nano mgu feb15-2013-1

NANOSCIENCE AND NANOTECHNOLOGY IN MEDICINE :

Pre-clinical studies on targeted tumour therapy with iron oxide

nanoparticles

C.K.K.Nair

Dean of Research, Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla 689101.

S ymp o s i um o n Nanoscience and Nanotechnology: Fundamentals and Recent Trends o n 1 5 F e b r u a r y 2 0 1 3

Nanometer• One billionth (10-9) of a meter• Hydrogen atom 0.04 nm• Proteins ~ 1-20 nm• Feature size of computer chips 90 nm

(in 2005)• Diameter of human hair ~ 10 µm

Nanotechnology is the creation of USEFUL/FUNCTIONAL materials, devices and systems (of any useful size) through control/manipulation of matter on the nanometer length scale and exploitation of novel phenomena and properties which arise because of the nanometer length scale:

• Physical• Chemical• Electrical• Mechanical• Optical• Magnetic••

NANOTECHNOLOGY

• Quantum size effects result in unique mechanical, electronic, photonic, and magnetic properties of nanoscale materials

• Chemical reactivity of nanoscale materials greatly different from more macroscopic form, e.g., gold

• Vastly increased surface area per unit mass, e.g., upwards of 1000 m2 per gram

• New chemical forms of common chemical elements, e.g., fullerenes, nanotubes of carbon, titanium oxide, zinc oxide, other layered compounds

Unique Properties of Nanoscale MaterialsUnique Properties of Nanoscale Materials

Source: Nanoscale Materials in Chemistry, Ed. K.J. Klabunde, Wiley, 2001

• Spherical iron nanocrystals

• J. Phys. Chem. 1996, Vol. 100, p. 12142

• Adsorption is like absorption except the adsorbed material is held near the surface rather than inside

• In bulk solids, all molecules are surrounded by and bound to neighboring atoms and the forces are in balance. Surface atoms are bound only on one side, leaving unbalanced atomic and molecular forces on the surface. These forces attract gases and molecules Van der Waals force, physical adsorption or physisorption

• At high temperatures, unbalanced surface forces may be satisfied by electron sharing or valence bonding with gas atoms chemical adsorption or chemisorption

- Basis for heterogeneous catalysis (key to production of fertilizers, pharmaceuticals, syntheticfibers, solvents, surfactants, gasoline, other fuels, automobile catalytic converters…)

- High specific surface area (area per unit mass)

Trifluoperazine

CH2CH2

N+N+Cl-Cl-

Antidepressent and antipsychotic drug. It is known to give severe side effects such as photosensitization of skin and eye tissues

TFP pKa 4 and 8

Dimensions:TFP 25 Å SiO2 60 Å

Biophysical Chemistry, 109, 113-119, 2004

Spleen ce llstreated with TFP/SiO2

Three different cells from three different tissues of mice : Spleen, liver and

fibrosarcoma tumor were incubated with TFP and

TFP/SiO2

Biophysical Chemistry, 109, 113-119, 2004.

Energy• More Efficient And Cost Effective Technologies For Energy Production− Solar Cells− Fuel Cells− Batteries− Bio Fuels

Consumer Goods• Foods and beverages−Advanced packaging materials, sensors, and lab-on-chips for food quality testing

• Appliances and textiles−Stain proof, water proof and wrinkle free textiles

• Household and cosmetics− Self-cleaning and scratch free products, paints, and better cosmetics

NANOTECHNOLOGY APPLICATIONS

Information Technology

Smaller, faster, more energy efficient and powerful computing and other IT-based systems

Medicine/Health Care

•Cancer treatment•Bone treatment•Dentistry•Drug delivery•Appetite control•Drug development

•Tissue engineering/

•Regenetaive medicine

•Medical tools•Diagnostic•Imaging

MEDICAL APPLICATIONS

• Cancer Nanotechnology (i) Dignosis using Quantum Dots (ii) Tumor Targeted Delivery (iii) Imaging (iv) Cancer Gene Therapy

• DNA Vaccines for parasitic, bacterial and viral diseases

• Oral and pulmonary routes for systemic delivery of proteins and peptides

• Nanotechnology in Tissue Engineering

NANOTECHNOLOGY IN DRUG DELIVERY - ADVANTAGES

• Prevention of drug from biological degradation

• Effective Targeting• Patient Compliance• Cost effectiveness• Product life extension

Biosensor

• High specificity• Direct, fast

response• High sensitivity• Single molecule

and cell signal capture and

detection

Ru bPy 3

• Probe molecules for a given target can be attached to CNT tips for biosensor development

• Electrochemical approach: requires nanoelectrode development using PECVD grown vertical nanotubes

• The signal can be amplified with metal ionmediator oxidation catalyzed by Guanine.

