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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
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                          •
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
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)
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
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.
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
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
• 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
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.
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.
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
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).
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).
Probe
       ssDNA           Target ssDNA




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


    GeneChip                       Biotin                      Hybridized Array
                                Labeled cRNA
                                                                          L
                                                           L
                                                                      L
                                                   L
                                     L
                        +   L                                    L




                                                       +
                    L
     L
                L
L
          L
                                             SAPE
                                         Streptavidin-
                                         phycoerythrin
DNA Sequencing
Using Nanopores
   Goal: Very rapid gene sequencing
-       Nanopore     in membrane
-         DNA in buffer
-         Voltage clamp                  (~2nm diameter)
-          Measure current


α-hemolysin pore     (very first, natural
                     pore)




    Axial View               Side View
• 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
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
Nano mgu feb15-2013-1
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
Spontaneous Blocking Events with
      Smaller NASA Pores
                 +200 mV

        100 pA
0.5 s
C
                   C             C
                                 C
                           TT
               G
               A       G
                       A        G G
                                A A
          A
          GG           A
                       G




Present        Future
• 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.
• 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 of
growth characterized by excessive
proliferation of cells without apparent
relation to the physiological demand
 of the organ involved.
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.
Tobacco              Alcohol




                                           Lack of Nutrients


  RISK FACTORS FOR CANCER


Environmental Factors             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/    1.68
                     Brain
2.74    Tonsil       Lung/Other     1.51
                     Tongue
2.59    Brain        Lymp.Leuk      1.43
Nano mgu feb15-2013-1
900
                                                                                                                                            876.7
            Age Specific Incidence Rates/lakh
800
                 Population for all Sites                                                                                753.3
                                                                                                                                   767.9


700
                          1998

600


                                                                                                                                                      508.1
500                                                                                                            479.7

                                                                                                                                        416.4           406.3

400                                                                                                    372.8                  365.5

                                                                                                                     318.3
300                                                                                            288.8       292.8
                                                                                          245.4                                                               217.7

200                                                                                189 178.2
                                                                           149.4
                                                                         102.2
100                                                               76.2
                                                                64.5
                                                        38 37
      10.8
                 3.5 9.2 4.6
              10.3             8.2 8.5 13.6 12 2220.8
          5.5
  0
       0-


              5-




                                       20-


                                               25-




                                                                                    45-


                                                                                            50-




                                                                                                                                      70-


                                                                                                                                                75-
                       10-


                               15-




                                                        30-


                                                                 35-


                                                                           40-




                                                                                                         55-


                                                                                                                   60-


                                                                                                                             65-




                                                                                                                                                          80+
                                               MALE                    FEMALE
CANCER INCIDENCE & MORTALITY WORLD WIDE
Nano mgu feb15-2013-1
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 %)
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
Oral cancer
• 50 – 70% of all cancers diagnosed in india
• Risk factors
          Tobacco
           Alcohol
           Precancerous lesions
            Cultural patterns
Prevention
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
Stomach Cancer
Lung cancer

• Most common cancer in the   • Worlds second most
  world                         common cancer
• Risk factors                • Risk Factors
• Prevention                  • prevention
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
Dr Suwas Darvekar
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
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
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
•    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
• 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
Oral cancer
• 50 – 70% of all cancers diagnosed in india
• Risk factors
          Tobacco
           Alcohol
           Precancerous lesions
            Cultural patterns
Prevention
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
Stomach Cancer
Lung cancer

• Most common cancer in the   • Worlds second most
  world                         common cancer
• Risk factors                • Risk Factors
• Prevention                  • 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
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
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.
Nano mgu feb15-2013-1
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
Major types of cancers
• Over 200 types of cancer are known grouped
  into major categories
  Carcinomas
  Sarcomas
  Melanomas
  Teratomas
  Leukemias and Lymphomas
Nano mgu feb15-2013-1
Nano mgu feb15-2013-1
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 factor
  e.g. SIS oncogene (PDGF)
Types of proto-oncogene

• Nuclear transcription factors
  e.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
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
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
                    Desirable
       100         Dose Range



        80


% with 60
Maximal
 Effect 40                      Adverse
                                Effects
        20


         0
               Log Dose
DOXYRUBICIN. HCL – MWT 580
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)
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.
Nano mgu feb15-2013-1
• 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     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.
• 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.
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.
Water dispersible Fe3O4 Nanoparticles Carrying Doxorubicin for Cancer Therapy: Journal of Nanoscience
and Nanotechnology, 2009, 9, 6344-6348.
Nano mgu feb15-2013-1
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Nano mgu feb15-2013-1
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Nano mgu feb15-2013-1

  • 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
  • 4. Source: Nanoscale Materials in Chemistry, Ed. K.J. Klabunde, Wiley, 2001
  • 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
  • 28. DNA Sequencing Using Nanopores Goal: Very rapid gene sequencing
  • 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
  • 34. Spontaneous Blocking Events with Smaller NASA Pores +200 mV 100 pA 0.5 s
  • 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
  • 38. Future Possiblities: Oxygen Selective Pump http://www.foresight.org/Nanomedicine/
  • 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
  • 46. 900 876.7 Age Specific Incidence Rates/lakh 800 Population for all Sites 753.3 767.9 700 1998 600 508.1 500 479.7 416.4 406.3 400 372.8 365.5 318.3 300 288.8 292.8 245.4 217.7 200 189 178.2 149.4 102.2 100 76.2 64.5 38 37 10.8 3.5 9.2 4.6 10.3 8.2 8.5 13.6 12 2220.8 5.5 0 0- 5- 20- 25- 45- 50- 70- 75- 10- 15- 30- 35- 40- 55- 60- 65- 80+ MALE FEMALE
  • 47. CANCER INCIDENCE & MORTALITY WORLD WIDE
  • 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
  • 72. cancer is a disease of the cell cycle
  • 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.
  • 80. oncogenes • Cellular oncogene c-onc • Viral oncogene v-onc • Proto-oncogene, activated by mutation to c-onc
  • 82. Types of proto-oncogene • Growth factor e.g. SIS oncogene (PDGF)
  • 83. Types of proto-oncogene • Nuclear transcription factors e.g. MYC
  • 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
  • 85. Transformation is a multistep process
  • 86. Transformation is a multistep process
  • 87. The environment: Some environmental agents associated with cancer are: • Viruses • Tobacco smoke • Food • Radiation • Chemicals • Pollution
  • 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
  • 90. Cost of cancer per year in U.S.
  • 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.

Hinweis der Redaktion

  1. 3
  2. Cells either grow and divide with restraint ...or not! The many kinds of malignant growth that the term &quot;cancer&quot; represents all have one lethal attribute in common: The cells of the malignancy go through the cell cycle without restraint. These cells &quot;defy&quot; 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.
  3. The Myc gene was originally identified in the avian myelocytomatosis virus.
  4. 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.
  5. 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.