Nanomaterials and their applications in healthcare

Sangeeta N. KaleFergusson College, Pune, India

ICS-UNIDO, SISSA workshop on Computer Design and Discovery of Potential D s f D l i C t i s 8 12 J 2009

Talk LayoutIntroduction to Nanotechnology

Possible Applications envisaged / realised(in biomedicine and other healthcare sectors )

Types of Nanomaterials

The Approach towards therapeutic applications

Case Studies for nanomaterials in:Cancer-hyperthermiaAlpha-amylase inhibitionDrug delivery and targetingSustained drug release

Case Studies of role of nanomaterials for:Water purificationToxic 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 nmWBC ~10,000 nm

Hydrogen atom:~ 0.1 nm

Bacteria: ~1,000 to 10,000 nm

Viruses: ~ 75 to 100 nmProteins: ~5 to 50 nmDNA (width) is 2 nm

Fine arrangement of atoms and molecules and control over them can do wonders..

To probeTo manipulateTo arrangeTo controlReplicate..

?

Bulk materials

nanomaterials

Atoms or molecules

Top-down approach

Bottom-up approach

Surface to volume ratioincreases

Biosensors

Drug targeting agents

Sustained drug delivery

Drug encapsulators

drugs

MRI contrast agents

Cell labeling and imaging

Cancer treatments

Nanoreactors

Dendrimers for drug attachements

Therapeutic Applications

superabsorbants

Tissue repairing

Cosmetics

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 conjugationprevent the oxidation of the core

gold or silver core with silica shell: used in fluorescence imaging

Silver, gold, silver core- gold shell

Types, Forms and applications envisagedCarbon 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 bioconjugationIn retinasFor cell labeling

Dendrimers :Polymersied macromolecules

Highly branched structures - molecular "hooks" - to attach cell identification tags, fluorescent dyes, enzymes

Types, Forms and applications envisaged

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

Lipid-based nanoparticles -pharmaceutical and cosmetic industries – their ability to fuse with the cell membrane and deliver molecules inside the cells

Types, Forms and applications envisaged

polymer–protein –to enhance protein stabilitypolymer–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)

PhysicalCharacterization:– Size– Size distribution– Molecular weight– Morphology– Surface area– Porosity– Solubility– Surface charge density– Purity– Sterility– Surface chemistry– Stability

In Vitro:– Binding– Pharmacology– Blood contactproperties– Cellular uptake– Cytotoxicity

In Vivo:– Absorption– Pharmacokinetics– Serum half-life– Protein binding– Tissue distribution– Metabolism– Excretion– Safety

Synthesis:Physical properties

Chemical properties

QualityPurityStability

The Approach

Therapeutic Benefits:

Solubility (for insoluble drugs)

Carrier for hydrophobic entities

Multifunctional capability

Active and passive targeting

Ligands; size exclusion

Reduced toxicity

Solubility

Stability

Specificity

Toxicity

Efficacy

Nanomaterials impart:

• 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

Case Studies …

ZnO nanoparticles as alpha-amylase inhibitorsFor anti-diabetic action

S. Dhobale etal, JAP 2008

• 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)

ZnO nanoparticles as alpha-amylase inhibitors

Glucose & maltose (simple reducing sugars which are easily absorbed & assimilated)

a- amylase enzyme

(Salivary amylase)

Starch (Complex

polysaccharide)

3, 5- dinitrosalicylic acid (DNS)

Reducing sugars+

3-amino-5- nitrosalicylic acid

Colour intensity of product

Concentration of reducing sugars in the sampleα

Control

Test

(Salivary Amylase + Starch + DW + DNS)

(Salivary Amylase + Starch + Inhibitor + DNS)

Difference in colour intensity indicates less sugar production in the presence of inhibitors (ZnO nanoparticles).