• CNT tips are at the scale close to molecules

• Dramatically reduced background noise

Traditional Macro- or Micro- Electrode

NanoelectrodeArray

Nanoscale electrodes create a dramatic improvement in signal detection over traditional electrodes

Electrode

• Scale difference between macro-/micro- electrodes and molecules is tremendous

• Background noise on electrode surface is therefore significant

• Significant amount of target molecules required

• Multiple electrodes results in magnified signal and desired redundance for statistical reliability.

• Can be combined with other electrocatalytic mechanism for magnified signals.

Nano-Electrode

Insulator

Electrochemical Detectionof DNA Hybridization

- by AC Voltammetry

1st, 2nd, and 3rd scan in AC voltammetry 1st – 2nd scan: mainly DNA signal2nd – 3rd scan: Background

2nd and 3rd

Lower CNT Density Lower Detection Limit J. Li, H.T. Ng, A. Cassell, W. Fan, H. Chen, J. Koehne, J. Han, M. Meyyappan, NanoLetters, 2003, Vol. 3, p. 597.

30 dies on a 4” Si wafer

200 m300 m

Potential applications:(1) Lab-on-a-chip applications(2) Early cancer detection(3) Infectious disease detection(4) Environmental monitoring(5) Pathogen detection

Target Molecule

1. Chen, G.Y., Thundat, T. Wachter, E. A., Warmack, R. A., “Adsorption-induced surface stress and its effects on resonance frequency of microcantilevers,” J. Appl. Phys 77, pp. 3618-3622 (1995).

2. Ratierri, R. et al., “Sensing of biological substances based on the bending of microfabricated cantilevers,” Sensors and Actuators B 61, 213-217 (1999).

3. Fritz, J. et al. “Translating Biomolecular Recognition into Nanomechanics,” Science 288, 316-318 (2000).

4. Wu, G. et al. “Origin of nanomechanical cantilever motion generated from biomolecular interactions,” PNAS 98(4), 1560-1564 (2001).

Thiolated ssDNA

5’-HS ATCCGCATTACGTCAATC

TAGGCGTAATGCAGTTAG-5’(Complementary Strand)

AuSelf-Assembly of ssDNA

PB = Sodium Phosphate Buffer

Wu, G. et al. “Origin of nanomechanical cantilever motion generated from biomolecular interactions,” PNAS 98(4), 1560-1564 (2001).

Probe ssDNA Target ssDNA

Wu, G. et al. “Origin of nanomechanical cantilever motion generated from biomolecular interactions,” PNAS 98(4), 1560-1564 (2001).

Time [min]

0 60 120 180 240

Injections

[HSA] = 1 mg/ml[fPSA]

6 ng/ml

60 ng/ml

No PSA Ab ([fPSA] = 60 g/ml)

HP only ([HP] = 1 mg/ml)

No PSA

Time [min]

0 60 120 180 240 300

[BSA] = 1 mg/ml

Injections

60 g/ml

6 g/ml

60 ng/ml

6 ng/ml

No fPSANo PSA Ab

([fPSA] = 60 g/ml)

[fPSA]

AuDTSSP

Rabbit Anti-Human PSA

Analyte

AuDTSSP

Rabbit Anti-Human PSA

Analyte

Wu, G. et al., “Bioassay of Prostate Specific Antigen (PSA) Using Microcantilevers,”

Nature Biotechnology (Sept., 2001)

HSA: Human Serum Albumin

HP: Human Plasminogen

fPSA: free PSA

cPSA: complex PSA

DNA microarrays can be manufactured by:• Photolitography (Affymetrix, Febit,

Nimblegen)• Inkjet (Agilent, Canon)• Robot spotting (many providers)

Nanochip− A lab on chip integrates one or more

laboratory operation on a single chip− Provides fast result and easy operation− Applications: Biochemical analysis

(DNA/protein/cell analysis) and bio-defense

DNA MICROARRAYS CONSIST OF 100 - 1 MILLION DNA PROBES ATTACHED TO A SURFACE OF 1 CM BY 1 CM (CHIP).

