Biomaterials 30 (2009) 60656075

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Gold nanoparticles with a monolayer of doxorubicin-conjugated amphiphilic

block copolymer for tumor-targeted drug delivery
Mani Prabaharan a, Jamison J. Grailer b, Srikanth Pilla a, Douglas A. Steeber b, Shaoqin Gong a, c, *
a

Department of Mechanical Engineering, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA

Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA
c
Department of Materials, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 6 June 2009
Accepted 24 July 2009
Available online 12 August 2009

Gold (Au) nanoparticles (NPs) stabilized with a monolayer of folate-conjugated poly(L-aspartate-doxorubicin)-b-poly(ethylene glycol) copolymer (Au-P(LA-DOX)-b-PEG-OH/FA) was synthesized as a tumortargeted drug delivery carrier. The Au-P(LA-DOX)-b-PEG-OH/FA NPs consist of an Au core, a hydrophobic
poly(L-aspartate-doxorubicin) (P(LA-DOX)) inner shell, and a hydrophilic poly(ethylene glycol) and
folate-conjugated poly(ethylene glycol) outer shell (PEG-OH/FA). The anticancer drug, doxorubicin
(DOX), was covalently conjugated onto the hydrophobic inner shell by acid-cleavable hydrazone linkage.
The DOX loading level was determined to be 17 wt%. The Au-P(LA-DOX)-b-PEG-OH/FA NPs formed stable
unimolecular micelles in aqueous solution. The size of the Au-P(LA-DOX)-b-PEG-OH/FA micelles were
determined as 2452 and 1025 nm by dynamic light scattering (DLS) and transmission electron
microscopy (TEM), respectively. The conjugated DOX was released from the Au-P(LA-DOX)-b-PEG-OH/FA
micelles much more rapidly at pH 5.3 and 6.6 than at pH 7.4, which is a desirable characteristic for
tumor-targeted drug delivery. Cellular uptake of the Au-P(LA-DOX)-b-PEG-OH/FA micelles facilitated by
the folate-receptor-mediated endocytosis process was higher than that of the micelles without folate.
This was consistent with the higher cytotoxicity observed with the Au-P(LA-DOX)-b-PEG-OH/FA micelles
against the 4T1 mouse mammary carcinoma cell line. These results suggest that Au-P(LA-DOX)-b-PEGOH/FA NPs could be used as a carrier with pH-triggered drug releasing properties for tumor-targeted
drug delivery.
2009 Elsevier Ltd. All rights reserved.

Keywords:
Gold nanoparticles
pH-sensitive
Drug delivery
Tumor-targeted
Cellular uptake
Cytotoxicity

1. Introduction
Gold (Au) NPs have received considerable attention during the
past decade due to their potential applications in catalysis, chemical sensing, electronics, optics, and biology [1]. Particularly,
monolayer-protected Au NPs have recently emerged as an attractive candidate for delivering various therapeutic agents such as
drugs, peptides, proteins, and nucleic acids into their targets [2,3].
Recent progress in monolayer-protected Au NPs involves using
thiolated compounds to stabilize Au NPs through thiol linkages [2].
The monolayer ranges from small organic compounds to macromolecules and can be further functionalized in various ways to
improve their drug delivery functionalities. For example, Corbierre
et al. reported polystyrene-functionalized Au NPs by the covalent

* Corresponding author. Department of Mechanical Engineering, University of

Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. Tel.: 1 414 229 5946; fax: 1
414 229 6958.
E-mail address: sgong@uwm.edu (S. Gong).
0142-9612/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2009.07.048

attachment of thiol-terminated polystyrene prepared by anionic

polymerization [4]. Au NPs with tetra(ethylene glycol)ylated
cationic ligands and uorogenic ligands were synthesized for
hydrophobic drug delivery [5]. Thomas and Klibanov synthesized
Au NPs with branched polyethylenimine monolayer to provide
hybrid Au NP-polymer transfection vectors [6]. Wang et al. recently
fabricated effective binders for DNA by anchoring b-cyclodextrin on
the periphery of oligo(ethylenediamino)-modied Au NPs [7].
Monolayer-protected Au NPs are one of the most promising
candidates for cancer treatment because of their functional versatility. Functionalized Au NPs with an outer PEG shell can offer
excellent stability in physiological conditions because PEG has
excellent solubility in water [8]. The PEG shell can also greatly
reduce the interaction between the Au NPs and the plasma protein,
thereby greatly minimizing the uptake of the Au NPs by the reticuloendothelial system (RES), which can lead to a much longer
circulation time in the bloodstream. This will allow the Au NPs to
preferentially accumulate in the tumor sites through the leaky
tumor neovasculature by the enhanced permeability and retention
(EPR) effect. In addition, due to their intense light scattering power,

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M. Prabaharan et al. / Biomaterials 30 (2009) 60656075

