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
2. NANOTECHNOLOGY
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
Nanometer • Chemical
• One billionth (10-9) of a meter • Electrical
• Hydrogen atom 0.04 nm • Mechanical
• Proteins ~ 1-20 nm • Optical
• Feature size of computer chips 90 nm • Magnetic
(in 2005) •
• Diameter of human hair ~ 10 µm •
3. Unique Properties of Nanoscale Materials
• 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
5. • Spherical iron nanocrystals
• J. Phys. Chem. 1996,
Vol. 100, p. 12142
6. • 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)
7. S
N CF3
CH 2
CH 2 -
- Cl
Cl
α ω
CH 2 N+ N+ CH3
H H
Trifluoperazine
Antidepressent and antipsychotic drug. It is known to give
severe side effects such as photosensitization of skin and
eye tissues TFP pK 4 and 8
TFP+ a
Dimensions:TFP 25 Å
TFP+ SiO2 TFP+ SiO2 60 Å
TFP+
Biophysical Chemistry, 109, 113-119, 2004
8. Three different cells from
three different tissues of
mice : Spleen, liver and
fibrosarcoma tumor were
incubated with TFP and
TFP/SiO2
Spleen ce lls
treated with TFP/SiO2
Biophysical Chemistry, 109, 113-119, 2004.
9. NANOTECHNOLOGY APPLICATIONS
Medicine/Health Care Energy
• More Efficient And Cost
•Cancer treatment
Effective Technologies
•Bone treatment For Energy Production
•Dentistry − Solar Cells
− Fuel Cells
•Drug delivery − Batteries
•Appetite control − Bio Fuels
•Drug development
•Tissue engineering/ Consumer Goods
•Regenetaive medicine • Foods and beverages
−Advanced packaging materials, sensors, and
•Medical tools
lab-on-chips for food quality testing
•Diagnostic • Appliances and textiles
•Imaging −Stain proof, water proof and wrinkle free
textiles
Information Technology • Household and cosmetics
Smaller, faster, more − Self-cleaning and scratch free products,
energy efficient and paints, and better cosmetics
powerful computing and
other IT-based systems
10. 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
11. NANOTECHNOLOGY IN DRUG DELIVERY - ADVANTAGES
• Prevention of drug from
biological degradation
• Effective Targeting
• Patient Compliance
• Cost effectiveness
• Product life extension
13. • 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 ion [(P ]
u )
Rby
2
3
+
mediator oxidation catalyzed by Guanine.
2+ • High specificity
2+
• Direct, fast
3+
response
• High sensitivity
• Single molecule
and cell
3+ signal
e capture and
detection
14. Nanoscale electrodes create a dramatic improvement in signal
detection over traditional electrodes
Traditional Macro- or Nanoelectrode
Electrode Micro- Electrode Array
Insulator
Nano-
• Scale difference between • CNT tips are at the scale Electrode
macro-/micro- electrodes and close to molecules
molecules is tremendous
• Dramatically reduced
• Background noise on electrode background noise
surface is therefore significant • Multiple electrodes results in magnified
• Significant amount of target signal and desired redundance for
molecules required statistical reliability.
• Can be combined with other
electrocatalytic mechanism for
magnified signals.
15. Electrochemical Detection
of DNA Hybridization
- by AC Voltammetry
1st #1-#2
2nd and 3rd
#2-#3
1st, 2nd, and 3rd scan in AC voltammetry 1st – 2nd scan: mainly DNA signal
2nd – 3rd scan: Background
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.
16. 300 µm
200 µm
30 dies on a 4” Si wafer Potential applications:
(1) Lab-on-a-chip applications
(2) Early cancer detection
(3) Infectious disease detection
(4) Environmental monitoring
(5) Pathogen detection
17. 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).
18. Self-Assembly of ssDNA
Thiolated ssDNA Au -
-+
-
5’-HS ATCCGCATTACGTCAATC + - -+ +
+ -- +
TAGGCGTAATGCAGTTAG-5’
(Complementary Strand) - +
PB = Sodium Phosphate Buffer
Wu, G. et al. “Origin of nanomechanical cantilever motion generated from biomolecular interactions,”
PNAS 98(4), 1560-1564 (2001).
19. Probe
ssDNA Target ssDNA
Wu, G. et al. “Origin of nanomechanical cantilever motion generated from biomolecular interactions,”
PNAS 98(4), 1560-1564 (2001).