By hybridisation, they can detect DNA or RNA:

If the hybridised DNA or RNA is labelled fluorescently it can be quantified by scanning of the chip.

Fabrication via Printing DNA sequence stuck

to glass substrate DNA solution pre-

synthesized in the lab Fabrication In Situ

Sequence “built” Photolithographic

techniques use light to release capping chemicals

365 nm light allows 20-m resolution

FabricationFabrication

DNA Microarrays• Each probe consists of thousands of strands of

identical oglionucleotides– The DNA sequences at each probe represent important

genes (or parts of genes)• Printing Systems

– Ex: HP, Corning Inc.– Printing systems can build lengths of DNA up to 60

nucleotides long– 1.28 x 1.28+ cm glass wafer

• Each “print head” has a ~100 m diameter and are separated by ~100 m. ( 5,000 – 20,000 probes)

• Photolithographic Chips– Ex: Affymetix – 1.28 x 1.28 cm glass/silicon wafer

• 24 x 24 m probe site ( 500,000 probes)– Lengths of DNA up to 25 nucleotides long– Requires a new set of masks for each new array type

GeneChip

PRACTICAL APPLICATION OF DNA MICROARRAYS

• DNA Microarrays are used to study gene activity (expression)– What proteins are being actively produced by a group of cells?

• “Which genes are being expressed?”• How?

– When a cell is making a protein, it translates the genes (made of DNA) which code for the protein into RNA used in its production

– The RNA present in a cell can be extracted– If a gene has been expressed in a cell

• RNA will bind to “a copy of itself” on the array• RNA with no complementary site will wash off the array

– The RNA can be “tagged” with a fluorescent dye to determine its presence

• DNA microarrays provide a high throughput technique for quantifying the presence of specific RNA sequences

The Process

CellsPoly-ARNA

10% Biotin-labeled UracilAntisense cRNA

Fragment (heat, Mg2+)

Labeledfragments

Hybridize Wash/stain Scan

(In-vitro Transcription)

Hybridization and Staining

GeneChip BiotinLabeled cRNA

SAPEStreptavidin-phycoerythrin

Hybridized Array

DNA SequencingUsing Nanopores

Goal: Very rapid gene sequencing

(~2nm diameter)

- Nanopore in membrane

- DNA in buffer

- Voltage clamp

- Measure current

-hemolysin pore

Axial View Side View

(very first, natural pore)

Open nanopore

DNA translocation event

• When there is no DNA translocation, there is a background ionic current

• When DNA goes through the pore, there is a drop in the background signal

• The goal is to correlate the extent and duration of the drop in the signal to the individual nucleotides

After a decade of using protein pores, efforts are underway in many groups to develop synthetic pores (such as in Si3N4)

• Interaction with single nuclotides

- ~20 nucleotides in HL simultaneously

• Slower translocation

- 1-5 s /nucleotide in HL

• Resistance to extreme conditions

- Temperature

- Voltage

• - hemolysin is toxic and hard to work with

• Voltage-clamp amplifier designed to measure pA level currents

• Fast (up to 1GHz) data acquisition• Software for automatic blocking event

detection and recording

Voltage ClampAmplifier

nanopore chip

KClKCl

Data Acquisition

Spontaneous Blocking Events with Smaller NASA Pores

+200 mV

GGA A A A

Present Future

A AG G G G

• Tree-like polymers, branching out from a central core and subdividing into hierarchical branching units

- Not more that 15 nm in size, Mol. Wt very high- Very dense surface surrounding a relatively

hollow core (vs. the linear structure in traditional polymers)

• Dendrimers consist of series of chemical shells built on a small core molecule

- Surface may consist of acids or amines means to attach functional Groups control/modify properties

- Each shell is called a generation (G0, G1, G2….)- Branch density increases with each generation- Contains cavities and channels can be used to trap guest

molecules for various applications.