monolayer-protected Au NPs targeted to cancer tissue may improve

surgeons ability to identify metastatic lesions. Specically, the
tunability of Au NPs optimal scattering wavelength (plasmon
resonance) may provide a means to dramatically enhance the
apparent optical contrast of lesions relative to neighboring tissue.
For instance, the optical properties of the Au NPs can greatly
enhance the contrast of X-ray computed tomography (CT) imaging
[9]. Finally, monolayer-protected Au NPs can be used to destroy the
tumor cells without damaging the surrounding tissue by photothermal therapy since they can efciently convert the absorbed
laser light energy into localized heat [10].
Au NPs with an end-functionalized monolayer, regarded as
a macroinitiator, can be used to prepare Au NPs covalently conjugated with polymer monolayer using surface-initiated polymerization. We recently synthesized amphiphilic Au NPs with
a polycaprolactone-methoxy poly(ethylene glycol) monolayer by
the ring-opening polymerization of 3-caprolactone using mercaptoundecanol stabilized Au NPs as the macroinitiator, followed by
esterication with carboxyl terminated methoxy poly(ethylene
glycol) as the drug deliver carrier [11]. In this work, we describe the
synthesis of folate (FA)-conjugated amphiphilic Au NPs with
a poly(L-aspartate-doxorubicin)-b-poly(ethylene glycol) monolayer
(Au-P(LA-DOX)-b-PEG-OH/FA) (cf. Fig. 1) by the ring-opening
polymerization of b-benzyl L-aspartate N-carboxyanhydride (BLA
NCA) using amine functionalized Au NPs as a macroinitiator,
followed by coupling reactions with a-hydroxy-u-carboxyl poly(ethylene glycol) (HOPEGCOOH) and subsequently folate for
tumor-targeted drug delivery. The DOX molecules were conjugated
to the hydrophobic poly(L-aspartate) segment by pH-sensitive
hydrazone linkage. Au-P(LA-DOX)-b-PEG-OH/FA NPs can form
stable unimolecular micelles in aqueous solutions due to their
unique amphiphilic multi-arms and globular architecture. In
contrast to conventional polymeric micelles that require the
aggregation of multiple linear amphiphilic molecules to achieve the
micellar behavior above the critical micelle concentration, unimolecular micelles formed by individual multi-arm amphiphilic

molecules exhibit excellent in vivo stability that is independent of

the polymer concentration or some other environmental uctuation such as temperature or pH due to its covalent nature [12].
Due to the presence of FA, Au-P(LA-DOX)-b-PEG-OH/FA NPs
offer active tumor-targeting ability in addition to their passivetargeting ability, which is attributed to the EPR effect exhibited by
the tumor tissue [13,14]. Conjugation of DOX onto the NPs is
expected to improve the drug loading level and greatly reduce the
chance of premature drug release during circulation in the bloodstream. Furthermore, since the hydrazone linkage between the
DOX and polymer is prone to hydrolysis in an acidic condition, the
release rate of DOX from the NPs will increase in the acidic environment of the endosomal intracellular compartments after the
Au-P(LA-DOX)-b-PEG-OH/FA NPs are internalized by the tumor
cells via the folate-mediated-endocytosis, thereby providing
a sufcient concentration of DOX in the tumor cells within a short
period of time (cf. Fig. 1) [1517]. The structure of the Au-P(LADOX)-b-PEG-OH/FA NPs was characterized by 1H NMR spectroscopy. The micellar properties of the Au-P(LA-DOX)-b-PEG-OH/FA
NPs were determined by dynamic light scattering (DLS) and
transmission electron microscopy (TEM) analyses. The in vitro DOX
release studies were performed at pH 5.3, 6.6, and 7.4 at 37  C. The
cellular uptake and cytotoxicity of the Au-P(LA-DOX)-b-PEG-OH/FA
micelles against 4T1 mouse mammary carcinoma cells were
assessed using confocal laser scanning microscopy (CLSM), ow
cytometry, and the MTT assay.
2. Materials and methods
2.1. Materials
Tetrachloroauric acid (HAuCl4), 2-aminoethanethiolHCl (AETHCl), b-benzyl
(BLA), and triphosgene were purchased from SigmaAldrich. Anhydrous
hydrazine, sodium borohydride (NaBH4), 4-dimethylamino pyridine (DMAP),
N-hydroxy succinimide (NHS) and 1,3-dicyclohexylcarbodiimide (DCC) were
purchased from Acros and used without further purication. HOPEGCOOH with
an Mw of 3000 was purchased from RAPP Polymere GmbH, Germany. FA was
obtained from bio-WORLD, USA. Triethylamine (Alfa Aesar) was distilled before use.
L-aspartate

DOX conjugated via

a pH-sensitive hydrazone
bond
O

OH

NHN
OH
OH

OCH3 O
H3C

OH
O
OH NH2

[H+]

OH

OH

OH

OCH3 O
H3C

OH
O
OH

NH2

DOX

NP
Au core
Hydrophobic poly(L-aspartate) segment
Hydrophilic PEG segment

DOX conjugated by hydrazone bond

FA ligand

Fig. 1. Schematic illustration of the Au-P(LA-DOX)-b-PEG-OH/FA NP and its pH-triggered drug release.

M. Prabaharan et al. / Biomaterials 30 (2009) 60656075

6067

Anhydrous DMF was purchased from Fisher Scientic Company. The model drug,
doxorubicinHCl (DOXHCl), was supplied by Tecoland Corporation, USA, and used
as supplied. NIH-3T3 and 4T1 cell lines were purchased from ATCC, USA. All other
chemicals used were of analytical reagent grade.

under vacuum and the product was puried by recrystallization using a mixed
solvent of THF and hexane. The formation of BLANCA was conrmed by 1H NMR
(300 MHz, CDCl3): 7.457.3 (5H, m, Ar-H), 6.2 (1H, s, NH), 5.2 (2H, s, CH2), 4.61 (1H, t,
CH) and 2.85 (2H, t, CH2) ppm.

2.2. Synthesis of amine functionalized Au NPs

2.4. Synthesis of Au-poly(b-benzyl L-aspartate) (AuPBLANH2)

Fig. 2 shows the reaction scheme for the synthesis of Au-P(LA-DOX)-b-PEG-OH/

FA. Au NPs were rst synthesized by the NaBH4 reduction method [18]. In a typical
experiment, 100 mL of 104 M aqueous solution of HAuCl4 was reduced by 0.01 g of
NaBH4 under vigorous stirring at room temperature in the dark. After 12 h of
reaction, the excess amount of NaBH4 was removed by dialysis against distilled
water for 4 h using a dialysis tubing (molecular weight cut-off of 2 kDa). The number
of Au NPs in aqueous solution was determined as 5.81 1015 g/L by atomic
absorption spectroscopy (AAS). Thereafter, 2.41 108 M of AETHCl was treated
with 100 mL of aqueous solution containing the Au NPs at room temperature in
order to functionalize 25 molecules of AETHCl per Au NP surface. After 6 h, the
resulting thiol stabilized Au NPs were treated with 2.41 108 M of triethylamine for
30 min followed by dialysis against deionized water for 24 h to obtain the amine
functionalized Au NPs.

