract
Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. Their
unique size-dependent properties make these materials superior and indispensable in many areas
of human activity. This brief review tries to summarise the most recent developments in the field
of applied nanomaterials, in particular their application in biology and medicine, and discusses their
commercialisation prospects.
Introduction Out of plethora of size-dependant physical properties

Nanotechnology [1] is enabling technology that deals available to someone who is interested in the practical
with nano-meter sized objects. It is expected that nanote- side of nanomaterials, optical [7] and magnetic [8] effects
chnology will be developed at several levels: materials, are the most used for biological applications.
devices and systems. The nanomaterials level is the most
advanced at present, both in scientific knowledge and in The aim of this review is firstly to give reader a historic
commercial applications. A decade ago, nanoparticles prospective of nanomaterial application to biology and
were studied because of their size-dependent physical and medicine, secondly to try to overview the most recent
chemical properties [2]. Now they have entered a com- developments in this field, and finally to discuss the hard
mercial exploration period [3,4]. road to commercialisation. Hybrid bionanomaterials can
also be applied to build novel electronic, optoelectronics
Living organisms are built of cells that are typically 10 µm and memory devices (see for example [9,10]). Neverthe-
across. However, the cell parts are much smaller and are less, this will not be discussed here and will be a subject of
in the sub-micron size domain. Even smaller are the pro- a separate article.
teins with a typical size of just 5 nm, which is comparable
with the dimensions of smallest manmade nanoparticles. Applications
This simple size comparison gives an idea of using nano- A list of some of the applications of nanomaterials to biol-
particles as very small probes that would allow us to spy ogy or medicine is given below:
at the cellular machinery without introducing too much
interference [5]. Understanding of biological processes on - Fluorescent biological labels [11-13]
the nanoscale level is a strong driving force behind devel-
opment of nanotechnology [6]. - Drug and gene delivery [14,15]
Page 1 of 6
(page number not for citation purposes)
Journal of Nanobiotechnology 2004, 2 http://www.jnanobiotechnology.com/content/2/1/3
- Bio detection of pathogens [16]
- Detection of proteins [17]
- Probing of DNA structure [18]
- Tissue engineering [19,20]
- Tumour destruction via heating (hyperthermia)[21]
- Separation and purification of biological molecules and

cells [22]
- MRI contrast enhancement [23]
- Phagokinetic studies [24]
As mentioned above, the fact that nanoparticles exist in

the same size domain as proteins makes nanomaterials Figure
Typical
to medical
configurations
1 or biologicalutilised
problems
in nano-bio materials applied
Typical configurations utilised in nano-bio materials applied
suitable for bio tagging or labelling. However, size is just
to medical or biological problems.
one of many characteristics of nanoparticles that itself is
rarely sufficient if one is to use nanoparticles as biological
tags. In order to interact with biological target, a biological
or molecular coating or layer acting as a bioinorganic
interface should be attached to the nanoparticle. Exam-
ples of biological coatings may include antibodies, The core particle is often protected by several monolayers
biopolymers like collagen [25], or monolayers of small of inert material, for example silica. Organic molecules
molecules that make the nanoparticles biocompatible that are adsorbed or chemisorbed on the surface of the
[26]. In addition, as optical detection techniques are wide particle are also used for this purpose. The same layer
spread in biological research, nanoparticles should either might act as a biocompatible material. However, more
fluoresce or change their optical properties. The often an additional layer of linker molecules is required to
approaches used in constructing nano-biomaterials are proceed with further functionalisation. This linear linker
schematically presented below (see Figure 1). molecule has reactive groups at both ends. One group is
aimed at attaching the linker to the nanoparticle surface
Nano-particle usually forms the core of nano-biomaterial. and the other is used to bind various moieties like bio-
It can be used as a convenient surface for molecular compatibles (dextran), antibodies, fluorophores etc.,
assembly, and may be composed of inorganic or poly- depending on the function required by the application.
meric materials. It can also be in the form of nano-vesicle
surrounded by a membrane or a layer. The shape is more Recent developments
often spherical but cylindrical, plate-like and other shapes Tissue engineering
are possible. The size and size distribution might be Natural bone surface is quite often contains features that
important in some cases, for example if penetration are about 100 nm across. If the surface of an artificial bone
through a pore structure of a cellular membrane is implant were left smooth, the body would try to reject it.
required. The size and size distribution are becoming Because of that smooth surface is likely to cause produc-
extremely critical when quantum-sized effects are used to tion of a fibrous tissue covering the surface of the implant.
control material properties. A tight control of the average This layer reduces the bone-implant contact, which may
particle size and a narrow distribution of sizes allow creat- result in loosening of the implant and further inflamma-
ing very efficient fluorescent probes that emit narrow light tion. It was demonstrated that by creating nano-sized fea-
in a very wide range of wavelengths. This helps with creat- tures on the surface of the hip or knee prosthesis one
ing biomarkers with many and well distinguished colours. could reduce the chances of rejection as well as to stimu-
The core itself might have several layers and be multifunc- late the production of osteoblasts. The osteoblasts are the
tional. For example, combining magnetic and lumines- cells responsible for the growth of the bone matrix and are
cent layers one can both detect and manipulate the found on the advancing surface of the developing bone.
particles.
Page 2 of 6
The effect was demonstrated with polymeric, ceramic and, sensitive to the daylight exposure. This effect can last for
more recently, metal materials. More than 90% of the up to six weeks.
human bone cells from suspension adhered to the nanos-
tructured metal surface [27], but only 50% in the control To avoid this side effect, the hydrophobic version of the
sample. In the end this findings would allow to design a dye molecule was enclosed inside a porous nanoparticle
more durable and longer lasting hip or knee replacements [28]. The dye stayed trapped inside the Ormosil nanopar-
and to reduce the chances of the implant getting loose. ticle and did not spread to the other parts of the body. At
the same time, its oxygen generating ability has not been
Titanium is a well-known bone repairing material widely affected and the pore size of about 1 nm freely allowed for
used in orthopaedics and dentistry. It has a high fracture the oxygen to diffuse out.
resistance, ductility and weight to strength ratio. Unfortu-
nately, it suffers from the lack of bioactivity, as it does not Multicolour optical coding for biological assays [29]
support sell adhesion and growth well. Apatite coatings The ever increasing research in proteomics and genomic
are known to be bioactive and to bond to the bone. generates escalating number of sequence data and
Hence, several techniques were used in the past to pro- requires development of high throughput screening tech-
duce an apatite coating on titanium. Those coatings suffer nologies. Realistically, various array technologies that are
from thickness non-uniformity, poor adhesion and low currently used in parallel analysis are likely to reach satu-
mechanical strength. In addition, a stable porous structure ration when a number of array elements exceed several
is required to support the nutrients transport through the millions. A three-dimensional approach, based on optical
cell growth. "bar coding" of polymer particles in solution, is limited
only by the number of unique tags one can reliably pro-
It was shown that using a biomimetic approach – a slow duce and detect.
growth of nanostructured apatite film from the simulated
body fluid – resulted in the formation of a strongly adher- Single quantum dots of compound semiconductors were
ent, uniform nanoporous layer [19]. The layer was found successfully used as a replacement of organic dyes in vari-
to be built of 60 nm crystallites, and possess a stable nan- ous bio-tagging applications [7]. This idea has been taken
oporous structure and bioactivity. one step further by combining differently sized and hence
having different fluorescent colours quantum dots, and
A real bone is a nanocomposite material, composed of combining them in polymeric microbeads [29]. A precise
hydroxyapatite crystallites in the organic matrix, which is control of quantum dot ratios has been achieved. The
mainly composed of collagen. Thanks to that, the bone is selection of nanoparticles used in those experiments had
mechanically tough and, at the same time, plastic, so it 6 different colours as well as 10 intensities. It is enough to
can recover from a mechanical damage. The actual nano- encode over 1 million combinations. The uniformity and
scale mechanism leading to this useful combination of reproducibility of beads was high letting for the bead
properties is still debated. identification accuracies of 99.99%.
An artificial hybrid material was prepared from 15–18 nm Manipulation of cells and biomolecules [30]
ceramic nanoparticles and poly (methyl methacrylate) Functionalised magnetic nanoparticles have found many
copolymer [20]. Using tribology approach, a viscoelastic applications including cell separation and probing; these
behaviour (healing) of the human teeth was demon- and other applications are discussed in a recent review [8].
strated. An investigated hybrid material, deposited as a Most of the magnetic particles studied so far are spherical,
coating on the tooth surface, improved scratch resistance which somewhat limits the possibilities to make these
as well as possessed a healing behaviour similar to that of nanoparticles multifunctional. Alternative cylindrically
the tooth. shaped nanoparticles can be created by employing metal
electrodeposition into nanoporous alumina template
Cancer therapy [30]. Depending on the properties of the template, nano-
Photodynamic cancer therapy is based on the destruction cylinder radius can be selected in the range of 5 to 500 nm
of the cancer cells by laser generated atomic oxygen, while their length can be as big as 60 µm. By sequentially
which is cytotoxic. A greater quantity of a special dye that depositing various thicknesses of different metals, the
is used to generate the atomic oxygen is taken in by the structure and the magnetic properties of individual cylin-
cancer cells when compared with a healthy tissue. Hence, ders can be tuned widely.
only the cancer cells are destroyed then exposed to a laser
radiation. Unfortunately, the remaining dye molecules As surface chemistry for functionalisation of metal sur-
migrate to the skin and the eyes and make the patient very faces is well developed, different ligands can be selectively
attached to different segments. For example, porphyrins
Page 3 of 6
Table 1: Examples of Companies commercialising nanomaterials for bio- and medical applications.
Company Major area of activity Technology
Advectus Life Sciences Inc. Drug delivery Polymeric nanoparticles engineered to carry anti-
tumour drug across the blood-brain barrier
Alnis Biosciences, Inc. Bio-pharmaceutical Biodegradable polymeric nanoparticles for drug
delivery
Argonide Membrane filtration Nanoporous ceramic materials for endotoxin
filtration, orthopaedic and dental implants, DNA and
protein separation
BASF Toothpaste Hydroxyapatite nanoparticles seems to improve
dental surface
Biophan Technologies, Inc. MRI shielding Nanomagnetic/carbon composite materials to shield
medical devices from RF fields
Capsulution NanoScience AG Pharmaceutical coatings to improve solubility of drugs Layer-by-layer poly-electrolyte coatings, 8–50 nm
Dynal Biotech Magnetic beads
Eiffel Technologies Drug delivery Reducing size of the drug particles to 50–100 nm
EnviroSystems, Inc. Surface desinfectsant Nanoemulsions
Evident Technologies Luminescent biomarkers Semiconductor quantum dots with amine or carboxyl
groups on the surface, emission from 350 to 2500 nm
Immunicon Tarcking and separation of different cell types magnetic core surrounded by a polymeric layer
coated with antibodies for capturing cells
KES Science and Technology, Inc. AiroCide filters Nano-TiO2 to destroy airborne pathogens
NanoBio Cortporation Pharmaceutical Antimicrobal nano-emulsions
NanoCarrier Co., Ltd Drug delivery Micellar nanoparticles for encapsulation of drugs,
proteins, DNA
NanoPharm AG Drug delivery Polybutilcyanoacrylate nanoparticles are coated with
drugs and then with surfactant, can go across the
blood-brain barrier
Nanoplex Technologies, Inc Nanobarcodes for bioanalysis
Nanoprobes, Inc. Gold nanoparticles for biological markers Gold nanoparticles bio-conjugates for TEM and/or
fluorescent microscopy
Nanoshpere, Inc. Gold biomarkers DNA barcode attached to each nanoprobe for
identification purposes, PCR is used to amplify the
signal; also catalytic silver deposition to amplify the
signal using surface plasmon resonance
NanoMed Pharmaceutical, Inc. Drug delivery Nanoparticles for drug delivery
Oxonica Ltd Sunscreens Doped transparent nanoparticles to effectively
absorb harmful UV and convert it into heat
PSiVida Ltd Tissue engineering, implants, drugs and gene delivery, Exploiting material properties of nanostructured
bio-filtration porous silicone
Smith & Nephew Acticoat bandages Nanocrystal silver is highly toxic to pathogenes
QuantumDot Corporation Luminescent biomarkers Bioconjugated semiconductor quantum dots
with thiol or carboxyl linkers were simultaneously Protein detection [31]

attached to the gold or nickel segments respectively. Thus, Proteins are the important part of the cell's language,
it is possible to produce magnetic nanowires with spa- machinery and structure, and understanding their func-
tially segregated fluorescent parts. In addition, because of tionalities is extremely important for further progress in
the large aspect ratios, the residual magnetisation of these human well being. Gold nanoparticles are widely used in
nanowires can be high. Hence, weaker magnetic field can immunohistochemistry to identify protein-protein inter-
be used to drive them. It has been shown that a self-assem- action. However, the multiple simultaneous detection
bly of magnetic nanowires in suspension can be control- capabilities of this technique are fairly limited. Surface-
led by weak external magnetic fields. This would enhanced Raman scattering spectroscopy is a well-estab-
potentially allow controlling cell assembly in different lished technique for detection and identification of single
shapes and forms. Moreover, an external magnetic field dye molecules. By combining both methods in a single
can be combined with a lithographically defined mag- nanoparticle probe one can drastically improve the multi-
netic pattern ("magnetic trapping"). plexing capabilities of protein probes. The group of Prof.
Mirkin has designed a sophisticated multifunctional
Page 4 of 6
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nanoparticles are coated with hydrophilic oligonucle- ment of nanomaterials is to make them multifunctional
otides containing a Raman dye at one end and terminally and controllable by external signals or by local environ-
capped with a small molecule recognition element (e.g. ment thus essentially turning them into nano-devices.
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Page 6 of 6
Chemical synthesis of magnetic nanoparticles
Taeghwan Hyeon
National Creative Research Initiative Center for Oxide Nanocrystalline Materials and School of Chemical
Engineering, Seoul National University, Seoul 151-744, Korea. E-mail: thyeon@plaza.snu.ac.kr
Received (in Cambridge, UK) 9th September 2002, Accepted 12th November 2002
First published as an Advance Article on the web 3rd December 2002
Recent advances in the synthesis of various magnetic nano- applications in ferrofluids, magnetic refrigeration systems,
particles using colloidal chemical approaches are reviewed. contrast enhancement in magnetic resonance imaging, magnetic
Typically, these approaches involve either rapid injection of carriers for drug targeting and catalysis.4 In the current article,
reagents into hot surfactant solution followed by aging at high I will discuss recent advances made in the synthesis of various
temperature, or the mixing of reagents at a low temperature and magnetic nanoparticles using colloidal chemical synthetic
slow heating under controlled conditions. Spherical cobalt procedures. A general review on the synthesis and under-
nanoparticles with various crystal structures have been synthe- standing of nanostructured magnetic materials up to 1996 is
sized by thermally decomposing dicobalt octacarbonyl or by given in the article by Leslie-Pelecky and Rieke.5 The materials
reducing cobalt salts. Nanoparticles of Fe–Pt and other related covered in the current article are colloidal systems composed of
iron or cobalt containing alloys have been made by simultane-
isolated particles with nanometer-sized dimensions that are
ously reacting their constituent precursors. Many different
ferrite nanoparticles have been synthesized by the thermal
stabilized by surfactant molecules and dispersed in solvent
decomposition of organometallic precursors followed by oxida- media. In the ideal case, these non-interacting systems derive
tion or by low-temperature reactions inside reverse micelles. their unique magnetic properties mostly from the reduced size
Rod-shaped iron nanoparticles have been synthesized from the of the isolated nanoparticles, and contributions from inter-
oriented growth of spherical nanoparticles, and cobalt nanoparticle interactions are negligible. The surfactant coating on
disks were synthesized from the thermal decomposition of magnetic nanoparticles prevents clustering due to steric repul-
dicobalt octacarbonyl in the presence of a mixture of two sion. Dynamic adsorption and desorption of surfactant mole-
surfactants. cules onto particle surfaces during synthesis enables reactive
species to be added onto the growing particles. These
nanoparticles can be dispersed in many organic solvents and can
be retrieved as powder forms by removing the solvent.
Introduction The followings are several key issues of nanoparticle
The development of uniform nanometer sized particles has been synthesis.
intensively pursued because of their technological and funda- 4 Particle size distribution (uniformity): Can we synthesize
mental scientific importance.1 These nanoparticulate materials monodisperse nanocrystals?
often exhibit very interesting electrical, optical, magnetic, and 4 Particle size control: Can we control the particle size of
chemical properties, which cannot be achieved by their bulk nanocrystals in a reproducible manner?
counterparts.2 The synthesis of discrete magnetic nanoparticles 4 Crystallinity and crystal structure: Can we obtain materials
with sizes ranging from 2 to 20 nm, is of significant importance, with satisfactory high crystallinity and the desired crystal
because of their applications in multi-terabit in22 magnetic structure?
storage devices.3 Such magnetic nanoparticles could also find 4 Shape-control: Can we synthesize non-spherical and aniso-
tropic nanoparticles?
4 Alignment: For device applications, we should also consider
Taeghwan Hyeon received his B. S. (1987) and M. S. (1989) in the alignment of nanoparticles on substrates.
Chemistry from Seoul National University, Seoul, Korea. He
obtained his Ph.D. from the University of Illinois at Urbana- In the present article, I am especially interested in the
Champaign (1996) under the supervision of Professor Kenneth synthesis of monodisperse magnetic nanoparticles. Mono-
S. Suslick. After conducting postdoctoral research with Pro- disperse nanoparticles are generally considered to be samples
fessor Wolfgang M. H. Sachtler at Northwestern University, he with standard deviations s @ 5% diameter for spherical
joined the faculty of the School of Chemical Engineering of particles. Often nanocrystals are referred to as well-defined
Seoul National University in September 1997. He has received crystalline materials, whereas nanoparticles is a term used more
several awards including the T. S. Piper Award (University of generally for particles with diameters of 2–50 nm with variable
Illinois, 1996), Korean Young Scientist Award (Korean Govern- crystallinity.
ment, 2002), KCS–Wiley Young Chemist Award (Korean Chemical methods have been widely used to produce
Chemical Society, 2001), and The Scientist of the Month Award nanostructured materials due to their straightforward nature and
(Korean Science and Engineering Foundation, 2002). In July their potential to produce large quantities of the final product.
DOI: 10.1039/b207789b
2002, he became the director of the National Creative Research Realizable particle sizes range in size from nanometers to
Initiative Center for Oxide Nanocrystalline Materials sup- micrometers, by controlling particle size during synthesis by
ported by the Korean Ministry of Science and Technology. His using competition between nucleation and growth. There are
current research interests include synthesis of metallic and several synthetic procedures for synthesizing monodisperse
oxide nanoparticles, synthesis of nanoporous carbon materials, nanoparticles. It is well known that a short burst of nucleation
and catalytic applications of nanostructured materials. followed by slow controlled growth is critical to produce
monodisperse particles. The following describe two representa-
This journal is © The Royal Society of Chemistry 2003 CHEM. COMMUN., 2003, 927–934 927
tive synthetic procedures. In the first synthetic procedure (Fig. Short-chain alkylphosphines allowed faster growth, and re-
1), the rapid injection of the reagents, often organometallic sulted in the bigger particles, while bulkier surfactants reduced
particle growth and favored production of smaller nano-
particles. For example, when bulky trioctylphosphine was
applied in the synthesis, 2–6 nm sized particles were generated.
In contrast, when tributylphosphine was used as a stabilizer
7–11 nm sized nanoparticles were produced. A further narrow-
ing of particle size distribution was proceeded using a size
selective precipitation by the gradual addition of alcohol (e.g.
ethanol) into a hydrocarbon (e.g. hexane) dispersion containing
nanoparticles with wide size distribution. Monodisperse cobalt
nanoparticles organize into two- and three-dimensional super-
lattices. Fig. 2 shows a 2D assembly of 9 nm cobalt
Fig. 1 Generalized synthesis of monodisperse nanoparticles by the injection

of reagents into hot surfactant solution followed by aging and size-selective
process.
compounds, into hot surfactant solution induces the simultane-

ous formation of many nuclei.6 Alternatively, reducing agents
are added to metal salt solutions at high temperature. In the
second procedure, reagents are mixed at low temperature and
the resulting reaction mixtures are slowly heated in a controlled
manner to generate nuclei. Subsequently, particle growth occurs
by the addition of reactive species. Particle size can be also
increased by aging at high temperature by Oswalt ripening, in
which smaller nanoparticles dissolve and deposit on the bigger
nanoparticles. The growth of nanoparticles can be stopped by
rapidly decreasing the reaction temperature. Nanoparticles
Fig. 2 TEM images of 9 nm e-Co nanoparticles (inset, high-resolution TEM
produced using these synthetic procedures often have particle
image).9a Reprinted with permission from reference 9(a). Copyright 1999
size distributions with s ~ 10%. Further size-selection proc- American Institute of Physics.
esses can narrow the particle size distribution below s = 5%.
The most frequently applied size-selection involves the addition nanoparticles. A high-resolution transmission electron micro-
of a poor solvent to precipitate the larger particles. When poor graph of a single nanoparticle, shown in the inset of Fig. 2,
solvent is added to a mixture containing nanoparticles of reveals its highly crystalline nature. The X-ray diffraction
various sizes, the biggest particles flocculate first because of pattern of these nanoparticles reveals a complex cubic structure
their greatest van der Waals attraction. This precipitate can be related to the beta phase of elemental manganese (e-Co).
retrieved by centrifugation. The precipitate can be re-dissolved Heating the e-Co nanoparticles at 300 °C generated hcp Co
in solvent, and further size-selection process can be performed nanoparticles. The Bawendi group have reported on the
to yield particles with narrower particle size distribution. Very synthesis of e-Co nanoparticle powders from the thermal
recently, Klabunde and coworkers reported the fabrication of decomposition of Co2(CO)8 in TOPO.10 The Alivisatos group
extremely monodisperse gold nanoparticles through digestive have reported on the synthesis of monodisperse e-Co nano-
ripening.7 particles by the rapid pyrolysis of dicobalt octacarbonyl in the
The particle sizes of nanoparticles can be controlled by presence of a surfactant mixture composed of oleic acid, lauric
systematically adjusting the reaction parameters, such as time, acid and trioctylphosphine.11 They could synthesize 3–17 nm
temperature, and the concentrations of reagents and stabilizing sized cobalt nanoparticles by controlling the precursor/surfac-
surfactants. In general, particle size increases with increasing tant ratio, the reaction temperature, and the injection time. Black
reaction time, because more monomeric species are generated, et al. later demonstrated spin-dependent electron transport using
and with increasing reaction temperature because the rate of self-assembled 10 nm e-Co nanoparticles over 100 nm wide
reaction is increased. electrodes.12 Single electron tunneling was clearly observed for
the two-dimensional hexagonal arrays of cobalt nanoparticles.
Similar single electron tunneling in cobalt nanoparticles was
Nanoparticles of iron, cobalt and nickel also reported by Pileni and workers.13
The Murray group at the IBM Watson research center has Hcp cobalt nanoparticles were synthesized by using the so-
studied the synthesis of monodisperse metallic magnetic called polyol process, in which high boiling alcohol is applied
nanoparticles intensively.8,9 They have synthesized cobalt as both a reductant and a solvent.8 In a typical synthesis,
nanoparticles with several different crystal structures using 1,2-dodecanediol is added into hydrated cobalt acetate solution
different synthetic procedures. High temperature reduction of dissolved in diphenyl ether containing oleic acid and trioctyl-
cobalt chloride was employed to synthesize e-phase cobalt phosphine at 250 °C. Nanoparticles were isolated by size-
nanoparticles.9a The injection of superhydride (LiBEt3H) selective precipitation, and particle size was controlled by
solution in dioctyl ether into a hot cobalt chloride solution in changing the relative concentration of precursor and stabilizer.
dioctyl ether (200 °C) in the presence of oleic acid and For example, when a 1+1 molar ratio of cobalt acetate and oleic
trialkylphosphine induced the simultaneous formation of many acid was used in the synthesis, 6–8 nm sized cobalt nano-
small metal clusters, which acted as nuclei for nanoparticle particles were produced, while increasing the concentration of
formation. Continued heating at 200 °C induced the growth of stabilizing surfactants by a factor of two yielded smaller 3–6 nm
these clusters to the nanoparticle level. Particle size was nanoparticles. Particle size could be also varied by controlling
controlled by the steric bulkiness of the stabilizing surfactants. the steric bulkiness of the phosphine stabilizers, for example,
928 CHEM. COMMUN., 2003, 927–934