Courtesy of: http://www.uea.ac.uk/cap/wmcc/anc.htm

• Desired features of effective drug delivery- Targeted delivery, controlled release (either timed or in response to

an external signal)

• Desirable characteristics of dendrimers- Uniform size - Water Solubility- Modifiable surface functionality - Availability of internal cavity- Control of molecular weight - Control of the surface and

internal structure

• Number of different drugs can be encapsulated in dendrimers and injected into the body for delivery

- Incorporating sensors would allow release of drugs where needed

• Gene Therapy- Current problem is getting enough genes into enough cells to make a

difference. Using viruses for this triggers immune reactions. Dendrimers provide an alternative without triggering immune response

• Cancer Therapy; Antimicrobial and Antiviral Agents

Future Possiblities: Oxygen Selective Pump

http://www.foresight.org/Nanomedicine/

Respirocytes: A Mechanical Artifical Red Blood Cell

•Bloodborne spherical 1-micron diamondoid 1000-atm pressure vessel •Active pumping powered by endogenous serum glucose•Able to deliver 236 times more oxygen to the tissues per unit volume than natural red cells and to manage carbonic acidity

http://www.foresight.org/Nanomedicine/Respirocytes.html

Cancer• Cancer is one of the most common diseases

in the developed world:• 1 in 4 deaths are due to cancer• 1 in 17 deaths are due to lung cancer• Lung cancer is the most common cancer in

men• Breast cancer is the most common cancer

in women• There are over 100 different forms of

cancer• The division of normal cells is precisely

controlled. New cells are only formed for growth or to replace dead ones.

• Cancerous cells divide repeatedly out of control even though they are not needed, they crowd out other normal cells and function abnormally. They can also destroy the correct functioning of major organs.

Cancer

Defined as the disturbance Defined as the disturbance of growth characterized by of growth characterized by excessive proliferation of cells excessive proliferation of cells without apparent relation to without apparent relation to the physiological demand of the physiological demand of the organ involved. the organ involved.

Newspapers, magazines, radio, and television are reporting discoveries and breakthroughs attributing one form of cancer or another to a specific gene.

Cancer of the breast, colon, prostate, and many other sites in the body are being connected to specific genes... But the meaning of this isn't always clear. Cancer is one of the most common and severe problems of clinical medicine. Cancer is not a single disease but rather a name applied to a great variety of malignant tumors that are formed by the same basic process of uncontrolled growth. Cell proliferation results in a mass that invades neighboring tissues and may metastasize to more distant sites. Some cancers, however, such as blood cancers, do not form tumors. Many aspects of cell function are controlled by a balance of positive and negative signals received from inside and outside the cell. In normal tissues, there is a balance between cell proliferation and cell death. In tumor, this balance is lost. CANCER HAS BOTH GENETIC AND ENVIRONMENTAL CAUSES.

Cancer

RISK FACTORS FOR CANCER

Environmental Factors

Tobacco Alcohol

Lack of Nutrients

Sociodemographic Factors

Ten Leading Sites of Cancer (1998)

AAR SITE SITE AAR

10.04 Lung Cervix Uteri 26.11

8.02 BOT Breast 19.37

5.8 Py.Fossa Esophagus 4.52

5.75 Esophagus Ovary 4.15

4.88 Larynx Myel.Leuk 3.21

4.15 Oth. Mouth Vagina 2.03

3.7 Myel.Leuk Hypopharyx 2

2.8 Hypopharyx Oth. Mouth/ Brain

2.74 Tonsil Lung/Other Tongue

2.59 Brain Lymp.Leuk 1.43

10.85.5 10.33.5 9.24.6 8.2 8.5 13.6 12 2220.8

38 3764.576.2

189 178.2

0- 5- 10-

MALE FEMALE

Age Specific Incidence Rates/lakh

Population for all Sites1998

Estimated cancer incidence of males in five cities - 2008

CANCER INCIDENCE & MORTALITY WORLD WIDE

Year wise total cancer prevalence in India [ICMR, 2006; ICMR, 2009].

Cancer prevalence in five metropolitan cities of India [Marimuthu, Projection of cancer incidence in five cities and cancer mortality in India. Indian J Cancer 45, 4-7.2008].

