Michelson Challenge Application Process Grant Proposal 2012

TITLE Acid sensitive copolymer micelle with liposome for controlled drug delivery of indazole-3-carboxylic acid and an immunocontraceptive agent, porcine zona pellucida (PZP) Applicant: Rahim Jindani, Department of Textile Engineering, Chemistry & Science, College of Textiles, Room 3320 North Carolina State of University, Raleigh, NC 27695-8301, USA E-mail: rjindan@ncsu.edu, Mobile: 248-821-0923

Student Advisor / Principal Investigator: Martin W. King, Professor of Biotextiles Department of Textile Engineering, Chemistry & Science, College of Textiles, Room 3305 North Carolina State of University, Raleigh, NC 27695-8301, USA E-mail: martin_king@ncsu.edu, Office: 919-515-2563 Mobile: 919-291-2563

Project (Abstract): Gonadotropes, follicle stimulating hormone (FSH) and luteinizing hormone (LH) are generated from the hypothalamus, which stimulates the formation of sperm and ova. [4,2] Derivatives of indazole-3-carboxylic acid are used to inhibit aerobic glycolysis in cancer cells, and they can also be used as an antispermatogenic drug in both males and females. A glycoprotein called porcine zona pellucida (PZP) which is extracted from the ovaries of pigs can surround the plasma membrane of a female oocyte, and is directly involved in preventing the binding of spermatozoa.[2] Stimuli responsive polymers are required which can be cross-linked with liposomes and polysaccharides that can be triggered at specific pH ranges of the male and female reproductive systems, and can cause controlled release of indazole-3-carboxylic acid and porcine zona Ppllucida as required in both sexes. [10,11] By introducing a combination of drugs that can be triggered using specific pH and temperature ranges, thermal responsive polymers will ensure the controlled drug release with liposomes & polysaccharides to ensure a minimum immunological response in vivo. These responsive polymers will be able to solublize lipophilic drugs. Anionic liposomes are used to work in a narrow pH range. These polymers can also be used with elastin like polymers [(VPGVG) 2-VPGEG-(VPGVG) which can convert thermal into mechanical energy if required to trigger the reaction. We plan to use the properties of pH responsive polymers accordingly to ensure that contraception in both sexes takes place effectively at a range of different pHs and temperatures. [5, 6, 14]

Statement of Research: This proposal addresses the need to provide a single dose sterilization technique for both sexes of cats and dogs by targeting the testes and ova in specific pH ranges. The study will also ensure that the desired effectiveness is achieved with minimal drug dosage.

Introduction: There is need for a single dose drug delivery mechanism to sterilize male/female cats and dogs. This mechanism needs to have the capacity to deliver the drug in a controlled way at a predictable concentration over an extended period. An effective way of controlling this drug release is by utilizing polymer systems that can respond to different pH and temperature ranges within male and female cats and dogs.

Problems & Objectives: The reproductive systems of males and females vary significantly in their function and physiology. There are various considerations relevant to all breeds of dogs and cats including their size, biological system, eating habits as well as the duration and period of puberty and menstrual cycle. Usually male testicies require a pH of 6.5 to 8 for the sperm to survive, whereas female ova require a pH of 3 to 6 for sustenance of the ovaries. The temperature range within which sperm can survive is between 33 to 36 degrees centigrade while the temperature range for ova is between 12 to 32 degrees centigrade.

Keeping these factors in mind the objective of the study is to have controlled drug release on targeted areas, namely the testes and the ova, that can be activated at specific pH levels and temperatures, and can be triggered to achieve sterility with a minimum inflammatory and immune response in both male and female cats and dogs.

Hypotheses: 1) To test if the polymer carrier N-isopropylacrylamide (NIPAM) and carboxylic acid with liposomes are able to respond within the specified pH ranges. 2) To determine if drug release occurs in a controlled manner in both sexes of dogs and cats, at specific pH and temperature ranges. Visualization of the drug movement will use green fluorescent protein (GFP) for tagging.