using tributylphosphine, 10–13 nm Co nanoparticles were nanoparticles were monodisperse and the electron diffraction
generated. Using hydrated nickel acetate as a metal precursor, pattern showed that the nanoparticles were nearly amorphous.
nickel nanoparticles with particle sizes in the range 8–13 nm The XRD pattern of the sample after being heat-treated in an
were obtained. Co/Ni alloy nanoparticles were also produced argon atmosphere at 500 °C revealed a bcc a-iron structure. The
using a mixture of cobalt acetate and nickel acetate through a particle size could be controlled from 4 to 11 nm by using
similar synthetic procedure.8b Diehl et al. used ferromagnetic different molar ratios of iron pentacarbonyl to oleic acid. Fig.
resonance (FMR) techniques to investigate the detailed mag- 3(a) and (b) show TEM images of iron nanoparticles with
netic characteristics of cobalt nanoparticles with different
crystalline structures.9b The structural characterization of an
ordered assembly of cobalt nanoparticles using high-resolution
transmission electron microscopy was reported by Wang et al.9c
They synthesized multiply twinned fcc cobalt nanocrystals from
the thermal decomposition of dicobalt octacarbonyl in the
presence of a surfactant mixture composed of oleic acid and
tributylphosphine. An HRTEM image of the nanoparticles
revealed complicated interference patterns.8
Reverse micelles, which are water-in-oil droplets stabilized
by a monolayer of surfactant, have been applied as nanoscale
reactors for the synthesis of various nanoparticles.14 Chen et al.
and later the Pileni group reported on the synthesis of relatively
uniform cobalt nanoparticles by the reduction of cobalt salt
inside reverse micelles.15 Chen et al. synthesized cobalt
nanoparticles with particle size in the range 1.8–4.4 nm by
reducing CoCl2 with NaBH4 in reverse micelles formed using
didodecyldimethylammonium bromide (DDAB). The Pileni
group reported the synthesis of cobalt nanoparticles from the
reaction between a micellar solution containing NaAOT
(sodium bis(2-ethylhexyl)sulfosuccinate) and Co(AOT)2, and
NaBH4 dissolved in NaAOT solution.16 Collisions followed by Fig. 3 TEM images of iron nanoparticles: (a) three-dimensional array of 7
exchange between micellar droplets induce chemical reaction of nm Fe nanoparticles and (b) 11 nm Fe nanoparticles.
cobalt ions and sodium borohydride to generate cobalt nano-
particles. The as-synthesized nanoparticles were poorly crystal- particle sizes of 7 and 11 nm, respectively, which were prepared
line while XRD, after heating at 500 °C, revealed the using 1+2 and 1+3 molar ratios of Fe(CO)5+oleic acid. In order
characteristic pattern of fcc cobalt. The average particle size to produce nanoparticles larger than 11 nm, a reaction mixture
measured using TEM data was 6 nm with a particle size with a 1+4 molar ratio of Fe(CO)5 and oleic acid was used
distribution s = 9%. A size selection process involving the during the synthesis. However, the particle size of the resulting
extraction of nanoparticles from reverse micelles significantly nanoparticles was still around 11 nm. We could produce iron
reduced the polydispersity of the particle size distribution and nanoparticles with particle sizes bigger than 11 nm by adding
these uniform particles self-assembled to generate two-dimen- more iron oleate complex to previously prepared 11 nm iron
sionally hexagonally ordered arrays.17 nanoparticles, and then aging at 300 °C. Using this synthetic
Relatively very little work has been done to synthesize procedure, we were able to tune the particle sizes of the
uniform iron nanoparticles. Suslick et al. reported the synthesis nanoparticles from 11 to 20 nm.
of iron nanoparticles from the sonochemical decomposition of Using a similar synthetic procedure, we prepared moderately
iron pentacarbonyl in the presence of polyvinylpyrrolidone monodisperse cobalt nanoparticles and successfully applied
(PVP) or oleic acid.18 The sonochemical decomposition of them to recyclable catalysts for Pauson–Khand reactions, which
volatile organometallic compounds has been applied to synthe- involve the cycloaddition of alkynes, alkenes and carbon
size various nanostructured materials.19 Transmission electron monoxide to synthesize cyclopentenones.22 We also used
micrographs showed that the iron particles ranged in size from aqueous colloidal cobalt nanoparticles stabilized by sodium
3 to 8 nm. Electron diffraction revealed that the particles as- dodecyl sulfate, which were synthesized by the reverse micelle
formed are amorphous, and that after in situ electron beam method described above, for use as recyclable aqueous-phase
heating they crystallized to bcc iron. These iron nanoparticles Pauson–Khand catalysts.23
were readily oxidized to FeO when exposed to air. Gedanken
and coworkers reported the synthesis of amorphous cobalt
nanoparticles by sonicating Co(CO)3(NO) in decane solution in
Nanoparticles of alloys of cobalt and iron
the presence of oleic acid.20 Spherical 5–10 nm sized amor- Sun et al. synthesized monodisperse iron–platinum nano-
phous nanoparticles were produced. One drawback of the particles by the simultaneous reduction of platinum acet-
sonochemical process in the synthesis of nanoparticles is its ylacetonate (Pt(acac)2) and the thermal decomposition of iron
inability to control particle size. pentacarbonyl (Fe(CO)5) in the presence of oleic acid and oleyl
Very recently, our research group synthesized monodisperse amine.24a The composition was adjusted by changing the molar
iron nanoparticles from the high temperature (300 °C) aging of ratio of the two precursors. Using a 3+2 molar ratio of Fe(CO)5
an iron–oleic acid metal complex, which was prepared by the to Pt(acac)2, Fe48Pt52 nanoparticles were produced, while using
thermal decomposition of iron pentacarbonyl in the presence of 4+1 mixtures Fe70Pt30 nanoparticles were formed. The particle
oleic acid at 100 °C.21 We were able to synthesize monodisperse size could be varied from 3 to 10 nm by adding more precursors
nanoparticles with sizes ranging from 4 to 20 nm without using to previously synthesized 3 nm particles, which acted as nuclei.
any size selection process. Initially, the iron oleate complex was When the solvent was evaporated slowly, three-dimensional
prepared by reacting Fe(CO)5 and oleic acid at 100 °C. Iron superlattices are generated, and by controlling the chain lengths
nanoparticles were then generated by aging the iron complex at of the stabilizing surfactants, inter-particle spacing could be
300 °C. A TEM image of the iron nanoparticles revealed that the tuned. For example, 6 nm Fe48Pt52 nanoparticles were arranged
CHEM. COMMUN., 2003, 927–934 929

with an interparticle spacing of 4 nm using oleic acid and oleyl Moreover, these nanoparticles self-assemble to generate super-
amine as stabilizers, while the same sized particles stabilized lattices, and when annealed the nanoparticles were transformed
with hexanoic acid and hexyl amine exhibited ~ 1 nm spacing. to the tetragonal phase.
Fig. 4 shows the TEM images of 6 nm sized Fe50Pt50 Park and Cheon synthesized nanoparticles of Co–Pt alloys
and CocorePtshell via transmetalation reactions.28 The injection
of 0.25 mmol of Co2(CO)8 into hot toluene solution containing
0.5 mmol of Pt(hfac)2 and oleic acid produced CoPt3 alloy
nanoparticles with a particle size of 1.8 nm (s = 0.1 nm).
Reaction of Pt(hfac)2 with previously prepared 6.33 nm sized
Co nanoparticles produced moderately monodisperse CocorePt-
shell nanoparticles with a particle size of 6.27 nm (s = 0.58 nm).
The Pt shell thickness and the Co core size were estimated to be
1.82 and 4.75 nm, respectively.
Chaudret and coworkers reported the synthesis of 1–2 nm
sized bimetallic Co–Pt nanoparticles from the reaction of
Co(h3-C8H13)(h4-C8H12) and Pt2(dba)3 (dba = dibenzylidenea-
cetone) under dihydrogen.29 The composition of the nano-
particles could be varied by changing the initial molar ratio of
the two organometallic precursors. XRD and TEM analysis
revealed that the Pt rich particles adopted a fcc crystalline
structure while the cobalt rich particles adopted a poly-
tetrahedral arrangement.
Nanoparticles of ferrites
Transition metal oxides constitute one of the most fascinating
classes of inorganic solids, as they exhibit a very wide variety of
structures, properties, and phenomena.30 Oxide materials have
been employed in many important advanced technology areas
due to their many interesting physical properties, including
magnetic, ferroelectric, superconducting, ionic and electrical
Fig. 4 TEM images of 6 nm sized Fe50Pt50 nanoparticles stabilized with
oleic groups (a) and hexanoic groups (b).24a Reprinted with permission from conducting characteristics. Nanoparticles of magnetic oxides,
reference 24(a). Copyright 2000 American Association for the Advance- including most representative ferrites, have been studied for
ment of Science. many years for their applications as magnetic storage media and
as ferrofluids. However, the synthesis of monodisperse mag-
nanoparticles stabilized with oleic groups (Fig. 4(a)) and netic oxide nanoparticles was only reported very recently.
hexanoic groups (Fig. 4(b)), respectively. XRD analysis The Alivisatos group reported the synthesis of g-Fe2O3
revealed that the as-synthesized nanoparticles showed a dis- nanoparticles from the thermal decomposition of iron Cupfer-
ordered face centered cubic (fcc) crystal structure and that this ron complexes (Cup: N-nitrosophenylhydroxylamine,
was transformed to an ordered face centered tetragonal structure C6H5N(NO)O2).31 Particle sizes were controlled by either
when heated at 500 °C. Magnetic studies on 4 nm sized Fe52Pt48 varying the reaction temperature or by using a varying amount
nanoparticles showed that the as-synthesized nanoparticles of the complex. Relatively uniform 4–10 nm sized g-Fe2O3
were superparamagnetic at room temperature and that they nanoparticles were synthesized. Using different metal Cupfer-
became ferromagnetic after heating at 500 °C. A write/read ron complexes, nanoparticles of Mn3O4 and Cu2O were also
experiment demonstrated that the annealed 120 nm thick generated.
superlattice of 4 nm Fe48Pt52 nanoparticles supported magneti- Very recently, our group reported on the synthesis of
zation reversal transitions at moderate linear densities. Dai et al. monodisperse and highly-crystalline maghemite nanoparticles,
reported the detailed structural characterization of Fe–Pt which did not involve a size selection process.21 The overall
nanoparticles during annealing using high-resolution transmis- synthetic procedure is depicted in the Fig. 5. Monodisperse iron
sion electron microscopy.24b Very recently, Rogach and nanoparticles were first synthesized, and then they were
coworkers grew micrometer-sized colloidal crystals of Fe–Pt transformed to monodisperse g-Fe2O3 nanocrystals by con-
nanoparticles using a three-layer technique based on the slow trolled oxidation using trimethylamine N-oxide ((CH3)3NO), as
diffusion of a non-solvent into a concentrated solution of a mild oxidant. In the current synthetic process, the size
nanoparticles.25 uniformity was determined during the synthesis of metallic
Using a similar synthetic procedure, Chen and Nikles nanoparticles. Transmission electron microscopic images of the
reported the synthesis of FePd and CoPt nanoparticles.26 7 nm particles showed two- and three-dimensional assembly of
sized Co48Pt52 nanoparticles were synthesized by the simultane- particles, demonstrating the uniformity of these nanoparticles.
ous reduction of platinum acetylacetonate and the thermal Electron diffraction, X-ray diffraction, and high-resolution
decomposition of cobalt tricarbonylnitrosyl (Co(CO)3(NO)) in transmission electron microscopy proved the highly crystalline
the presence of oleic acid and oleyl amine. 11 nm Fe50Pd50 nature of the g-Fe2O3 structures. XPS and Raman spectroscopic
nanoparticles were produced using a similar synthetic method results confirmed the g-Fe2O3 structures. Particle size could be
employing palladium acetylacetonate and iron pentacarbonyl as varied from 4 to 20 nm by altering the experimental parameters.
precursors. They also synthesized FexCoyPt1002x2y nano- The dominating size controlling factor was the molar ratio of
particles from the simultaneous reduction of cobalt acet- iron pentacarbonyl to oleic acid. Fig. 6(a) and (b) show the TEM
ylacetonate and platinum acetylacetonate and the thermal images of maghemite nanoparticles with sizes of 7 and 11 nm
decomposition of iron pentacarbonyl in the presence of oleic that were synthesized by using reaction mixtures with [Fe-
acid and oleyl amine.27 The average particle diameter was 3.5 (CO)5]+[oleic acid] molar ratios of 1+2 and 1+3, respectively.
nm and the particle size distribution was very narrow. As described in the synthesis of iron nanoparticles, nano-
930 CHEM. COMMUN., 2003, 927–934

Fig. 5 Synthetic procedure for monodisperse g-Fe2O3 nanoparticles.
by mild oxidation allowed better control of particle size and

reproducibility.
Using a similar procedure, based on the thermal decomposi-
tion of a metal–surfactant complex followed by mild oxidation,
we synthesized highly crystalline and monodisperse cobalt
ferrite (CoFe2O4) nanocrystals.32 In this synthesis, uniform
iron–cobalt alloy nanoparticles were first generated from the
thermal decomposition of a metal–oleate complex, and then
further oxidized to yield cobalt ferrite nanocrystals. A single
molecular precursor, (h5-C5H5)CoFe2(CO)9, was employed in
the synthesis of the mixed-metal–oleate complex. The as-
synthesized poorly crystalline metallic nanoparticles were then
transformed into cobalt ferrite nanocrystals by oxidizing them
with the mild oxidant, trimethylamine N-oxide. Cobalt ferrite
nanoparticles with particle diameters of 6 nm were synthesized
using starting reaction mixtures with a metal precursor to oleic
acid molar ratio of 1+3. The uniformity of the nanocrystals was
further demonstrated by the formation of a three-dimensional
close-packed superlattice assembly. Larger 9 nm sized nano-
crystals, as shown in Fig. 7, were produced when lauric acid was
Fig. 7 TEM images of 9 nm sized cobalt ferrite nanoparticles.32 Reprinted

with permission from reference 32. Copyright 2002 American Chemical
Society.
used in combination with oleic acid at a molar ratio of [metal

precursor]+[oleic acid]+[lauric acid] = 1+1+2. Monodisperse
Fig. 6 TEM images of g-Fe2O3 nanoparticles: (a) 7 nm, (b) 11 nm, (c) 13 nanocrystals were obtained without a further size selection
nm.21 Reprinted with permission from reference 21. Copyright 2001 process. Nanoscale elemental analysis of ten 9 nm cobalt ferrite
American Chemical Society.
nanocrystals by energy-dispersive X-ray spectroscopy (EDS)
showed that an average molar atomic ratio of Fe to Co was 2.3
particles larger than 11 nm were synthesized by a seeded growth with a standard deviation of 20%. The EDS data well matched
process, employing addition of more iron–oleate complex to the the bulk elemental analysis results obtained by inductively
11 nm sized particles followed by aging at 300 °C. The resulting coupled plasma atomic emission spectrometry (ICP-AES).
larger iron nanoparticles were then transformed to iron oxide Moumen and Pileni reported on a synthesis of cobalt ferrite
nanocrystals by oxidizing with trimethylamine N-oxide. We nanoparticles using a microemulsion method,33 which had been
could also synthesize monodisperse g-Fe2O3 nanocrystals by introduced earlier by Klabunde and coworkers.34 Aqueous
directly injecting Fe(CO)5 into a solution containing both the methylamine (CH3NH3OH) was added to sodium dodecyl
surfactant and trimethylamine N-oxide. Fig. 6(c) shows a TEM sulfate solution containing cobalt(II) dodecyl sulfate (Co(DS)2)
image of 13 nm sized g-Fe2O3 nanocrystals synthesized through and iron(II) dodecyl sulfate (Fe(DS)2) to generate a precipitate.
the direct injection method. However, the first method employ- The size of the nanoparticles was controlled from 2 to 5 nm by
ing the synthesis of monodisperse iron nanoparticles followed changing the sodium dodecyl sulfate concentration and by
CHEM. COMMUN., 2003, 927–934 931

maintaining constant concentrations of Co(DS)2, Fe(DS)2, and nanocrystals by controlling experimental conditions such as the
methylamine. The particles were not well-isolated and aggre- ratio of surfactants, the injection volume, and the time-
gated into networks, and the particle size was not uniform with dependent monomer concentration.40 They explained the for-
a standard deviation of about 30%. Later, detailed structural and mation of anisotropic rod-shaped nanoparticles based on the
magnetic characterizations of these cobalt ferrite nanoparticles relative differences between the growth rates of different crystal
were reported by Zhang and coworkers.35 From neutron faces at high monomer concentration. Cheon and coworkers
diffraction studies, they calculated that 78% of tetrahedral sites described the synthesis of CdS nanorods and of related
(the A site in normal spinel structure with the general formula of nanostructures in pure hexadecylamine using a single-source
AB2O4) were occupied by Fe3+ cations. The 12 nm nano- molecular precursor, Cd(S2CNEt2)2.41 The Peng group con-
particles had a blocking temperature of 320 K. The same ducted detailed mechanistic studies on the shape-controlled
research group also prepared magnesium ferrite nanoparticles synthesis of CdSe nanoparticles, and explained the growth of
using a similar microemulsion method, and the magnetic anisotropic CdSe nanocrystals on the basis of diffusion
properties were compared with cobalt ferrite nanoparticles.36 control.42 Wang and coworkers synthesized gold nanorods
The blocking temperature of cobalt ferrite was found to be at using an electrochemical method.43 In this synthesis, the
least 150 K higher than that of the same sized magnesium ferrite electrochemical cell consisted of a gold anode and platinum
nanoparticles. cathode and the electrolyte solution was a solution of hex-
Markovich and coworkers synthesized magnetite (Fe3O4) adecyltrimethylammonium bromide and tetradecylammonium
nanoparticles by reacting aqueous ammonia with an aqueous bromide. The aspect ratios of the nanorods were controlled by
solution containing FeCl3 and FeCl2 at a molar ratio of 2+1.37 surfactant molar ratios.
The aqueous nanoparticles were dispersible in hexane after Anisotropic magnetic nanoparticles are expected to offer
being coated with oleic acid. After several cycles of size- many advantages because shape anisotropy would exert a
selective precipitation, uniform nanoparticles were obtained. tremendous influence on their magnetic properties. The ten-
The Langmuir–Blodgett (LB) technique was used to produce dency for the magnetization to align in a particular direction in
two-dimensional (2D) arrays of these uniform nanoparticles. a specimen is denoted magnetic anisotropy. Magnetic aniso-
Very recently, Sun and Zeng synthesized monodisperse tropy generally originates from crystal symmetry, shape, stress,
magnetite nanoparticles from a high temperature reaction of or directed atomic pair ordering.3d,5,44 Of these, crystalline
iron(III) acetylacetonate (Fe(acac)3).38 For example, 4 nm sized anisotropy and shape anisotropy are common forms of aniso-
magnetite nanoparticles were synthesized by refluxing a tropy in magnetic materials. Symmetrically shaped nano-
reaction mixture composed of 2 mmol of Fe(acac)3, 20 mL of particles, such as spheres, do not have any net shape anisotropy.
diphenyl ether, 10 mmol of 1,2-hexadecanediol, 6 mmol of oleic However, rod-shaped nanoparticles have shape anisotropy in
acid, and 6 mmol of oleyl amine. A seed-mediated growth addition to crystalline anisotropy, which will increase the
method was used to synthesize larger nanoparticles. Smaller coercivity. Submicrometer-sized acicular (rod-shaped) mag-
sized nanoparticles ( < 4 nm) were mixed with more precursor netic particles have been used in commercial particulate
materials and the resulting mixture was refluxed to produce the magnetic recording media.
larger nanoparticles. By controlling the quantity of the nano- Relatively few anisotropic magnetic nanoparticles have been
particle seeds, various sized larger nanoparticles were gen- reported until 2000. Particle dimensions of the anisotropic
erated. Fig. 8 shows the TEM image of 16 nm Fe3O4 magnetic nanoparticles are too big to exhibit any nanosize effect
and these materials were often extensively agglomerated.45
Gibson and Putzer reported on the synthesis of disk-shaped
cobalt nanoparticles of width 100 nm and thickness 15 nm by
sonicating an aqueous solution of Co2+ and hydrazine.45 The
nanoparticles obtained were single-domain particles. The
particles were not uniform and agglomerated.
Our group reported the synthesis of nearly uniform rod-
shaped iron nanoparticles from the controlled growth of
monodisperse spherical nanoparticles.46 In the first step of the
synthesis, monodisperse 2 nm spherical iron nanoparticles were
prepared by the thermal decomposition of an organometallic
precursor (Fe(CO)5) in the presence of a stabilizing surfactant,
trioctylphosphine oxide (TOPO). An aliquot of Fe(CO)5 in
Fig. 8 TEM images of three-dimensional array of 16 nm Fe3O4 trioctylphosphine (TOP) was added to the resulting spherical
nanoparticles.38 Reprinted with permission from reference 38. Copyright
2002 American Chemical Society.
nanoparticles in TOPO, at 320 °C, and the resulting black
solution was aged for 30 min at 320 °C. The nanoparticles so
nanoparticles. The as-synthesized magnetite nanoparticles were produced were retrieved by precipitation. The precipitate was
transformed to either maghemite (g-Fe2O3) or iron (a-Fe) by dissolved in 19 mL of pyridine containing 0.5 g of didodecyldi-
annealing in an oxygen or in an argon atmosphere, re- methylammonium bromide (DDAB), and the resulting solution
spectively. was refluxed for 12 h. The precipitate formed during the reflux
was removed by centrifugation and the supernatant was
vacuum-dried to yield a black powder. A TEM image of the
Shape control of nanoparticles sample (Fig. 9) revealed nearly monodisperse rod-shaped
Recently, the shape control of nanoparticles to generate particles of dimensions 2 nm (width) 3 11 nm (length) with a
anisotropic nanoparticles was recognized as a very important standard deviation of 5.7% in the length. The electron
issue. The Alivisatos group reported the synthesis of rod-shaped diffraction pattern showed the bcc structure of a-Fe. When the
CdSe nanocrystals from the high temperature reaction between DDAB concentration in pyridine was increased, rod-shaped
dimethylcadmium and selenium in the presence of a mixed particles with higher aspect ratios were obtained. The width of
surfactant solution composed of trioctylphosphine oxide and these nanorods (2 nm) was almost unchanged, but the length
hexylphosphonic acid.39 Later the same group reported on the increased to 22 nm (standard deviation of 5.9%) and 27 nm
synthesis of rod-, arrow-, teardrop- and tetrapod-shaped CdSe (standard deviation of 5.5%). The transformation of nano-
932 CHEM. COMMUN., 2003, 927–934

plained by the high surface tension that reduces the surface-to-
volume ratio. The size of nanoparticles could be controlled by
the ratio of surfactant to precursor, which is consistent with
observations in the synthesis of CdSe and other nanoparticles.
The use of two different surfactants seemed to be important for
the synthesis of disk-shaped nanoparticles by modulating the
relative growth rates of different faces. At a fixed oleic acid
concentration, the length of the nanorods (nanodisks) was found
to be proportional to the concentration of TOPO. High
resolution transmission electron microscopic images showed
that the long direction of the nanorods (nanodisks) is parallel to
the (101) planes, demonstrating that TOPO selectively stabi-
lizes the (101) face of Co and decreases its relative growth rate.
The size uniformity was demonstrated by the spontaneous self-
assembly of the nanocrystals and by the formation of super-
structures such as ribbons of nanorods (nanodisks). A TEM
image of self-assembled 4 nm by 25 nm hcp Co nanorods
(nanodisks) is shown in Fig. 10.
Fig. 9 TEM images of rod-shaped 2 nm (width) 3 11 nm (length) iron
nanoparticles.46 Reprinted with permission from reference 46. Copyright
2000 American Chemical Society.
spheres to nanorods seems to be due to the so-called oriented