CANCER – THE SECOND LEADING CAUSE OF DEATH

World• Every year- 10 million diagnosed 6 million die

Worldwide - Lung Cancer (12.3 %) Breast Cancer(10.4%) Colorectal Cancer(9.4%)

Death from cancer - Lung (17.8%) Stomach (10.4%) Liver (8.8 %)

India3.4 % of all deaths – cancer7 lakh new cases detected every year

Males - mouth/oropharynx oesophagus stomach Lower respiratory tract Females - Cervix Breast mouth/oropharynx oesophagus

Oral cancer

• 50 – 70% of all cancers diagnosed in india• Risk factors Tobacco Alcohol Precancerous lesions Cultural patternsPrevention

Cancer cervix• Most common cancer of women –developing countries• Causative agent - HPV• Risk Factors• Prevention and control

Breast Cancer• commonest cause of death of middle aged women-developed

countries (35 – 50 yrs)• Risk factors• Prevention

Lung cancer Stomach Cancer

• Most common cancer in the world

• Risk factors• Prevention

• Worlds second most common cancer

• Risk Factors• prevention

Environmental Genetic• Tobacco• Alcohol• Dietary Factors• Occupational exposures• Viruses• Parasites• Customs ,habits, Lifestyles• Others – sunlight, pollution, drugs

Eg• Retinoblastoma in Children• Leukemia in Mongols

CAUSES OF CANCER

Dr Suwas Darvekar

Common sites of oral cancerThe most common sites of the oral cancer is the tongue and the floor of the mouth. The other common sites are buccal vestibule, buccal mucosa, gingiva and rarely hard and soft palate. Cancer of bucco-pharyngeal mucosa is common in smokers.

PRECANCEROUS LESIONSLuekoplakia Erythroplakia Sub mucous fibrosis

CANCERS OF Tongue, Cheek, Subbuccal mucosa, Buccal vestibule

CANCER CONTROLPrimary Prevention Secondary Prevention• Control of tobacco &

alcohol consumption• Personal Hygiene• Radiation• Occupational Exposures• Immunisation• Foods & drugs• Air pollution• Treatment of precancerous

lesion• Legislation• Cancer Education

• Cancer Registration Hospital based registries Population based registries• Early detection of cases• Treatment

Danger Signals• A lump or a hard area in breast• A change in wart or mole• A persistent change in bowel habits• A persistent cough or hoarseness• Excessive loss during menstrual periods or loss of

blood outside usual dates.• Blood loss from any natural orifice• A swelling that does not get better• Unexplained loss of weight

Cancer Screening

Methods• Mass Screening by comprehensive cancer detection examination• Mass Screening at single sites• Selective screening - for those at special risk

• Pre malignant lesion can be identified• Most cancers are localized in initial stages• 75% occurs at accessible body sites

• Screening of cancer cervix – Pap Smear

• Screening of breast cancer Breast self examination(BSE) Palpation Thermography MammographyScreening of lung cancer Chest radiograph Sputum Cytology

Oral cancer

• 50 – 70% of all cancers diagnosed in india• Risk factors Tobacco Alcohol Precancerous lesions Cultural patternsPrevention

Cancer cervix• Most common cancer of women –developing countries• Causative agent - HPV• Risk Factors• Prevention and control

Breast Cancer• commonest cause of death of middle aged women-developed

countries (35 – 50 yrs)• Risk factors• Prevention

Lung cancer Stomach Cancer

• Most common cancer in the world

• Risk factors• Prevention

• Worlds second most common cancer

• Risk Factors• prevention

What causes cancer?• Cancer arises from the mutation of a normal

gene.• Mutated genes that cause cancer are called

oncogenes.• It is thought that several mutations need to

occur to give rise to cancer• Cells that are old or not functioning properly

normally self destruct and are replaced by new cells.

• However, cancerous cells do not self destruct and continue to divide rapidly producing millions of new cancerous cells.

Carcinogens• Ionising radiation – X Rays, UV light

• Chemicals – tar from cigarettes

• Virus infection – papilloma virus can be responsible for cervical cancer.

• Hereditary predisposition – Some families are more susceptible to getting certain cancers. Remember you can’t inherit cancer its just that you maybe more susceptible to getting it.

GROWTH OF NORMAL CELLS• Growth means size increase and proliferation• • Not all adult cells can proliferate• • Special reserve cells retain proliferation potenitial• – Embryonic stem cells can make any cell in the body• – Although, many stem cells are committed and

have limited potential. i.e. can produce all the intestinal epithelial cells.

