Nanomaterials and their applications in

healthcare
Sangeeta N. Kale
Fergusson College, Pune, India

ICS-UNIDO, SISSA workshop on Computer Design and Discovery of Potential

D sf D l i C t i s 8 12 J 2009
 Talk Layout
9 Introduction to Nanotechnology

9 Possible Applications envisaged / realised

(in biomedicine and other healthcare sectors )

9 Types of Nanomaterials

9 The Approach towards therapeutic applications

9 Case Studies for nanomaterials in:

™ Cancer-hyperthermia
™ Alpha-amylase inhibition
™ Drug delivery and targeting
™ Sustained drug release

9 Case Studies of role of nanomaterials for:

™Water purification
™Toxic gas sensing (industrial pollutants)
 What is ‘nano’?
Nano -a Greek word, meaning “dwarf”!
A nanometer is one thousandth of a micron or a billionth of a meter
Nanoparticles range from 1 to 100 nm

RBC ~7,000 nm
WBC ~10,000 nm

Bacteria: ~1,000 to
10,000 nm
Viruses: ~ 75 to 100 nm
Proteins: ~5 to 50 nm
DNA (width) is 2 nm
Hydrogen atom:~ 0.1 nm
 Fine arrangement of
atoms and molecules
and control over them
can do wonders..

To probe
To manipulate
To arrange
To control
Replicate..
 Bulk materials

Top-down approach

Surface to
volume ratio
increases
nanomaterials

Bottom-up approach

Atoms or molecules
 Cell labeling and imaging

Drug encapsulators

Biosensors
Therapeutic
Applications
Nanoreactors MRI contrast agents
Drug targeting agents Tissue repairing

Sustained drug delivery

drugs

Cosmetics superabsorbants

Cancer treatments Dendrimers for drug attachements

 Types, Forms of nanomaterials - applications
envisaged
Metallic particles: Superparamagnetic nps
- cancer-hyperthermia
- selective magnetic bioseparations
-coated with antibodies to cell-specific antigens,
for separation from the surrounding matrix
- membrane transport studies
- drug delivery
- MRI contrast agents

Gold shell nanoparticles:

To improves solubility of drugs
Reduce toxicity
Permits further conjugation
prevent the oxidation of the core

gold or silver core with silica shell: Silver, gold,

used in fluorescence imaging silver core- gold shell
 Types, Forms and applications envisaged
Carbon nanomaterials
(fullerenes and nanotubes)
Functionalized CNT-
for the therapeutic delivery

Quantum dots: tag multiple

biomolecules to monitor complex
cellular changes and events
associated with disease -future
pharmaceuticals and therapeutics

Quantum dots:
For bioconjugation
In retinas
For cell labeling
 Types, Forms and applications envisaged
Dendrimers :
Polymersied macromolecules

Highly branched structures - molecular

"hooks" - to attach cell identification
tags, fluorescent dyes, enzymes

Additionally,

Creation of interior cavities or channels

with properties different from those on
the exterior:
serve as vessels or hosts for guest
molecules.
…targeted drug and gene delivery
agents or nanoscale reactors for
catalysis
 Types, Forms and applications envisaged
Lipid-based nanoparticles
-pharmaceutical and cosmetic industries
– their ability to fuse with the cell
membrane and deliver molecules inside
the cells

polymer–protein –
to enhance protein stability
polymer–drug – drug delivery,
tumour targeting

Ceramic nanoparticles: inorganic

systems – Drug vehicles (if porous and
biocompatible), used in cosmetic
applications (ZnO,TiO2) – water
treatments (photocatalytic converters)
 The Approach

Synthesis:
Physical
properties
Physical
Chemical Characterization:
properties – Size
– Size distribution
Quality – Molecular weight In Vitro:
Purity – Morphology – Binding In Vivo:
Stability – Surface area – Pharmacology – Absorption
– Porosity – Blood contact – Pharmacokinetics
– Solubility properties – Serum half-life
– Surface charge density – Cellular – Protein binding
– Purity uptake – Tissue distribution
– Sterility – Cytotoxicity – Metabolism
– Surface chemistry – Excretion
– Stability – Safety
Therapeutic Benefits: Nanomaterials
impart:
Solubility (for insoluble drugs)
Solubility
Carrier for hydrophobic entities
Stability
Multifunctional capability
Specificity

Active and passive targeting

Toxicity

Ligands; size exclusion Efficacy

Reduced toxicity
 Case Studies …
• ZnO as alpha-amylase inhibitor

• Co-Ni-O as drug targeting and hyperthermia

agent

• Crosslinked polymer structures for sustained

drug release

• SnO2 nanoparticles for hazardous gas sensing

• ZnO as effective industrial dye-degrading

catalyst
ZnO nanoparticles as alpha-amylase inhibitors
For anti-diabetic action

S. Dhobale etal, JAP 2008

 ZnO nanoparticles as alpha-amylase inhibitors
• Amylase inhibitors, also known as starch blockers, contain substances
that prevent dietary starches from being absorbed by the body via
inhibiting breakdown of complex sugars to simpler ones - diabetes
control

• zinc oxide nanoparticles as possible alpha-amylase inhibitors.

