RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BANGALORE

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DESSERTATION

1. / Name of the candidate & Address / : /

UPASANI AMOL SATISH

10,SHRIKRUPA,VIDYANAGAR
DONDAICHA, DIST:-DHULE
(MH) 425408
2. / Name of the institution / : / H.K.E’S COLLEGE OF PHARMACY,
SEDAM ROAD, GULBARGA - 585105
3. / Course of the study and subject / M.PHARM
(PHARMACOLOGY)
4. / Date of Admission to the Course / 24.06.08
5. / Title of the topic / pharmacoLOGICAL screening of funtionalized noble (SILVER and gold) nanoparticles.
6. / Brief resume of the intended work
6.1 Need of the study:
Nanotechnology is expected to open some new aspects to fight and prevent diseases using atomic scale tailoring of materials. The ability to uncover the structure and function of bio-systems at the nanoscale stimulates research leading to improvement in biology, biotechnology, healthcare and pharmacology. The size of nanomaterials is similar to that of most biological molecules and structures; therefore, nanomaterials can be useful for both in vivo and in vitro biomedical research and applications. In all the nanomaterials with different pharmacological properties, metallic nanoparticles are the best. Nanoparticles increase chemical activity due to crystallographic surface structure with their large surface to volume ratio. In this dissertation we are working on the pharmacological screening of bio and polymer functionalized Ag and Au nanoparticles with regard to wound healing and in cancer chemotherapy which further has promoted the research in the well known activity of respective ions and noble metal based compounds, nanoparticles.
Among the inorganic antibacterial and wound healing agents, bulk Ag (Silver) has been known most extensively since ancient times to fight infections and control spoilage. The antibacterial and antiviral actions of Ag, Ag+ ions and Ag compounds have been thoroughly investigated1-3. It is well known that Ag ion and Ag-based compounds are highly toxic to microorganisms4, showing strong bactericide effect against as many as 16 species of bacteria, including Escherichia coli5. Ag nanoparticles have been tested in various fields of biological sciences, viz. drug delivery, and woundtreatment. Ag nanoparticles inhibit bacterial growth at a low concentration than antibiotics and have no side effects6. Apart from this excellent application, till today the mode of anti-bacterial effect and wound healing activity of Ag nanoparticle is still not clearly understood. The further clarity in mechanism Ag nanoparticles i.e., the bacterial cell receptor and the functionalized Ag nanoparticle (bio and polymer) study will enable us to develop a strong theory of co-relation of nanoparticles and its wound healing effect.
Cancer is currently the second leading cause of death in world, while it shows probably the highest clinical complexity7. Nanobiotechnology could enable the parallel in vitro measurements of many chemical/bio-chemical particles at the same time, while keeping the test itself simple, sensitive, reliable, and inexpensive8. In addition Nanopharmacology provides the tools to discover novel particle methods, enhancing reliability and accuracy of diagnosis, due to their enhanced scattering and photothermal properties.9 Nanoparticles can then be used as targeted deliver agents (a recent form of drug administration to the cancer patients).
Au nanoparticles displaying novel properties of metal nanoconjugates have wider and more effective biological and medical applications, for example, these building blocks have potential for fabricating biological labels, biological particles, bioanalysis and biodiagnosis technologies, and monitoring of diseases, drug discovery, environmental detection of biological reagents, and even medical clinical diagnosis and therapy 10-11. At present, the most active area in developing Au based pharmaceuticals is their investigation as potential anti tumor agents and for other medical treatments.