1 g of BLANCA (4.016 mmol) was dissolved in 50 mL of anhydrous DMF under

stirring at room temperature. Then, 100 mg of amine functionalized Au NPs in 10 mL
of DMF was added to the BLANCA solution. Thereafter, the reaction mixture was
stirred at 55  C in a steam of dry nitrogen. After 48 h, the reaction mixture was
poured into a 10-fold volume of diethyl ether at 0  C, and a precipitate was collected
by ltration and washed with diethyl ether, followed by drying in vacuum.
2.5. Synthesis of Au-poly(b-benzyl L-aspartate)-b-poly(ethylene glycol) (Au-PBLA-bPEG-OH/FA)
Au-PBLA-b-PEG-OH/FA was prepared using a two-step reaction procedure. First,
Au-PBLA-b-PEG-OH was prepared by reacting 0.5 g of AuPBLANH2 with 1 g of HO
PEGCOOH (0.33 mmol) using DCC (0.07 g, 0.33 mmol) and NHS (0.04 g, 0.33 mmol)
as the condensing agents in 50 mL of anhydrous DMF, as shown in Fig. 2. The
reaction was allowed for 12 h at room temperature under constant stirring. After the
by-product, dicyclohexylurea, was removed by ltration, the product Au-PBLA-bPEG-OH was dialyzed against deionized water using a dialysis tubing (molecular
weight cut-off of 12 kDa) for 48 h and freeze-dried. Thereafter, 0.5 g of Au-PBLA-bPEG-OH reacted with 1.77 mg of FA in presence of DCC and DMAP as the catalysts in
anhydrous DMF for 24 h to obtain the product Au-PBLA-b-PEG-OH/FA.

2.3. Synthesis of BLANCA

13 g (60 mmol) of BLA was suspended in 100 mL of anhydrous THF. With this
solution, 9 g (30 mmol) of triphosgene in 10 mL anhydrous THF was slowly added
over a period of 30 min. Then, the mixture was stirred at 55  C under nitrogen for
about 3 h until a clear solution was observed. Thereafter, the solvent was removed

H
N

O
O
NaBH4

HAuCl4

HS-CH2-CH2-NH3Cl

Au

Au

NH2

BLA-NCA
25

TEA
Gold NPs

O
H
N

Au

NH

O
HOOC

Au

OH

O
n

OC

m OH

25

25

H
N

NH

NHS/DCC

DMAP/DCC
O
HN

N N
NH2

Au

H
N

NH

O
n

OC

OH/FA
25

O COOH

NH2NH2

NH
FA

COOH

N
H

H
N

Au

NH

O
n

OC

DOX

OH/FA

Au

NH

NHNH2

O
n

OC

OH

m OH/FA
25

25

H
N

NHN
OH
OH

OCH3 O
OH
H3C
O
OHNH2

Fig. 2. Reaction scheme for the synthesis of Au-P(LA-DOX)-b-PEG-OH/FA.

O
Au-P(LA-DOX)-b-PEG-OH/FA

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M. Prabaharan et al. / Biomaterials 30 (2009) 60656075

2.6. Synthesis of Au-P(LA-DOX)-b-PEG-OH/FA

Au-P(LA-DOX)-b-PEG-OH/FA was synthesized by using a two-step reaction
procedure. In the rst step, the benzyl groups of Au-PBLA-b-PEG-OH/FA were
substituted with hydrazide groups by reacting 0.5 g of Au-PBLA-b-PEG-FA with
excess of anhydrous hydrazine (20 mg) in 50 mL of dry DMF at 40  C for 24 h.
Thereafter, the formed product, Au-P(LA-hydrazide)-b-PEG-OH/FA, was dialyzed
against distilled water for 48 h and followed by freeze-drying. In the next step,
0.25 g of Au-P(LA-hydrazide)-b-PEG-OH/FA was dissolved in 25 mL of DMF, and an
excess amount of DOXHCl (75 mg, 0.137 mmol) was added. The mixed solution was
stirred at room temperature for 24 h. Thereafter, the reaction mixture was dialyzed
against alkaline water (pH 8) for 48 h to obtain Au-P(LA-DOX)-b-PEG-OH/FA. After
freeze-drying, the product was puried using LH20 gel column to completely
remove the unreacted DOX. The absence of free DOX in the product was conrmed
by thin layer chromatography (TLC).

cells were cultured in FA-free RPMI medium supplemented with 10% fetal calf
serum, penicillin/streptomycin, and L-glutamine (complete media, all from Invitrogen Corp., Grand Island, NY) at 37  C with 5% CO2. Then, 4T1 cells were cultured
and lifted as above before being seeded (104) into 96-well plates and incubated for
24 h. The medium was then replaced with fresh medium containing the free DOX
(34 mg/mL), Au-P(LA-DOX)-b-PEG-OH/FA and Au-P(LA-DOX)-b-PEG-OH micelles
(DOX concentration 34 mg/mL) and incubated for 48 h. Thereafter, the wells were
washed three times with warm phosphate buffer solution and incubated again for
another 4 h with FA-free RPMI containing 250 mg/mL of MTT. After discarding the
culture medium, 150 mL of DMSO was added to dissolve the precipitates, and the
resulting solution was measured for absorbance at 570 nm with a reference wavelength of 690 nm using a microplate reader (Molecular Devices).