attachment mechanism, which is initiated by the relatively
strong binding of DDAB surfactant on the central region of the
growing nanoparticles. After the fusion of two nanospheres, a
third nanosphere will bind on the edge (instead of the central
region where the DDAB is strongly bound), thus generating a
catenated structure. The continued attachment of nanospheres
onto the ends of the growing nanoparticles generates the Fig. 10 TEM images of 4 nm (width) 3 25 nm (length) disk-shaped cobalt
unidirectional nanorods. In some parts of TEM image, catenated nanoparticles.49a Reprinted with permission from reference 49(a). Copy-
nanospheres, which seem to be of the intermediate structure, right 2001 American Association for the Advancement of Science.
were observed. Magnetic studies revealed that the blocking
temperatures of 2 nm 3 11 nm rod-shaped nanoparticles and 2 Very recently, the same research group reported the synthesis
nm spherical nanoparticles were 110 and 12 K, respectively. of hcp-Co nanodisks, which were thought to be nanorods as
These results are consistent with classical micromagnetic described above.49b The disk-shape of the nanocrystals was
theory, which predicts that the anisotropy energy is proportional verified by tilting experiments in TEM. The same workers
to the volume of a single particle and the anisotropy constant. developed a much easier procedure to synthesize cobalt
The magnetic anisotropy constant (K) was deduced from the nanodisks, employing a linear amine instead of TOPO. Thermal
blocking temperature using the equation K = 25kbTB/V, where decomposition of dicobalt octacarbonyl in the presence of oleic
kb is the Boltzman constant and V is the volume of single acid and a linear amine followed by aging generated disk-
nanoparticle.47 The magnetic anisotropy constant of 2 nm shaped nanoparticles along with spherical nanoparticles. A pure
nanospheres was calculated to be 9.1 3 106 erg cm23, and that fraction of hcp Co nanodisks could be obtained after magnetic
of the rod-shaped particles as 1.6 3 107 erg cm23. By treating separation. The length and diameter can be controlled, primarily
the rod-shaped particles as prolate spheroids, the shape by variation of the reaction time following nucleation as well as
anisotropy constant of 2 nm 3 11 nm rod-shaped nanoparticles by varying the precursor to surfactant ratios. The average size of
was calculated to be 7.9 3106 erg cm23. When this shape the smaller disks was about 2 3 4 nm and about 4 3 90 nm for
anisotropy constant was added to the magnetocrystalline the largest.
anisotropy constant of the spheres, the overall value was found Chaudret and coworkers reported on the synthesis of nickel
to agree well with the experimentally determined anisotropy nanorods from the reduction of Ni(COD)2 (COD = cycloocta-
constant of the rod-shaped particles.48 The longer iron nanorods 1,5-diene) in the presence of hexadecylamine.50 They reported
with lengths of 22 nm, 27 nm, and 37 nm were found to be that increasing concentrations of amine induced the formation
readily oxidized to FeO. of nanorods. Magnetic studies revealed that these nanorods
The Alivisatos group extended the synthetic procedure showed a saturation magnetization close to the bulk value,
developed for CdSe nanorods to synthesize cobalt nanorods,49a demonstrating the absence of the influence of amine coordina-
which are now confirmed to be nanodisks as will be discussed tion on magnetic properties of the nanorods. Gedanken and
below.49b In this synthesis, dicobalt octacarbonyl, Co2(CO)8, in coworkers reported the fabrication of the magnetite nanorods by
o-dichlorobenzene was injected into hot (185 °C) o-di- the sonication of aqueous iron(II) acetate in the presence of b-
chlorobenzene solution containing oleic acid and trioctylphos- cyclodextrin.51
phine oxide (TOPO). Quenching of the reaction mixture shortly
after this injection then generated relatively uniform sized
cobalt nanorods (nanodisks) with a hcp crystal structure. Upon
Conclusion and outlook
further thermal aging of several minutes, the high-energy hcp In the past few years, considerable progress has been made in
rod (disk)-shaped particles were transformed to monodisperse the synthesis of monodisperse and well-defined structured
spherical e-Co nanocrystals. The change of crystal structure at magnetic nanoparticles with sizes ranging from 2 to 20 nm. The
the relatively low temperature of < 200 °C can be explained by outlook of such magnetic nanoparticles is very promising
the facile diffusion and phase transitions for nanometer sized because these materials will find many important industrial
particles. The transformation to spherical particles was ex- applications including ultrahigh-density magnetic storage
CHEM. COMMUN., 2003, 927–934 933

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934 CHEM. COMMUN., 2003, 927–934

Published on Web 08/28/2004
Shape-Controlled Synthesis and Shape-Induced Texture of MnFe2O4

Nanoparticles
Hao Zeng,† Philip M. Rice,‡ Shan X. Wang,§ and Shouheng Sun*,†
IBM T. J. Watson Research Center, Yorktown Heights, New York 10598, IBM Almaden Research Center,
650 Harry Road, San Jose, California 95120, and Department of Materials Science and Engineering,
Stanford UniVersity, Stanford, California 94305
Received July 8, 2004; E-mail: ssun@us.ibm.com
The shape is one of the most important factors in determining

the structural, physical, and chemical properties of a nanoparticle
and an assembled array of the particles.1 Shape-controlled synthesis
of nanoparticles has become a recent focus, as different shapes of
the particles can introduce electronic, optical, and magnetic
properties that are different from those observed in their spherical
counterparts. Semiconductor nanoparticles have been made in
various nonspherical shapes of triangle, rod, cube, arrow, and
tetrapod,2 which are interesting for quantum confinement studies
and important for potential sensor, transistor, and solar cell
applications. As the restoring force on the conduction electrons is
extraordinarily sensitive to particle curvature, nonspherical metallic
Au or Ag nanoparticles, produced via various approaches,3 have
been found to shift their surface plasmon frequency drastically,4
making them useful as multicolor diagnostic labels and for other
optical devices. Controlling the shape of a magnetic nanoparticle
is even more appealing as the shape may lead to anisotropic
magnetic properties of the nanoparticles.5 Although recent progress Figure 1. TEM images of the as synthesized (A) 12 nm cubelike and (B)
12 nm polyhedron-shaped MnFe2O4 nanoparticles. HRTEM images of a
in shape-controlled synthesis of magnetic nanoparticles has led to part of a single (C) cubelike and (D) polyhedron-shaped MnFe2O4
nanorods,6 nanocubes,7 and other variously shaped Fe2O3 nano- nanoparticles.
particles,8 there is still no example of using nanoparticles with
different shapes as building blocks to control the crystal orientation nanoparticles could become useful building blocks for the construc-
of the nanoparticles in an assembly. As the magnetic easy axis of tion of functional nanostructures.
a particle correlates closely with its crystal structure,9 the shape- The size of the MnFe2O4 particles is tuned by varying the
induced crystal orientation of each nanoparticle in an assembly concentration of the precursors, while the shape is controlled by
would lead to an aligned magnetic easy axis. Such alignment is the amount of stabilizers added to the reaction mixture. For a
often a necessity for a nanoparticle assembly to be suitable for reaction containing 2 mmol of Fe(acac)3, 1 mmol of Mn(acac)2,
various magnetic applications in such as data storage and advanced 10 mmol of 1,2-hexadecandiol, 6 mmol of oleic acid, 6 mmol of
magnets.10 oleylamine, 20 mL of benzyl ether gave 12 nm MnFe2O4, while
Here we report shape-controlled synthesis of MnFe2O4 nano- 22 mL or 25 mL of benzyl ether yielded 10- or 8 nm particles,
particles and shape-induced texture of the particles in a self- respectively. When the surfactant/Fe(acac)3 ratio is smaller than
assembled superlattice. We recently reported that reaction of 3:1, the particles are nearly spherical with no well-defined facets.12
Fe(acac)3 and M(acac)2 with 1,2-hexadecanediol, oleic acid, and Increasing the ratio to 3:1 yields cubelike particles.13 If the particles
oleylamine in a high-boiling ether solvent could lead to mono- were prepared using the seed-mediated growth as reported before,11
disperse MFe2O4 nanoparticles (M ) Fe, Co, Mn, Mg, etc.). The polyhedron-shaped particles were obtained. Figure 1 shows the
composition of the particles was controlled by the initial molar ratio representative TEM images of the MnFe2O4 nanoparticles with
of Fe(acac)3 and M(acac)2, and the size was tuned by varying the different morphologies, with 1A being cubelike and 1B polyhedron-
reaction conditions or via seed-meditated growth.11 Our further shaped particles. The HRTEM image of a part of a single cubelike
experiments indicated that the size of the MFe2O4 particles (M ) MnFe2O4 nanoparticle (Figure 1C) shows lattice fringes with the
Mn in this work) could be tuned more conveniently through a one- interfringe distance measured to be 0.214 nm, and that of a
pot reaction by changing the concentration of the reactants. More polyhedron-shaped particle (Figure 1D) shows the interfringe
importantly, this one-pot reaction could also lead to the particles distance to be 0.257 nm, close to the lattice spacing of the {400}
with controlled shapes. Slow evaporation of the particle dispersion planes at 0.213 nm and {311} planes at 0.256 nm in the cubic
yielded nanoparticle superlattices. The nanoparticles within the spinel-structured MnFe2O4. HRTEM further reveals that the {400}
superlattice exhibited preferred alignment of crystal orientations lattice fringes are parallel to the edges of the cube, while the {220}
correlated with their shape. With this texture control, the shaped fringes (spacing 0.301 nm) run face-diagonally in the cube,12
indicating that the cubelike particles are terminated with {100}
† IBM T. J. Watson Research Center.
‡ IBM Almaden Research Center.
planes. The TEM tilted angle analyses of the polyhedron-shaped
§ Stanford University. particles indicated that the hexagon shape shown in Figure 1B likely
11458 9 J. AM. CHEM. SOC. 2004, 126, 11458-11459 10.1021/ja045911d CCC: $27.50 © 2004 American Chemical Society
COMMUNICATIONS
that chemical process can be used to regulate the growth of a particle

and control the crystal orientation of a particle in a superlattice
assembly. Such capability of control will be significant in future
fabrication of high-performance magnetic nanodevices. A robust
magnetic nanoparticle superlattice array with an aligned magnetic
easy axis and controlled magnetic interactions between the neigh-
boring particles may revolutionize the information storage technol-
ogy with unprecedented sensitivity and density.
Acknowledgment. The work was supported in part by DARPA/
ONR under Grant No. N00014-01-1-0885.
Supporting Information Available: A typical experimental pro-
cedure for making 12 nm cubelike MnFe2O4 nanoparticles, and further
characterization data for MnFe2O4 nanoparticles and their assemblies.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 2. TEM images of 12 nm MnFe2O4 nanoparticle superlattices of
(A) cubelike and (B) polyhedron-shaped nanoparticles. XRD (Co KR λ ) References
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particle’s spinel lattice within the cubic superlattice. This means (10) (a) Sun, S.; Murray, C. B.; Weller, D.; Folks, L.; Moser, A. Science 2000,
that the incident beam is parallel to the 〈100〉 orientation, further 287, 1989-1992. (b) Zeng, H.; Li, J.; Wang, Z. L.; Liu, J. P.; Sun, S.
confirming the XRD observation shown in Figure 2C. Similarly, Nature 2002, 420, 395-398.
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for the polyhedron-shaped particle assembly, both XRD in Figure S.; Zeng, H.; Robinson, D. B.; Raoux, S.; Rice, P. M.; Wang, S. X.; Li,
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(12) See the Supporting Information.
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induce texture in a self-assembled nanoparticle superlattice. observations that more surfactant facilitates thermodynamical growth of
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J. AM. CHEM. SOC. 9 VOL. 126, NO. 37, 2004 11459

1
1
Biosensing using Carbon Nanotube Field-effect
Transistors
Padmakar D. Kichambare and Alexander Star
1.1
Overview
This chapter covers recent advances in biodetection using single-walled carbon

nanotube field-effect transistors (NTFETs). In particular, we describe fabrication of
NTFET devices and their application for electronic detection of biomolecules. A
typical NTFET fabrication process consists of combination of chemical vapor depo-
sition (CVD) and complementary metal oxide semiconductor (CMOS) processes.
The NTFET devices have electronic properties comparable to traditional metal
oxide semiconductor field-effect transistors (MOSFETs) and readily respond to
changes in the chemical environment, enabling a direct and reliable pathway for
detection of biomolecules with extreme sensitivity and selectivity. We address the
challenges in effective integration of carbon nanotubes into conventional electron-
ics for biosensor applications. We also discuss in detail recent applications of
NTFETs for label-free electronic detection of antibody–antigen interactions, DNA
hybridization, and enzymatic reactions.
1.2
Introduction
The interplay between nanomaterials and biological systems forms an emerging

research field of broad importance. In particular, novel biosensors based on
nanomaterials have received considerable attention [1–4]. Integration of one-
dimensional (1D) nanomaterials, such as nanowires, into electric devices offers
substantial advantages for the detection of biological species and has significant
advantages over the conventional optical biodetection methods [5]. The first advan-
tage is related to size compatibility: Electronic circuits in which the component
parts are comparable in size to biological entities ensure appropriate size compati-
bility between the detector and the detected biological species. The second advan-
tage to developing nanomaterial based electronic detection is that most biological
processes involve electrostatic interactions and charge transfer, which are directly
Nanotechnologies for the Life Sciences Vol. 8

Nanomaterials for Biosensors. Edited by Challa S. S. R. Kumar
Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-31388-4
2 1 Biosensing using Carbon Nanotube Field-effect Transistors
detected by electronic nanocircuits. Nanowire-based electronic devices, therefore,

eventually integrate the biology and electronics into a common platform suitable
for electronic control and biological sensing as well as bioelectronically driven
nanoassembly [6].
One promising approach for the direct electrical detection of biomolecules uses
nanowires configured as field-effect transistors (FETs). FETs readily change their
conductance upon binding of charged target biomolecules to their receptor linked
to the device surfaces. For example, recent studies by Lieber’s group have demon-
strated the use of silicon nanowire FETs for detecting proteins [7], DNA hybrids
[8], and viruses [9]. This biodetection approach may allow in principle selective de-
tection at a single particle levels [10, 11]. Nanowires hold the possibility of very
high sensitivity detection owing to the depletion or accumulation of charge car-
riers, which are caused by binding of a charged biomolecules at the surface. This
surface binding can affect the entire cross-sectional conduction pathway of these
nanostructures. For some nanowires, such as hollow carbon nanotubes, every
atom is on the surface and exposed to the environment; even small changes in
the charge environment can drastically change their electrical properties. Thus,
among different nanomaterials, carbon nanotubes have a great potential for
biosensing.
Among numerous applications of carbon nanotubes [12–14], carbon nanotube
based sensing technology is rapidly emerging into an independent research field.
As for any new research field, there is no yet consensus in the literature about the
exact sensing mechanism. In this chapter, in addition to selected examples of
carbon nanotube based sensors, we address the controversial carbon nanotube
sensing mechanism.
To date, sensor applications of carbon nanotubes have been summarized and
discussed in several excellent review articles [15–17], which primarily focus on
carbon nanotube based electrochemical sensors. This chapter covers only recent
advances in biodetection using carbon nanotube field-effect transistors (NTFETs).
It is divided into two large sections: NTFET fabrication and their sensor applica-
tions. Section 1.3 gives a detailed description of NTFET device structure, its fabri-
cation method and introduces device characteristics. This section also addresses
technical challenges in effective integration of carbon nanotubes into CMOS elec-
tronics. Section 1.4, which focuses on sensor applications of NTFETs, is divided
into several subsections. Before discussing NTFET application for biological detec-
tion we describe the effect of environmental conditions on NTFET device character-
istics. We give selected examples of NTFET sensitivity for small molecules, mobile
ions, and water (relative humidity). The effect of these factors should be well
understood before NTFET biodetection is reviewed. We also briefly describe the
operation of NTFETs in conducting media, which is particularly important for bio-
sensor applications. Then we briefly summarize interactions of carbon nanotubes
with biomolecules (e.g., polysaccharides, DNA and proteins) to set a stage for the
subsequent subsections that describe in great details recent applications of NTFETs
for label-free electronic detection of proteins, antibody–antigen interactions, DNA
hybridization, and enzymatic reactions.
1.3 Carbon Nanotube Field-effect Transistors (NTFETs) 3
1.3
Carbon Nanotube Field-effect Transistors (NTFETs)
1.3.1
Carbon Nanotubes
Since their discovery by Iijima over a decade ago [18], interest in carbon nanotubes
has grown considerably [19]. Recent advances in the synthesis and purification of
carbon nanotubes have turned them into commercially available materials. Subse-
quently, several experiments have been undertaken to study the physical and elec-
trical properties of carbon nanotubes on the individual and macroscopic scale [20–
23]. On the macroscopic scale, spectroscopic and optical absorption measurements
have been carried out to test the purity of the carbon nanotubes [24, 25]. For elec-
tronic transport measurements it is particularly interesting to perform experiments
on isolated, individual carbon nanotubes. The properties of carbon nanotubes de-
pend strongly on physical aspects such as their diameter, length, and presence of
residual catalyst [12]. The properties measured from a large quantity of nanotubes
could be an average of all nanotubes in the sample, so that the unique character-
istics of individual carbon nanotubes could be shadowed. Experiments on individ-
ual nanotubes are very challenging due to their small size, which prohibits the
application of well-established testing techniques. Moreover, their small size also
makes their manipulation rather difficult. Specialized techniques are needed to
mount or grow an individual carbon nanotube on the electrode with sub-micron
precision.
Carbon nanotubes are hollow cylinders made of sheets of carbon atoms and
can be divided into single-walled carbon nanotubes (SWNTs) and multi-walled car-
bon nanotubes (MWNTs). SWNTs possess a cylindrical nanostructure with a high
aspect ratio, formed by rolling up a single graphite sheet into a tube (Fig. 1.1).
SWNTs are, typically, a few nanometers in diameter and up to several microns
long. MWNTs consist of several layers of graphene cylinders that are concentrically
nested like rings of a tree trunk, with an interlayer spacing of 3.4 Å [26]. Because of
their unique properties, carbon nanotubes have become a material that has gener-
ated substantial interest on nanoelectronic devices and nanosensors [27, 28]. These
properties are largely dependent upon physical aspects such as diameter, length,
presence of catalyst and chirality. For example, SWNT can be metallic or semicon-
ducting, depending upon the intrinsic band gap and helicity [29]. Semiconducting
SWNTs can be used to fabricate FET devices, as demonstrated by Dekker and co-
workers [30]. In addition, semiconducting SWNTs exhibit significant conductance
changes in response to the physisorption of different gases [24, 31, 32]. Therefore,
SWNT-based nanosensors can be fabricated based on FET layout, where the solid-
state gate is replaced by adsorbed molecules that modulate the nanotube conduc-
tance [33]. Since semiconducting SWNTs have a very high mobility and, because
all their atoms are located at the surface, they are the perfect nanomaterial for sen-
sors. These sensors offer several advantages for the detection of biological species.
First, carbon nanotubes form the conducting channel in a transistor configura-
Fig. 1.1. A seamlessly rolled-up single or semiconducting, depending on their helicity

graphite sheet forms a single-walled carbon and diameter. Semiconducting SWNTs are
nanotube (SWNT). SWNTs are, typically, a few used for the fabrication of nanotube field-effect
nanometers in diameter and up to several transistors (NTFETs). (Adapted with
micrometers long. They can be either metallic permission from Ref. [4], 8 Wiley-VCH Verlag).
tion. Second, the nanotubes are typically located on the surface of the supporting
substrate and are in direct contact with the environment. This device geometry
contrasts with traditional metal oxide semiconductor field-effect transistors
(MOSFETs) where the conducting channel is buried in the bulk material in which
the depletion layer is formed. Lastly, all of the electrical current flows at the surface
of nanotubes. All these remarkable characteristics lead to a FET device configura-
tion that is extremely sensitive to minute variations in the surrounding environ-
ment.
1.3.2
Nanotube Synthesis
Several synthesis methods are used to produce carbon nanotubes [34]. The three
most commonly used methods are the arc discharge, laser ablation, and chemical
vapor deposition (CVD) techniques. While the arc and laser methods can produce
large quantities of carbon nanotubes they lead to resilient contaminants, including
pyrolytic and amorphous carbon [35, 36], which are difficult to remove from the
sample. Such impurities result in low recovery yield for the carbon nanotube prod-
uct. However, recent advances in scaling up these methods, as well as development
new fabrication methods such as high pressure carbon monoxide (HiPCO), have
created commercial supplies of carbon nanotubes with more than 90% purity
with competitive prices. In contrast, the less scalable CVD process offers the best
chance of obtaining controllable routes for the selective production of carbon nano-
tubes with defined properties [37]. CVD is catalytically driven, wherein a metal cat-
alyst is used in conjunction with the thermal decomposition of hydrocarbon feed-
stock gases to produce carbon nanotubes. In most cases, the resultant growth of
nanotubes occurs on a fixed substrate within the process. Figure 1.2 illustrates a
typical CVD process for the generation of SWNTs. SWNTs are synthesized by the
Fig. 1.2. Schematic of a chemical vapor deposition (CVD)

reactor that uses a two-zone furnace. Carbon nanotubes grow
on the substrate placed inside the quartz tube. (Reprinted with
permission from Ref. [34], 8 2001, CRC Press).
reaction of a hydrocarbon (e.g., CH4 ) vapor over a dispersed Fe catalyst. The syn-
thesis apparatus consists of a quartz tube reactor inside a combined preheater and
furnace set-up. The preheat section is operated at @200 C. The catalysts are depos-
ited and then hydrocarbon vapors are carried into the reaction zone of the furnace.
An Ar/(10%)-H2 carrier gas is used that controls the partial pressure inside the
quartz tube reactor. Reaction temperatures are typically in the range 900–1000 C.
The SWNTs grow on the substrates (Fig. 1.3) and form thick mats that are readily
harvested. This process produces highly pure SWNTs at a yield approaching 50%
conversion of all hydrocarbon feedstock into carbon nanotube product. Similarly,
a CVD processes sometimes utilize a feed of hydrocarbon-catalyst liquid for the
production of nanotubes, and for this purpose a syringe pump is used to allow
the continuous injection of this solution into a preheat section. Various gaseous
feed-stocks are used to produce nanotubes, ranging from CH4 to C2 H2 . A wide
range of transition metals and rare earth promoters have been investigated for the
synthesis of SWNTs by CVD. In general, a transition metal is the major compo-
nent in the catalyst particles used regardless of the catalyst support. The most com-
mon metals found to be successful in the growth of SWNTs are Fe, Co, and Ni [38,
39]. However, bimetallic catalysts consisting of Fe/Ni, Co/Ni, or Co/Pt [40] are re-
Fig. 1.3. Transmission electron microscopy (TEM) image of an