Nanoparticle-based contrast agents that are designed to allow improved and significantly faster diagnostics; Ag and Au nanoparticles are successfully in early identification and destruction of cancer cells. Nano-cancer therapy in which tiny iron particles, Ag Au and platinum are injected into the tumor tissue or directed there with a magnet before being stimulated to generate heat by means of an alternating magnetic field; this method is currently being clinically tested. The selective absorption of magnetic bioactive nanoparticles in tumor cells allows the killing of tumor cells based on targeted local heating without significant negative impact on healthy tissue; proceeds in an uncontrolled manner and that vital gene functions can be negatively impacted in the process. The enhanced scattering property in imaging is used in detecting single cancer cell once nanoparticles are conjugated to cancer cell antibodies.The enhanced absorbed light energy is rapidly converted into heat in one picosecond12. This causes rapid temperature rise that leads to heating the surrounding, to melting the surrounding cells, to melting the nanoparticles themselves or to ablating atoms from the nanoparticles. These photothermal properties will be shown to be useful in many applications such as making nano-motors and when conjugated to antibodies, they can be used in selective laser photothermal therapy of cancer.
Nanopharmacology involves no special risks in the area of usage of bioactive nanoparticles and allied pharmacy fields. Nanobiotechnological and pharmacological methods are already being used in the medical and pharmaceutical field today.
In this regard we have made an effort to synthesize Au and Ag chemico bio-nanoparticles from different microorganisms and aqueous plant extract and exposed to molecular, cellular, tissue cultured, spectroscopic, scanner based, radiological and pathological Antineoplastic (Anticancer) screening. Our strong belief is that the so obtained nano-functionalized particles can be scaled up in a much easier way. We are also seeking best possible methods to develop and scale up the nano particles in an economic mode with a green pollution free chemistry involved. Some initial success has been achieved on the synthesis and stabilization of Ag nanoparticles9. The future idea is to detect manifestation of cancer it its premature state itself and destroy them.
LIST OF REFERENCES:
1. Oka, M. T., Tomioka, Tomita, K., Nishino, A. and Ueda, S., Inactivation of enveloped viruses by a Ag-thiosulfate complex. Metal-based Drugs, 1994, 1, 511.
2. Oloffs, A., Crosse-Siestrup, C., Bisson, S., Rinck, M., Rudolvh, R. and Gross, U., Biocompatibility of Ag-coated polyurethane catheters and Ag-coated Dacron material. Biomaterials, 1994, 15, 753–758.
3. Tokumaru, T., Shimizu, Y. and Fox, C. L., Antiviral activities of Ag sulfadiazine and ocular infection. Res. Commun. Chem.Pathol. Pharmacol., 1984, 8, 151–158.
4. Slawson, R. M., Van Dyke, M. I., Lee, H. and Trevors, J. T., Germanium and Ag resistance, accumulation, and toxicity in microorganisms.
Plasmid, 1992, 27, 72–79.
  1. Spadaro, J. A., Berger, T. J., Barranco, S. D., Chapin, S. E. and Becker, R. O, Antibacterial effects of Ag electrodes with weak direct current. Microb. Agents Chemother., 1974, 6, 637.
  2. Lok, C. N. et al., Proteomic analysis of the mode of antibacterial action of Ag nanoparticles. J. Proteo. Res., 2005, 5, 916–924.
  3. Mostafa A. El-Sayed., “Why are gold Nanoparticles more precious than pretty gold: Properties and applications in making Nano-Motors and in cancer Diagnostics and Laser selective Photo Thermal Therapy., Laser Dynamic Laboratory”., Georgia Tech Atlanta., GA 30332 USA
  4. Bethesda, Maryland Harold P. Freeman, M.D., Chairman; Paul Calabresi, M.D.; Frances M. Visco, J.D. National Cancer Institute Division of Extramural Activities President's Cancer Panel Meeting, Meeting Minutes The Real Impact of the Reduction in Cancer Morality Research September 29, 1997
  5. Nanomedicine nanotechnology for health November 6th Europian technology platform on Nanomedicine Page 9: Magnetic nanoparticles and automated image analysis software delineate malign from benign lymph nodes in a cancer patient © Harisinghani, M., MGH, Boston Scanning Force Microscopy of collagen. The fiber's helical structure with a period of only 67 nm is clearly visible. © van der Werf, K.O., Subramaniam, V., University of Twente Scanning Force Micrograph of a red blood cell © nanoAnalytics GmbH, Germany
  6. Hacker, G.W.; Gu, J. Gold and Silver Staining: Techniques in Molecular Morphology. CRC Press: Boca Raton, 2002.
  7. Jin, R. C.; Cao, Y. W.; Mirkin, C. A.; Kelly, K. L.; Schatz G. C.; Zheng J. G. Photoinduced conversion of silver nanospheres to nanoprisms. Science 2001, 294, 1901-1903.
  8. 4Wang, Z. P.; Hu, J. Q.; Jin, Y.; Yao, X.; Li, J. H. In situ amplified chemical luminescent detection of DNA and immunoassay of IgG using special-shaped gold nanoconjugatess as label. Clin. Chem. 2006, 52, 1958-1961.