3. Results and discussion

2.7. Preparation of micelles from the Au-P(LA-DOX)-b-PEG-OH/FA NPs

3.1. Synthesis and characterization of Au-P(LA-DOX)-b-PEG-OH/FA

Au-P(LA-DOX)-b-PEG-OH/FA micelles were prepared using the membrane

dialysis method. Briey, 50 mg of Au-P(LA-DOX)-b-PEG-OH/FA was dissolved in
5 mL of DMF under stirring. With this solution, 15 mL of Millipore water was added
dropwise. Thereafter, the polymer solution was dialyzed against Millipore water
using a dialysis tubing (molecular weight cut-off 2 kDa) for 96 h followed by freezedrying. In order to determine the drug conjugation level, a weighed quantity (25 mg)
of Au-P(LA-DOX)-b-PEG-OH/FA was treated with 0.1 N HCl solutions at room
temperature for 48 h under uniform stirring. After centrifugation, the DOX in the
supernatant was assayed by UVvisible spectrophotometer at a wavelength of
485 nm. All the experiments were carried out in triplicate.

The reaction scheme for the synthesis of Au-P(LA-DOX)-b-PEGOH/FA NPs is shown in Fig. 2. The amine functionalized Au NPs
were rst synthesized by using a two-step procedure. In the rst
step, Au NPs were prepared by reducing the Au salt, HAuCl4, using
NaBH4 in aqueous solution at room temperature. In the second step,
Au NPs were functionalized with the amine groups by reacting
AETHCl on the monolayer of Au NPs through thiol linkers in the
presence of triethylamine. The number of amine functionality on
the Au NPs was maintained as 25 by controlling AETHCl mole ratio
to the number Au NPs during the reaction. Thereafter, AuPBLA
NH2 was prepared by the ring-opening polymerization of BLANCA
using amine functionalized Au NPs as an initiator in anhydrous
DMF. The formation of AuPBLANH2 was conrmed by the 1H
NMR spectrum, as shown in Fig. 3A. The peaks at 7.3 ppm and
5.1 ppm are ascribed to the protons of benzyl and methylene
groups in the PBLA side chains, respectively. The signal at 2.7 ppm
corresponds with the methylene group of the side chain, which
connects the main chain of PBLA (CHCH2COO), was also
identied. The peaks at 2.9 and 3.0 ppm were observed due to the
methylene protons of AETHCl. These results conrm the formation
of AuPBLANH2.
To obtain Au-PBLA-b-PEG-OH, the amino end groups of Au
PBLANH2 were coupled with the carboxylic acid terminal groups
of HOPEGCOOH by the amide linkage. The coupling reaction was
carried out in dry DMF at room temperature in the presence of DCC
and NHS as the activating agent for the carboxylic group. In the 1H
NMR spectrum of Au-PBLA-b-PEG-OH (Fig. 3B), in addition to the
characteristic peaks of PBLA, the peak at 3.6 ppm was observed due
to the methylene protons of oxyethylene units of PEG. These results
clearly indicate the formation of Au-PBLA-b-PEG-OH. From the 1H
NMR spectra (Fig. 3B), the number average molecular weight (Mn)
of the PBLA-b-PEG-OH segment was determined as 4975 by
calculating the relative intensity ratio of the methylene proton of
the PEG chain (OCH2CH2, d 3.6) and the methylene proton near
the benzyl group of the PBLA chain (COOCH2C6H5, d 5.1).
Au-PBLA-b-PEG-OH/FA was synthesized by reacting g-carboxyl
group of FA with some of the terminal hydroxyl groups of Au-PBLAb-PEG-OH by ester forming reaction using DMAP and DCC as the
catalysts. The presence of FA in the product, Au-PBLA-b-PEG-OH/FA,
was conrmed by the appearance of weak signals at 6.78.7 ppm,
which corresponded with the aromatic protons of FA (Fig. 3C). To
conjugate DOX onto Au-PBLA-b-PEG-OH/FA, the benzyl groups of
Au-PBLA-b-PEG-OH/FA were rst substituted with hydrazide
groups by esteramide exchange aminolysis reaction. The hydrazide
groups of resulting product were then conjugated with DOX through
an acid-sensitive hydrazone bond to obtain Au-P(LA-DOX)-b-PEGOH/FA, as shown in Fig. 2. The absence of aromatic protons peak at
7.3 ppm and benzyl methylene proton at 5.1 ppm in the 1H NMR
spectrum of Au-P(LA-DOX)-b-PEG-OH/FA (Fig. 3D) proved the
complete debenzylation of the poly(L-aspartate) (PLA) segments of

2.8. Characterization
1
H NMR spectrum of the samples was recorded on a Bruker DPX 300 spectrometer using CDCl3 and DMSO as the solvents at 25  C. Absorbance measurements
were carried out using Varian Cary 100 Bio UVvisible spectrophotometer. The
calibration curve of absorbance against different concentrations of DOX was made at
485 nm. The particle size was determined by DLS using a Beckman Coulter PCS
submicron particle size analyzer with angle detection at 90 . For TEM studies, a drop
of particle or micelle solutions (0.05 mg/mL) containing 0.8 wt% phosphotungstic
acid was deposited onto a 200 mesh copper grid coated with carbon and dried at
room temperature. The shape and size of the particles were observed at 75 kV with
a Hitachi H-600 TEM.