20. 200
PSA [BSA] = 1 mg/ml [fPSA]
150 60 µg/ml
Analyte
Deflection, h [nm]
6µg/ml
Rabbit Anti- 100 Injections
Human PSA
50 60 ng/ml
DTSSP
Au 6 ng/ml
0
SiNx No PSA Ab
No fPSA ([fPSA] = 60 µg/ml)
Glass -50
0 60 120 180 240 300
Time [min]
HSA: Human Serum Albumin 80
[f P S A ]
[H S A ] = 1 m g / m l
60
HP: Human Plasminogen
60 n g/m l
Deflection, h [nm]
40
fPSA: free PSA I n je c t io n s 6 n g /m l
20
cPSA: complex PSA 0
Wu, G. et al., “Bioassay of Prostate Specific -20 No PSA Ab H P o n ly No PSA
µ
Antigen (PSA) Using Microcantilevers,”
( [f P S A ] = 6 0 g /m l) ( [ H P ] = 1 m g /m l)
-40
Nature Biotechnology (Sept., 2001) 0 60 120 1 80 240
T im e [m in ]
21. 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
22. 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.
23. Fabrication
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-µm resolution
24. 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 GeneChip
• Each “print head” has a ~100 µm diameter and are
separated by ~100 µm. (≈ 5,000 – 20,000 probes)
• Photolithographic Chips
– Ex: Affymetix
– 1.28 x 1.28 cm glass/silicon wafer
• 24 x 24 µm probe site (≈ 500,000 probes)
– Lengths of DNA up to 25 nucleotides long
– Requires a new set of masks for each new array type
25. 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
26. The Process
Poly-A 10% Biotin-labeled Uracil
Cells cDNA
RNA Antisense cRNA
IVT
AAAA L L L
(In-vitro
Transcription)
Fragment (heat, Mg2+)
Labeled
fragments
Hybridize Wash/stain Scan
L
L
L
27. Hybridization and Staining
GeneChip Biotin Hybridized Array
Labeled cRNA
L
L
L
L
L
+ L L
+
L
L
L
L
L
SAPE
Streptavidin-
phycoerythrin
29. - Nanopore in membrane
- DNA in buffer
- Voltage clamp (~2nm diameter)
- Measure current
α-hemolysin pore (very first, natural
pore)
Axial View Side View
30. • When there is no DNA translocation,
there is a background ionic current
Open nanopore
• 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
DNA translocation event
31. 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 αHL simultaneously
• Slower translocation
- 1-5 µs /nucleotide in αHL
• Resistance to extreme conditions
- Temperature
- pH
- Voltage
• α - hemolysin is toxic and hard to work with
33. nanopore
chip
Voltage Clamp
Amplifier
AgCl
AgCl
KCl KCl
Data Acquisition
• Voltage-clamp amplifier designed to measure
pA level currents
• Fast (up to 1GHz) data acquisition
• Software for automatic blocking event
detection and recording
35. C
C C
C
TT
G
A G
A G G
A A
A
GG A
G
Present Future
36. • 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 Courtesy of: http://www.uea.ac.uk/cap/wmcc/anc.htm
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.
37. • 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
39. 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
40. 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.
41. Cancer
Defined as the disturbance of
growth characterized by excessive
proliferation of cells without apparent
relation to the physiological demand
of the organ involved.
42. Cancer
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.
43. Tobacco Alcohol
Lack of Nutrients
RISK FACTORS FOR CANCER
Environmental Factors Sociodemographic Factors
44. 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/ 1.68
Brain
2.74 Tonsil Lung/Other 1.51
Tongue
2.59 Brain Lymp.Leuk 1.43
49. Year wise total cancer prevalence in India
[ICMR, 2006; ICMR, 2009].
50. 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].
51. 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 %)
52. India
3.4 % of all deaths – cancer
7 lakh new cases detected every year
Males - mouth/oropharynx
oesophagus
stomach
Lower respiratory tract
Females - Cervix
Breast
mouth/oropharynx
oesophagus
53. Oral cancer
• 50 – 70% of all cancers diagnosed in india
• Risk factors
Tobacco
Alcohol
Precancerous lesions
Cultural patterns
Prevention
54. 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
55. Stomach Cancer
Lung cancer
• Most common cancer in the • Worlds second most
world common cancer
• Risk factors • Risk Factors
• Prevention • prevention
56. CAUSES OF CANCER
Environmental Genetic
• Tobacco Eg
• Alcohol • Retinoblastoma in Children
• Dietary Factors • Leukemia in Mongols
• Occupational exposures
• Viruses
• Parasites
• Customs ,habits, Lifestyles
• Others – sunlight, pollution,
drugs
58. Common sites of oral cancer
The 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.