SWNT synthesized by chemical vapor deposition (CVD).
(Reprinted with permission from Ref. [37], 8 2001, The
American Chemical Society).
ported to give the best yield of nanotubes, in addition to some rare earth metals
that have also been studied [41].
Despite challenges, the understanding the growth mechanism of SWNTs is cru-
cial for, ultimately, tailoring the production of SWNTs with known lengths, diame-
ters, helical structures and placement of SWNTs at the desired location. Presently,
efforts are underway to understand the mechanism of catalytic growth of SWNTs
on surfaces and the role of impurities and to increase nanotube yield by varying
the substrate, catalyst, and growth conditions [42]. Directional growth of SWNTs
has been achieved by electric fields [43, 44], gas flow [45], lattice directions [46],
and atomic steps [47].
1.3.3
Fabrication of NTFETs
To date carbon nanotubes have been used to fabricate various devices, including
nanotube-based mechanical devices [48] and field emission devices [13]. This sec-
tion focuses specifically on fabrication of carbon nanotube field-effect transistors
(NTFETs). Figure 1.4(a) shows a schematic drawing of NTFET. A semiconducting
carbon nanotube is contacted by source and drain electrodes while the gate elec-
Fig. 1.4. (a) Schematic representation of a SWNT. (c) Scanning electron microscopy
nanotube field-effect transistor (NTFET) device (SEM) image of a typical NTFET device
with a semiconducting SWNT contacted by two consisting of a random array of carbon
Ti/Au electrodes, representing the source (S) nanotubes. (d) Typical NTFET transfer
and the drain (D) with a Si back gate characteristic – dependence of the source-drain
separated by a SiO2 insulating layer in a conductance (GSD ) on the gate voltage (VG ) –
transistor-configured circuit. (b) Atomic force (i) maximum conductance, (ii) modulation,
microscopy (AFM) image of a typical NTFET (iii) transconductance, (iv) hysteresis, and (v)
device consisting of a single semiconducting threshold voltage.
trode, which is electrically insulated from the nanotube channel, is used to manip-
ulate the nanotube’s conductivity. Depending on the particular method of nano-
tube fabrication, a NTFET can be structured in different ways [49]. However, most
publications on nanotube transistors report the use of a degenerately doped Si-
substrate with a comparatively thick (100–500 nm) thermally grown oxide layer
[30, 50–53]. Silicon substrates are readily available and can be used with both
bulk-produced nanotubes and nanotubes grown directly on the Si-substrate by
CVD. If doped highly, the Si substrate stays conductive even at low temperatures,
making it usable as a so-called back-gate with the SiO2 as a stable, if low-k, gate
dielectric. Bulk produced nanotubes (laser ablation [54] or HiPCO [55]) are usually
purified and deposited onto Si substrates by suspending them in organic solvents
(e.g., chloroform, dichloroethane, etc.) and then spin-coating or drop casting on the
substrates. In this approach the nanotubes create a random network over the sub-
strate surface. Alignment of the nanotubes is possible, if an AC dielectric field is
applied during deposition [56]. Generally, two different configurations of NTFETs,
regarding source and drain contacts, are possible. By patterning the contacts before
nanotube deposition [30] one can contact nanotubes in bulk, whereas by deposit-
ing the contacts onto the nanotubes one contacts only the ends of nanotubes [57],
because depositing contact material on top of the nanotube normally destroys the
nanotubes underneath the contacts. The second configuration usually guarantees
a lower contact resistance than what is achievable in bulk-contacted devices
[58]. Room temperature NTFET manufacturing methods, while compatible with
CMOS, are limited by ability to disperse effectively carbon nanotubes in the solu-
tion and deposit them without further aggregation on device surfaces [59]. Nano-
tube bundle formation may decrease the semiconducting character of NTFET due
to the occasional presence of nanotube bundles containing metallic nanotubes.
For CVD grown carbon nanotubes metal contacts are deposited onto the nano-
tubes [60, 61], because typical contact materials cannot withstand CVD tempera-
tures, thus making it impossible to grow carbon nanotubes on CMOS structures.
Often, the metal contacts are annealed to lower contact resistance [62]. Several
studies have tried to optimize the material used for the contacts, including Cr/Au
[49] and Pt [30], but only the Cr/Au contacts have been used widely. In this type of
contact the chromium layer is a thin (1–3 nm) adhesion layer that facilitates adhe-
sion of the gold to SiO2 . An adhesion layer of Ti [63], especially when annealed,
allows deposition of smooth films of many metals onto carbon nanotubes because
Ti forms titanium carbide at the interface with the nanotube. For this reason,
Ti/Au-contacts are another frequently used combination of contact materials. Many
publications investigating Schottky barriers between a nanotube and its contacts
[61, 64] have employed such contacts. Palladium (Pd) is another material investi-
gated that wets nanotubes well and has been used recently to produce NTFETs
with ohmic contacts, i.e., contacts without Schottky barriers [52, 63].
Depending on the number of carbon nanotubes connecting the source and drain
electrodes, there are two different device architectures. In the first device architec-
ture, a single nanotube connects the source and drain (Fig. 1.4b). These devices
have been used for biosensing with excellent sensitivity. However, there is substan-
tial variation between the different devices that are fabricated and this variation is
reflected in the electronic characteristics of individual nanotubes. In addition, the
interface between the nanotube and the metallic contact may vary from device to
device. Specialized techniques are needed either to mount or grow an individual
carbon nanotube at a predetermined location. Placement is difficult and impracti-
cal for mass fabrication of NTFETs. For example, although the process of attaching
a carbon nanotube strand via arc-discharge or contact method to sharp metal probe
is fast, simple and economical it suffers from low yield. Therefore, it is difficult to
determine the quality of carbon nanotube strand attached to metal tip unless exam-
ined under SEM. When checked under SEM a large percentage of the metal probes
have multiple nanotubes attached or clusters of amorphous carbon accompanying
the carbon nanotubes [65]. Hence random networks of SWNTs have been explored
as an alternative [66].
Nanotube networks take up more space than individual SWNTs, but they are
much easier to fabricate and show great promise towards simple mass fabrication
of NTFETs. In this second configuration, the devices contain a random array of
nanotubes between source and drain electrodes (Fig. 1.4c). In this configuration,
current flows along several conducting channels that determine the overall device
resistance. The device characteristics depend on the number of nanotubes and
density of the nanotube network. It is reported that the conductance drops are as-
sociated with junctions formed by crossed semiconducting and metallic nanotubes.
Local conductance is more dependent on the number of connections to the specific
area; clusters of nanotubes with many paths to the electrode have significantly
higher conductance than those parts of the network connected through fewer
paths. Areas with low conductance typically only have two to three connections to
the network, thus it is likely that these connections are dominated by the presence
of highly resistive metallic/semiconducting junctions. When a sufficient back gate
voltage is applied to the sample, current flow through the semiconducting tubes is
suppressed. Using this technique, differences between metallic and semiconduct-
ing SWNT can be distinguished. This type of device configuration, containing a
network of conducting nanotube channels, is less sensitive than devices made of
single nanotubes. In both types of device configurations, the parameter used for
detection is the transfer characteristic – the dependence of either the source-drain
current (ISD ) or conductance (GSD ) (for a fixed source-drain voltage VSD ) on the
gate voltage (VG ) (Fig. 1.4d).
NTFETs can operate as p-type or n-type transistors. The mode of operation can
be changed from the pristine p-type to n-type by either adding electron donor mol-
ecules (n-doping) or removing adsorbed oxygen by annealing the contacts under
vacuum [67]. Polymer-gated NTFETs can also tune their modes of operation: a
change in the chemical group of the polymer changes the NTFET from p-type to
n-type [68, 69]. Oxygen doping was attributed to the fact that the oxygen interacts
with the nanotube–metal junction and causes the p-type characteristic for NTFETs
in air by pinning the metal’s Fermi level near the nanotube’s valance band maxi-
mum [33]. However, there is no apparent consensus in the literature about the
exact mechanism of chemical sensitivity of NTFETs.
1.4 Sensor Applications of NTFETs 9
1.4
Sensor Applications of NTFETs
Before discussing NTFET applications for biological detection we first describe

the effect of small molecules, relative humidity, and conductive liquid media on
NTFET devices characteristics. Effect of these factors should be well understood be-
fore NTFET biodetection is reviewed.
1.4.1
Sensitivity of NTFETs to Chemical Environment
Generally, the molecular species in the ambient environment have a significant

impact on the electrical properties of NTFETs. The conductance of semiconducting
SWNTs can be substantially increased or decreased by exposure to NO2 or NH3
[24]. Exposure to NH3 effectively shifts the valance band of the nanotube away
from the Fermi level, resulting in hole depletion and reduced conductance. In con-
trast, on exposure to NO2 molecules the conductance of nanotubes increases by
three orders of magnitude [70]. Here, exposure of the initially depleted sample to
NO2 resulted in the nanotube Fermi level shifting closer to the valence band. This
caused enriched hole carriers in the nanotube and enhanced sample conductance.
These results show that molecular gating effects can shift the Fermi level of semi-
conducting SWNTs and modulate the resistance of the sample by several orders of
magnitude.
The electronic properties of SWNTs are also extremely sensitive to air or oxygen
exposure [33]. Isolated semiconducting nanotubes can be converted into apparent
metals through room temperature exposure to oxygen. As the surrounding me-
dium was cycled between vacuum and air, a rapid and reversible change in the
SWNT resistance occurred in step with the changing environment. Initially, in a
pure atmospheric pressure oxygen environment, the thermoelectric power (TEP)
S was positive with a magnitude of nearly þ20 mV K1 . This relatively large posi-
tive TEP is consistent with that reported for pristine SWNTs near room tempera-
ture [71]. As oxygen was gradually removed from the chamber, the TEP changed
continuously from positive to negative, with a final equilibrium value of approxi-
mately 10 mV K1 . When oxygen was reintroduced into the chamber, the TEP
reversed sign and once again became positive. These dramatic 10–15% variations
in R and change in sign of the TEP demonstrate that SWNTs are exceptionally sen-
sitive to oxygen.
In the carbon nanotubes sensors mentioned above, chemical sensing experi-
ments have been conducted with devices in which both nanotubes and nanotube–
metal contacts were directly exposed to the environment. The sensing could be
dominated by the interaction of molecules with the metal contacts or the contact
interfaces. Adsorbed molecules would modify the metal work functions and, there-
by, the Schottky barrier [72, 73]. Heinze et al. [64] have assigned the effect of oxy-
gen to the Schottky barrier. Recently, a new device architecture has been studied in
which the interface between the metallic contacts and nanotubes is covered by a
Fig. 1.5. (a) AFM image of a contact passivated NTFET device

covered with poly(ethylene imine). (b) ISD –VG dependence for
the device in vacuum (center curve), as well as in NH3 and
NO2 gases. (Adapted with permission from Ref. [74], 8 2003
American Institute of Physics).
passivation layer, referred to as contact-passivated [74]. In this configuration, with

the junction isolated and only the central length of the nanotube channels exposed,
the contacts should be isolated from the effect of chemicals. At the same time, the
section of the device that is open to the environment can be doped via charge trans-
fer. NTFETs with such configuration have been investigated by measuring sensitiv-
ity to NH3 , NO2 , and poly(ethylene imine) (Fig. 1.5).
The NTFET devices were fabricated using SWNTs grown by CVD on 200 nm of
silicon dioxide on doped silicon from iron nanoparticles as described in Section
1.3.1. These particles were exposed to flowing hydrocarbon to grow carbon nano-
tubes, and after growth optical lithography was used to pattern electrical leads
(35 nm titanium capped with 5 nm gold) on top of the nanotubes. Contact passiva-
tion was achieved by 70 nm silicon monoxide layer. Source and drain electrodes
were separated by nearly a micrometer. The dependence of the source-drain cur-
rent (ISD ) as function of the gate voltage (VG ) was measured from þ10 to 10 V
using a semiconductor parameter analyzer in air/water/gas mixtures. The low con-
centrations of gas mixtures could be introduced to the devices by mixing different
proportions of air and gases. The contact-passivated devices demonstrated NH3
and NO2 sensitivity similar to regular NTFETs. Poly(ethylene imine) also produced
negative threshold shifts of tens of volts, despite being in contact with only the
center region of devices. Thus, the NTFET sensor character was preserved despite
isolating Schottky barriers.
Several groups have reported that NTFET fabricated on SiO2/Si substrates exhib-
its hysteresis in current versus gate-voltage characteristics and attributed the hyste-
resis to charge traps in bulk SiO2 , oxygen-related defect trap sites near nanotubes,
or the traps at the SiO2/Si interface. It is mentioned that thermally grown SiO2 sur-
face consists of Si-OH silanol groups and is hydrated by a network of water mole-
cules that are hydrogen bonded to the silanols. The CVD nanotube growth condi-
tion (900 C) may dehydrate the surface and condense to form SiaOaSi siloxanes.
When such a surface is exposed to and stored in ambient air, the surface siloxanes
on the substrate react with water and gradually revert to SiaOH, after which the
substrate becomes rehydrated. Heating under dry conditions significantly removes
water and reduces hysteresis in the transistors.
Kim et al. have reported that the hysteresis in electrical characteristics of NTFETs
is due to charge trapping by water molecules around the nanotubes, including
SiO2 surface-bound water proximal to the nanotubes [75]. They have demon-
strated that coating nanotube devices with PMMA can afford nearly hysteresis-
free NTFETs [75]. This passivation is attributed to two factors. First, the ester
groups of poly(methyl methacrylate) (PMMA) can hydrogen bond with silanol
group on SiO2 . Baking at 150 C combined with the polymer–SiO2 interaction
can significantly remove the silanol-bound water. Second, PMMA is hydrophobic
and can keep water in the environment from permeating the PMMA and adsorb-
ing on the nanotube in a significant manner.
Bradley et al. have attributed hysteresis in NTFET devices to cation diffusion
[76], based on the following experiments. First, NTFET devices that exhibit very
small hysteresis were fabricated. Subsequently, these devices were modified by
the addition of an electrolyte coating that created mobile ions on the surface of
the device and resulted in the large hysteresis. Experiments were also conducted
to explore possible mechanisms for cation-induced hysteresis by varying the
humidity that changes the hydration layer around the nanotubes, thus leading to
the increase of the ionic mobility. The hysteresis has been found to be sensitive to
humidity on sub-second time scales, showing promise as a humidity sensor [77].
Sensitivity of NTFETs to charges as well as NTFET operation in conducting
liquid media is important for biosensor design where the sensor should operate
in physiological buffers with complex mixtures of biomolecules. Figure 1.6 shows
a typical transfer characteristic of NTFET measured in air and water using the sili-
con and water as the gate electrode, respectively. The change in device characteris-
Fig. 1.6. (a) Detection in liquid with NTFET the liquid gate. Note the different x-scales for
devices by using either the back gate or liquid the back and liquid gates. (Adapted with
gate configuration. (b) NTFET transfer permission from Ref. [93], 8 2003, The
characteristics in air (solid line), using the American Physical Society.)
back gate, and in water (dashed line), using
tics upon exposure to a water/gas mixture is reflected in the transfer characteris-

tics. Saline or electrolytes can also gate NTFETs and give high transconductance
[62, 78].
1.4.2
Bioconjugates of Carbon Nanotubes
Numerous reports demonstrate the ability to chemically functionalize nanotubes

for biological applications [79, 80]. Such chemistry is readily transferable to many
applications, ranging from sensors [81, 82] to electronic devices [83]. SWNTs are
chemically stable and highly hydrophobic. Therefore, they require surface modifi-
cation to establish effective SWNT–biomolecule interaction.
So far, two methods of exohedral functionalization of SWNTs have been devel-
oped – namely covalent and noncovalent. While covalent modifications [84] are
often effective at introducing functionality, they impair the desirable mechanical
and electronic properties of SWNTs. Noncovalent modifications [85], however, not
only improve the solubility of SWNTs in water, but they also constitute non-
destructive processes, which preserve the primary structures of the SWNTs, along
with their unique mechanical and electronic properties.
Previously, it has been shown that polysaccharides such as starch [86, 82, 83],
gum Arabic [84], and the b-1,3-glucans, curdlan and schizophyllan [85], will solu-
bilize SWNTs in water. It has been proposed that at least some of these polymers
achieve their goal by wrapping themselves in helical fashion around SWNTs (Fig.
Fig. 1.7. Molecular model of SWNT wrapped in an amylose

coil. (Reprinted from Ref. [79], 8 2002, The American Chemical
Society.)
1.7). Solubilization of the SWNTs with cyclodextrins (CD), which are macrocyclic
polysaccharides, has been also investigated [86]. The observed aqueous solubility
of SWNTs with g-CD is unlikely due to encapsulation because the inner cavity di-
mensions of this CD are far too small to allow it to thread onto even the smallest
diameter SWNTs. More recently, however, it has been shown [87] that h-CD, which
has 12 a1,4-linked d-glucopyranose residues and therefore is large enough, does
thread onto SWNTs in water, not only solubilizing the NTs but also permitting
some partial separations according to their diameters.
Nucleic acids, such as single-stranded DNA, short double-stranded DNA, and
some total RNA can also disperse SWNTs in water [88, 89]. Molecular modeling
has shown [20] that the non-specific DNA–SWNT interactions in water are from
the nucleic acid–base stacking on the nanotube surface, resulting in the hydro-
philic sugar–phosphate backbone pointing to the exterior to achieve the solubility
in water. The mode of interaction could be helical wrapping or simple surface
adsorption. The charge differences among the DNA–SWNT conjugates, which are
associated with the negatively charged phosphate groups of DNA and the different
electronic properties of SWNTs, have allowed post-production preparation of sam-
ples enriched in metallic and semiconducting SWNTs.
Various proteins can also strongly bind to the nanotube exterior surface via
non-specific adsorption. Proteins such as streptavidin and HupR crystallize in heli-
cal fashion, resulting in ordered arrays of proteins on the nanotube surface [90].
Mechanistically, the non-specific adsorption of proteins onto the nanotube surface
may be more complicated than the widely attributed hydrophobic interactions.
Quite possibly, the observed substantial protein adsorption is, at least in part, asso-
ciated with the amino affinity of carbon nanotubes, as was demonstrated recently
by monitoring the conductance change in the carbon nanotube [91]. Also, inter-
molecular interactions involving aromatic amino acids, i.e., histidine and trypto-
phan, in the polypeptide chains of the proteins can contribute to the observed affin-
ity of the peptides to carbon nanotubes [92].
1.4.3
Protein Detection
Carbon nanotube interactions with proteins have been explored by NTFET devices
[91]. In NTFET devices, the ability to measure the electronic properties of the nano-
tube allowed to query the electronic state of the immobilization substrate. In that
work two types of measurements of the device transfer characteristics were per-
formed. In the first measurement, referred to as a substrate-gate transfer character-
istic, the current through the drain contact (at fixed source-drain bias) was moni-
tored while a variable gate voltage was applied through a metallic gate buried
underneath the SiO2 substrate. In the second measurement, referred to as liquid-
gate transfer characteristics, the device was immersed in a buffer solution and a
variable gate voltage was applied through a platinum electrode. The current was
passed through the drain contact and a silver reference electrode in the solution.
During these measurements, the assembly was shaken gently, using a lab rotator
at 3 Hz. The effect of protein adsorption was studied with both measurements. De-
vices were incubated with streptavidin (40 nm) in 15 mm phosphate buffer at 25 C.
Liquid-gate transfer characteristics were measured continually during the incuba-
tions. After 10 h, the devices were rinsed with distilled water and blown dry, and
the substrate-gated transfer characteristics of the dried devices were measured.
These results were discussed in terms of a simple model in which adsorbed
streptavidin coats the single-walled nanotube (Fig. 1.8). The gradual shift in the
threshold voltage is assumed to result from the slow accumulation of a full mono-
layer of adsorbed protein. This coverage-dependent threshold shift is analogous
Fig. 1.8. (a) Size comparison between a threshold voltage. (iii) After 10 h of incubation
carbon nanotube and a streptavidin molecule. with streptavidin. Arrows indicate the threshold
(b) Current versus gate voltage for a nanotube voltages for the three curves [the arrow for (i)
device; VSD ¼ 10 mV. (ii) In phosphate buffer is behind that for (ii)]. (Adapted with
before streptavidin addition. (i) same permission from Ref. [91], 8 2003, The
conditions, to measure the uncertainty in the American Chemical Society.)
to the concentration-dependent shift observed when such devices are exposed to

aqueous ammonia [93]. The protein adsorbate equilibrates over several hours so
that only the full monolayer can be conclusively determined. Such protein mono-
layers form under various conditions at interfaces that permit protein crystalliza-
tion, including sidewalls of MWNTs [90, 94]. The results support the proposal
that conductance changes are due to charge injection or field effects caused by pro-
teins adsorbed solely along the lengths of the nanotubes.
Protein adsorption on NTFET leads to appreciable changes in the electrical con-
ductance of the devices that can be exploited for label-free detection of biomole-
cules with a high potential for miniaturization. For example, Dai and coworkers
[95] have used a sensor design consisting of an array of four NTFET sensors on
SiO2/Si chips. Each NTFET consists of multiple SWNTs connected roughly in par-
allel across two closely spaced bridging metal electrodes. Three types of devices
with different surface functional groups were prepared for the investigation of the
biosensing: (1) unmodified as-made devices, (2) devices fabricated with mPEG-SH
SAMs formed on, and only on, the metal contact electrodes and, lastly, (3) devices
with mPEG-SH SAMs on the metal contacts and a Tween 20 coating on the carbon
nanotubes. Electrical conductance of these devices upon the addition of various
protein molecules was monitored. While device type 1 showed a significant con-
ductance change with protein adsorption, device type 2 with an mPEG-SH SAM
on the metal electrodes did not give any conductance change, except in the case of
the protein avidin. It was reported that the metal–nanotube interface or contact
region is highly susceptible to modulation by adsorbed species [64]. Modulation
of metal work function can alter the Schottky barrier of the metal–nanotube inter-
face, thus leading to a significant change in the nature of contacts and, conse-
quently, a change in the conductance of the devices.
In situ detection of a small number of proteins by directly measuring the elec-
tron transport properties of a single SWNT has been reported by Nagahara and
coworkers [96]. Cytochrome c (cytc) adsorption onto individual NTFET has been
detected via the changes in the electron transport properties of the transistors.
The adsorption of cytc induces a decrease in the conductance of the NTFET
devices, corresponding to a few tens of molecules. This experiment was carried
out by measuring the conductance versus electrochemical potential of the SWNT
with respect to a reference electrode inserted in the solution, and observed a nega-
tive shift in the conductance versus potential plot upon protein adsorption. The
number of adsorbed proteins has been estimated from this shift.
1.4.4
Detection of Antibody–Antigen Interactions
Specific sensitivity can be achieved by employing recognition layers that induce

chemical reactions and modify the transfer characteristics. In this two-layer archi-
tecture carbon nanotubes function as extremely sensitive transducers while the rec-
ognition layer provides chemical selectivity and prevents non-specific binding that
is common for complex biological samples.
Following this design, nanotubes have been functionalized to be biocompatible

and to be capable of recognizing proteins. This functionalization has involved
noncovalent binding between a bifunctional molecule and a nanotube to anchor a
bioreceptor molecule with a high degree of control and specificity. Star and co-
workers have fabricated [97] NTFET devices sensitive to streptavidin using a bio-
tin-functionalized carbon nanotube bridging two microelectrodes (source and
drain, Fig. 1.9a). The SWNT in the NTFET device was coated with a mixture of
two polymers, poly(ethyleneimine) and poly(ethylene glycol). The former provided
amino groups for the coupling of biotin–N-hydroxysuccinimidyl ester (Fig. 1.9b)
and the latter prevented the nonspecific adsorption of proteins on the functional-
ized carbon nanotube. Figure 1.9(c) shows an AFM image of the device after its
exposure to streptavidin labeled with gold nanoparticles (10 nm). Lighter dots rep-
resent gold nanoparticles and indicate the presence of streptavidin bound to the
Fig. 1.9. (a) Schematic of NTFET coated nanoparticles (10 nm in diameter). (d) Source-
with a biotinylated polymer layer for specific drain current dependence on gate voltage of
streptavidin binding. (b) Biotinylation reaction the NTFET device based on SWNTs functioned
of the polymer layer (PEI/PEG) on the side-wall with biotin in both the absence and presence
of the SWNT. (c) AFM image of the polymer- of streptavidin. (Adapted with permission from
coated and biotinylated NTFET device after Ref. [97], 8 2003, The American Chemical
exposure to streptavidin labeled with gold Society.)
biotinylated carbon nanotube. The source-drain current dependence on the gate