2.9. Drug release

The release studies were performed in a glass apparatus at 37  C in acetate
buffer (pH 5.3 and 6.6) and phosphate buffer (pH 7.4) solutions. First, 50 mg of AuP(LA-DOX)-b-PEG-OH/FA micelles was dispersed in 5 mL of medium and placed in
a dialysis bag with a molecular weight cut-off of 2 kDa. The dialysis bag was then
immersed in 95 mL of the release medium and kept in a horizontal laboratory shaker
maintaining a constant temperature and stirring (100 rpm). Samples (2 mL) were
periodically removed and the volume of each sample was replaced by the same
volume of fresh medium. The amount of released DOX was analyzed with a spectrophotometer at 485 nm. The drug release studies were performed in triplicate for
each of the samples.
2.10. Cellular uptake and cytotoxicity
The cellular uptake experiments were performed using ow cytometry and
CLSM. For ow cytometry, 4T1 cells (1 106) were seeded in 6-well culture plates
and grown overnight. The cells were then treated with free DOX (34 mg/mL) or AuP(LA-DOX)-b-PEG-OH/FA or Au-P(LA-DOX)-b-PEG-OH (i.e., both FA-conjugated and
FA-free) micelles (DOX concentration 34 mg/mL) for 30 and 120 min. Thereafter,
the cells were lifted using Cellstripper (Media Tech, Inc) and washed, and the DOX
uptake was analyzed using a FACSCalibur ow cytometer (BD Biosciences). A
minimum of 2  104 cells was analyzed from each sample with uorescence intensity shown on a four-decade log scale. For CLSM studies, 4T1 cells (1 106) were
seeded onto 22 mm round glass coverslips, placed in 6-well plates, and grown
overnight. Cells were treated with free DOX (34 mg/mL) or Au-P(LA-DOX)-b-PEG-OH/
FA or Au-P(LA-DOX)-b-PEG-OH micelles (DOX concentration 34 mg/mL) for 2 h.
Then, the cells were washed and xed with 1.5% formaldehyde. Coverslips were
placed onto glass microscope slides and DOX uptake was analyzed using a Leica TCS
SP2 Confocal System installed on an upright compound microscope (Leica, Wetzler,
Germany). Digital monochromatic images were acquired using Leica Confocal
Software (Version 2.61). To determine the DOX distribution in the nucleus and
cytoplasm of the 4T1 cells, the DOX mean uorescence intensity (MFI) in the CLSM
images was measured in a 4 mm2 area located in the nucleus or cytoplasm (n 10
cells) for each sample using ImageJ software (http://rsb.info.nih.gov/ij).
The cytotoxicity of Au-P(LA-DOX)-b-PEG-OH/FA and Au-P(LA-DOX)-b-PEG-OH
micelles against NIH-3T3 and 4T1 cells was assessed using the MTT assay [19]. The

M. Prabaharan et al. / Biomaterials 30 (2009) 60656075

Fig. 3. 1H NMR spectrum of (A) Au-PBLA-NH2; (B) Au-PBLA-b-PEG-OH; (C) Au-PBLA-b-PEG-OH/FA; and (D) Au-P(LA-DOX)-b-PEG-OH/FA.

6069

M. Prabaharan et al. / Biomaterials 30 (2009) 60656075

Au-P(LA-DOX)-b-PEG-OH/FA. Moreover, the conjugation of DOX

was conrmed by the presence of characteristic DOX peaks at 5.4,
5.3, 4.0, 2.1, 1.9, and 1.2 ppm in addition to the characteristic peaks of
PLA, PEG, and FA. The substitution level of DOX on the PLA backbone
was determined as 17 wt% by UVvisible spectrophotometer analysis at 485 nm, which corresponds with 47 DOX molecules per AuP(LA-DOX)-b-PEG-OH/FA NP on average.
The formation of Au, amine functionalized Au, and Au-PBLA-bPEG-OH/FA NPs was conrmed by UVvisible spectroscopy
analysis, as shown in Fig. 4. The UVvisible spectra of Au, amine
functionalized Au, and Au-PBLA-b-PEG-OH/FA NPs showed characteristic surface plasmon resonance (SPR) bands at 512, 525, and
565 nm, respectively. These results indicate the formation
and existence of Au NPs [20,21]. The width of the absorption band
and the position of the maximum absorption peak depend on the
morphology of the particles (size, shape, and uniformity), coagulation among the particles, and the dielectric environment [22,23].
The signicant red shift in SPR with peak broadening of Au-PBLA-bPEG-OH/FA and amine functionalized Au NPs compared to the Au
NPs suggests a linear increase in particle size consequent to the
surface modication [24].

A
80

Intensity, %

6070

60

40

20

0
-10

For drug nanocarriers, the size and stability are important

properties that inuence their in vivo performance. These two
factors will directly affect the biodistribution and circulation time
of the carriers. Stable and smaller particle sizes (<200 nm) can
reduce the uptake of the reticuloendothelial system (RES) and
provide efcient passive tumor-targeting ability via the enhanced
permeability and retention effects [25]. Au-P(LA-DOX)-b-PEG-OH/
FA NPs formed stable unimolecular micelles in aqueous solutions
because of its amphiphilic multi-arm and globular core/shell
architecture. In this study, the size and size distribution of micelles
made from Au-P(LA-DOX)-b-PEG-OH/FA NPs were examined by
DLS and compared with that of amine functionalized Au NPs.
Fig. 5A shows the particle size distribution histogram of amine
functionalized Au NPs. Amine functionalized Au NPs showed
a narrow size distribution ranging from 5 to 11 nm with an average
particle size diameter of 6.3 nm and a polydispersity index of 0.028.
In the case of micelles made from Au-P(LA-DOX)-b-PEG-OH/FA NPs,

60

Absorbance

0.5
0.4

A
C

0.3
B
0.2
0.1
0.0
300

400

500
600
Wavelength, nm

700

800

Fig. 4. UVvisible absorption spectra of (A) Au NPs; (B) amine functionalized Au NPs;
and (C) Au-PBLA-b-PEG-OH/FA NPs.