• Co-precipitation method : Zinc oxide nanoparticles -1-thioglycerol

surfactant - size ~18 nm, Wurtzite structure.

• Cytotoxicity studies demonstrated that upto a dose of 20 mg/ml, ZnO

nanoparticles were non-toxic to the cells.

• Alpha-amylase inhibitory activity - exhibit 49% glucose-inhibition at

neutral pH and 35oC temperature. This inhibitory activity was similar to
that obtained with Acarbose (a standard alpha-amylase inhibitor)
 The basic testing mechanism : DNS assay

Starch (Complex a- amylase enzyme Glucose & maltose (simple

reducing sugars which are
polysaccharide) (Salivary amylase) easily absorbed & assimilated)

Colour intensity Concentration of

+
Reducing of product α reducing sugars in
the sample
sugars

3, 5- dinitrosalicylic acid 3-amino-5- nitrosalicylic

(DNS) acid

Control = (Salivary Amylase + Difference in

Starch + DW + colour intensity
DNS) indicates less
sugar production
Test = (Salivary Amylase + in the presence of
Starch + Inhibitor + inhibitors (ZnO
DNS) nanoparticles).
probable binding of ZnO with TG at –SH
sites forming the respective
carbocations, which facilitate
the attack of −NH2 group of the
amylase. proposed that the reaction of
amylase with ZnO-TG takes place
through bonding of −NH2 with the
primary carbocation

human fibrosarcoma HT-1080 and skin

carcinoma A-431 cell lines, mouse
primary fibroblast cells
cell viability 20 μg/ml
 50
56

Percent inhibiton
40

49 30

Percent inhibition
20
42 10
0
35 16 24 32 40o 48
Temperature ( C)

28
21
14

3 4 5 6 7 8
pH

Collaborators:
S.L. Lavare (Biotechnology, FCP, Pune) J. Appl. Phys. (2008)
C.V. Rode (NCL, Pune)
R. Kaul-Ghanekar (IRSHA, Pune)
Magnetic Nanoparticles for Cancer Hyperthermia

Journal of Biomedical Nanotechnology, 2007

Ceramics International, 2007

Nanomedicine, 2007
Nanotechnology, 2008
 Magnetic Nanoparticles for Cancer Hyperthermia
Malignant tumor cells/cancer cells are: 
Altered self cells that have escaped normal growth regulation 
mechanism, leading to disease, characterized by uncontrolled 
growth and spread of abnormal cells.

Normal Cells Cancer Cells

™ Growth is very orderly and  ™ Keep on reproducing
precise.    ™ Do not  stick together
™ Good Cell adhesion ™ Do not  die if they move to   
™ Cannot be placed wrongly another part of the body 
™ Pre‐programmed to    ™ Stay immature and continue 
reproduce 50‐60 times,  to multiply
maximum ™ Die at 45‐48oC
™ withstand 52oC
 Magnetic Nanoparticles for Hyperthermia
Mn
100 (a)

% Viability
80

60 LSMO
BSA:LSMO
R,A 40 Dextran:LSMO

20 HT-1080

Néel losses 0
0 20 40 60 80 100
Brown losses Concentration (μg/ml)
Tunable Tc

60

40
Treated
LSMO
60 (3)
Untreated
LSMO

Temperature ( C)
20
(1)
M (emu/gm)

o
-20
T = 300K
50
-40
(2)
-60
-2000 -1000 0 1000 2000
H (Oe)

40

30
Journal of Biomedical Nanotechnology, 2007
0 50 100 150 200 250 300
Ceramics International, 2007 Time (sec.)
Functionalized Nickel cobaltite nps for drug delivery
and cancer hyperthermia
Nearly monodispersed, superparamagnetic nickel cobaltite nanoparticles
(NiCo2O4) (NCO) synthesized by combustion method . Functionalized using a
biocompatible coat, namely, Mercapto-propionic acid (MPA)

On subjecting the NCO:MPA nanoparticle dispersion (0.1mg/ml) to a radio-

frequency absorption of 20 MHz, the nanoparticles get heated till 75oC within
two minutes, suggesting it to be a promising cancer hyperthermia agent

Further two different amino acids, namely Cysteine and Lysine were conjugated
to the NCO:MPA system

Evaluate the potential of MPA:NCO nanoparticles as possible drug-delivery as

well as drug-targeting agent,

Nanomedicine, 2007
MPA has been shown to conjugate with
NCO nanoparticles at carboxyl site,
leaving the carboxyl end of another
molecule free for further conjugation.
The MPA-MPA interaction occurs to
form S-S bonds in between.