=

=

The basic testing mechanism : DNS assay

human fibrosarcoma HT-1080 and skin carcinoma A-431 cell lines, mouse primary fibroblast cellscell viability 20 μg/ml

probable binding of ZnO with TG at –SH sites forming the respective carbocations, which facilitatethe attack of −NH2 group of the amylase. proposed that the reaction of amylase with ZnO-TG takes place through bonding of −NH2 with theprimary carbocation

16 24 32 40 480

10

20

30

40

50

Per

cent

inhi

bito

n

Temperature (oC)

3 4 5 6 7 8

14

2128

35424956

Per

cent

inhi

bitio

n

pH

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

Magnetic Nanoparticles for Cancer Hyperthermia

Ceramics International, 2007 Journal of Biomedical Nanotechnology, 2007

Nanomedicine, 2007

Nanotechnology, 2008

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

Growth is very orderly and precise.   Good Cell adhesionCannot be placed wronglyPre‐programmed to   reproduce 50‐60 times, maximumwithstand 52oC

Cancer Cells

Keep on reproducingDo not  stick togetherDo not  die if they move to   another part of the body Stay immature and continue to multiplyDie at 45‐48oC

Magnetic Nanoparticles for Cancer Hyperthermia

Magnetic Nanoparticles for Hyperthermia

Néel lossesBrown lossesTunable Tc

R,A

O

Mn

-2000 -1000 0 1000 2000-60

-40

-20

0

20

40

60

H (Oe)

M (e

mu/

gm)

Treated LSMO

Untreated LSMO

T = 300K

Ceramics International, 2007 Journal of Biomedical Nanotechnology, 2007

0 50 100 150 200 250 300

30

40

50

60 (3)

(2)

(1)

Tem

pera

ture

(o C)

Time (sec.)

0 20 40 60 80 1000

20

40

60

80

100

HT-1080

(a)

% V

iabi

lity

Concentration (μg/ml)

LSMOBSA:LSMO Dextran:LSMO

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,

Functionalized Nickel cobaltite nps for drug delivery and cancer hyperthermia

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

Cysteine and Lysine amino acids -conjugated to MPA at - free carboxyl end via formation of a dimer or by an electrostatic interaction, respectively.

With amino-acids conjugation ..

200 300 400 500 600 700 800

1 5 0 30 0 4 50 60 0 75 0 9 0 05 0

6 0

7 0

8 0

9 0

10 0

Wei

gh

t (%

)

T e m p e ra tu re 0C

(e)

(d)(c)(b)

Abs

orba

nce

(a.u

.)

Wavelength (nm)

(a)

Collaborators:S.B. Ogale (NCL, Pune)S.D. Dhole (Pune Univ)S.D. Kulkarni (NCL, Pune)R. Kaul-Ghanekar (IRSHA, Pune)

a) NCO, b) MPA, c) NCO:MPA, d) NCO:MPA:cysteine e) NCO:MPA lysine.

TGAfor NCO:MPA sample: decomposition of sulphides and carboxylates at lower temperature and metal-carboxylates at higher temperature

Langmuir, under review

SCO

OS

COO

‐

NCOS

COO SCOO‐

S

COO

S

COO‐

SCOO

S COO ‐S

COO

S

COO‐

SCO

OS

COO

‐S

COOSCOO‐

S

COO

S

COO‐

S

COO

S

COO‐

The Bio-Chemistry …

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

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)

Preamble and Motivation

Cage:Crosslinked polymer structures -synthesized using polyvinyl alcohol and borax

Caged Entities:Manganite (magnetic nanoparticles)CdS (luminescent nanoparticles)Trypsin (a drug)

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.

Synthesis of Crosslinked-Polymer (LSMO:CL-PVA)

La0.7Sr0.3MnO3Prepared by Citrate gel route, Particle size ~20 nm

Borax 4 % (in water)

PVA 4% (in water)

Heat slowly lessthan 100 OC,

Mix liquid 1:1 ratioWith constant stirring

Mix LSMO powderWhile preparing CL-PVA

Sequential Aliquots extractedConstant stirring on shaker

PVA-water-soluble, non-toxic, biodegradable, Crosslinker, Boron is reported to improve strength and flexibility of the PVA matrix

Schematic of Interaction

The signature at 848-1050 cm-1

((b)) persist in (c), of B-O.

FTIR Analysis of Crosslinked-Polymer

B-O-B vibration frequency gets modified upon CL-PVA formation, due to borax working as a crosslinker to PVA matrix.

10 20 30 40 50 60

0

2

4

6

8

10

12

Mn

conc

entr

atio

n (p

pm)

Time (min.)