6.2Review of literature:
No work has been reported on the pharmacological screening of bio and polymer functionalized Ag and Au nanoparticles with respect to wound healing and in chemotherapy of cancer. This will be a special project in joint venture with Gulbarga University, Gulbarga. (Letter enclosed with hard copy). The synthesis, characterization and stability studies have been carried out at material science department, Gulbarga University Gulbarga.
Few reported works of Ag nanoparticles, as antibacterial agents are the local effects of Ag-coated polyurethane catheters and Dacron material were compared to uncoated polyurethane catheters and Dacron material in a long-term implantation test using rabbits. Ag-coated and uncoated materials displayed comparable signs of inflammation and tissue reaction1-4.Some possible mechanism of Ag resistance in microorganisms is accumulation of the metal ions in the cell is reported along with the biosynthesis5 and the effort on the interaction of Ag nanoparticles with HIV viruses was made6. Last couple of years good amount of interest has been generated among pharmacological and nanobiotechnological research community on the antibacterial effect of Ag nanoparticles7-10. The dose dependent pharmacological screening of given organic and inorganic materials are always a prime concern to understand the efficacy and safety. The biocompatibility of polymer based nanoparticles are also studied and further compared with biofuntionazed nanoaprticles.
In over 50 % of all cancer cases, radiation therapy is the standard form of therapy. This therapy however ignores the tumor’s inner structure consisting of sections being more or less sensitive to radiation.11 The imaging procedure will serve as an input to a better radiotherapy planning that puts higher doses on the radiation-resistant sections and lower doses on the radiation-sensitive sections, thereby reducing damage in the healthy neighborhood. The use of gold nanoparticles, as well as their potential therapeutic applications, in ancient and contemporary medicine has been reviewed periodically over the years 12-13. At present, the most active area in developing Au based pharmaceuticals is their investigation as potential anti tumor agents and for other medical treatments 14-15.
Few reported works of sivler nanoparticles as antibacterial agents are.
1.Oka, M. T., Tomioka, Tomita, K., Nishino, A. and Ueda, S., Inactivation of enveloped viruses by a Ag-thiosulfate complex. Metal-based Drugs, 1994, 1, 511.
2. Oloffs, A., Crosse-Siestrup, C., Bisson, S., Rinck, M., Rudolvh, R. and Gross, U., Biocompatibility of Ag-coated polyurethane catheters and Ag-coated Dacron material. Biomaterials, 1994, 15, 753–758.
3. Tokumaru, T., Shimizu, Y. and Fox, C. L., Antiviral activities of Ag sulfadiazine and ocular infection. Res. Commun. Chem.Pathol. Pharmacol., 1984, 8, 151–158.
4. Slawson, R. M., Van Dyke, M. I., Lee, H. and Trevors, J. T., Germanium and Ag resistance, accumulation, and toxicity in microorganisms. Plasmid, 1992, 27, 72–79.
5. Elchiguuerra, J. L., Burt, J. L., Morones, J. R., Camacho-Bragado, A., Gao, X., Lara, H. H. and Yacaman, M. J., Interaction of Ag nanoparticles with HIV-1. J. Nanobiotechnol., 2005, 3, 1477– 3155.
6. Jain, P. and Pradeep, T., Potential of Ag nanoparticle-coated polyurethane foam as antibacterial water filters. Biotechnol.Bioeng., 2005, 90, 59–63.
7. Son, W. K., Youk, J. H., Lee, T. S. and Park, W. H., Preparation of antimicrobial ultrafine cellulose acetate fibers with Ag nanoparticles. Macromol. Rapid Commun., 2004, 25, 1632–1637.
8. Li, P., Li, J., Wu, C., Wu, Q. and Li, J., Synergistic antibacterial effects of β-lactam antibiotic combined with Ag nanoparticles. Nanotechnology, 2005, 16, 1912–1917.
9. Lok, C. N. et al., Proteomic analysis of the mode of antibacterial action of
Ag nanoparticles. J. Proteo. Res., 2005, 5, 916–924.
10. Baker, C., Pradhan, A., Pakstis, L., Pochan, D. J. and Shah, S. I., Synthesis
and antibacterial properties of Ag nanoparticles. J.Nanosci. Nanotechnol.,
2005, 5, 244–249.
  1. Daniel MC, Astruc D. Gold nanoconjugatess: assembly, supramolecular
chemistry, quantum size-related properties, and applications toward biology,
catalysis and nanotechnology. Chem Rev. 2004; 104:293–346.