Intensity, %

70

0.6

10

20

30 40 50 60
Diameter, nm

70

80

90

10

20

30 40 50 60
Diameter, nm

70

80

90

3.2. Particle size analysis

0.7

50
40
30
20
10
0
-10

Fig. 5. Size distribution of (A) amine functionalized Au NPs; and (B) Au-P(LA-DOX)-bPEG-OH/FA NPs.

the size distribution was relatively broad, ranging from 24 to

52 nm, and the average micelle size was 34 nm with a polydispersity index of 0.029 (Fig. 5B). The increased size and size
distribution of micelles formed from Au-P(LA-DOX)-b-PEG-OH/FA
NPs might be due to the presence of a fairly thick P(LA-DOX)-bPEG-OH/FA monolayer on the surface of Au NPs. These results
clearly indicate that the Au-P(LA-DOX)-b-PEG-OH/FA NPs could
form core/shell unimolecular micelles and that the monolayer of
amphiphilic P(LA-DOX)-b-PEG-OH/FA copolymer stabilizes the Au
NPs in aqueous solutions.
The size and morphology of amine functionalized Au and AuP(LA-DOX)-b-PEG-OH/FA NPs were further evaluated by TEM. As
shown in Fig. 6A, amine functionalized Au NPs are spherical with an
average diameter of 6 nm. Furthermore, it was observed that amine
functionalized Au NPs are well-separated from each other (i.e., no
aggregates), indicating AET effectively stabilize the Au NPs. Fig. 6B
shows a TEM image of the micelles made from Au-P(LA-DOX)-bPEG-OH/FA NPs. The morphology of these micelles appeared as
well-dened Au core and P(LA-DOX)-b-PEG-OH/FA shell structures
with a diameter in the range of 1025 nm. In addition, no aggregation between the Au-P(LA-DOX)-b-PEG-OH/FA micelles was

M. Prabaharan et al. / Biomaterials 30 (2009) 60656075

6071

Fig. 6. TEM images of (A) amine functionalized Au NPs; and (B) Au-P(LA-DOX)-b-PEG-OH/FA NPs.

3.3. Drug release

The drug release behavior of the Au-P(LA-DOX)-b-PEG-OH/FA
micelles was investigated under a simulated physiological condition (PBS, pH 7.4) and in an acidic environment (acetate buffer, pH
5.3, and 6.6) at 37  C to assess the feasibility of using Au-P(LADOX)-b-PEG-OH/FA NPs as an anticancer drug delivery carrier. As
shown in Fig. 7, the rate and amount of DOX release from the AuP(LA-DOX)-b-PEG-OH/FA NPs were strongly dependent on the pH
of the medium. Au-P(LA-DOX)-b-PEG-OH/FA NPs showed a much
faster DOX release at pH 5.3 and 6.6 than at pH 7.4. At pH 5.3 and
6.6, Au-P(LA-DOX)-b-PEG-OH/FA micelles released out about 34
and 27%, respectively, of the conjugated DOX for the rst 2 h, and
liberated 94 and 83%, respectively, after 45 h; however, at pH 7.4,
the micelles released only 5% of DOX for 2 h and less than 15% after
45 h. This result shows that the release of DOX from the Au-P(LADOX)-b-PEG-OH/FA NPs in an acidic environment was governed by
the acid-cleavable characteristic of the hydrazone linkage between
the DOX molecules and the NPs. An acid-cleavable hydrazone
linkage can undergo hydrolysis under acidic conditions; thus,
unmodied DOX can be released from the Au-P(LA-DOX)-b-PEGOH/FA NPs by hydrolysis of the hydrazone linkage at the 13-keto
position of the DOX molecules (cf. Fig. 1). The DOX release proles
from the Au-P(LA-DOX)-b-PEG-OH/FA NPs agree with those
reported previously with DOX-conjugated polymeric micelles
[27,16].

The strong, pH-dependent DOX release prole is highly desirable for effective treatment of cancer. The very slow DOX release
rate observed at pH 7.4, mimicking the physiological conditions of
the bloodstream, ensures minimal DOX release during blood
circulation. It is expected that the Au-P(LA-DOX)-b-PEG-OH/FA
micelles will accumulate in the tumor tissue preferentially through
the EPR effect. Once in the tumor tissue, these Au-P(LA-DOX)-bPEG-OH/FA micelles will be internalized by the tumor cells, largely
via folate-receptor-mediated endocytosis, and will be located in the
acidic endosomal compartments where DOX could be cleaved from
the Au NPs and subsequently diffuse into the cytosol and later into
the nucleus [28,29]. Therefore, the much higher release rate
observed at pH 6.6 and 5.3, mimicking the physiological environments of the tumor tissue and the endocytic compartments of the
tumor cells, respectively, can help to quickly provide a sufcient
level of DOX in the tumor tissue/cells, thereby greatly enhancing
the efcacy of cancer therapy.

100

pH 5.3
pH 6.6
pH 7.4

80
Release, %

observed in the TEM image, conrming that the monolayer of

amphiphilic P(LA-DOX)-b-PEG-OH/FA copolymer on the Au core
prevents the NPs from aggregation. The diameter of micelles made
from the Au-P(LA-DOX)-b-PEG-OH/FA NPs observed by TEM is
smaller than their diameter obtained from the DLS experiment. The
larger value from the DLS measurement relative to TEM is most
likely due to the existence of a swollen PEG corona around the Au
core. The size range of Au-P(LA-DOX)-b-PEG-OH/FA micelles
determined by the DLS and TEM experiments could be suitable for
an injectable anti-cancer drug delivery carrier because it allows for
extravasation at tumor sites due to the EPR effect, and the FAreceptor-mediated endocytosis process that leads to the preferred
accumulation of drug-conjugated micelles within tumors [26].

60

40

20

0
0

10

20

30

40
50
Time, h

60

70

80

Fig. 7. DOX release prole from the Au-P(LA-DOX)-b-PEG-OH/FA NPs at 37  C.

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M. Prabaharan et al. / Biomaterials 30 (2009) 60656075

Au-P(LA-DOX)-bPEG-OH/FA

Au-P(LA-DOX)-bPEG-OH

Au-P(LA-DOX)-bPEG-OH/FA

Au-P(LA-DOX)-bPEG-OH

Free DOX

Control

Count

Control

Free DOX

100

101
102
103
Fluorescence Intensity

104 100

101

102

103

104

Fig. 8. Flow cytometry results of 4T1 cells that were incubated with free DOX, Au-P(LA-DOX)-b-PEG-OH, and Au-P(LA-DOX)-b-PEG-OH/FA micelles for (A) 30 min and (B) 120 min.
(DOX concentration: 34 mg/mL).