CANCERS OF Tongue, Cheek, Subbuccal mucosa, Buccal vestibule
PRECANCEROUS LESIONS
Luekoplakia Erythroplakia Sub mucous fibrosis
59. CANCER CONTROL
Primary Prevention Secondary Prevention
• Control of tobacco & • Cancer Registration
alcohol
Hospital based registries
consumption
Population based registries
• Personal Hygiene
• Early detection of cases
• Radiation
• Treatment
• Occupational Exposures
• Immunisation
• Foods & drugs
• Air pollution
• Treatment of precancerous
lesion
• Legislation
• Cancer Education
60. 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
61. Cancer Screening
• Pre malignant lesion can be identified
• Most cancers are localized in initial stages
• 75% occurs at accessible body sites
Methods
• Mass Screening by comprehensive cancer detection
examination
• Mass Screening at single sites
• Selective screening - for those at special risk
62. • Screening of cancer cervix – Pap Smear
• Screening of breast cancer
Breast self examination(BSE)
Palpation
Thermography
Mammography
Screening of lung cancer
Chest radiograph
Sputum Cytology
63. Oral cancer
• 50 – 70% of all cancers diagnosed in india
• Risk factors
Tobacco
Alcohol
Precancerous lesions
Cultural patterns
Prevention
64. 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
65. Stomach Cancer
Lung cancer
• Most common cancer in the • Worlds second most
world common cancer
• Risk factors • Risk Factors
• Prevention • prevention
66. 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.
67. 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.
68. 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
69. 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
70. INHERITED CANCERS - a small percentage of many
cancers
• Breast cancer
– ~3% cases between 36 and 45 years of
age
have 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
71. Types of genes which may mutate to cause cancer:
• Tumour suppressor
genes
• oncogenes
• DNA repair genes
• telomerase
• p53
73. 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.
75. Hallmarks of Cancer
Six 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
76. Major types of cancers
• Over 200 types of cancer are known grouped
into major categories
Carcinomas
Sarcomas
Melanomas
Teratomas
Leukemias and Lymphomas
79. 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.
84. 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
88. Viruses
Viruses—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
89. MODALITIES OF CANCER TREATMENT
LOCAL THERAPEUTIC MODALITIES
SURGERY
RADIOTHERAPY
SYSTEMIC FORMS OF TREATMENT
CHEMOTHERAPY
ENDOCRINE THERAPY
IMMUNOTHERAPY
GENE THERAPY
91. Targeted drug delivery to
tumour using magnetic
nanoparticles:
Preclinical studies with Fe3O4 –
doxorubicin nanoparticles
92. 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
93. Dose-Effect Curve
Desirable
100 Dose Range
80
% with 60
Maximal
Effect 40 Adverse
Effects
20
0
Log Dose
95. Doxorubicin (Adriamycin)
intercalation of DNA
Prevent DNA replication
Common Side Effects:
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)
96. 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.
98. • 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.
99. Water dispersible Fe3O4 nanoparticlescarring doxorubicin for cancer therapy
O.D.Jayakumar, R.Ganguli, A.K.Tyagi , D.K.Chandraseharan and C.K.K.Nair
Journal of Nanosciene and Nanotechnology, 9, 6344-6348, 2009.
100. • 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.
Water dispersible Fe3O4 Nanoparticles Carrying Doxorubicin for Cancer Therapy: Journal of Nanoscience
and Nanotechnology, 2009, 9, 6344-6348.
101. TEM images of Fe3O4-PVP-
POES nanoparticles and
Fe3O4-PVP-POES-Doxo
complex.
(a, b):Fe3O4-PVP-POES
(c, d):Fe3O4-PVP-POES-Doxo
Water dispersible Fe3O4 Nanoparticles Carrying Doxorubicin for Cancer Therapy: Journal of Nanoscience
and Nanotechnology, 2009, 9, 6344-6348.
102. Water dispersible Fe3O4 Nanoparticles Carrying Doxorubicin for Cancer Therapy: Journal of Nanoscience
and Nanotechnology, 2009, 9, 6344-6348.