• – Proliferation requires the cell cycle• G0,G1,S,G2, and M phase

CAUSES OF CANCER• DNA Mutations– Radiation – other environmental (tobacco, alcohol, radon, asbestos, etc)– Random somatic mutations– Inherited germ line mutations• Genetic predisposition-– Rb, p53, APC, CDKN2A, BRCA1, BRCA2» Will discuss these later in a pathway context• Infectious agents– Viral• HPV – cervical cancer• Hepatitis – liver cancer– Vaccines have been developed and are extremely effective – not available– Bacterial• H. pylori – stomach cancer

INHERITED CANCERS - a small percentage of many cancers

• Breast cancer– ~3% cases between 36 and 45 years of agehave a BRCA1 mutation– ~3% cases between 36 and 45 years of age have a BRCA2 mutation• 1/500 people have a BRCA1 mutation–There may be other breast cancer genes– and many cancers are random

Types of genes which may mutate to cause cancer:

• Tumour suppressor genes

• oncogenes• DNA repair genes• telomerase• p53

cancer is a disease of the cell cycle

Tumour Growth or neoplasia

Pathways that control colorectal tumorigenesis. Mutations in the APC/b-catenin pathway initiate the neoplastic process, resulting in small benign tumors (adenomas). These tumors progress, becoming larger and more dangerous, as mutations in other growth-controlling pathway genes (such as K-Ras, B-RAF, PIK3CA, or p53) accumulate. The process is accelerated by mutations in stability genes. The top line indicates potential clinical applications of knowledge of these pathways.

Hallmarks of CancerSix changes for cancer – found in most, if not all, cancers

1. Self-sufficiency in growth signals

2. Insensitivity to growth-inhibitory signals

3. Evasion of apoptosis

4. Limitless replicative capacity

5. Sustained angiogenesis

6. Tissue invasion and metastasis

Major types of cancers

• Over 200 types of cancer are known grouped into major categoriesCarcinomas SarcomasMelanomasTeratomasLeukemias and Lymphomas

Tumour suppressor genes• The gene’s normal function is

to regulate cell division. Both alleles need to be mutated or removed in order to lose the gene activity.

• The first mutation may be inherited or somatic.

• The second mutation will often be a gross event leading to loss of heterozygosity in the surrounding area.

oncogenes

• Cellular oncogene c-onc• Viral oncogene v-onc• Proto-oncogene, activated by mutation

to c-onc

Proto-oncogene activation

Types of proto-oncogene

• Growth factore.g. SIS oncogene (PDGF)

Types of proto-oncogene

• Nuclear transcription factorse.g. MYC

p53• suppresses progression

through the cell cycle in response to DNA damage

• initiates apoptosis if the damage to the cell is severe

• acts as a tumour suppressor

• is a transcription factor and once activated, it represses transcription of one set of genes (several of which are involved in stimulating cell growth) while stimulating expression of other genes involved in cell cycle control

Transformation is a multistep

process

Transformation is a multistep process

The environment:

Some environmental agents associated with cancer are:

• Viruses • Tobacco smoke • Food • Radiation • Chemicals • Pollution

VirusesViruses—mostly in the form of DNA viruses—have been causally linked to cancer.

• human papillomaviruses—primarily types 16 and 18, which are sexually transmitted—have been linked to cervical cancer;

• more than 25 other types of papillomaviruses have been linked to cancer as well

• hepatitis B and C—linked to cancer of the liver • human immunodeficiency virus (HIV)—linked

to Kaposi's sarcoma and lymphoma • retroviruses—linked to cancers in animals

other than humans

MODALITIES OF CANCER TREATMENT LOCAL THERAPEUTIC MODALITIES

SURGERY RADIOTHERAPY

SYSTEMIC FORMS OF TREATMENT

CHEMOTHERAPY ENDOCRINE THERAPY IMMUNOTHERAPY

GENE THERAPY

Cost of cancer per year in U.S.

Targeted drug delivery to tumour using

magnetic nanoparticles:

Preclinical studies with Fe3O4 – doxorubicin nanoparticles

Nanoparticles for targeting drugs to tumour

- Fe2O3 nanoparticles coated with polymer – polyoxyethylene 25 propylene glycol stearate - Binding Drugs - Doxyrubicin , Sanazole

- Effect of the complexes in vitro on EAC cells and DLA cells

- Administration to mice bearing DLA solid tumour on hind limbs for 7 days.