The cytotoxicity studies showed cell

viability of ~ 100% upto 40μg/ml on SiHa
and B16F10 cell lines and on mouse
primary fibroblasts.
On subjecting the NCO:MPA nanoparticle dispersion (0.1mg/ml) to a
radio-frequency absorption of 20 MHz, the nanoparticles get heated
till 75oC

Nanotechnology, 2008
With amino-acids Cysteine and Lysine amino acids -
conjugated to MPA at - free carboxyl
conjugation ..
end via formation of a dimer or by an
electrostatic interaction, respectively.
 a) NCO, b) MPA, c) NCO:MPA,
d) NCO:MPA:cysteine
e) NCO:MPA lysine.
100

90
Absorbance (a.u.)

TGAfor NCO:MPA sample:

W eig h t (% )
decomposition of sulphides
80

70 and carboxylates at lower

temperature and metal-
60
carboxylates at higher
temperature
50
150 300 450 600 750 900
0
T e m p e ra tu r e C
(e)

(d)
(b) (c)
(a)
200 300 400 500 600 700 800
Wavelength (nm) Collaborators:
S.B. Ogale (NCL, Pune)
S.D. Dhole (Pune Univ)
Langmuir, under review S.D. Kulkarni (NCL, Pune)
R. Kaul-Ghanekar (IRSHA, Pune)
 COO ‐
‐
O
CO
‐

S
O

S S
CO

S
S S

COO
O
CO
O
S CO
S
COO‐ COO S COO‐
NCO COO S
O
The Bio-Chemistry … CO CO
S S O S

COO
COO
‐ S CO
O O‐
CO

S
S

S
COO

COO
‐

‐
Crosslinked polymer structures for sustained –
release of drugs / nanomaterials
Nanoscale polymer capsules can be designed to break down
and release drugs at controlled rates, to allow
differential release in certain environments, such as an
acid medium, and to promote uptake in tumors versus
normal tissues
 Preamble and Motivation

Traditional drug delivery - oral and intravenous routes largely inefficient due
to exposure of complete metabolic system Specificity needed –

Nanoparticles drug delivery demands are:

a) should be mesoporous b) responsive to some stimuli, to initiate the release
c) non-toxic d) should not obstruct functionality of the released molecules

Few initial intiatives:

DC Gupta etal 1998 Polymer Inter.

(nanocapsules of polysiloxanes releasing 2-pyridine aldoxime chloride)

Arrona G. etal 1998 Minerva Stomatol

(slow-release of antibiotic materials through polymers )

Wei Jia etal 2004 Journal Advances in Therapy

(Indapamide-based slow-release pellets)

W Wu etal 2007 J. Magn. Magn. Mater. (polysterene beads)

Jie Lu etal 2007 Small (mesoporous silica)

 Introduction
Particles released in sustained fashion could be fungicide, bactericide, insecticide,
herbicide, or even organic molecules and drugs, thereby hinting applications as novel
pesticides, fertilizers, food preservatives, drugs, dust-free and corrosion
free coatings and microbial-resistant materials.

Cage: Caged Entities:

Crosslinked polymer structures - Manganite (magnetic nanoparticles)
synthesized using polyvinyl alcohol CdS (luminescent nanoparticles)
and borax Trypsin (a drug)
 Synthesis of Crosslinked-Polymer
(LSMO:CL-PVA)
La0.7Sr0.3MnO3
Borax 4 % PVA 4% (in water)
Prepared by Citrate gel route,
(in water)
Particle size ~20 nm
Heat slowly less
than 100 OC,

Mix LSMO powder

Mix liquid 1:1 ratio
While preparing CL-PVA
With constant stirring

Constant stirring on shaker

Sequential Aliquots extracted
 Schematic of Interaction
PVA-water-soluble, non-toxic, biodegradable, Crosslinker, Boron is reported to
improve strength and flexibility of the PVA matrix
 FTIR Analysis of Crosslinked-Polymer

The signature at
848-1050 cm-1
((b)) persist in
(c), of B-O.