104. Cytotoxic effect on DLA cells
Fe3O4-PVP-POES-Doxo
exhibited more
toxicity towards
DLA cells
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 Fe 3O4-PVP-POES.
Water dispersible Fe3O4 Nanoparticles Carrying Doxorubicin for Cancer Therapy: Journal of Nanoscience
and Nanotechnology, 2009, 9, 6344-6348.
105. Induction of apoptosis Apoptotic index of Fe3O4-PVP-POES-
Doxorubicin was higher than that of doxorubicin or Fe3O4-PVP-POES.
Characteristic morphology
of normal cell (A) and
apoptotic cell (B)
A
B
For Doxorubicin, the concentrations – A and B – corresponding to 50 and 100 micrograms/ml; for the Fe 3O4-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 Fe O -PVP-POES.
106. 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.
Water dispersible Fe3O4 Nanoparticles Carrying Doxorubicin for Cancer Therapy: Journal of Nanoscience and
Nanotechnology, 2009, 9, 6344-6348.
107. 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.
Water dispersible Fe3O4 Nanoparticles Carrying Doxorubicin for Cancer Therapy: Journal of Nanoscience
and Nanotechnology, 2009, 9, 6344-6348.
108. Effect on cardio toxicity
• 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.
n s indicate not significant and *** indicate p <0.001 when compared with untreated control.
109. Effect on cardio toxicity
• 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.
n s indicate not significant, * indicate p <0.05 and *** indicate p <0.001 when compared with
untreated control.
110. OXIDATIVE ENXYME THERAPY USING MAGNETIV NANOPARTICLES
D-Alanine --- (DAO) ------------→ Pyruvic acid + H2O2
FTIR spectra of Fe2O3 magnetic nanoparticles FTIR spectra of Fe2O3 -DAO complex
XRD pattern of Fe2O3 nanoparticles coated with PVP XRD pattern of Fe 2O3 -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.
111. OXIDATIVE ENXYME THERAPY OF TUMOUR
USING MAGNETIV NANOPARTICLES
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 Fe 2O3-
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.
112. COMET ASSAY OF DLA CELLS TREATED
WITH FE2O3-DAO AND D-ALANINE Apoptoic index in dla cells treated
with fe2o3-dao in presence of d-alanine.
Treatments Apoptoic index (%)
Control 0
0.2M D-Alanine + DAO 85.2± 5.55 a
(0.36U)
0.2 M D-Alanine + Fe2O3- 95.5±3.53a
DAO (0.36U)
0.2M D-Alanine 6.5± 2.12b
Fe2O3-DAO (0.36U) 11.0± 1.41a
Apoptoic index in DLA cells treated with
Representative images of DLA cells after comet assay. Fe2O3-DAO in presence of D-alanine. (‘a’
a) The untreated DLA cells. b) DLA cells treated with represents, p<0.001 compared to
Fe2O3-DAO and D-alanine showing Fan-like comets respective control, ‘b’ represents, p<0.05
indicative of apoptosis c). DLA cells treated with compared to respective control)
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.
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.
113. 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.
Cells either grow and divide with restraint ...or not! The many kinds of malignant growth that the term "cancer" represents all have one lethal attribute in common: The cells of the malignancy go through the cell cycle without restraint. These cells "defy" the control mechanisms that lie with them. What Is the Connection Among Cancer, the Cell Cycle, and Genetics? There are many protein molecules involved in the cell cycle, each is the product of a single gene. When there is a mutation in one of these genes, it can: increase the likelihood that a cell will become cancerous and eventually, through repeated, unrestrained division, overtake the normal cells, become malignant; and possibly spread, or metastasise throughout the body. Cancer can develop at almost any stage in life. There are some forms of cancer that develop very early, such as retinoblastoma (a cancer of the eye); others tend to develop in childhood, such as various forms of leukaemia, a cancer of the blood; and, of course, there are many forms that develop during adulthood. In each case, cancer is the result of a mutated gene, or a series of mutated genes, that lead to unregulated cell growth and haphazard controls over cell proliferation.
The Myc gene was originally identified in the avian myelocytomatosis virus.
Dose-effect relationship for the individual patient. For most drugs the dose-response curve is not linear and at some point the dose-response curve approaches a plateau and there is a diminishing return for further increases in the dose With this minimal added benefit, there may be significantly greater risk for toxicity.
mechanism of action intercalation of DNA, causing inhibition of DNA and RNA synthesis leads to double strand breaks, mediated either by topoisomerase II or the generation of free radicals.