3.4. Cellular uptake

The cellular uptake of Au-P(LA-DOX)-b-PEG-OH/FA micelles by
the folate receptor positive cell line, 4T1 cells, was studied by ow

20 m

20 m

cytometry and compared with free DOX and FA-free Au-P(LADOX)-b-PEG-OH micelles to understand the effect of FA on the
cellular uptake. Since DOX itself is uorescent, it was used directly
to measure cellular uptake without additional markers. Therefore,

20 m

90
Nucleus

80

Cytoplasm

70

DOX MFI

60
50
40
30
20
10
0

Free Dox

Non-targeted

FA-Targeted

Fig. 9. CLSM images of 4T1 cells incubated with (A) free DOX, (B) Au-P(LA-DOX)-b-PEG-OH, and (C) Au-P(LA-DOX)-b-PEG-OH/FA micelles at 37  C for 2 h. (D) DOX mean uorescence intensity (MFI) in the nucleus and cytoplasm of the 4T1 cells incubated with free DOX, FA-targeted and non-targeted unimolecular micelles shown in (A), (B) and (C). (DOX
concentration: 34 mg/mL).

M. Prabaharan et al. / Biomaterials 30 (2009) 60656075

the uorescence intensity is proportional to the amount of DOX

internalized by the cells. Fig. 8 shows the ow cytometry histograms of cell-associated DOX uorescence for 4T1 cells. Cells
without any DOX treatment were used as a negative control and
showed only the auto-uorescence of the cells. For 4T1 cells incubated with equivalent DOX concentration in each formulation and
incubated for the same time, the Au-P(LA-DOX)-b-PEG-OH/FA
micelles showed higher uorescence intensity than Au-P(LA-DOX)b-PEG-OH micelles. This result directly conrms that the cellular
uptake of the micelles can be enhanced by attaching FA on their
surface and the Au-P(LA-DOX)-b-PEG-OH/FA micelles were transported into the 4T1 cells by a FA-receptor-mediated endocytosis
process. The cellular uptake of free DOX was greater than that of
Au-P(LA-DOX)-b-PEG-OH and Au-P(LA-DOX)-b-PEG-OH/FA NPs,
indicating that free DOX was rapidly transported into cells via
a passive diffusion mechanism [30].
The cellular uptake of free DOX, Au-P(LA-DOX)-b-PEG-OH, and
Au-P(LA-DOX)-b-PEG-OH/FA micelles by 4T1 cells were comparatively estimated by a CLSM, as shown in Fig. 9. As such, 4T1 cells
treated with free DOX for 2 h showed that DOX was predominantly
accumulated in the nucleus (Fig. 9A and D, p < 0.0005). The intense
DOX accumulation in the nucleus for free DOX occurred because
intracellular free DOX molecules in the cytosol were rapidly
transported to the nucleus and avidly bound to the chromosomal
DNA [31].

1. Control
2. Au-P(LA-DOX)-b-PEG-OH
3. Au-P(LA-DOX)-b-PEG-OH/FA
4. Free DOX

Cell Viability, % of Control

100
80
60
40
20
0
1

3.5. Cytotoxicity
The cytotoxic effects of free DOX, Au-P(LA-DOX)-b-PEG-OH, and
Au-P(LA-DOX)-b-PEG-OH/FA micelles against the cultured NIH-3T3
and 4T1 cells were evaluated using the MTT assay [19]. NIH-3T3
cells are healthy mouse embryonic broblast cells without overexpressed FA receptors; 4T1 cells are mouse mammary carcinoma
cells with over-expressed FA receptors. Cells were treated with free
DOX or DOX-conjugated micelles with an equivalent concentration
of DOX (34 mg/mL). Fig. 10A shows the NIH-3T3 cell viability in the
presence of free DOX, Au-P(LA-DOX)-b-PEG-OH, and Au-P(LADOX)-b-PEG-OH/FA micelles. The results showed no signicant
difference in the viability of NIH-3T3 cells when cultured in the
presence of Au-P(LA-DOX)-b-PEG-OH and Au-P(LA-DOX)-b-PEGOH/FA micelles, which indicates the cytotoxicity of DOX-conjugated
micelles with or without FA was similar against NIH-3T3 cells. This
observation clearly conrms that the FA molecule present on the
surface of the DOX-conjugated micelles does not have any effect on
the NIH-3T3 cellular uptake.
Fig. 10B shows the cytotoxic effect of free DOX, Au-P(LA-DOX)-bPEG-OH, and Au-P(LA-DOX)-b-PEG-OH/FA micelles against 4T1
cells. The cell viability in the presence of Au-P(LA-DOX)-b-PEG-OH/
FA micelles was lower than that in the presence of Au-P(LA-DOX)-

1. Control
2. Au-P(LA-DOX)-b-PEG-OH
3. Au-P(LA-DOX)-b-PEG-OH/FA
4. Free DOX

100
Cell Viability, % of Control

In the case of the DOX-conjugated micelles, less intense red

uorescence was observed in the cytoplasm and nucleus, indicating
that the DOX-conjugated micelles were initially located within the
endosomal intracellular compartments, releasing cleaved DOX in
the cytosol region in a sustained manner. However, consistent to
the ow cytometry ndings, the internalization of the Au-P(LADOX)-b-PEG-OH/FA micelles was much higher compared with the
Au-P(LA-DOX)-b-PEG-OH micelles, resulting in a stronger uorescence intensity. In addition, there was signicantly more DOX
uorescence in the nucleus compared to the cytoplasm in the AuP(LA-DOX)-b-PEG-OH/FA treated cells (Fig. 9C and D, p < 0.0005),
while the Au-P(LA-DOX)-b-PEG-OH treated cells had uniform
DOX uorescence in the nucleus and cytoplasm (Fig. 9B and D,
p 0.47). These results clearly indicate that the cellular uptake of
the Au-P(LA-DOX)-b-PEG-OH/FA micelles were facilitated by
a folate-receptor-mediated endocytosis process while the Au-P(LADOX)-b-PEG-OH micelles were transported into cells through
a non-specic endocytosis mechanism.