B-O-B vibration frequency gets modified upon CL-PVA formation, due

to borax working as a crosslinker to PVA matrix.
 Release Studies
For Manganites

12
(a) (b) (c) (d) (e) (f)
Mn concentration (ppm)

10
8
6
4
2
0
10 20 30 40 50 60
Time (min.)

For CdS nanoparticles

PL (at 542 nm, excitation at 365 nm)
Release Studies: For Trypsin enzyme
The Flow-Chart
Enzyme Action

Precursors used: Percloric acid,Trypsin (for standard OD), BSA from bovine pancreas

Take Supernatent Solution of 1 ml trypsin enzyme + CL-PVA

and
add 1 ml of Phosphate Buffer pH =7.4

Add BSA in 0.5ml

Keep it for 10 min. at Room Temp. and add in 10 ml water

Add 1ml of Percloric acid 20 %

Centrifuge the precipitate and take supernatent

Enzyme action Completed. Take OD of Enzyme at =280nm

 Release Studies: For Trypsin enzyme
UV absorption study - released enzyme :BSA

BSA as a protein did not denatured rapidly: indirect bio-safe nature

 Conclusions

¾ CL-PVA structures have been used to trap nanoparticles or a

trypsin for sustained - release/delivery applications.

¾ A magnetic system, namely La0.7Sr0.3MnO3, a luminescent

system, namely CdS and a bioenzyme, namely trypsin has
been studied to evaluate the candidature.

¾ AAS has been used to qualify the released manganite

nanoparticles and PL has been used to characterize the released
CdS nanoparticles.

¾ Colorimetric test has been done by interacting BSA with

released trypsin enzyme to confirm the released enzyme.

¾ We find a systematic release of these nanoparticles/enzyme

out of the CL-PVA. It is hence envisaged that CL-PVA would
be excellent carriers of nanomaterials/drugs that could be
released in a controlled fashion.
Mater Sc. Engg. C, under review
 Nanoparticle-based Drug Delivery to Localized Prostate
Cancer (Targeted Drug Delivery)

The ligands (green triangles) on

New generation binders for paints and
the surface of the nanoparticle fit
coatings
into the cell receptors, allowing
Corrosion - causes rust and the encapsulated drug molecules to
deterioration of metals and other enter the tumor cell after binding
materials.
SnO2 nanoparticles for sensing toxic sugar industry
gas pollutants
Work with inputs from Vasant Dada Sugar Institute, Pune

J. Phys. D: Appl. Phys (submitted)

 Introduction
Gas moities
Gas moities
Ag Ag

SnO2 SnO2

Tin oxide (SnO2) thin film for sensing Sulfur dioxide (SO2)

SO2 - major pollutant evolved from Sugar Industries.

Chemical spray pyrolysis technique - prepare SnO2 (Tin oxide) thin

film. which was either:

1. subjected to annealing by low energy irradiation

using Pulsed Laser source (248 nm, 3 Hz, 20 ns)
2. or furnace annealed at 600oC for 2 hrs.
Laser Annealing parameters

¾ Distance Between Sample and

Laser: 46 cm

¾ Time of radiation:1 min.

¾ Repetition Rate: 3 Hz

¾ Energy Density of Laser

:132 -146 mJ/cm2

¾ Wavelength of Laser: 248 nm

 90

(110)
(b)

(101)
(a) 80
(ii)

(211)
70
Intensity (a.u.)

(200)
(i)

( δR / R % )
60
(iii) 50
40
30
20
(ii)
10
0
100 150 200 250 300
(i) Temperature (K)

20 30 40 50 60 70 80
2θ (degree)

Valence band spectroscopy

measurement reveal that laser
annealing treatment introduces
band gap states due to
preferential removal of oxygen
species from the surface.
 Laser Annealed

As it is Furnace
Annealed

(a) (b) (c)

(d) (e)
 SnO2 as SO2 sensor at Room Temperature

Sample removed from gas chamber

2.02

1 :1

Sample exposed to Gas

2.00

1.98

1.96
2.5
1.94
(a)
(ii)
Rt Kohm

1.92
2.0

Sensitivity (S)
1.90

1.88

1.86
1.5
1.84

0 50 100 150 200

1.0
T im e (S e c .)

Modification of oxygen vacancies in (i)

0.5
the SnOx film upon exposing them to
oxidizing or reducing gases
0.0

Perfectly stoichiometric SnO2 will 0 5 10 15 20 25 30 35

exhibit large resistance with poor Time (sec.)
sensing property.