  1. 12. Turner, A. P. F. Biosensors-sense and sensitivity. Science 2000, 290,
  2. 1315-1317.
  3. 13. Biosensing: New probes offer much faster results Nature Nanotechnology
  4. 2,746 - 748 (01 Dec 2007), doi: 10.1038/nnano.2007.398, News and
  5. Views J. Lewis, D. T. Walz, Prog. Med. Chem. 1982, 19, 1
  6. 14 Elizabeth Singer, Ph.D., and Steven Smith, Ph.D., New Approaches Target
  7. Nanoparticles to Cancer cells Physorg.com Published: 16:38 EST, May
  8. 22, 2006 Willis, R.A., : The Spread of Tumors in the Human Body.
  9. London, Butterworth & Co, 1952.
  10. 15 Natalia, C., Tansil and Zhiqiang Gao., Nanoparticles in biomolecular
  11. detection, Nanotoday, Volume 1, Issue 1, February 2006, Pages 28-37.
National & International paper published in the Nan biotechnology and Pharmacology.
1.Extracellular biosynthesis of Ag nanoparticles using the fungus Fusarium semitectum, S Basavaraj, SD Balaji, Arunkumar Lagashtty, AH Rajasab, A Venkataraman, Mater Res Bull, 43 (2008) 1164-1170: doi: 10.1016/j.materresbull.2007.06.20.
2.Biosynthesis and Stabilization of Au and Au-Ag Alloy Nanoparticles by Fungus, Fusarium semitectum, DS Balaji, S Basavaraj, Raghunandan Deshpande, Mahesh D Bedre, A Venkataraman, Sci Technol Adv Mater 9 (2008) 035012; doi: 10.1088/1468-6996/9/3/035012.
3.D.S. Balaji, S. Basavaraj, Mahesh D Bedre, B.K. Prabhakar, A. Venkataraman, Extracellular biosynthesis of functionalized Ag nanoparticles by strains of Cladosporium cladosporioides fungus Colloids and Surfaces B: Biointerfaces, In press; doi: 10.1016/j.colsurfb.2008.09.22.
4.D.S. Balaji, S. Basavaraj, Mahesh D Bedre, D. Raghundan, B. K. Parabhakar, A.Venkataraman, Bio-stabilization of functionalized Ag-Au alloy nanoparticles from the fungus strains of Cladosporium cladpsporioides Pharmakine, 1 (2008) 12..
5.D B Mahesh, S Basavaraj, D S. Balaji, Shivakumar, L Arunkumar and A Venkataramana Preparation, characterization of polyaniline and polyniline Ag Nanocomposites via interfacial polymerization method, polymer Composite, In press.
Our strong belief is that the so obtained nanofuctionalised particles can be scaled up in a much easier way. We are also seeking best possible methods to develop and scale up the nano particles in an economic mode with a green pollution free chemistry involved. Some initial success has been achieved on the synthesis and stabilization of Ag nanoparticles9. The future idea is to detect manifestation of cancer it in premature state itself and destroy them. Our team, we are working hard to build a concept to develop user friendly, compact, economical method or a device to have a regular, prophylactic check in the malignant abnormalities.
6.3Objectives of the study.
Ag nanoparticles are known to be good antibiotic agents. Microorganisms (bacteria, yeast and fungi) play an important role in toxic metals remediation through reduction of metal ions; this was considered interesting as nanofactories. Recently, it was found that aqueous Ag nitrate solution may be reduced extracellularly using micro-organisms and plant extracts, to generate extremely stable Ag nanoparticles in water. These particles can be incorporated in materials and cloth becoming them sterile. The sterile materials are important in hospital, where often wounds are contaminated with different gram positive and gram negative microorganisms, in particular, Staphylococcus aureus. A new generation of dressing incorporating antimicrobial agents like Ag was developed to reduce or prevent infections. Extracellular production of Ag nanoparticles which was developed by bio route and by Ag nanoparticle synthesized by bio route and by interfacial polymerization is taken in different concentrations further screened on wound healing activity. The wound healing effect of these Ag nanoparticles synthesized naturally and synthetically in different concentration to know its dose dependent activity on different microorganisms and the probable mechanism of action is studied.. Moreover, these particles could have innumerable applications, in different areas as receptors, catalysis, biolabelling and others.