voltage of the NTFET shows a significant change upon the streptavidin binding
to the biotin-functionalized carbon nanotube (Fig. 1.9d). The experiments reveal
the specific binding of the streptavidin, which occurs only at the biotinylated
interface.
The mechanism of the biodetection was explained in terms of the effect of
the electron doping of the carbon nanotube channel upon the binding of the
charged streptavidin molecules. Dai and coworkers [98] have also analyzed specific
antigen–antibody interactions using NTFET devices. In particular, they have
studied the affinity binding of 10E3 mAbs antibody (a prototype target of the auto-
immune response in patients with systematic lupus erythematosus and mixed con-
nective tissue disease) to human auto antigen U1A.
1.4.5
DNA Detection
DNA biosensors based on nucleic acid recognition processes are quickly being
developed towards the goal of rapid, simple and inexpensive testing of genetic
and infectious diseases. To date, there are several reports on the electrochemical
detection of DNA hybridization using multi-walled carbon nanotube (MWNT) elec-
trodes [99]. Whereas electrochemical methods rely on the electrochemical behavior
of the labels, measurements of the direct electron transfer between SWNTs and
DNA molecules paves the way for label-free DNA detection (Fig. 1.10). To illustrate
the practical utility of this new nanoelectronic detection method, an allele-specific
assay to detect the presence of SNPs using NTFETs has been recently developed
[100]. This DNA assay targeted the H63D polymorphism in the human HFE
gene, which is associated with hereditary hemochromatosis, a common and easily
treated disease of iron metabolism [101, 102].
DNA sensing mechanism using NTFETs has been recently explored by selective
attachment of DNA molecules at different device segments. Tang et al. [103] have
found that DNA hybridization on gold electrodes rather than on SWNT sidewalls is
mainly responsible for NTFET detection due to Schottky barrier modulation. In
another approach, DNA hybridization occurs on the surface at the gate of NTFET
[104]. As a result, the conductance in SWNTs was changed through the gate insu-
lators. In the work, the 5 0 end-amino modified peptide nucleic acid (PNA) oligonu-
cleotides were covalently immobilized onto the Au surfaces of the back gate of
NTFETs. PNA is a synthetic analog of DNA, in which both the phosphate and the
deoxyribose of the DNA backbone are replaced by a polypeptide. PNA mimics the
behavior of DNA and hybridizes with complementary DNA or RNA sequences,
thus enabling PNA chips to be used in biosensors. The micro-flow chip was fabri-
cated by using poly(dimethylsiloxane) (PDMS) prepolymer. The NTFET nano-
sensor array was placed onto the PDMS chip in such a way that the PNA probe-
modified Au side was positioned to face the open chamber for the introduction of
solutions and the electrical measurements. A PNA probe with the base sequence
5 0 -NH2 -ACC ACC ACT TC-3 0 , which was fully complementary to the tumor necro-
Fig. 1.10. Label-free detection of DNA responses in SNP detection assays. (d)
hybridization using NTFET devices. (a) G–Vg Fluorescence microscopy image of the NTFET
curves after incubation with allele-specific wild- network device, with the electrodes 10 mm
type capture probe and after challenging the apart, after incubation with Cy5-labeled DNA
device with wild-type synthetic HFE target molecules. (Adapted with permission from
(50 nm). (b) G–Vg curves in the experiment Ref. [100], 8 2006, The National Academy of
with mutant capture probe. (c) Graph with Sciences of the USA.)
electronic ð1 G=G0 Þ and fluorescent
sis factor-a (TNF-a) gene sequence, was used as a model system. The base se-
quence for full complementary target DNA was 5 0 -GGT TTC GAA GTG GTG
GTC TTG-3 0 while the non-complementary DNA oligonucleotide sequence was
5 0 -CCC TAA GCC CCC AA-3 0 .
The electrical properties of the NTFET devices were measured at room tempera-
ture in air. First, the blank PBS solution was introduced into the PDMS-based
micro flow chip, revealing that no substantial change in the source-drain current
of NTFET was obtained. The current increased dramatically while monitoring in
real time for about 3 h. The increase in conductance for the p-type NTFET device
was consistent with an increase in negative surface charge density associated with
binding of negatively charged oligonucleotides at the surface. DNA hybridization
can be detected by measuring the electrical characteristics of NTFETs, and SWNT
based FET can be employed for label-free, direct real time electrical detection of
biomolecule binding.
1.4.6
Enzymatic Reactions
SWNTs can be made water soluble by wrapping in amylose (linear component of

starch) [86]. These SWNT solutions are stable for weeks, provided nobody spits on
them. Indeed, the addition of saliva, which contains a-amylase, precipitates the
nanotubes as the enzyme breaks amylose down into smaller carbohydrate frag-
ments, finally resulting in the formation of glucose. The enzymatic degradation of
starch has been recently monitored electronically using NTFETs [105]. Figure
1.11(a) shows the experimental setup used for this study. NTFET devices display
transconductance and source-drain current–voltage characteristics typical of the
p-type device behavior. The device characteristics, i.e., the source-drain current ISD
as a function of the gate voltage VG , were measured to evaluate the effect of starch
deposition and the subsequent enzymatic degradation of the starch layer on the
carbon nanotubes.
Starch was deposited onto the FET by soaking the silicon wafer in a 5% aqueous
starch solution and the device characteristics were found to be shifted by approxi-
Fig. 1.11. (a) NTFET device for electronic vapor. (c) NTFET device characteristics in the
monitoring of the enzymatic degradation of form of ISD –VG curves measured from þ10 to
starch with amyloglucosidase (AMG) to 10 V gate voltage with a þ0:6 V bias voltage
glucose. (b) High-resolution transmission before (bare) and after starch deposition, as
electron microscopy (HRTEM) image of a well as after hydrolysis with AMG. (Adapted
SWNT (2.0 nm diameter) after treatment with with permission from Ref. [104], 8 2004, The
a drop of a 1% of an aqueous solution of American Chemical Society.)
starch. The starch had been stained with RuO4
mately 2 V toward more negative gate voltages. The direction of the shift equates
with electron doping of the nanotube channel by the polysaccharide. Quantitatively
similar doping effects have been observed when carbon nanotube FET devices were
exposed to NH3 gas, amines [106], poly(ethylene imine) (PEI) [107], and proteins
[91]. After the enzyme-catalyzed reaction had been performed on the starch-
functionalized devices and washed with buffer, the ISD vs. VG characteristics recov-
ered almost completely to the trace recorded before starch deposition (Fig. 1.11).
This indicates that, during the enzyme-catalyzed reaction, nearly all the starch
deposited on the surface of the nanotube device is hydrolyzed to glucose which is
washed off by the buffer prior to the electronic measurements.
1.4.7
Glucose Detection
The diagnosis and management of diabetes mellitus requires a tight monitoring

of blood glucose levels. Dekker and coworkers have demonstrated the use of indi-
vidual semiconducting SWNT as a versatile biosensor [108]. The redox enzyme
glucose oxidase (GOx ) that catalyses the oxidation of b-d-glucose (C6 H12 O6 ) to d-
glucono-1,5-lactone (C6 H10 O6 ) has been studied. The redox enzymes go through
a catalytic reaction cycle where groups in the enzyme temporarily change their
charge state and conformational changes occur in the enzyme that can be detected
using NTFET devices.
In addition to pH sensitivity, GOx -coated semiconducting SWNTs appeared to be
sensitive to glucose, the substrate of GOx . Figure 1.12 exhibits real-time measure-
Fig. 1.12. Real time electronic response of the a second device where the conductance was a
NTFET sensor to glucose, the substrate of factor of 10 lower; (b) the same measurement
glucose oxidase (GOx ). The conductance of a on a semiconducting SWNT without GOx ; no
semiconducting SWNT with immobilized GOx conductance increase is observed in this case.
is measured as a function of time in 5 mL milli- (Reprinted with permission from Ref. [107],
Q water. The conductance of the GOx -coated 8 2003, The American Chemical Society.) (B)
SWNT increases upon addition of glucose to Schematic of GOx immobilized on SWNT for
the liquid. Inset: (a) the same measurement on electronic glucose detection.
References 21
ments where the conductance of a GOx-coated semiconducting SWNT in milli-Q

water has been recorded in the liquid (left-hand arrow in each graph in Fig. 1.12).
No significant change in conductance was observed as a result of water addition.
When 0.1 m glucose in milli-Q water was added to the liquid (right-hand arrow in
each graph), however, the conductance of the tube increased by about 10%. A sim-
ilar 10% conductance change was observed for another device (Fig. 1.12a inset),
which had a factor 10 lower conductance. Glucose did not change the conductance
of the bare SWNT but did increase the device conductance after GOx was immobi-
lized. Inset (b) of Fig. 1.12 shows such measurement on a bare semiconducting
SWNT. These measurements clearly indicate that the GOx activity is responsible
for the observed increase in conductance upon glucose addition, thus rendering
such nanodevices as feasible enzymatic-activity sensors.
1.5
Conclusion and Outlook
Recent advances in the rapidly developing area of biomolecule detection using car-
bon nanotube systems have been summarized here. SWNTs appear as structurally
defined components for various electronic devices. The semiconductive properties
of SWNTs are of special interest as these SWNTs have been applied to fabricate
FETs for sensing applications. This area requires further development, particularly
related to the fabrication of FETs based on individual SWNTs. The use of carbon
nanotubes as nanocircuitry elements is particularly interesting. Biomaterials linked
to nanotubes may be used as binding elements for the specific linkage of the nano-
tube to surface in the form of addressable structures.
Important chemical means to functionalize SWNTs with other electronic materi-
als such as conductive polymers or nanoparticles is anticipated to generate materi-
als of new properties and functions. The localized nanoscale contacts of SWNTs
with bio-surfaces will be a major advance in understanding and exploring the new
applications. The use of nanodevices to monitor various biologically significant
reactions is envisioned. In future, it should be possible to connect the living cells
directly to these nanoelectronic devices to measure the electronic responses of liv-
ing systems. The combination of the unique electronic properties of SWNTs and
catalytic features of biological system could provide new opportunities for carbon
nanotubes based bioelectronics.
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Infoshare A
Comprehensive
knowledge
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Source
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Dec 2008
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Why
f are we here ?
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We are here to serve the students and
knowledge seekers throughout the world.
Contact: Infoshare.silverforum.net@gmail.com
IMPLEMENTING NANO ROBOTS
IN
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VENTILATION
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&
IN BLOOD SUGAR DETECTION (mobile phones) u m

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ABSTRACT
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Nano-biotechnology is now becoming an emerging field that is
going to bring a lot of changes in the current century of technological revolution. It is a
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one part of NANO-TECHNOLOGY. Apart from its participation in all fields, the part of
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nanos in human science and medicine is large. Nanomedicine is the process of
diagnosing, treating, preventing disease and traumatic injury, of relieving pain, and of
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preserving and improving human health, using molecular tools and molecular knowledge
h
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of the human body.
Most symptoms such as fever and itching have specific biochemical
n fcauses that can also be managed, reduced, and eliminated using the appropriate injected
I nanorobots.
Our paper mainly concentrated on implementing Nano robots in
detecting human physiology. This paper mainly concentrates on implementing nano
robots in medical field. In this paper we have two ideas.
One is using nano robots to exhale oxygen and carbon dioxide according
to the human pressure. The nano robots are called as artificial red cells.
The second part of our paper deals with introducing nanosensors and
nanorobots in detecting Human blood sugar level. These nanorobots are Embedded
with mobile phones and the status of the patient can be read from remote places. These
nano particles that reduce the size of microelectronic components will become a major
part in human medicines, which may make this entire world to hide in a single chip.
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APPLICATIONS:
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* We could then hold our breath for nearly 4 hours if sitting quietly
at the bottom of a swimming pool.
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minutes before we had to take a breath!
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* If we were sprinting at top speed, we could run for at least 15
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INTRODUCTION:
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The term “nanotechnology” generally refers to engineering and manufacturing
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at the molecular or nanometer length scale. (A nanometer is one-billionth of a meter,
about the width of 6 bonded carbon atoms.) . Nanotechnology will have given us
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specially engineered drugs which are nanoscale cancer-seeking missiles, a molecular
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technology that specifically targets just the mutant cancer cells in the human body, and
leaves everything else blissfully alone. To do this, these drug molecules will have to be
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big enough – thousands of atoms – so that we can code the information into them of
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where they should go and what they should kill. They will be examples of an exquisite,
human-made nanotechnology of the future. It is most useful to regard the emerging field
n fof nanomedicine as a set of three mutually overlapping and progressively more powerful
I technologies. First, in the relatively near term, nanomedicine can address many
important medical problems by using nanoscale-structured materials that can be
manufactured today. This includes the interaction of nanostructures materials with
biological systems. Second, over the next 5-10 years, biotechnology will make possible
even more remarkable advances in molecular medicine and biorobotics (microbiological

robots), some of which are already on the drawing boards. . Third, in the longer term,
perhaps 10-20 years from today, the earliest molecular machine systems and nanorobots
may join the medical armamentarium, finally giving physicians the most potent tools
imaginable to conquer human disease, ill-health, and suffering. Our paper concentrates
mainly on our dream system that user nano sensor in mobile phones to detect human
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blood sugar level and also nano robots in respiratory process. Most broadly,
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nanomedicine is the process of diagnosing, treating, and preventing disease and traumatic
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injury, of relieving pain, and of preserving and improving human health, using molecular
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tools and molecular knowledge of the human body. “Over the past century we have
learned about the workings of biological nanomachines to an incredible level of detail,
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and the benefits of this knowledge are beginning to be felt in medicine. In coming
r
of life.”
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decades we will learn to modify and adapt this machinery to extend the quality and length
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MAKING NANO ROBOTS:
h
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The typical medical nanodevice will probably be a micron-scale robot assembled from
nanoscale parts. These parts could range in size from 1-100 nm (1 nm = 10-9 meter), and
n fmight be fitted together to make a working machine measuring perhaps 0.5-3 microns (1
micron = 10-6 meter) in diameter. Three microns is about the maximum size for blood
I borne medical nanorobots, due to the capillary passage requirement. Carbon will likely be
the principal element comprising the bulk of a medical nanorobot, probably in the form
of diamond or diamonded/fullerene nanocomposites largely because of the tremendous
strength and chemical inertness of diamond. Many other light elements such as hydrogen,
sulfur, oxygen, nitrogen, fluorine, silicon, etc. will be used for special purposes in
nanoscale gears and other components.
APPEARANCE OF NANO ROBOTS:
It is impossible to say exactly what a generic nanorobot would look like.
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Nanorobots intended to travel through the bloodstream to their target will probably be
500-3000 nanometers (1 nanometer = 10-9 meter) in characteristic dimension. Non-blood

. n
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borne tissue-traversing nanorobots might be as large as 50-100 microns, and alimentary
u
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or bronchial-traveling nanorobots may be even larger still. Each species of medical
nanorobot will be designed to accomplish a specific task, and many shapes and sizes are
possible.
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In most cases a human patient who is undergoing a nanomedical treatment
v
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is going to look just like anyone else who is sick. The typical nanomedical treatment (e.g.
to combat a bacterial or viral infection) will consist of an injection of perhaps a few cubic
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centimeters of micron-sized nanorobots suspended in fluid (probably a water/saline
suspension). The typical therapeutic dose may include up to 1-10 trillion (1 trillion =
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1012) individual nanorobots, although in some cases treatment may only require a few
h a
million or a few billion individual devices to be injected. Each nanorobot will be on the
order of perhaps 0.5 micron up to perhaps 3 microns in diameter. (The exact size depends
o s
on the design, and on exactly what the nanorobots are intended to do.) The adult human
n fbody has a volume of perhaps 100,000 cm3 and a blood volume of ~5400 cm3, so adding
a mere ~3 cm3 dose of nanorobots is not particularly invasive. The nanorobots are going
I to be doing exactly what the doctor tells them to do, and nothing more (barring
malfunctions). So the only physical change you will see in the patient is that he or she
will very rapidly become well again. Most symptoms such as fever and itching have
specific biochemical causes which can also be managed, reduced, and eliminated using
the appropriate injected nanorobots. Major rashes or lesions such as those that occur
when you have the measles will take a bit longer to reverse, because in this case the
broken skin must also be repaired.
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ARTIFICIAL RED CELL:
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We named this Nanorobot as ventilons.The ventilons measures about 1
micron in diameter and just floats along in the bloodstream. It is a spherical nanorobot
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made of 18 billion atoms. These atoms are mostly carbon atoms arranged as diamond in a
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porous lattice structure inside the spherical shell. The ventilons is essentially a tiny
pressure tank that can be pumped full of up to 9 billion oxygen (O2) and carbon dioxide
o s
(CO2) molecules. Later on, these gases can be released from the tiny tank in a controlled
n fmanner. The gases are stored onboard at pressures up to about 1000 atmospheres.
(Ventilons can be rendered completely nonflammable by constructing the device
I internally of sapphire, a flame proof material with chemical and mechanical properties
otherwise similar to diamond.). The surface of each ventilons is 37% covered with 29,160
molecular sorting rotors that can load and unload gases into the tanks. There are also gas
concentration sensors on the outside of each device. When the nanorobot passes through
the lung capillaries, O2 partial pressure is high and CO2 partial pressure is low, so the
onboard computer tells the sorting rotors to load the tanks with oxygen and to dump the
CO2. When the device later finds itself in the oxygen-starved peripheral tissues, the
sensor readings are reversed. That is, CO2 partial pressure is relatively high and O2 partial
pressure relatively low, so the onboard computer commands the sorting rotors to release
O2 and to absorb CO2.
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Ventilons mimic the action of the natural hemoglobin-filled
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red blood cells. But a ventilons can deliver 236 times more oxygen per unit volume than
a natural red cell. This nanorobot is far more efficient than biology, mainly because its
r
diamonded construction permits a much higher operating pressure. (The operating
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pressure of the natural red blood cell is the equivalent of only about 0.51 atm, of which
only about 0.13 atm is deliverable to tissues.) So the injection of a 5 cm3 dose of 50%
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ventilons aqueous suspension into the bloodstream can exactly replace the entire O2 and
CO2 carrying capacity of the patient's entire 5,400 cm3 of blood! Ventilons will have
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pressure sensors to receive acoustic signals from the doctor, who will use an
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ultrasound-like transmitter device to give the ventilons commands to modify their
behavior while they are still inside the patient's body. For example, the doctor might
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order all the ventilons to just stop pumping, and become dormant. Later, the doctor might
a
order them all to turn on again.
h
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n f
I
ARROW indicates
high pressure of
1000 atm.
CIRCLE indicates
nano particles.
CARBON-DI- e t
OXIDE
&
OXYGEN
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APPLICATION:
v e
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By adding 1 liter of ventilons into our bloodstream, we could then hold
s
our breath for nearly 4 hours if sitting quietly at the bottom of a swimming pool. Or if we
.
r e
were sprinting at top speed, we could run for at least 15 minutes before we had to take a
breath! It is clear that very "simple" medical nanodevices can have extremely useful
a
abilities, even when applied in relatively small doses. Other more complex devices will
h
o s
have a broader range of capabilities. Some devices may have mobility the ability to swim
through the blood, or crawl through body tissue or along the walls of arteries. Others will
n fhave different shapes, colors, and surface textures, depending on the functions they must
I perform. They will have different types of robotic manipulators, different sensor arrays
and so forth. Each medical nanorobot will be designed to do a particular job extremely
well, and will have a unique shape and behavior.
OUR NANOSYSTEM TO DETECT HUMAN PHYSIOLOGY:

Currently operate with micron sized active regions and offer the ability to do
thousands of measurements individual gene activities. Such arrays will allow hundreds of
thousands of human genes to be monitored throughout a mission and will allow the
determination of the effects of microgravity on human physiology in ways that are not
imagined at present, as well as providing early warning of cancer or other disease states.
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systems will be able to apply preventative care at the earliest possible point.
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By determining which genes are activated or inhibited, rack-mounted intelligent medical
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Comprehensive cellular protein analysis and enzyme assays are equally feasible and
u
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instructive. Nanotech-based gas chromatograph/mass spectrometer similar technologies,
such as a nanotech-based MS/MS, will allow the characterization and quantification of
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multitudes of substances in a single small biological sample. In many cases, sensors will
r
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be integrated with on-chip signal processing and data acquisition along with micro
fluidics and other sample transport and preparation technologies. Systems for sensing
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biological and inorganic substances of interest in both aqueous and gaseous phases are
needed. Technologies such as micro-machined ion-mobility spectrometers, ion trap mass
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spectrometers, calorimetric spectrometers, micro lasers and optics, on-chip separators,
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optical spectrometers (e.g., UV, visible, and infrared), ultra sensitive acoustic wave
detectors, polymerase chain reaction (PCR) gene sequencing instrumentation (including
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restriction enzyme digestion and PCR amplification) and many others could potentially
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reside on the same chip or in close proximity allowing minute quantities of sample to
provide a wealth of information. The advantages of a laboratory on a chip include device
n fminiaturization for the space and volume restrictions of space travel, lower power
I consumption, nearly instantaneous response times for near-real time results, conservation
of reagents, and ease of operation by non-laboratory personnel, such as astronauts. As
with many advances in nanotechnology, the chief difficulty may be in integrating these
many different units into functioning systems and interfacing them to the macro real
world.
NANOSENSORS IN MOBILEPHONES
DISPLAY
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NANO SENSORS TO
DETECT PULSE RATE Pins to inject
robots . n
&
CORPUSCLES
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MOBILE
COMPONENTS o r
NANO ROBOTS TO
EXTRACT GLUCOSE CELLS
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IN BLOOD
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System demonstration:
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 Our mobile system has small pins attached to the mobile
phones.
r e  These pins help in taking samples of glucose.
h a  From these samples the corpuscles are readed using the
small specific nanorobots inside the mobile.
o s  Nano-chromatrons separate the glucose molecules which
n f cause diabetes.
o The molecules inhibited are read and compared with the other section and
I the approximation is made about the sugar level.
o These sugar levels are compared with compressed DB, s and precautions
are displayed.
o By having sound sensors it may possible to calculate heartbeats & pulse
rates there by calculating the BP level.