When the gas is of reducing type, such as SO2, a reduction reaction occurs and
there is a increase in oxygen vacancies, which decreases the SnO2 resistance.
Nanotechnology for water purification and waste
water treatment

R. Kitture et.al. Mater Sc. And Engg:C (submitted)

Rapid Photocatayltic industrial dye degradation studies: a
comparison betwee ZnO and TiO2 nanoparicles

OH• Radicals formed due to photogenerated holes and superoxides (O2 •)

formed due to photogenerated electrons further degrade the dye
adsorbed on the catalyst surface.
 Materials Used

• Methyl Orange , an dye - used in textiles, foodstuffs, pulp and

paper, and leather industry

• ZnO, TiO2 procured from CDH, Delhi, India were used as catalyst
without further purification

• P 25, procured from Degussa, was also used without further

purification

• For comparison, ZnO was synthesized by citrate gel method

Methyl Orange structure

UV-Visible Absorbance of degradation of Methyl Orange
after 20 minutes

MO
1.0 TiO2 bulk 100 % MO
TiO2
P 25
ZnO ZnO
bulk bulk CG
P25
ZnO bulk
0.8
Intensity (a.u.)

ZnO CG

0.6
46.7 %
0.4 46.7 %

0.2 21.7 %
6.7 %
0.0

300 400 500 600 700 800

Wavelength (nm)
ZnO bulk shows ~93% degradation within a short time span of 20 minutes,
whereas P 25, very well known catalyst shows ~78% degradation
UV-Visible Absorbance of degradation of Methyl Orange
after 20 minutes

1.0
a
0.8
Absorbance

0.6 46%
b 58%
0.4
c
0.2

0.0
d 99%

300 400 500 600 700 800

Wavelength (nm)
UV-Visible Absorbance of degradation of Methylene Blue
after 20 minutes

With With
MB @ MB @
1.0 0 min 20 min
ZnO
nano @
ZnO
bulk @
20 min 20 min

0.8
Absorbance

0.6 a
49%

0.4
b 77%
0.2
c
0.0
d 99%
300 400 500 600 700 800
Wavelength (nm)
 UV-Visible Absorbance of Catalysts

Work in progress..
Intensity (a.u.)

ZnObulk
ZnOCG
TiO2bulk
P25

300 400 500 600 700 800

Wavelength (nm)
• Methyl Orange, and Methylene Blue was degraded (decolorized) using
various oxide semiconductors in aqueous medium with the help of solar light.
• ZnO bulk showed highest degradation of 93% within short time period of 20
min
• This may be due to the wide absorbance of ZnO that extends over visible
range of solar spectrum.
 Nanotechnology holds great promise for the diagnosis and
treatment of human disease.
But.. Issues to be worried about:
toxicological and pharmacological profiles of nanomaterials

Risk : toxicity x exposure

Size and charge of most nanoparticles preclude their efficient clearance
from the body - <5.5 nm resulted in rapid and efficient elimination (Hak
Soo Choi et.al. Nature Biotech. 25, 2007)

Depending on the functional groups, toxicity varies

from very toxic to Benific

The biocompatibility and biodistribution of CNT appears to be dramatically

dependent on the surface functionalisation of these nanostructures,
making f-CNT viable material for a variety of biomedical applications (L.
Lacerda etal: NSTI conference, 2008)
 Final Conclusions and Comments
In a nutshell, oxide nanomaterials can yield
interesting manifestations in the domain
of healthcare:

• ZnO nps can be used as good alpha-

amylase inhibitor

• Metal oxide nanoparticles (Co-Ni-O) can

be fairly non-toxic and could be used as
hyperthermia and drug delivery agent

• Novel polymer-embedded nanoparticles

can work as good drug delivery and
sustained release cage structures.

• Nanoparticles of SnO2 could be used as

effective hazardous gas sensors for
industrial gas pollutants

• Industrial water can be effectively

treated with inorganic oxides for clean
water solutions
 Collaborators:
Thank you… ¾ B.B. Kale (C-MET, Pune)
¾ Ding Jun (NUS, Singapore)
¾ Samuel Lofland (Rowan
University, USA)
¾ S.D. Dhole (Pune
University)
¾ R. Kaul-Ghanekar (IRSHA)
¾ Beatrice H. (Rouen Univ,
France)

UGC-DAE Consortium, Indore

¾ Ram Janay Choudhary
¾ D.M. Phase
¾ R.R. Rawat
¾ V. Ganesan

National Chemical Laboratory

¾ S.B. Ogale
¾ I.S. Mulla
¾ C.V. Rode

For queries/suggestions:
sangeetakale2004@gmail.com