The studies of the properties of self-assembled nanostructured materials ranging from molecular to macroscopic length scales with a goal of developing the bottom-up paradigm to assemble virtually any kind of a functional system ranging from integrated photonics and electronics to biological, chemical or electrochemical particles is today’s need. In nanobiotherapeutics, nanobiodiagnostics, nanobiopharmaceuticals, the nanoparticles are prepared in an aqueous medium through reduction of an ionic moiety by a reducing agent. The conditions in the aqueous medium are such that nano biochemico particles are formed having mean diameters of from about 1 to about 100 nanometers. Ligand, micelle or capping can then be associated with the nanoparticles thus formed, which ligand is specific for a biological target. The nanoparticles and compositions can then be delivered to a biological system, organism, patient, animal, tissue, organ or cell preparation where the particles rapidly associate with the biological target. Strikingly different chemical and electrochemical behaviors of these particles are elaborately studied for their different diseased condition predominantly in microbiological and in malignancy as anti-neoplastic agent. The optical, chemical and electrochemical features are not only established in imaging the abnormalities in the different diseased condition and the optoelectrical and chemicothermal properties are used in scavenging the cancer cells.
The essence of the work is to pharmacologically screen the bio- functionalized gold and silver nanoparticles for wound healing and anti-malignant activities.
7. / MATERIALS AND METHODS
7.1Sources of Data:
a)Internet
b)Helinet
c)RGUHS Library, Bangalore
d)International Pharmacology, biotechnology and microbiology abstracts.
e)Gulbarga University
  1. Material Science Department
  2. Biotechnology Department
  3. Biochemistry Department
  4. Microbiology Department
f) Peripheral cancer Institute, Gulbarga
7.2Methods:
Different functionalized Ag nanoparticles will be collected from Material Science Department, Gulbarga University, Gulbarga. The human cancer cells will be procured from Peripheral Cancer Institute, Gulbarga. The functionalized nanoparticles will be formulated as patches for wound healing activity. The pure control antibiotic drug will be obtained from Alkem Laboratories & Aurobindo Pharma. The FESEM, TEM and AFM studies are done at JNCASR, Bangalore.
a)Checking the SPR of Ag: UV-Visible spectrometry is used to confirm the presence and concentration of the Ag and Au nanoparticles.
b)Method of zone of inhibition is carried out for checking the anti- microbial activity.
c)SEM studies (Scanning electron microscopy studies): Formulation of the patch and the zone of inhibition
d)Wound healing activity is monitored by SEM.
e) Special laser compound microscopy and Transmission Electron microscopy is used for understanding the accumulation of nanoparticles in the cells in IIT, Mumbai and JNCASR Bangalore.
f)Atomic Force microscopy is used for Singe cell-receptor-nanoparticle interaction done Veeco laboratories JNCASR Bangalore.
g)Electron Microscopic Study: the zone of inhibition, wound healing activity and anti-cancer activity of natural and synthetically developed nanoparticles effect is described using electron microscopy.
7.3Does the study require any investigation or invention to be conducted on patients or other human or animals? If so please describe briefly.
-----Yes-----
7.4Has the ethical clearance been obtained from your institution in case of 7.3
-----Yes-----
8. /

Signature of Candidate

/ [UPASANI AMOL SATISH]
9. / Remarks of the Guide
10. / Name & Designation of
(In Block letters)
10.1Guide / sRI. nEELKANTH REDDY PATIL
M.Pharm.
ASST, PROFESSOR
H.K.E’S. COLLEGE OF PHARMACY, SEDAM ROAD, GULBARGA. 585105
10.2Signature
10.3 Co-Guide / sRI. raghunandan deshpande
M.Pharm
Lecturer.
H.K.E’S. COLLEGE OF PHARMACY, SEDAM ROAD, GULBARGA. 585105
10.4Signature
10.5Head of the Department /

Dr. K. Prasad

M.Pharm Phd

Professor

H.K.E’S. COLLEGE OF PHARMACY, SEDAM ROAD, GULBARGA. 585105
10.6Signature
11. / 11.1Remarks of the Chairman & Principal / The present study was permitted for executing the work in the Institution & IAEC permission has been granted.
11.2Signature / (Dr. S. Appala Raju)

Application for Permission for animal experiments.