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Nano robots used in our mobile phones
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CONCLUSION:
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Nanomedicine will eliminate virtually all common diseases of
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the 20th century, virtually all medical pain and suffering, and allow the extension of
human capabilitiesmost especially our mental abilities. Consider that a nanostructured
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data storage device measuring ~8,000 micron3, a cubic volume about the size of a
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single human liver cell and smaller than a typical neuron, could store an amount of
information equivalent to the entire Library of Congress. If implanted somewhere in
o s
the human brain, together with the appropriate interface mechanisms, such a device
n fcould allow extremely rapid access to this information.
I A single nanocomputer CPU, also having the volume of just
one tiny human cell, could compute at the rate of 10 teraflops (1013 floating-point
operations per second), approximately equalling (by many estimates) the
computational output of the entire human brain. Such a nanocomputer might produce
only about 0.001 watt of waste heat, as compared to the ~25 watts of waste heat for
the biological brain in which the nanocomputer might be embedded.
But perhaps the most important long-term benefit to human society as a
whole could be the dawning of a new era of peace. We could hope that people who
are independently well-fed, well-clothed, well-housed, smart, well-educated, healthy

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and happy will have little motivation to make war. Human beings who have a
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reasonable prospect of living many "normal" lifetimes will learn patience from
experience, and will be extremely unlikely to risk those "many lifetimes" for any but
the most compelling of reasons.
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Available online at www.sciencedirect.com
Toxicology Letters 176 (2008) 1–12
Mini review
Nanosilver: A nanoproduct in medical application

X. Chen ∗ , H.J. Schluesener
Institute of Brain Research, University of Tuebingen Calwer Str. 3, D-72076 Tuebingen, Germany
Received 11 April 2007; received in revised form 8 October 2007; accepted 9 October 2007
Available online 16 October 2007
Abstract
Nanotechnology is a most promising field for generating new applications in medicine. However, only few nanoproducts are
currently in use for medical purposes. A most prominent nanoproduct is nanosilver. Nanosilver particles are generally smaller than
100 nm and contain 20–15,000 silver atoms. At nanoscale, silver exhibits remarkably unusual physical, chemical and biological
properties. Due to its strong antibacterial activity, nanosilver coatings are used on various textiles but as well as coatings on certain
implants. Further, nanosilver is used for treatment of wounds and burns or as a contraceptive and marketed as a water disinfectant
and room spray. Thus, use of nanosilver is becoming more and more widespread in medicine and related applications and due
to increasing exposure toxicological and environmental issues need to be raised. In sharp contrast to the attention paid to new
applications of nanosilver, few studies provide only scant insights into the interaction of nanosilver particle with the human body
after entering via different portals. Biodistribution, organ accumulation, degradation, possible adverse effects and toxicity are only
slowly recognized and this review is focusing on major questions associated with the increased medical use of nanosilver and related
nanomaterials.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Silver; Nanoparticles; Rout of entry; Toxicity; Mechanism
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. General characteristics of silver nanoparticles and their entry portals into human body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. Nanosilver’s interactions with tissues and routes of exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1. Respiratory system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.2. Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.3. Gastrointestinal tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4. Impacts of nanosilver on other tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5. Possible mechanisms of cytotoxicity of silver nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1. Introduction
∗ Corresponding author. Tel.: +49 7071 2984882;

Silver is a white and brilliant metallic element, posi-
fax: +49 7071 294846. tioned 47th in the periodic chart with Ag, meaning
E-mail address: mornsmile@yahoo.com (X. Chen). “argentum”, as its chemical symbol. Pure silver is ideally
0378-4274/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2007.10.004
2 X. Chen, H.J. Schluesener / Toxicology Letters 176 (2008) 1–12
ductile and malleable and has the highest electrical and the healthcare sector have been and are being heatedly
thermal conductivity of any metal as well as the lowest explored. “Silver nanoparticles are emerging as one of
contact resistance (Nordberg et al., 1988). Along with the fastest growing product categories in the nanotech-
gold, another rare and precious metal, silver has been nology industry”, according to a market research report
widely utilized for thousands of years in human history, (http://www.bourneresearch.com). Still, the remarkably
applications including jewellery, utensils, monetary cur- strong anti-microbial activity is a major direction for
rency, dental alloy, photography or explosives. Among development of nanosilver products. A wide category
silver’s many applications, those exploiting its disin- of products in this respect has already been available on
fectant property for hygienic and medicinal purposes the market. In medical arena, there are wound dress-
are time honoured and prominent, though the mecha- ings, contraceptive devices, surgical instruments and
nism is not yet fully understood. Silver vessels were bone prostheses all coated or embedded with nanosil-
used in ancient times to preserve water and wine and ver (Cheng et al., 2004; Chen et al., 2006b; Muangman
silver powder was believed by Hippocrates, father of et al., 2006; Cohen et al., 2007; Lansdown, 2006; Zhang
modern medicine, to have beneficial healing and anti- et al., 2007c). In daily life, consumers may have nanosil-
disease properties and listed as a treatment for ulcers. ver containing room spays, laundry detergents, water
But it is mainly silver compounds that actually entered purificants and wall paint (Cheng et al., 2004; Zhang
medical practice. Silver compounds were major weapon and Sun, 2007b). Silver nanoparticles are also incorpo-
against wound infection in World War I until the advent rated into textiles for manufacture of clothing, underwear
of antibiotics. In 1884 German obstetrician C.S.F. Crede and socks (Lee et al., 2007; Vigneshwaran et al., 2007).
introduced l% silver nitrate as an eye solution for pre- There are washing machines, which employs nanosil-
vention of Gonococcal ophthalmia neonatorum, which is ver (http://www.samsung.co.za/silvernano/silvernano/
perhaps the first scientifically documented medical use wash faq popup.html). More nanosilver products are
of silver (Russell and Hugo, 1994). Further, topically in the pipeline. It is estimated that of all the nano-
used silver sulfadiazine cream was standard antibacte- materials in medical and healthcare sector, nanosilver
rial treatment for serious burn wounds and is still widely application has the highest degree of commercialization.
used in burn units. Therefore, exposure to nanosilver in the body is becom-
Irreversible pigmentation of the skin and/or the ing increasingly widespread and intimate. Consequently,
eye, i.e. argyria or argyrosis, due to silver deposition, silver in the form of nanoparticles has gained an increas-
may develop after prolonged exposure to silver or sil- ing access to tissues, cells and biological molecules
ver compounds (Van de Voorde et al., 2005; Eturska within the human body. The traditional belief is that
and Obreshkova, 1979; Spencer et al., 1980). Due to except for argyria or argyrosis and some minor prob-
this problem and together with the advent of antibi- lems, silver is relatively non-toxic to mammalian cells.
otics like penicillins and cephalosporins, silver’s lustre Silver poisoning only occurs among workers who have
largely faded away as an anti-infection agent. However, chronic history of silver exposure. Drake and Hazel-
advancement of modern science has helped silver renew wood have reviewed health effects of silver and silver
its lost lustre. Metallic silver is subjected to new engi- compounds from a perspective of occupational exposure.
neering technologies with resultant extraordinarily novel Metallic silver was viewed to be a minimal health risk
morphologies and characteristics. Instead of being made (Drake and Hazelwood, 2005). However, once reach-
“big”, metallic silver is engineered into ultrafine particles ing nanoscale, certain materials do exhibit significant
whose size is measured in nanometres (nm). When these toxicity to mammalian cells even if they are biochem-
particles have at least one dimension, which is less than ically inert and biocompatible in bulk size, e.g. carbon
100 nm, they are named nanoparticles (Oberdorster et al., (Magrez et al., 2006; Shvedova et al., 2005). When it
2005b; Warheit et al., 2007). Upon reaching nanoscale, comes back to silver nanoparticles, in sharp contrast
like other nanomaterials and primarily by virtue of to the attention paid to their applications, only a few
extremely small size, silver particles exhibit remark- studies have provided very limited insights into such
ably unusual physicochemical properties and biological aspects as their entry portals into human body, biodis-
activities. Great research efforts have been committed to tribution, organ accumulation as well as their potential
this respect and yielded exciting and encouraging results interactions with tissues, cells and molecules and their
(Lee and El-Sayed, 2006; Evanoff and Chumanov, 2005; relevant toxicological implications. It is our opinion
Elechiguerra et al., 2005; Liu et al., 2006; Makarava that these are questions that need to be imperatively
et al., 2005). As a consequence, applications of engi- answered before people rush to indulge into the nanosil-
neered silver nanoparticles (nanosilver) especially in ver boom.
X. Chen, H.J. Schluesener / Toxicology Letters 176 (2008) 1–12 3
2. General characteristics of silver nanoparticles 2003; Lee and El-Sayed, 2006). Thus, systemic admin-
and their entry portals into human body istration is also a potential route of entry. Nanoparticles
that have been systemically administered or translocated
Silver nanoparticles have been synthesized through from other portals have direct contact with blood compo-
an array of methods, e.g. spark discharging, electrochem- nents, and the cardiovascular system, and are distributed
ical reduction, solution irradiating and cryochemical throughout the body.
synthesis, to name a few (Sun and Xia, 2002; Zhang
et al., 2002; Bogle et al., 2006; Sergeev et al., 1999; 3. Nanosilver’s interactions with tissues and
Pyatenko et al., 2004). Particle morphologies include routes of exposure
spheres, rods, cubes, wires and multifacets, normally
within a size range of <100 nm. As is the case with 3.1. Respiratory system
all nanomaterials, the principle characteristic of silver
nanoparticles is their ultrasmall size. Ultrasmall parti- The respiratory system serves as a major portal for
cle size leads to ultralarge surface area per mass where ambient particulate materials. Pathologies resulting from
a large proportion of atoms are in immediate contact airborne particle materials, e.g. quartz, asbestos and
with ambiance and readily available for reaction. Unique carbon, have long been topics thoroughly researched
interactions with bacteria and virus have been demon- in occupational and environmental medicine (Alfaro-
strated of silver nanoparticles of certain size ranges and Moreno et al., 2007; Kanj et al., 2006; Gillissen et al.,
shapes (Elechiguerra et al., 2005; Jose et al., 2005; Lok 2006; Lam et al., 2006; Ovrevik et al., 2005; Warheit,
et al., 2006). Small size also confers greater particle 2001a; Donaldson et al., 2001; Parks et al., 1999). Usu-
mobility both in the environment and in the body. Fur- ally the sizes of these particles are at micron level.
ther, nanoparticles produced through different processes Recently, the pathogenic effects and pathology of inhaled
and for different purposes may vary in surface charge manufactured nanoparticles received attention (Nel et
and agglomeration state. Some silver nanoparticles have al., 2006; Oberdorster et al., 2005a; Donaldson et al.,
coatings and others are hybridized with other materials 2006; Lam et al., 2006). Being different than micron and
to form nanocomposites (Kobayashi et al., 2005; Lesniak above level particles that are largely trapped and cleared
et al., 2005). In addition, nanoparticle colloids may need by upper airway mucocilliary escalator system, parti-
different stabilizers. All these combined may probably cles less than 2.5 ␮m can get down to the alveoli. The
modify the intrinsic physiochemical properties of silver deposition of inhaled ultrafine particles (aerodynamic
and may therefore give rise to modify cellular uptake, diameter <100 nm) mainly takes place in the alveolar
interaction with biological macromolecules and translo- region. Healthcare and hygiene spray products contain-
cation within the human body. Adverse reactions can ing silver nanoparticles have entered daily use. Most
occur that would not otherwise be seen with silver in commercialized silver nanoparticles are usually less than
bulk form. 100 nm, way far under the 2.5 ␮m size. Of great con-
The human body has several semi-open interfaces cern are silver nanoparticle aerosol directly applied into
for direct substance exchange with the environment, the nasal or oral cavity, as concentrated nanoparticles
i.e. the respiratory tract, gastrointestinal tract (GIT) and can be channelled into the lungs. At the alveolar region,
skin. In view of nanosilver’s diverse forms of existence, particles will first encounter and be submersed into the
they are also the principle routes of exposure to silver surfactant lining of the alveoli before having contact
nanoparticles. Female genital tract is also an entryway with any cell. The submersion process seems to take
of potential importance since nanosilver has been incor- place regardless of particle type and nature of the sur-
porated into maternal hygiene products (Cheng et al., face (Geiser et al., 2003; Gehr et al., 2000). Results of
2004; Zhang and Sun, 2007b). At these sites, nanoparti- studies using different types of particles suggests that
cles can undergo a series of processes like binding and surfactant dipalmitoylphosphatidylcholine (DPPC) and
reacting with proteins, phagocytosis, deposition, clear- surfactant protein (SP-D) are absorbed to the particle
ance and translocation. On the other hand nanoparticles surface, which may be a mediation mechanism for toxi-
can elicit a spectrum of tissue responses such as cell city of particulate matters (Liu et al., 1998; Kendall et al.,
activation, generation of reactive oxygen species (ROS), 2004; Gerber et al., 2006). The surface structures of silica
inflammation and cell death (Chen et al., 2006a; Xia particles are found to interact with the lining fluid layer
et al., 2006; Ahn et al., 2005). Additionally, colloidal and produce surface radicals and ROS which are associ-
noble metal nanoparticles have been exploited for diag- ated with silica particle’s specific toxicity (Fubini, 1997)
nostic imaging or therapeutic purposes (West and Halas, and particle–surfactant lining interaction influences sub-
sequent particle–cell interaction (Fubini, 1997; Emerson linked to ultrafine particles’ small sizes and large sur-
and Davis, 1983; Bridges et al., 2000; Peters et al., 2006). face area with associated free radical generating systems
Toxicological data on silver nanoparticles in this respect (Xia et al., 2006; Sayes et al., 2006). Both inhalation
is scarcely available. But an important fact must be noted and instillation experiments have shown that ultrafine
that silver, a member of the transition metal family, has an silver particles are taken up by alveolar macrophages and
oxidation state, which makes it useful as a catalyst. Actu- aggregated silver particles persist there at least for up to
ally high catalysis activity for oxidation has been demon- 7 days (Takenaka et al., 2001). And aggregated silver
strated of novel silver nanoparticles based catalysts (Zhai nanoparticles and some other nanomaterials have been
et al., 2006; Shen et al., 2004). In some recent studies reported to be cytotoxic to alveolar macrophage cells
exposure of nanoparticles of various chemical composi- as well as epithelial lung cells (Soto et al., 2007). Yet
tion were found or indicated to induce oxidative stress in the impact mechanisms of silver nanoparticles on alve-
lung epithelial cells (Kaewamatawong et al., 2006; Koike olar macrophages’ function have yet to be elucidated.
and Kobayashi, 2006; Limbach et al., 2007; Sharma et The respiratory impacts of inhaled nanoparticles need to
al., 2007). Such damaging effects of nanoparticles were be addressed in a more complicated context with regard
associated with catalytic activity (Limbach et al., 2007). to a morbid respiratory system with basal inflammatory
It is sound to assume, that in such a highly pro-oxidative conditions, e.g. asthma, chronic obstructive pulmonary
environment as the intra-alveolar space, the enormous diseases or respiratory infection. Since there is ample
surface area of silver nanoparticles may serve as an effi- evidence that inhalation of particles (engineered ultra-
cient facilitator of generation of radicals and ROS. fine particles included) induces/aggravates respiratory
The lungs have defence mechanisms to eliminate inflammations and epithelial damage, in which altered
inhaled solid particles so that particle deposition nor- macrophage responses could play an important role
mally will not achieve a lung burden that is sufficient to (Renwick et al., 2004; Dick et al., 2003; Lundborg et al.,
produce respiratory pathogenic effects. This is true when 2006; Schaumann et al., 2004; Ken-Ichiro et al., 2006;
the inhaled particles do not interfere with the elimination Inoue et al., 2006, 2007).
function (Lippmann et al., 1980). Particles in the alve- Significant alveolar-capillary translocation via endo-
olar region are eliminated through several major routes. cytosis and transcytosis has been suggested by animal
The first is mucocilliary escalator transport along the and human studies (Takenaka et al., 2001; Kreyling et
tracheobronchial tree, the second translocation into the al., 2002; Oberdorster et al., 2002; Kato et al., 2003;
lymphatic system and the third is particle dissolution Shimada et al., 2006) and the impact of inhaled particles
with subsequent transfer into the blood (Oberdorster, on other organs has received recognition. For instance,
1988; Takenaka et al., 2000). Alveolar macrophages are respiratory exposure to single-wall carbon nanotubes has
a key component of the clearance mechanisms. They are been suggested to provoke not only pulmonary toxic-
the chief cells that are recruited to confront deposited ity but vascular effects in laboratory mice (Zheng et
particles and their capacity for phagocytosis as well as al., 2007). Cardiovascular events such as coagulation
their response to the phagocytic stimulus determines the and cardiac rhythm disturbances were associated with
fate of the particles (Brain, 1992). Deposited nanopar- inhalation of ambient ultrafine particles (Nurkiewicz
ticles, partly through interaction with lung epithelial et al., 2006; Yeates and Mauderly, 2001; Brook et
cells, could induce rapid and increased recruitment of al., 2004). Takenaka et al. (2001) examined the pul-
macrophages (Renwick et al., 2004; Barlow et al., 2005). monary and systemic distribution of inhaled and instilled
In addition, it has been shown that inhaled ultrafine ultrafine silver particles (14.6 ± 1.0 nm) in rats. Their
particles, such as carbon and TiO2 , impair the phago- experiment showed that the silver content in the lung
cytic function of alveolar macrophages (Renwick et al., decreases rapidly with time after inhalation of a rela-
2001, 2004; Lundborg et al., 2001; Möller et al., 2002; tively low concentration of ultrafine silver which was
Winfried et al., 2005). Functionally impaired alveo- subsequently detected in blood and other organs such as
lar macrophages could favor retention of particles in heart, liver, kidney, and even brain. These results provide
the lungs, resulting in building up of a toxic dose; evidence for penetration and circulation of inhaled silver
inflammation could be stimulated (Warheit et al., 1997). nanoparticles. As circulating nanoparticles can be taken
Further, phagocytosis of particles can lead to activation up by other organs, many systemic effects might occur.
of macrophages and release of chemokines, cytokines, The authors especially mentioned that at each checkpoint
ROS, and other mediators which may result in sustained a significant portion (9–21%) of silver was observed in
inflammation (Kang et al., 2005; Hubbard et al., 2002; the liver. This indicates that liver might play a major role
Brown et al., 2004). High inflammogenicity has been in clearance of circulatory silver nanoparticles.
3.2. Skin with localized argyria has been reported (Kakurai et al.,
2003). In vitro mast cells were found to be activated
Through various techniques, textile fibers can be by silver salts (Suzuki et al., 2001; Yoshimaru et al.,
coated or impregnated with silver nanoparticles (Lee et 2006). Epidermal keratinocytes have also been shown
al., 2007; Vigneshwaran et al., 2007). These textiles are to be capable of phagocytosing nanoparticles and set-
called “smart textiles” which are claimed to have advan- ting off inflammatory responses (Monteiro-Riviere et al.,
tages over normal textiles having the ability to inhibit 2005). Mechanical irritation and interference with der-
growth of bacteria and mold. These anti-microbially mal resident microflora by nanosilver-based fibers might
active textiles have been employed for manufacturing also pose potential problems.
of underwear, lingerie, socks as well as hospital and Disposition of transdermally penetrated nanoparti-
laboratory gowns and clothes. Nanosilver is gaining in cles is not clearly understood. Yet there is evidence that
textiles and there is an increase in interest due to its intradermal nanoparticles could gain access to systemic
close contact with human skin. One of skin’s major distribution through subcutaneous lymphatics. Gopee et
roles is to provide protection to the underlying organs. al. (2007) intradermally injected hairless mice with UV
It consists of an outer epidermis and dermis. The stra- fluorescent quantum dots which, within minutes fol-
tum corneum layer of the epidermis is a strict barrier lowing injection, could be observed moving from the
allowing limited penetration of particulate materials. injection sites through the lymphatic duct system to
This aspect has potential to serve as novel route for regional lymph nodes. While intact skin is shown to
drug delivery and has attracted enormous pharmaceu- be pervious to nanoparticles, skin wounds may give
tical research interests (Shim et al., 2004; Lopez et al., rise to easier translocations. Dressings and bandages
2005; Kohli and Alpar, 2004). Transdermal penetration embedded or coated with silver nanocrystallines are used
of fine particles has been documented. For example, for prevention of sepsis of skin wounds like burns and
TiO2 particles (micron-sized), a common ingredient in ulcers. Close contact may allow nanoparticles to pene-
sunscreen products, are described to get through the trate through compromised skin barrier and gain access
human stratum corneum to epidermis and even reach to the dermal capillaries. One clinical report described
dermis (Lademann et al., 1999). It was also reported that abnormal elevation of blood silver level and argyria-
flexing movement of normal skin facilitated the pen- like symptoms following the use of nanosilver coated
etration of micrometer-size fluorescent beads into the dressings for burns (Trop et al., 2006). Absorption of
dermis (Tinkle et al., 2003). Yet, data on nanosilver nanosilver into the circulation has been indicated.
are few to none. For each kind of nanosilver-based tex- Though nanosilver-based dressings and surgical
tile, the release of nanoparticles from the textile fibers sutures have received approval for clinical application
under various conditions, e.g. sweating, repetitive attri- and good control of wound infection is achieved, their
tion and laundering needs to be investigated. Appropriate dermal toxicity is still a topic of dispute and concern.
models should be employed to assess the possibility Despite laboratory and clinical studies confirming the
of transdermal penetration of silver nanoparticles since dermal biocompatibility of nanosilver-based dressings
several recent studies have reported transdermal penetra- (Wright et al., 2002; Supp et al., 2005; Chen et al.,
tion of nanoparticles. Ryman-Rasmussen et al. (2006) 2006b; Muangman et al., 2006) several other researches
demonstrated that quantum dots with diverse physico- have demonstrated the cytotoxicity of these materials.
chemical properties could penetrate the intact stratum Paddles-Ledinek et al. exposed cultured keratinocytes
corneum barrier and localize within the epidermal and to extracts of several types of silver containing dress-
dermal layers. Fullerene-based peptides were also shown ings. The results showed that extracts of nanocrystalline
to be capable of penetrating intact skin and mechanical coated dressings are among those, which are the most
stressors could facilitate their traversion into the dermis cytotoxic. Keratinocyte proliferation was significantly
(Rouse et al., 2007). Intradermal nanoparticles could inhibited and cell morphology affected (Paddle-Ledinek
enter subcutaneous lymphatics (Gopee et al., 2007). et al., 2006). In vitro, keratinocyte viability also pre-
Damaged skin also allows for micron-sized particles to cipitously dropped after direct contact with one type
gain access to the dermis and regional lymph nodes (Kim of nanosilver dressing (Lam et al., 2004). In a third
et al., 2004). This along with the observation that parti- study, nanosilver crystallines released from a commer-
cles in the skin can be phagocytized by macrophages and cially available dressing were found to be toxic to both
Langerhans cells could possibly lead to perturbations of keratinocytes and fibroblasts, and fibroblasts appeared
the immune system (Tinkle et al., 2003). A case of acti- to be more sensitive to silver than keratinocytes (Poon
vation of mast cells by silver nanoparticles in a patient and Burd, 2004). These obvious discrepancies might
be attributable to difference in laboratory conditions 1987). Particle characteristics like size, surface charge,
and techniques employed. Thus, the establishment of and coatings can modify the translocation process.
a set of integrated and standardized evaluation pro- Particles once in the submucosal region are able to
tocols is necessary. Further, it has to be noted that enter both lymphatics and capillaries. Lymphatic absorp-
most experiments have been carried out by vitro mod- tion may give rise to immune response, for instance the
els in which cells may behave differently from in mucosal secretory immune function may probably be
vivo conditions. In clinical situations, wound exuda- affected. At the same time those particles entering capil-
tion might provide as a barrier against nanosilver’s laries become circulatory and will soon encounter their
adverse effect on wound healing. The high content of first pass, i.e. liver. Based on the extent to which col-
protein exudates may probably neutralize nanosilver’s loidal nanosilver is orally used, it is a logical assumption
tissue toxicity. Still, critical observations through rigor- that ingested silver nanoparticles might have impact on
ously designed clinical studies can provide substantial the liver since the liver serves as the first checkpoint for
and matter-of-fact data for comprehensive toxicological everything absorbed through GIT before becoming sys-
assessments. temic. As has been shown in the study by Takenaka et al.,
It is worth noting that some other types of nanoparti- 2001 the liver seems to be a major depository of circu-
cles, i.e. single-/multi-wall carbon nanotubes, quantum latory ultrafine silver particles. Further, toxic effects of
dots with surface coating, and nanoscale Titania, silver nanoparticles to liver cells have been reported from
have been shown to have toxical effects on epidermal an in vitro experiment (Hussain et al., 2005). In addition,
keratinocytes and fibroblasts or be capable of altering at least one clinical report has associated impaired liver
their gene/protein expression (Ding et al., 2005; Sarkar function to silver nanoparticles released from a wound
et al., 2007; Ryman-Rasmussen et al., 2007; Zhang et dressing (Trop et al., 2006). As stated earlier, however,
al., 2007a; Tian et al., 2006; Monteiro-Riviere et al., systemic toxicity of ingested nanosilver is scarcely seen.
2005; Witzmann and Monteiro-Riviere, 2006; Christie This situation may probably be accounted for by the
et al., 2006). presence in the GIT of a complex mixture of compounds
including ingested food, digestive enzymes, electrolytes,
3.3. Gastrointestinal tract and intestinal microbial flora, etc. Ingested nanoparti-
cles can have interactions with these compounds, which
All materials given orally are in close contact with the might change reactivity and toxicity of the particles. This
gastrointestinal tract (GIT) which has an overall surface is of particular relevance to silver, as it is well known for
area up to 200 m2 for nutrient exchange. Gastrointesti- its high affinity for the thiol groups of proteins. It has
nal ingestion is probably the most common voluntary been described that medium high protein concentration
route of exposure for nanosilver since numerous col- could lessen the in vitro cytotoxicity of nanoparticles
loidal silver nanoparticle products are publicly peddled and nanosilver’s antibacterial activity could be blocked
as so called “health maintainers” or “immuno-boosters”; by thiol containing agents. Apart from that, GIT ingested
most of them are used orally. Silver nanoparticles are particles will undergo sequential pH stress from gastric
also employed in products for water disinfection and acid and intestinal fluids. Particle surface characteris-
food stabilisation (Zhang and Sun, 2007b). Besides, par- tics may be modified by the shift in ambient pH, which
ticles discharged by respiratory mucocilliary escalation leads to altered solubility and ion state of the particles.
can end up in GIT. But despite extensive GIT expo- The renewal of the epithelium also hinders nanoparticles
sure, apart from occasional cases of systemic argyria penetration through the intestinal wall.
due to prolonged ingestion and disturbance of intestinal Considering the rare occurrence of system adverse
function, reports on local or systemic adverse effects of effects of orally ingested silver nanoparticles, more
orally ingested nanosilver are remarkably few. Neverthe- research is still needed on their kinetics in the GIT for a
less, the occurrence of systemic argyria after ingestion of better safety assessment of GIT as a major route of expo-
colloidal nanosilver in itself is an evidence that transloca- sure. It is noteworthy that recently nano- and micro-sized
tion of silver nanoparticles from the intestinal tract takes particles were found to induce granulomas in different
place. The kinetic mechanism of nanosilver translocation organs and tissues. In an histological analysis reported
is unclear, but it has been demonstrated that the intestinal by Gatti et al., inorganic particles, heterogeneous in
lymphatic tissue (Peyer’s patches) can take up intestinal nature but homogeneous in size (nano- to micro-size)
particles (Smith et al., 1995). Uptake can also take place were identified in livers and kidneys with “granulomas
trans-cellularly via normal enterocytes and through para- of unknown origin” (Gatti and Rivasi, 2002), and colon
cellular pathways (Jani et al., 1990; Aprahamian et al., tissues affected by cancer and Crohn’s disease were also
found to contain micro- and nanoparticles (Gatti et al., tations like drastic reduction of mitochondrial function,
2004). Most of the particles are debris of materials tradi- increased membrane leakage, necrosis and induction of
tionally considered to be biocompatible, silver included. apoptosis. The findings are of significant practical impli-
But clearly the presence of these particles is linked to cations because silver nanoparticles are now able to
pathologies. The authors suggest that material which access human sperms via a variety of commercialized
is accepted in bulk form can become less biocompati- products like contraceptive devices and maternal hygiene
ble when its size is reduced below a certain “critical” items. Fertility problems may occur. In addition, as a fair
threshold. extrapolation, another question emerged: what will they
do to egg cells?
4. Impacts of nanosilver on other tissues Liver appears to be a major accumulation site of cir-
culatory silver nanoparticles (Takenaka et al., 2001).
Haemo-compatibility is a top safety concern for sys- Like germ line stem cells, similar patterns of cytotox-
temically administrated materials. Nanosized inorganic, icity of silver nanoparticles (decrease of mitochondria
non-biodegradable particles were detected in blood function, LDH leakage and abnormal cell morpholo-
and were associated with thrombosis and activation of gies) were observed with in vitro BRL 3A rat liver cells,
immunological reactions (Gatti et al., 2005). Further, but to a lesser extent (Hussain et al., 2005). In another
exposure to ambient ultrafine particles has been associ- study by the same researchers, a neuronendocrine cell
ated with cardiovascular morbidity and mortality (Peters line (PC-12 cells) was exposed to silver nanoparti-
et al., 2004; Pope et al., 2004; Samet et al., 2000). Studies cles as a control against Mn nanoparticles and Mn2+
have provided evidence that exposure to ambient ultra- (Hussain et al., 2006). Experimental results showed that
fine particles elicits inflammatory responses in vascular silver nanoparticles were more toxic to mitochondria
endothelial cells, blood cells, i.e. leukocytes, platelet and than Mn nanoparticles and Mn2+ . These findings are
induces changes in other blood components (Andrea et of importance, because considerable amount of silver
al., 2007; Mark et al., 2006; Regina et al., 2007). For sil- could be detected in rat brain following inhalation of sil-
ver, interactions of metal colloids with erythrocytes were ver nanoparticles (Takenaka et al., 2001). The scope of
studied by Garner et al. (1994). It was observed that treat- the concerns raised goes beyond the possibility of sil-
ment of intact erythrocyte samples with citrate coated ver nanoparticles penetrating across blood brain barrier
colloidal silver particles induced a large depletion of since results of experiments using different nanoparticles
intracellular glutathione through a mechanism different have suggested that inhaled nanoparticles are very likely
than oxidization shown with silver nitrate. In cell lysates, to deposit in the olfactory mucosa of the nasopharyn-
solute species exchanged glutathione with citrate on the geal region and subsequently be translocated into brain
nanosilver particle surface. The specific mechanism of via the olfactory nerve (Oberdorster et al., 2004; Elder
interaction between haemoglobin and silver nanoparti- et al., 2006). A re-examination of Takenaka et al. (2001)
cles is not known. But a recent study might provide report revealed that both immediately and 1 day follow-
some clues for further investigation: Gan et al. (2004) ing inhalation Ag concentration in olfactory region was
revealed that silver nanoparticles could greatly enhance much higher than in the rest of brain. Whether inhaled
the electron-transfer reactivity of myoglobin which is silver nanoparticles could enter brain through transloca-
closely related to haemoglobin and often used as a model tion via the olfactory nerve is a topic of interest awaiting
for study on heme proteins. Interactions with other blood validation. The neurological toxicity of silver is not clin-
components such as platelets and plasma proteins have ically ascertained, however, several seizure cases have
also to be examined before silver nanoparticles were been related to exposure to silver or silver compounds
intravascularly injected, especially for diagnostic imag- (Mirsattari et al., 2004; Ohbo et al., 1996; Iwasaki et al.,
ing purposes. 1997).
Recently the identification of cytotoxicity of nanopar-
ticles towards mammalian germline stem cells has 5. Possible mechanisms of cytotoxicity of silver
aroused great concern over the biosafety of nanoma- nanoparticles
terials (Braydich-Stolle et al., 2005). In their study,
Braydich-Stolle et al. utilized a cell line with spermato- After reviewing the literature a fact is noticeable
gonial stem cell characteristics to test the in vitro toxicity that for the limited number of cell lines tested (C18-4
of several types of nanoparticles. The results showed germ cell line, BRL 3A liver cell line and PC-12 neu-
that of all the tested materials (Ag, MoO3 and Al), roendocrine cell line) exposure to silver nanoparticles
silver nanoparticles were the most toxic with manifes- significantly decreased the function of mitochondria.
This is a shared characteristic of all cellular responses, intracellular organelles. It is known that thiol-group con-
and apoptosis or apoptosis-like change of cell mor- taining proteins are abundant in the cell membrane.
phology also occurred in all three cell lines following Detrimental protein–nanosilver interactions are highly
exposure (Braydich-Stolle et al., 2005; Hussain et al., possible and lipoperoxidation may play some role. The
2005, 2006). It has been well established that dysfunc- mechanisms mentioned above seem to be shared by
tion of mitochondria is an early and key step towards Ag+ . Truly, at least Ag+ could be released from silver
apoptosis. Thus, mitochondria seem to be sensitive tar- nanoparticles. Yet, silver’s catalysis potential should not
gets of cytotoxicity of silver nanoparticles. However, the be underestimated. In view of the tremendous per mass
mechanism of silver nanoparticles’ action on mitochon- surface area of nanoparticles, it is arbitrary to decide that
drion is yet to be elucidated. In the study with BRL nanosilver would not churned out plethora of ROS. For
3A liver cell line, depletion of GSH level and increased example, the proinflammatory effects of nanoparticles
ROS was found in association with mitochondrial per- in the lung are directly related to their surface area and
turbation, suggesting that oxidative stress might mediate ROS-generating capability (Oberdorster et al., 2005b;
the cytotoxicity of silver nanoparticles. Researches on Donaldson and Tran, 2002; Donaldson et al., 2004).
Ag+ seem to be able to lend some support to this view, Taking into account their unique physicochemical prop-
concerning mitochondrial oxidative stress. As a well- erties, it is unlikely that nanoparticles do not possess
known thiol group reactive cytotoxin, Ag+ has been unique toxicity mechanisms. It remains to be determined
reported to be able to cause mitochondria perturbation whether silver nanoparticles as well as other nanomateri-
and disrupt mitochondrial function (Kone et al., 1988; als will introduce new mechanisms of injury from which
Chappell and Greviller, 1954). Recently, it has been new pathologies may result. Finally for silver, whether
found that Ag+ seems to perturb mitochondria through nano-sized or not, there is always the problem of argyia.
interactions with thiol groups of the mitochondrial inner
membrane. As these effects of Ag+ could be completely
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NeuroRx威: The Journal of the American Society for Experimental NeuroTherapeutics
Drug Transport to Brain with Targeted Nanoparticles
Jean-Christophe Olivier
Faculty of Medicine and Pharmacy, University of Poitiers, 86000 Poitiers, France
Summary: Nanoparticle drug carriers consist of solid biode- PLGA) have a good safety profiles and provide drug-sustained
gradable particles in size ranging from 10 to 1000 nm (50 –300 release. The availability of functionalized PEG-PLA permits to
nm generally). They cannot freely diffuse through the blood- prepare target-specific nanoparticles by conjugation of cell sur-
brain barrier (BBB) and require receptor-mediated transport face ligand. Using peptidomimetic antibodies to BBB transcy-
through brain capillary endothelium to deliver their content into tosis receptor, brain-targeted pegylated immunonanoparticles
the brain parenchyma. Polysorbate 80-coated polybutylcyano- can now be synthesized that should make possible the delivery
acrylate nanoparticles can deliver drugs to the brain by a still of entrapped actives into the brain parenchyma without induc-
debated mechanism. Despite interesting results these nanopar- ing BBB permeability alteration. This review presents their
ticles have limitations, discussed in this review, that may pre- general properties (structure, loading capacity, pharmacokinet-
clude, or at least limit, their potential clinical applications. ics) and currently available methods for immunonanoparticle
Long-circulating nanoparticles made of methoxypoly(ethylene preparation. Key Words: Nanoparticle, immunonanoparticle,
glycol)- polylactide or poly(lactide-co-glycolide) (mPEG-PLA/ brain targeting, blood brain barrier, transcytosis, PEG.
INTRODUCTION ble 1 summarizes the ideal nanoparticle properties re-