Parameter studied: Tumour volume reduction

Dose-Effect Curve

Log Dose

% with Maximal

Effect Adverse Effects

Desirable Dose Range

DOXYRUBICIN. HCL – MWT 580

Doxorubicin (Adriamycin)Doxorubicin (Adriamycin)

Prevent DNA replicationPrevent DNA replication

hair loss, mouth sores, n/v, lowered blood counts (WBCs, RBCs and platelets),

skin damage if drug leaks out of vein during infusion (necrosis)*

damage to the heart muscle (cardiotoxicity from free radicals)

Common Side Effects:

intercalation of DNA

Cancer chemotherapy - the agent exerts anticancer action through cytotoxic mechanisms

Common problems encountered: – Poor selectivity toward the target cancer tissue– Harm normal cells as well– Sub-therapeutic drug levels at tumor site due to varied

biodistribution, fail to eradicate the tumour, stimulate overgrowth of resistant malignant cells

– Possess irreversible toxic side effects. Cardio, neuro, renal toxicities

• Specific targeting to tumour improves the anticancer potential of chemotherapeutic agents

• Targeting by magnetic particles - a unique opportunity to treat tumors due to magnetic responsiveness.

• Antitumor activity of water dispersible Fe3O4 nanoparticles (coated with Poly Vinyl Pyrolidone (PVP) and

Poly oxy ethylene 25- propylene glycol stearate (POES)) (Fe3O4-PVP-POES) complexed with Doxorubicin (Fe3O4-PVP-POES-Doxo).

• Effect of complexing Doxorubicin with Fe3O4-PVP-POES on its cardio toxic properties.

Water dispersible Fe3O4 nanoparticlescarring doxorubicin for cancer therapyO.D.Jayakumar, R.Ganguli, A.K.Tyagi , D.K.Chandraseharan and C.K.K.NairJournal of Nanosciene and Nanotechnology, 9, 6344-6348, 2009.

Water dispersible Fe3O4 Nanoparticles Carrying Doxorubicin for Cancer Therapy: Journal of Nanoscience and Nanotechnology, 2009, 9, 6344-6348.

• XRD pattern of Fe3O4 nanoparticles coated with PVP-POES (a) and PVP + POES and complexed with Doxorubicin (b).

• Inset shows the DC magnetization vs field of Fe3O4 nanoparticles coated with PVP measured at room temperature.

• XRD pattern of Fe3O4 nanoparticles coated with PVP-POES (a) and PVP + POES and complexed with Doxorubicin (b).

• Inset shows the DC magnetization vs field of Fe3O4 nanoparticles coated with PVP measured at room temperature.

TEM images of Fe3O4-PVP-POES nanoparticles and Fe3O4-PVP-POES-Doxo complex.

(a, b):Fe3O4-PVP-POES

(c, d):Fe3O4-PVP-POES-Doxo

TEM images of Fe3O4-PVP-POES nanoparticles and Fe3O4-PVP-POES-Doxo complex.

(a, b):Fe3O4-PVP-POES

(c, d):Fe3O4-PVP-POES-Doxo

For Doxorubicin, the concentrations - A, B and C – corresponding to 20, 50 and 100 micrograms/ml; for the Fe3O4-PVP-POES nanoparticles the concentrations were 100, 250 and 500 micrograms/ml; and for the nanoparticle-doxorubicin complex the concentrations were 20, 50 and 100 micrograms/ml with respect to doxorubicin and 100, 250 and 500 micrograms/ml with respect to Fe3O4-PVP-POES.

Fe3O4-PVP-POES-Doxo exhibited more toxicity towards DLA cells

Cytotoxic effect on DLA cells

Induction of apoptosis Apoptotic index of Fe3O4-PVP-POES-Doxorubicin was higher than that of doxorubicin or Fe3O4-PVP-POES. Induction of apoptosis Apoptotic index of Fe3O4-PVP-POES-Doxorubicin was higher than that of doxorubicin or Fe3O4-PVP-POES.

For Doxorubicin, the concentrations – A and B – corresponding to 50 and 100 micrograms/ml; for the Fe3O4-PVP-POES nanoparticles the concentrations were 250 and 500 micrograms/ml; and for the nanoparticle-doxorubicin complex the concentrations were 50 and 100 micrograms/ml with respect to doxorubicin and 250 and 500 micrograms/ml with respect to Fe3O4-PVP-POES.

Characteristic morphology of normal cell (A) and apoptotic cell (B)

Effect on tumor growth • Daltons Lymphoma solid

tumor on hind limbs of mice.

• The treatments were started on the 13th day after tumor tranplantation.

• Targeting Doxorubicin - Fe3O4 nanoparticles to tumor site - by an external magnetic field - keeping a magnet at the tumor site for 15 minutes - after the oral administration of the complex, for 7 consecutive days.