80
60
40
20
0

1. Control
2. Au-P(LA-DOX)-b-PEG-OH/FA
(FA free medium)
3. Au-P(LA-DOX)-b-PEG-OH/FA
(Free FA, 0.5 mg/mL)
4. Au-P(LA-DOX)-b-PEG-OH/FA
(Free FA, 1 mg/mL)

100
Cell Viability, % of Control

6073

80

60

40

20

0
1

Fig. 10. Cytotoxicity of free DOX, Au-P(LA-DOX)-b-PEG-OH, and Au-P(LA-DOX)-b-PEGOH/FA NPs against (A) NIH-3T3 and (B) 4T1 cells (incubation time, 48 h).

Fig. 11. Effect of free FA on the viability of 4T1 cells incubated with Au-P(LA-DOX)-bPEG-OH/FA NPs for 48 h (DOX concentration: 34 mg/mL).

6074

M. Prabaharan et al. / Biomaterials 30 (2009) 60656075

b-PEG-OH micelles. This result indicates that FA-targeting ligands

present on the surface of Au-P(LA-DOX)-b-PEG-OH/FA micelles
played an important role in enhancing the cytotoxic effect by
binding the micelles with the over-expressed receptor located on
the surfaces of the 4T1 cells and subsequently increasing their
intracellular uptake as a result of the receptor-mediated endocytosis. In the presence of free DOX, the cell viability of the cultured
4T1 cells dramatically decreased, indicating that the cytotoxicity of
free DOX was much higher than that of the DOX-conjugated
micelles. As previously discussed, the fast diffusion of free DOX into
the cell nuclei might be the reason for this observation.
The presence of free FA in the medium may hamper the binding
between FA-conjugated micelles and folate receptors through
competitive interaction. To estimate the role of free FA on the
cellular uptake of Au-P(LA-DOX)-b-PEG-OH/FA micelles, 4T1 cells
were incubated with Au-P(LA-DOX)-b-PEG-OH/FA micelles (DOX
concentration: 34 mg/mL) in a culture medium containing
increasing concentrations of free FA. The cell viability in the presence of Au-P(LA-DOX)-b-PEG-OH/FA micelles increased with
increasing free FA concentration, indicating that the cytotoxicity of
Au-P(LA-DOX)-b-PEG-OH/FA micelles against 4T1 cells was inhibited by excess free FA (Fig. 11). The cell viability of Au-P(LA-DOX)-bPEG-OH/FA micelles was approximately 10% in the presence of
FA-free medium, but it was about 28 and 45% in the presence of
medium containing 0.5 and 1 mg/mL free FA, respectively. These
results conrm that free FA molecules inhibited the cellular uptake
of the Au-P(LA-DOX)-b-PEG-OH/FA micelles by competitive binding
to the folate receptors on the cancer cell surface.
4. Conclusions
FA-conjugated amphiphilic Au-P(LA-DOX)-b-PEG-OH/FA NPs
was synthesized as a tumor-targeted, anticancer drug delivery
nanocarrier. The anticancer drug DOX was conjugated onto the
hydrophobic inner shell of the NPs via an acid-labile hydrazone
linkage. The amount of DOX-conjugated onto the NPs was found to
be 17 wt%. Due to its amphiphilic multi-arm and globular structure,
Au-P(LA-DOX)-b-PEG-OH/FA NPs formed stable unimolecular
micelles composed of an Au core, P(LA-DOX) inner shell, and PEGOH/FA outer shell in aqueous solution. The size of micelles made
from the Au-P(LA-DOX)-b-PEG-OH/FA NPs was determined as 24
52 and 1025 nm by DLS and TEM, respectively. The release of DOX
from the Au-P(LA-DOX)-b-PEG-OH/FA NPs depended strongly on
the pH values of the medium. It was found that DOX released rapidly
at acidic pHs such as those encountered in tumor tissue and the
endocytic compartments of the tumor cell due to the hydrolysis of
hydrazone linkage. Flow cytometry and confocal image analysis
revealed that the extent of 4T1 cellular uptake for Au-P(LA-DOX)-bPEG-OH/FA micelles was greater than FA-free Au-P(LA-DOX)-bPEG-OH micelles due to the folate-receptor-mediated endocytosis
mechanism. The cytotoxicity of Au-P(LA-DOX)-b-PEG-OH/FA
micelles to 4T1 cells was higher than that of FA-free Au-P(LA-DOX)b-PEG-OH micelles, indicating that the FA-conjugated micelles have
the ability to selectively target to cancer cells. These results indicate
that the Au-P(LA-DOX)-b-PEG-OH/FA NPs could be a promising
anticancer nanomedicine to achieve better efcacy for chemotherapy. Finally, the Au-P(LA-DOX)-b-PEG-OH/FA NPs potentially
can be used for photothermal cancer therapy and contrast agent for
various imaging modalities (e.g., computed tomography (CT)
imaging), thereby making targeted cancer theranostics possible.
Acknowledgement
We acknowledge the nancial support from the University of
Wisconsin-Milwaukee.

Appendix
Figures with essential colour discrimination. Certain gures in
this article, in particular Figs. 1 and 9, are difcult to interpret in
black and white. The full colour images can be found in the on-line
version, at doi:10.1016/j.biomaterials.2009.07.048.

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