quired for drug brain delivery.4 One particularly
Nanoparticles are solid colloidal matrix-like particles
interesting application of nanoparticule could be the drug
made of polymers1 or lipids.2 Generally administered by
brain delivery, accompanied with the local sustained re-
the intravenous route like liposomes, they have been
lease, of the new large molecule therapeutics now avail-
developed for the targeted delivery of therapeutic or
able to treat the CNS: peptides, proteins, genes, antisense
imaging agents. Their main advantages over liposomes
drugs. Due to their poor stability in biological fluids,
are the low number of excipients used in their formula-
rapid enzymatic degradation, unfavorable pharmacoki-
tions, the simple procedures for preparation, a high phys-
netic properties, and lack of diffusion toward the CNS,
ical stability, and the possibility of sustained drug release
that may be suitable in the treatment of chronic diseases. they may be advantageously formulated in brain-targeted
Until the mid 1990s, their development as drug carriers protective nanocontainers.5 Compared with conventional
was seriously limited by the lack of long-circulating drugs, they possess a high intrinsic pharmacological ac-
properties.3 Therefore, in contrast to liposomes and de- tivity. The small dose requested for therapeutic effi-
spite the abundance of experimental works and achieve- ciency could easily fit the loading capacity of nanopar-
ments in the field of nanoparticle technology, no nano- ticles and would not require the administration of large
particle-based drug formulation has been marketed so amount of potentially toxic nanoparticle excipient. Be-
far. Due to their size ranging from 10 to 1000 nm (gen- cause of the large variety of the nanoparticles developed
erally 50 –300 nm), and like liposomes, they are unable so far, this review will focus on nanoparticles investi-
to diffuse through the blood-brain barrier (BBB) to reach gated for brain delivery. Nanoparticles made of polybu-
the brain parenchyma. Based on general parenteral for- tylcyanoacrylate (PBCA, FIG. 1) have been intensely
mulation considerations and specific BBB features, Ta- investigated since the first papers in 1995 showing that
when coated with the nonionic surfactant polysorbate 80
they permitted to deliver drugs to the brain.6,7 Despite
interesting results, PBCA nanoparticles have limitations,
Address correspondence and reprint requests to Jean-Christophe discussed in this review, that may preclude, or at least
Olivier, Ph.D., Faculty of Medicine and Pharmacy, University of Poitiers,
34rueduJardindesPlantes,86000Poitiers,France.E-mail:Jean.Christophe. limit, their potential clinical applications. Nanoparticles
Olivier@univ-poitiers.fr. made of polylactide homopolymers (PLA) or poly(lac-
108 Vol. 2, 108 –119, January 2005 © The American Society for Experimental NeuroTherapeutics, Inc.
BRAIN-TARGETED NANOPARTICLES 109
TABLE 1. Ideal Properties of Nanoparticles for Drug Phase I trials were therefore carried out with poly(iso-
Brain Delivery hexyl cyanoacrylate) nanoparticles that have the best
safety profile and an appropriate degradation rate.17 Lack-
● Nontoxic, biodegradable, and biocompatible
● Particle diameter ⬍ 100 nm ing stealth properties, poly(alkylcyanoacrylate) nanopar-
● Physical stability in blood (no aggregation) ticles administered intravenously are rapidly cleared from
● Avoidance of the MPS (no opsonization), prolonged the blood stream by the monuclear phagocyte system
blood circulation time (MPS) and mainly accumulate in liver and spleen,18 –20
● BBB-targeted and brain delivery (receptor-mediated together with the entrapped compounds.21–23 Only pegy-
transcytosis across brain capillary endothelial cells)
● Scalable and cost-effective manufacturing process lated polyalkylcyanoacrylate nanoparticles have lower
● Amenable to small molecules, peptides, proteins, or MPS uptake and prolonged blood circulation in vivo.24
nucleic acids
● Minimal nanoparticle excipient-induced drug alteration Brain delivery with PBCA nanoparticles
(chemical degradation/alteration, protein denaturation) Adsorbed onto polysorbate 80-coated PBCA nanopar-
● Possible modulation of drug release profiles ticles administered intravenously compounds with poor
brain diffusion as diverse as doxorubicin,25,26 loperam-
ide,27 tubocurarine,28 the hexapeptide dalargin6,7 were
tide-co-glycolide) heteropolymers (PLGA) may be a successfully delivered to the brain, where they induced a
promising alternative. In the mid 1990s, long-circulating pharmacological effect (for review, see Kreuter29). The
pegylated PLA or PLGA nanoparticles have been made chemical nature of the overcoating surfactant is of im-
available that opened great opportunities for drug target- portance, because only polysorbates, not poloxamers
ing.3 Pegylated nanoparticles are made of methoxypoly- (184, 188, 388, or 407), poloxamine 908, Cremophors
(ethylene glycol)-PLA/PLGA (mPEG-PLA/PLGA, FIG. (EZ or RH40) or polyoxyethylene(23)-laurylether, led to
1), i.e., esters of PLA or PLGA with PEG of various a CNS pharmacological effect of dalargin.30 As the
molecular weights. More recently, the synthesis of func- mechanism of action, it was hypothesized that polysor-
tionalized pegylated PLA/PLGA nanoparticles opened bate-coated nanoparticles were transported across the
new perspectives for targeted drug delivery in general, BBB via endocytosis by the brain capillary endothelial
and for drug brain targeting in particular. This review cells.29 This endocytosis would be triggered by a serum
will present their general properties and will propose protein, apolipoprotein E, reported to adsorb on polysor-
preparation methods of brain-targeted pegylated nano- bate 20, 40, 60, or 80-coated nanoparticles after a 5-min
particles.
PBCA NANOPARTICLES
General considerations
Nanoparticles made of poly(alkylcyanoacrylate) poly-
mers (FIG. 1) were first described in 19778 and were
recently the subject of a comprehensive review of their
properties, preparation methods and potential therapeutic
applications.9 They are generally prepared from (iso)bu-
tylcyanoacrylate or (iso)hexylcyanoacrylate monomers
by emulsion anionic polymerization in an acidic aqueous
solution of a colloidal stabilizer such as dextran 70,
polysorbates, and poloxamers. Inclusion of drug can be
made during the polymerization process or by adsorption
onto preformed nanoparticles. Using the first method,
chemical reactions may occur between drugs and mono-
mers.10 Alternatively, the interaction between adsorbed
drugs and the nanoparticle may lack stability, especially
when a surfactant is subsequently added to the prepara-
tion11 or, once the nanoparticles are dispersed in blood,
by a combined effect of serum protein competition and
polymer degradation.12 The length of the alkyl pendant
governs degradation rates13,14 and toxicity15,16 of poly- FIG. 1. Structure of poly(alkylcyanoacrylate), methoxypoly(eth-
ylene glycol)-polylactide [or poly(lactic acid)] (mPEG-PLA) and
(alkylcyanoacrylate) nanoparticles, which decrease in the methoxypoly(ethylene glycol)-poly(lactide-co-glycolide) [or poly-
order methyl⬎ethyl⬎butyl/isobutyl⬎hexyl/isohexyl. (lactic-co-glycolic acid)] (mPEG-PLGA).
NeuroRx威, Vol. 2, No. 1, 2005

110 JEAN-CHRISTOPHE OLIVIER
incubation in citrate-stabilized plasma at 37°C, but not effective at delivering dalargin to the brain.11 In a con-
on nanoparticles coated with poloxamers 338, 407, Cre- text of general toxicity induced by the high dose of
mophor EL, or RH 40.29 Despite numerous arguments PBCA nanoparticles and associated to the synergistic
listed by Kreuter,29 this hypothesis raises questions, BBB permeabilization effect of polysorbate 80, major
based on the following observations. 1) Apolipoprotein E damage to the BBB cannot be excluded. Beyond the
adsorption is not specific of polysorbate 80-coated sur- ongoing controversy about their mechanism of action,
faces because it was shown to adsorb onto pegylated polysorbate 80-coated PBCA nanoparticles should be
PLA nanoparticles.31,32 2) Polysorbate 80-coated poly- evaluated in term of benefit/risk ratio and of innovative
(methylmethacrylate) nanoparticles are not distributed therapeutics. In addition to the toxicity issue, the short
into the brain after IV administration.33 3) Replacing duration of the pharmacological effect observed after
polysorbate 80-coated PBCA nanoparticles with polysor- administration of drugs formulated with this carrier (210
bate 80-coated polystyrene nanoparticles completely min at the best39) would probably necessitate daily in-
abolished dalargin brain delivery.11 4) The pharmacoki- travenous administrations, a perspective not suitable for
netic profile of polysorbate 80-coated nanoparticles is the treatment of chronic brain diseases.
not favorable to brain distribution, due to a massive
uptake by the MPS resulting in liver and spleen accu-
PEGYLATED PLA OR PLGA
mulation.33 5) Polysorbate 80 and serum protein compe-
NANOPARTICLES
tition, as well as the rapid nanoparticle degradation in
serum/plasma, were shown to induce desorption of com- General considerations
pounds adsorbed onto PBCA nanoparticles within a few Among the few biodegradable polymers, polymers de-
minutes.11,12 As an evidence of this desorption, blood rived from glycolic acid and from D,L-lactic acid enanti-
pharmacokinetic profiles of drugs adsorbed onto poly- omers are presently the most attractive compounds be-
sorbate 80-coated PBCA nanoparticles administered in- cause of their biocompatibility and their resorbability
travenously were actually similar to free solutions,25,34,35 through natural pathways.40,41 They are widely used for
and not at all typical of drugs associated to nonstealth the preparation of biodegradable medical devices and of
colloidal drug carriers.21,22,23,36 Therefore, as an alterna- drug-sustained release microspheres or implants mar-
tive to the brain uptake of nanoparticles, we hypothe- keted in Europe, Japan, and the U.S.42 Degradation of
sized a nanoparticle-induced nonspecific BBB permeabi- PLA or PLGA occurs by autocatalytic cleavage of the
lization.11 It has been known for a long time that ester bonds through spontaneous hydrolysis into oli-
polysorbate 80 causes BBB disturbance at intravenous gomers and D,L-lactic and glycolic acid monomers.43
systemic doses as low as 3 mg/kg37 (25–100 mg/kg Lactate converted into pyruvate and glycolate enter the
polysorbate 80 doses were used in brain targeting exper- Krebs’ cycle to be degraded into CO2 and H2O. After
iments7,25,27). Recently, Calvo et al.36 showed that a intravenous administration of 14C-PLA18000 radiolabeled
polysorbate 80 intravenous dose of 20 mg/kg in rats nanoparticles to rats, 90% of the recovered 14C was
dramatically increased BBB permeability to sucrose. In eliminated within 25 days, among which 80% was as
rats treated with polysorbate 80-coated PBCA nanopar- CO2.44 Degradation rate depends on four basic parame-
ticles (polysorbate 80: 25 mg/kg, nanoparticles: 50 mg/ ters: hydrolysis rate constant (depending on the molec-
kg) inulin spaces increased by 10% (not significant) after ular weight, the lactic/glycolic ratio, and the morphol-
10 min and by 99% (significant) after 45 min compared ogy), amount of water absorbed, diffusion coefficient of
with control.38 Because apparently no brain uptake was the polymer fragments through the polymer matrix, and
observed with control drug-polysorbate 80 solutions, the solubility of the degradation products in the surrounding
toxicity of PBCA nanoparticles was proposed as a syn- aqueous medium.40,41 All of these parameters are influ-
ergistic factor for BBB permeabilization.11 The nanopar- enced by temperature, additives (including drug mole-
ticle doses permitting brain delivery (100 –166 mg/kg cules), pH, ionic strength, buffering capacity, size and
generally) were close to the lethal dose 50% of PBCA processing history, steric hindrance etc. Despite a higher
nanoparticles (230 mg/kg in mice16). Polysorbate 80- water uptake the PLA or PLGA blocks of mPEG-PLA/
coated or uncoated PBCA nanoparticles (unloaded with PLGA block copolymers have similar degradation be-
drug) induced a dramatic decrease in mice locomotor haviors.45,46 mPEG blocks are released (10 –25% within
activity (associated with obvious signs of distress) at a 3 days and 30 –50% within 20 days at pH 7.4, 37°C) after
nanoparticle dose of 135 mg/kg and the permeabilization cleavage of the ester bonds,47– 49 and, in the range of
of an in vitro BBB model at a concentration of 10 ␮g/ml molecular weights of 1000 –20,000, are mainly excreted
(to be compared to the 1.5 mg/ml theoretical concentra- via the kidney.50 Up to an extensive PLA/PLGA polymer
tion reached in mice blood after dosing animals with a degradation, nanoparticle morphology and size are gen-
135 mg/kg nanoparticle dose).11 In contrast, the nontoxic erally preserved.48,51 Generally considered as biocom-
polysorbate 80-coated polystyrene nanoparticles were in- patible,41 PLA or PLGA microspheres have also a good