Effect on tumor growth• The growth of the

tumor decreased in all treated animals during the period of administration.

• Regression in tumor growth on administration with Doxorubicin -Fe3O4 nanoparticle complex (Fe3O4-PVP-POES-DOXO) in conjunction with magnetic treatment.

• Decrease in GSH - due to the oxidative stress induced by the drug.

• Magnet mediated targeting of the drug nanoparticle complex to the tumor site decreased the circulating levels of the drug complex - helped to maintain normal levels of GSH in heart tissue.

Effect on cardio toxicity

n s indicate not significant and *** indicate p <0.001 when compared with untreated control.

• Increase in the MDA level - due to oxidative stress induced by the drug.

• Magnet mediated targeting of the drug nanoparticle complex to the tumor site decreased the circulating levels of the drug complex - normal levels of MDA in heart tissue.

Effect on cardio toxicity

n s indicate not significant, * indicate p <0.05 and *** indicate p <0.001 when compared with untreated control.

OXIDATIVE ENXYME THERAPY USING MAGNETIV NANOPARTICLES

D-Alanine --- (DAO) ------------→Pyruvic acid + H2O2FTIR spectra of Fe2O3 magnetic nanoparticles FTIR spectra of Fe2O3 -DAO complex

XRD pattern of Fe2O3 nanoparticles coated with PVP XRD pattern of Fe2O3

-DAO complex

D-aminoacid oxidase-Fe2O3 nanoparticle complex mediated antitumor activity in Swiss Albino mice.

S.A.Divakaran, K.M.Sreekanth, K.V.Rao and C.K.K.Nair, Journal of Cancer Therapy, 2, 666-674 2011.

Effect of administration of Fe2O3 nanoparticles coated with PVP and complexed with DAO and magnetic targeting on DLA solid tumor growth on hind limb of mice. 1 represent animals on the initial day of commencement of the experiment, 1a & 1b represents untreated control animals, 2a & 2b represents animals treated with Fe2O3- DAO, 3a& 3b represents animals treated with D- alanine. 4a & 4b represents animals treated with Fe2O3-

DAO and D- alanine without magnetic treatment, 5a&5b represents animals treated with Fe2O3- DAO and D- alanine with magnetic treatment. The suffix ‘a’ indicates 4th day of treatment and ‘b’ indicates15th day of treatment

Journal of Cancer Therapy, 2, 666-674 2011.

OXIDATIVE ENXYME THERAPY OF TUMOUR USING MAGNETIV NANOPARTICLES

Representative images of DLA cells after comet assay. a) The untreated DLA cells. b) DLA cells treated with Fe2O3-DAO and D-alanine showing Fan-like comets indicative of apoptosis c). DLA cells treated with enzyme (DAO) and D-alanine showing DNA damaged and apoptotic comets and d) DLA cells treated with Fe2O3-DAO e) DLA cells treated with D-alanine.

Treatments Apoptoic index (%)

Control 0

0.2M D-Alanine + DAO (0.36U)

85.2± 5.55 a

0.2 M D-Alanine + Fe2O3-DAO (0.36U)

95.5±3.53a

0.2M D-Alanine 6.5± 2.12b

Fe2O3-DAO (0.36U) 11.0± 1.41a

Apoptoic index in DLA cells treated with Fe2O3-DAO in presence of D-alanine. (‘a’

represents, p<0.001 compared to respective control, ‘b’ represents, p<0.05 compared to respective control)

Apoptoic index in dla cells treated with fe2o3-dao in presence of d-alanine.

COMET ASSAY OF DLA CELLS TREATED WITH FE2O3-DAO AND D-ALANINE

D-aminoacid oxidase-Fe2O3 nanoparticle complex mediated antitumor activity in Swiss Albino mice. S.A.Divakaran, K.M.Sreekanth, K.V.Rao and C.K.K.Nair, Journal of Cancer Therapy, 2, 666-674 2011.

Conclusions

• Doxorubicin and the enzyme DAO can be complexed to magnetic Fe3O4

nanoparticles.

• This complexes can be targeted by means of an external magnetic field.

• By targeting nanoparticle bound anticancer drug or administering the ezyme substrate

• D-ala tumor growth can be controlled.

• The targeting enhanced the efficacy of the treatment - decrease the circulatory concentrations of the drug and helps to minimize its toxic side effects.

THANK YOU

nano mgu feb15-2013-1

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