Release StudiesFor Manganites

For CdS nanoparticlesPL (at 542 nm, excitation at 365 nm)

(a) (b) (c) (d) (e) (f)

Release Studies: For Trypsin enzymeThe Flow-Chart

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

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

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

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

CL-PVA structures have been used to trap nanoparticles or a trypsin for sustained - release/delivery applications.

A magnetic system, namely La0.7Sr0.3MnO3, a luminescent system, namely CdS and a bioenzyme, namely trypsin has been studied to evaluate the candidature.

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

Colorimetric test has been done by interacting BSA with released trypsin enzyme to confirm the released enzyme.

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.

Conclusions

Mater Sc. Engg. C, under review

Nanoparticle-based Drug Delivery to Localized Prostate Cancer (Targeted Drug Delivery)

The ligands (green triangles) on the surface of the nanoparticle fit into the cell receptors, allowing encapsulated drug molecules to enter the tumor cell after binding

New generation binders for paints and coatings

Corrosion - causes rust and the deterioration of metals and other materials.

SnO2 nanoparticles for sensing toxic sugar industry

gas pollutantsWork with inputs from Vasant Dada Sugar Institute, Pune

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

Introduction

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.

Gas moitiesGas moities

SnO2 SnO2

Ag Ag

Laser Annealing parameters

Distance Between Sample and Laser: 46 cm

Time of radiation:1 min.

Repetition Rate: 3 Hz

Energy Density of Laser :132 -146 mJ/cm2

Wavelength of Laser: 248 nm

100 150 200 250 3000

102030405060708090 (b)

(i)

(ii)

( δR

/ R

% )

Temperature (K)

20 30 40 50 60 70 80

(211

)

(200

)(1

01)

(110

)(iii)

(ii)

(i)

Inte

nsity

(a.u

.)

2θ (degree)

(a)

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

(a)

(d)

(c)

(e)

(b)

As it is

Laser Annealed

Furnace Annealed

SnO2 as SO2 sensor at Room Temperature

0 50 100 150 200

1.84

1.86

1.88

1.90

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1.94

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R t Koh

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T im e (Sec.)

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Sample exposed to GasSample removed from gas chamber

0 5 10 15 20 25 30 35

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(ii)

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itivi

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)

Time (sec.)

Modification of oxygen vacancies in the SnOx film upon exposing them to oxidizing or reducing gases

Perfectly stoichiometric SnO2 will exhibit large resistance with poor 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

300 400 500 600 700 800

0.0

0.2

0.4

0.6

0.8

1.0In

tens

ity (a

.u.)

Wavelength (nm)

6.7 %

21.7 %

46.7 %46.7 %

100 % MO TiO2 bulk P25 ZnO bulk ZnO CG

ZnO bulk shows ~93% degradation within a short time span of 20 minutes, whereas P 25, very well known catalyst shows ~78% degradation

MO TiO2bulk P 25 ZnO

bulkZnOCG

300 400 500 600 700 800

0.0

0.2

0.4

0.6

0.8

1.0

d

cb

a

58%

46%

99%

Abso

rban

ce

Wavelength (nm)

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

UV-Visible Absorbance of degradation of Methylene Blue after 20 minutes

300 400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

dcb

a49%

77%

99%

Abso

rban

ce

Wavelength (nm)

MB @ 0 min

MB @ 20 min

With ZnO nano @ 20 min

With ZnO bulk @ 20 min

UV-Visible Absorbance of Catalysts

300 400 500 600 700 800

Inte

nsity

(a.u

.)

Wavelength (nm)

ZnObulk ZnOCG TiO2bulk P25

• 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.

Work in progress..

Nanotechnology holds great promise for the diagnosis and treatment of human disease.

But.. Issues to be worried about: toxicological and pharmacological profiles of nanomaterials

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)

Risk : toxicity x exposure

Final Conclusions and CommentsIn 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

Thank you…

For queries/suggestions:sangeetakale2004@gmail.com

Collaborators:

B.B. Kale (C-MET, Pune)Ding Jun (NUS, Singapore)Samuel Lofland (Rowan University, USA)S.D. Dhole (Pune University)R. Kaul-Ghanekar (IRSHA)

Beatrice H. (Rouen Univ, France)

UGC-DAE Consortium, IndoreRam Janay ChoudharyD.M. PhaseR.R. RawatV. Ganesan

National Chemical LaboratoryS.B. Ogale I.S. MullaC.V. Rode