CNS biocompatibility.52,53 No mortality was reported solvent evaporation or diffusion, the PEG moieties mi-
with albumin-coated nanoparticles in mice with up to a grate toward the aqueous phase, whereas the hydrophobic
2000 mg/kg dose.44 However, PLA60000 nanoparticles PLA/PLGA moieties aggregate as the nanoparticle core.
stabilized with sodium cholate were much more toxic mPEG-PLA copolymers with relatively high PEG to PLA
with two of five deaths at a 220 mg/kg dose and five of weight ratio (e.g., mPEG5000-PLA2000-3000) may self-as-
five at a 440 mg/kg dose associated with marked clinical semble as polymeric micelles.59,64 – 66 Depending on the
signs (dyspnea, reduced locomotor activity), alteration of copolymer solubility in water, polymeric micelles may be
hematological and biochemical parameters and lung prepared either by self-dispersion in water (mPEG5000-
hemorrhage.54 This toxicity was attributed to a dissem- PLA1500-200065,67) or by the precipitation/solvent evapora-
inated intravascular coagulation and associated events tion technique using a classical solvent extraction procedure
related to the physical surface properties of the nanopar- (mPEG5000-PLA3000-11090067) or by dialysis.55 Self-dispers-
ticles rather than to the chemical toxicity of cholate or ing mPEG-PLA copolymers are also used as emulsion sta-
PLA. In contrast, mPEG2000-PLA30000 nanoparticles bilizers in the preparation of PLA nanoparticles.57 The
were shown to have a good safety profile, with no ap- size of mPEG-PLA nanoparticles prepared with constant
parent signs of toxicity at the highest studied dose of 440 PEG5000 was found to increase with the PLA block mo-
mg/kg in mice.54 lecular weight.59 With mPEG5000-PLA2000-30000 nano-
particle diameters (from 26 to 64 nm in diameter) were
Nanoparticle preparation shown to be independent of the copolymer concentration
Nanoparticles made of mPEG-PLA/PLGA copolymers in the organic phase, whereas with higher PLA block
are mainly prepared using the emulsion/solvent evapo- molecular mass (45,000 Da) nanoparticle size was de-
ration technique or the precipitation solvent diffusion pendent on the copolymer concentration in the organic
technique.1 In the first method, copolymers are dissolved phase.59 After preparation, nanoparticles can be freeze-
in an organic solvent immiscible to water (such as di- dried in the presence of appropriate cryoprotector for long-
chloromethane, chloroform, ethylacetate) and emulsified term preservation.51,62,63
in an aqueous phase generally containing an emulsifying
agent (mainly polyvinylalcohol and sodium cholate). Pegylated nanoparticle structure
Then the solvent is evaporated off under normal or low Nanoparticles prepared from mPEG-PLA/PLGA co-
pressure to form nanoparticles. Hydrophobic compounds polymers are constituted of a PLA/PLGA hydrophobic
(drug or else) to be incorporated are dissolved in the core surrounded by a hydrophilic PEG corona or outer
organic phase. Hydrosoluble compounds are first dis- shell. In mPEG5000-PLA2000-75000 nanoparticles, negligi-
solved in water and emulsified in the polymer-dissolving ble penetration of the PEG into the solid-like PLA core
organic phase. The primary water-in-oil emulsion thus was reported, whereas as much of 25% PEG is entrapped
formed is then processed like the organic polymer phase within the nanoparticle core in the case of mPEG5000-
described above. This variant of the first method is called PLA110000.59 It is likely that the [(water-in-oil) in water]
[(water-in-oil) in water] (or multiple emulsion) solvent solvent evaporation technique increases PEG entrapment,
evaporation technique. In the second method, polymers compared with the precipitation/solvent evaporation tech-
are dissolved in an organic solvent miscible to water nique.46 The water content of mPEG5000PLA45000 nanopar-
(such as acetone or ethanol) and dispersed in an aqueous ticles (200 nm diameter) is around 30% compared with
phase generally containing a colloid stabilizer. The al- around 10% for PLA nanoparticles.46 At room tempera-
most instantaneous diffusion of the organic solvent into ture and 37°C, a solid-like central core and more mobile
the aqueous phase results in the precipitation of the co- interfacial region coexist within the PLA core of nano-
polymers as nanoparticles. Finally, the solvent is evap- particles made of mPEG5000-PLA [glass transition tem-
orated off as above or extracted by dialysis against wa- perature of around 333K], whereas the PEG corona layer
ter.55 In principle, only compounds soluble in the organic situated on the nanoparticle surface is in the liquid
solvent can be incorporated using the second method. phase.67 The PLA chain packing density increases with
Both basic methods require formulation optimization de- the PLA molecular weights due to an increase in the
pending on the type of polymers/copolymers used, their number of attractive hydrophobic interactions between
molecular weights, the compound to be incorporated, the lactic acid units.59 In nanoparticles made of mPEG5000-
nanoparticle size to be achieved, etc.56 –59 Other less PLA2000 –3000, PLA chains possess some mobility.59 Be-
frequently used methods include the emulsion solvent cause of the relatively high critical micellar concentra-
diffusion in an oil phase60,61 and the salting out pro- tion, these nanoparticles may dissociate upon dilution in
cess.62,63 Because of their different water solubility, the blood.65 PEG conformation at the PLA-PEG nanopar-
hydrophobic PLA/PLGA and hydrophilic PEG blocks of ticle surface is of utmost importance for the opsonin-
the mPEG-PLA/PLGA copolymer tend to phase-separate repelling function of the PEG layer and has been exten-
in the presence of water. Therefore, during the organic sively studied.57–59,66,68 The PEG layer thickness

depends on the PEG molecular weight and surface den- PEG molecular weights of 5000 and above. Covalent
sity.57 Depending on their surface density, PEG blocks linkage of the PEG coating and sufficient PLA block
have brush-like (elongated coil, high density) or mush- molecular weight is essential to ensure a sufficient sta-
room-like (random coil, low density) conformations.66,68 bility and to avoid loss of the coating benefit by
PEG surfaces in brush-like and intermediate configura- desorption and/or displacement in vivo.57,72,73,80 In
tions reduced phagocytosis and complement activation, mice, blood circulation times of 111In-labeled mPEG-
whereas PEG surfaces in mushroom-like configuration PLGA5000-20000 nanoparticles (140 ⫾ 10 nm diameter)
were potent complement activators and favored phago- increased compared to PLGA ones with an advantage to
cytosis.32,47,69,70 Based on the Alexander-de Gennes the higher PEG molecular weight.81 Within 5 min, how-
model, the distance between PEG chains should be ever, ⬃50% (PEG20000) to 75% (PEG5000) of injected
around 1 nm to repel small globular proteins (approxi- nanoparticles (estimated from the blood clearance
mately 2 nm radius) and 1.5 nm to repel large ones (6-8 curves) had been cleared from the blood compartment
nm).32 Due to the large choice in the PLA or PEG (compared with 95% with control PLGA nanoparticles).
molecular weights available, the conformation of PEG In another study performed in rats, the blood half-lives of
blocks at the PEG-PLA nanoparticle surface is a com- [14C]PLA-labeled mPEG-PLA30,000 nanoparticles with
plex issue to be addressed. At nanoparticle surface, the PEG molecular weight of 200073 (205 nm diameter) or
area available per PEG chain at the outer boundary of the 500078 (140 ⫾ 60 nm) were markedly higher (6 h) and
shell is dependent on PEG to PLA molecular weight ratio independent of the PEG molecular weights. Less prolonged
that governs the PLA packing density and the surface blood circulation times were observed with PLGA nano-
curvature (linked to the nanoparticle size) of the assem- particles coated with PLA3000-PEG4000 (147 ⫾ 3.6nm) or
bly.57,58,71 As an example, an increase in the diameter of PLA3000-PEG5000 (161 ⫾ 3.7nm) (T[1/2] ⫽ 15 min and
nanoparticles made of mPEG5000-PLA45000 results in a T[1/2] ⫽ 1 h, respectively, estimations from the blood
lower surface curvature, thus in an apparent increase clearance curves).72 With nanoparticles made of
in PEG surface coverage59 and in an improved colloidal mPEG5000-PLA7000-125I (150 ⫾ 2nm diameter) or of
stability.58 mPEG14000-PLA6000-125I (35.8 ⫾ 0.5nm) blood half-
lives determined in rats were 29.9 ⫾ 12.4 and 42.3 ⫾
Pharmacokinetics 16.2 min respectively (no statistical difference).82 In rats,
Like any colloidal drug carrier not especially designed a blood half-life of 270.9 min was determined for 125I-
to escape from MPS uptake, PLA or PLGA nanoparticles BSA loaded in mPEG5000-PLGA45000 nanoparticles
are rapidly removed from the blood stream after vascular (around 200 nm diameter), compared with 13.6 min
administration and preferentially accumulate in liver and when formulated in PLGA nanoparticles.75 The large
spleen.44,72 Blood half-lives are generally around 2-3 variability in blood half-lives determined in those works,
min.44,73–75 After intravenous administration, the first even with the same PEG block molecular weight of
step of the process that leads to the nanoparticle uptake 5000, may be ascribed to the above discussed density-
by the MPS is the opsonization phenomenon. Opsonins, related PEG conformation in the coating layer. The poly-
including complement proteins, apolipoproteins, fi- dispersity of the PLA block molecular weights should be
bronectin, and Igs,31 interact with specific membrane also considered, which renders the pegylated nanopar-
receptors of monocytes and tissue macrophages, result- ticle system more complex than liposomes (the molecu-
ing in recognition and phagocytosis. It is generally ad- lar weight of the hydrophobic moieties of the pegylated
mitted that hydrophobic surfaces promote protein ad- phospholipids are constant and the fluidity of the lipidic
sorption and that negative surfaces are activators of the membrane permits a statistically homogeneous distribu-
complement system.76 Following the rule hydrophobic tion of pegylated phospholipids) and could lead to a
and negative PLA or PLGA nanoparticle surfaces57,58 surface heterogeneity pointed out by Gbadamosi et al.69
activate the complement system32 and coagulation fac- Such a surface heterogeneity may explain the rapid clear-
tors77 in vitro. In contrast, hydrophilic coating with PEG ance of a significant fraction of intravenously injected
sterically stabilizes PLA or PLGA nanoparticles and re- long-circulating nanoparticles by the MPS.72,81,83 Be-
duces opsonization and phagocytosis in vitro32 or ex cause of this polydispersity, space available for PEG
vivo,78 and uptake by neutrophilic granulocytes in vivo.79 block expansion is likely to be variable on nanoparticle
Compared with nonpegylated PLA nanoparticles, pegy- surface. Mushroom-like and brush-like conformations
lated nanoparticle surfaces have lower negative ␨ poten- may coexist within a single nanoparticle or among a
tial values, due to the surface shielding by the PEG population of polydispersed nanoparticles (the size of
corona.3,57,58 mPEG2000-PLA nanoparticles did not acti- micellar-like mPEG-PLA nanoparticles and therefore the
vate the complement47 and the coagulation77 systems in PEG conformation in the corona are dependent on the
vitro and did not alter coagulation parameters in vivo.54 PLA molecular weight, see above), thus explaining vari-
Gref et al.32 showed a maximum antiopsonic effect with ability observed in blood half-lives. Therefore, molecular

weights of PEG and PLA block, as well as polydispersity weight,89,91,103 water uptake by nanoparticles48 and drug
of copolymers, should be carefully selected in designing solubility in the biological medium. In most studies, in
long-circulating pegylated nanoparticles. vitro release profiles are characterized by an initial fast
release (burst) of drug close to or at the surface followed
Drug loading by a sustained release.91,92,103 Removing the low molec-
Conventional drugs and general principles. Various ular weight fraction from the polymer was shown to
kinds of conventional drugs were formulated as PLA, PLGA, reduce the initial burst of drug release.103 Depending on
or mPEG-PLA nanoparticles. Examples are savoxepine,84 formulations in vitro, drug releases last from a few
doxorubicin,85 irinotecan,86 paclitaxel,87,88antiestrogen hours84,91,92 or a few days87 to several weeks.61,84,88,90
RU58668, tyrphostin AG-1295, lidocaine,91 propranolol
89 90
Administered locally, betamethasone sodium phosphate-
hydrochloride,92 heparin,93 and enalaprilat.94 Basically, drug loaded PLGA nanoparticles were efficient at controlling
entrapment efficiency depends on the solid-state drug inflammation over at least 3 weeks in a rabbit model of
solubility in PLA/PLGA polymer (solid dissolution or arthritis, compared with one day for the solution.61
dispersion), which is related to the polymer composition Peptides, polypeptides, and protein drugs. Certainly
(lactic/glycolic ratio), the molecular weight, the drug- one of the most promising, and challenging, applications
polymer interaction and the presence of end-functional of nanoparticles in brain delivery are the sustained re-
groups (ester or carboxyl).95–99 The PEG moiety has no lease of therapeutic peptides and proteins. Due to their
or little effect on drug loading.91 Because PLA and hydrosolubility the preparation method is generally
PLGA are hydrophobic polymers, lipophilic drugs are based on the [(water-in-oil)-in water] solvent evapora-
easier to formulate (in dissolved state) in PLA/mPEG- tion technique.75,104,105 Entrapment efficiencies gener-
PLA nanoparticles, than hydrosoluble ones (segregation ally range from 10% to 90%,75,104,106 and nanoparticle
in separate domains). Despite the [(water-in-oil) in wa- contents from 1% or less99,107,108 to more than 15%.106
ter] solvent evaporation technique, the entrapment of Apart from formulation issues inherent to peptide chem-
hydrophilic drugs may be a challenge due to the drug ical instability or chemical reaction between peptides and
diffusion from the inner to the outer aqueous phases polymer degradation products,109 the formulation of pep-
promoted by the large surface area developed. Nanopar- tide-loaded nanoparticles is similar to conventional
ticle formulators have nevertheless several means to op- drugs.99,107,108 Proteins, however, are highly organized,
timize drug encapsulation: the selection of the prepara- complex structures that have to be preserved to maintain
tion procedure,61,84,87,100 the use of additives,96,97 the pH biological activity (receptor binding, antigenicity, enzy-
optimization of the aqueous phases,92 the use of union- matic activity, etc.). The general issues of the protein
ized base or acid form of drugs,84,86,96,97 the PLA/PLGA stability and assessment and stabilization methods in
block polymer molecular weight. The incorporation of PLA or PLGA delivery systems have been extensively
carboxylic groups to mPEG-PLA55 or the drug chemical reviewed.110 –112 Structural and chemical integrity are
conjugation via cleavable linkage101 may be interesting lost during nanoparticle preparation and storage by pro-
alternatives to improve drug loading efficiency and ad- tein exposure to damaging conditions, such as interfaces
just release rates. Early drug release during storage may (aqueous/organic in emulsions, hydrophobic surfaces of
be solved by freeze-drying. Drug entrapment efficiency polymers), elevated temperatures (e.g., by sonication),
can reach more than 80%84,92,91 and drug content up to shear force (e.g., sonication, vigorous stirring, extrusion,
50%.91 In most cases, however, drug contents are 5-10% high pressure homogenization process), surfactants,
(wt/wt) of nanoparticle weights86,88,94,96,97,102 or even (freeze-) drying etc.110 Moreover, upon administration,
less.87 Therefore, when formulating drug nanoparticles, proteins are exposed to physiological temperature and
it should always be kept in mind that generally as high as acidic by-products of PLA/PLGA polymer degradation
90% of the material to be administered will likely be within nanoparticles for long time periods that can also
nanoparticle excipients with their potential toxicity. affect their stability.110,112 The study of the physical and
Drugs with high intrinsic pharmacological activities chemical structure of the entrapped protein accompanied
should be preferred to avoid the administration of mas- with an appropriate evaluation of the biological activity
sive dose of nanoparticle material. Drug release from of the released material is the only way to confirm the
biodegradable polymeric nanoparticles depends on the maintenance of the protein integrity and activity.112 Each
Fickian diffusion through the polymer matrix and on the nanoparticle formulation of protein is unique and re-
degradation rate of the polymer. The prediction of the quires specific adaptation and evaluation. Improved pro-
release profile is complex because it results from a com- tein stability was achieved by altering preparation
bined effect of various parameters: solid-state drug poly- processes,113 by changing polymer/copolymer,105,114
mer solubility98 and drug-polymer interactions,55,91,92,100 by changing or mixing solvents,49,113 by adding protec-
polymer degradation rate,61 block copolymer molecular tive additives110,111 such as hydrophilic polymers
weight and polydispersity,103 PEG content and molecular (PEG115,116), surfactants (poloxamer 188104,117), pro-

teins (serum albumin,118 gelatin105), cyclodextrins118,119

to the inner aqueous phase. Such formulation optimiza-
tions permitted sustained release of active protein over
several weeks in vitro.105,114
Plasmid DNA, oligonucleotides. Plasmid DNA-
loaded nanoparticles are generally prepared using the
[(water-in-oil)-in water] solvent evaporation tech-
nique.120,121 The plasmid DNA loading, release rates,
and transfection efficiency were shown to be dependent
of the nature and the molecular weight of the polymer,122
the nanoparticle size121 and the colloid stabilizer.122 An
in vitro plasmid gene sustained release over several
weeks was achieved with PLGA123 or mPEG-PLA nano-
particles.120 PLGA nanoparticles were shown to be en-
docytosed by cells in vitro124. After endocytosis, PLGA
nanoparticles escape from the endolysosomal compart-
ment to the cytoplasm and gradually release their con-
tent, resulting in sustained gene expression.125,126 In a rat
osteotomy model, PLGA nanoparticles administered in
the bone-gap tissue permitted a plasmid gene expression
for at least 5 weeks demonstrating their sustained release
properties.123 Like PLGA nanoparticles with an impor-
tant poly(vinyl alcohol) coating,127 pegylated nanopar-
ticles may interact poorly with cells, which may result in
low, or even no gene expression. Such a problem may be
overcome with appropriate targeting ligands able to trig-
ger endocytosis.128
Oligonucleotides were successfully encapsulated
within PLA129 –131 or mPEG-PLA132 nanoparticles. In
vitro-sustained release and intracellular delivery were
demonstrated.131,133
Perspectives in brain targeting. The most achieved
work in the field of brain targeting with colloidal drug
FIG. 2. Structure of functionalized PEG-PLA. Biotin-PEG-PLA
carriers has been carried out with pegylated immunoli- (a); succinimidyl tartrate PEG-PLA (b), succinimidyl succinate
posomes that access the brain from blood via receptor- PEG-PLA (c), aldehyde-PEG-PLA (d), maleinimido propionate
mediated transcytosis and deliver their content (small PEG-PLA (e), and maleimide-PEG-PLA (f).
drug molecules, plasmid) into the brain parenchyma,
without damaging the BBB.134 –137 This requires the terminal groups, mPEG-PLA copolymers do not permit
presence of receptor-specific targeting ligands at the tip ligands to be tethered to the PEG chain. The covalent
of 1-2% of the PEG2000 strands. Targeting ligands are conjugation of protein ligands to pegylated nanoparticles
peptidomimetic monoclonal antibodies, i.e., able to trig- requires chemically reactive functions at the free end of
ger the activation of receptors (transferrin or insulin re- 1-2% of the PEG strands of the PEG corona. Several
ceptors) that are highly expressed on the brain capillary functionalized copolymers have been recently synthe-
endothelium.134,136,137 These antibodies directed against sized: the biotinylated,139 the amine-reactive64,140 and
external receptor epitopes do not interfere with the nat- the thiol-reactive copolymers141,142 that permit protein
ural ligand binding sites, thus avoiding competition. Col- chemical conjugation in nondenaturing conditions143
loidal carriers should have diameter less than 100 nm to (FIG. 2). They are generally synthesized by ring opening
fit the loading capacity of these transport systems. Be- polymerization starting from heterobifunctional PEG and
cause immunoliposomes are not able of sustained release lactide and/or glycolide.64,140 –142 Polymer block conju-
of transported compounds, as shown by the relatively gation is an alternative method.144 Biotinylated PEG-
short-lasting plasmid expression in brain,136 they require PLA nanoparticles may link biotinylated antibodies
frequent administrations to sustain a pharmacological through an avidin spacer145 (FIG. 3, panel 1a), or avidin-
effect.138 Pegylated PLA immunonanoparticles with sus- antibody conjugates146 (FIG. 3, panel 1b). Amine-reac-
tained release properties may offer an interesting alter- tive PEG-PLA (succinimide and aldehyde derivatives)
native. Because of the presence of unreactive methoxy can directly react with ⑀-amino groups of the lysine

Due to the lack of free thiol, antibody conjugation to

thiol-reactive functions (maleimide) requires the intro-
duction of thiol residues by reacting 2-iminothiolane
(Traut’s reagent) with ⑀-amino groups of the lysine res-
idues. The thiolation was shown not to interfere with
target recognition.150 In mild conditions that preserve
antibody reactivity, a stable thioether bond can be estab-
lished between maleimide and thiol (FIG. 3, panel 3).
Such a method was successfully applied to the prepara-
tion of brain-targeted immunoliposomes.134 In a recent
work, we used the same procedure to design brain-tar-
geted pegylated immunonanoparticles.142 Maleimide-
functionalized pegylated nanoparticles were prepared
with maleimide-PEG3500-PLA40000 and mPEG2600-
PLA40000 (according to a 1:40 molar ratio) using the
[(water-in-oil) in water] solvent evaporation technique.
Thiolated mouse OX26 anti-rat transferrin receptor
monoclonal antibodies were then successfully conju-
gated to the functionalized nanoparticles. The mean
number of antibodies per nanoparticles was determined
to be 67 and visualized at the nanoparticle surface by
transmission electron microscopy after labeling with an
anti-mouse IgG antibody gold conjugate (FIG. 4).
CONCLUSION
Even though being effective at delivering drug to the
brain by a still-debated mechanism, polysorbate 80-
coated PBCA nanoparticles may have limited clinical
applications due to a potential toxicity, BBB permeabi-
lization, and short lasting delivery. Technology now ex-
FIG. 3. Currently available conjugation techniques to prepare

pegylated PLA immunonanoparticles. For comments, see text.
residues of antibodies in mild conditions (FIG. 3, panel

2). An ␣-acetal-PEG-PLA block copolymer is required
to prepare aldehyde-functionalized PEG-PLA nanopar-
ticles64,147,148 (FIG. 3, panel 2b). After nanoparticle FIG. 4. Transmission electron micrograph of pegylated immu-
nonanoparticles negatively stained with phosphotungstic acid
preparation, the acetal groups are converted by mild acid solution. Antibodies conjugated to the nanoparticle are revealed
treatment (pH 2) into aldehyde functions that are reactive by binding with a 10-nm gold-labeled secondary antibody. The
with amine of peptidyl ligand at pH 7.147,149 Antibodies magnification bar is 15 nm. Reprinted with permission from
Olivier et al. Synthesis of pegylated immunonanoparticles.
may be chemically linked through Schiff base formation Pharm Res 19:1137–1143. Copyright 姝 2002, Kluwer Academic
and successive reductive amination using NaBH3CN.147 Publishers, with kind permission of Springer Science and Busi-
ness Media. All rights reserved.142

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In: Protein Analysis Techniques

SERUM PROTEOMICS FOR CANCER:

Rakesh Sharma, Bharati D Shrinivas

T able 1. C har acter istics of global analyses.

1. PROTEOMIC ANALYSIS OF SERUM/PLASMA IS EFFECTIVE

1.1. Characteristics of Global Analyses and Diagnostic Availability of

[23]. From these f indings, i t i s s uggested t hat S NPs a nd D NA m ethylation c ould be us ed

T able 2. Diagnostic availability of biomar ker s identified by global analyses.

1.2. Advantages of Proteomic Analysis in the Search for Diagnostic

Genomic, t ranscriptomic, pr oteomic, metabolomic a nalyses pr ovide i nformation a bout

in c ontrast t o o ther gl obal a nalyses. A lthough t he i dentification o f s mall molecular

Early d iagnosis is very i mportant t o r educe c ancer m ortality. P rotein bi omarkers i n

2. SEPARATION TECHNOLOGIES IN PROTEOMIC ANALYSIS

(a) First-dimensional separation; Isoelectric focusing (b) Second-dimensional separation; SDS-PAGE

pH3 IPG strip pH10 Isoelectrically focused proteins

Functional group Hydrophobic, cationic, anionic, Biomarker peak

2.2. ProteinChip Array®

ClinProt® (Bruker D altonics, Bremen, G ermany) is a p urification method ba sed on a ffinity

2.4. Shotgun Proteomics Using LC

LC i s a s eparation t echnology us ed c ommonly in t he i solation of v arious biological

A bl ock di agram of a ba sic MS i s s hown i n Figure 2. T he s ample i nlet, i on s ource, m ass

3.2. Ionization Methods

Ionization methods for biological macromolecules, such as proteins/peptides, were developed

Charged droplets Desolvated ions

Figure 3. Ionization methods in proteomic analysis.

(a) Linear type (b) Reflector type

Ions Trapping Detector

Endcap electrode Endcap electrode

3.3. Mass Analyzers

3.4. Types of MS Used in Proteomic Analysis

4. IDENTIFICATION OF PROTEINS/PEPTIDES USING MS

4.1. Protein Identification by Peptide Mass Fingerprinting

Peptide m ass f ingerprinting ( PMF) i s a n a nalytical t echnique f or pr otein i dentification

4.2. Peptide Identification by MS/MS Ion Search

5. CANCER BIOMARKER AND DIAGNOSIS

MALDI source TOF1 TOF2

ESI source TOF mass analyzer

Isolation of interest protein using

The m/z measurement of

Actual measurement values (m/z) of peptides

3. Actual measurement values of peptides derived from interest

Selection of interest peptides

Detection of fragment ions

Actual measurement values (m/z) of parent ion and fragment ions

Fragment ions ←Parent ion

T able 4. C ancer biomar ker s identified by pr oteomic analysis of ser um/plasma.

5.1. Characteristics of Proteomic Cancer Biomarkers and Diagnostic

been us ed i n a nalysis, t he 2D -DIGE–MS sy stem a nd P roteinChip a rray®–SELDI-TOF-MS

3444 m/z HCC

skeletal m uscle, a nd pe ripheral bl ood l eukocytes [ 109]; h owever, n o i ncrease o f i ts se rum

5.2. Early Diagnosis of HCC Using ProteinChip Array®

In cancer d iagnosis, ear ly detection b efore t he o nset o f cl inical s ymptoms i s very

5.3. Proteomic Strategy for Discovering Specific Cancer Biomarkers

5.4. From Biomarker Discovery to Clinical Application

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6. CANCER MALDI IMAGING TECHNIQUE AND DIAGNOSIS

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hepatocellular c arcinoma: na tional s urveillance pr ogram of c irrhotic a nd nonc irrhotic

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Demonstration of an in vivo generated sub-picomolar aﬃnity