Testable Objectives for Hypotheses: a. At pH < 4.5, carboxylic acid is able to release anionic liposomes attached to PZP as they become responsive. b. At pH > 7.2, N-isopropyl acrylamide will release cationic liposomes attached to indazole-3-carboxylic acid causing sperm counts to drop to zero ideally within 12 hours of injection. c. To observe liposomes and the subsequent drug reaction on the interaction with ova and testes by studying GFP tags.

Literature Review: pH-Sensitive Polymer--Liposome Systems Elizabeth R. Gillies and Jean M. J. Frechet, Development of acid sensitive co-polymer micelles for drug delivery, Pure Appl. Chem., Vol. 76, Nos. 7-8, pp.1295-1307, 2004. Responsive Polymer/Liposome Complexes: Design, Characterization and Application Franoise M. Winnik and Tania Principi, Department of Chemistry, Mc Master University, 1280 Main St W, Hamilton, Ontario Canada L8S 4M1. Contraceptive Vaccines for Wildlife Jay F. Kirkpatrick, Robin O. Lyda and Kimberly M. Frank, Contraceptive vaccines for wildlife: a review, The Science and Conservation Center, Zoo Montana, Billing, MT, USA Vaccines for contraception of wildlife animals have been tested and tried in various parts of the world. At this time a number of species have been studied, including 76 exotic species in various countries. [53] Contraception using PZP has been evaluated on various animals, and their use with multilamellar liposomes has caused effective contraception for 3 years in horses and deer. The only drawback of using multilamellar liposomes is that during preparation they cause the drug to have a high viscosity. Liposomes are known to reduce the immunological response of the body to foreign interactions. A better approach would be to use

small chains of liposomes with polymeric biomaterials that could help reduce the viscosity of the drug.[52, 53] Indazole-3-carboxylic acid has been used as a drug to kill cancer cells by inhibiting aerobic glycolysis. It is a commercial product named Lonidamine. It can also be used as a contraceptive drug since it is considered an anti-spermatozoa drug, which kills sertoli cells. For example, derivatives of indazole-3-carboxylic acid, such as KU-AS-272 developed by Dr, Joseph Tash and Dr. Kathy Roby, have shown significant effectiveness in clinical trials in both sexes, especially in the male reproductive system. Indazole-3carboxylic acid if used with liposomes can cause effective contraception in both sexes. The drug has already shown successful results in clinical trials on rats. The other useful part of using indazole-3carboxylic acid is that a minimum concentration is required to achieve an effective performance, and thus a 10 year release profile can be constructed easily and injected easily in the reproductive systems of both sexes.

The image of the proposed drug molecule will be similar to drug molecule presented in the following diagram with liposomes attached to it. Given that the drug is encapsulated in between two layers of polymeric biomaterials, its structure and release properties can be switched according to the specific pH.

Picture source: http://www.azonano.com/article.aspx?ArticleID=1538 The reproductive systems for males and females have different pH ranges and temperatures, which ensures the survival of both sperm and ovaries. By developing acid sensitive copolymer micelles for the controlled release of drugs at different specific pHs it will be easy to establish contraception in both sexes with delivery of a single dose. Liposomes containing polysaccharides are used to ensure a low immunogenic response. Various liposome concentrations, with different charges and effective pH ranges will be used as required to interact with ova and testes. Liposomes can be cross-linked with other polymer systems to create membranes that will hold the drug molecules prior to being released at specific pH values. GFPs will be attached to these liposomes so as to visualize how anionic and cationic liposomes assist in the drug release profile of both sexes and promote drug interactions in both reproductive systems. [12]

The mechanism of sperm and ovary production is controlled by the release of FSH and LH hormones. FSH and LH levels play important roles in the production of sperm and ovaries. During menstruation the FSH level is high, and it falls with the release of blood. Sperm remain alive in a temperature range of 2 to 7 degrees centigrade and in a pH range of 7.2 to 8.0. On the other hand, ovaries need to be exposed to a pH range of 3.8 - 4.5 and a temperature range of 4 to 38 degree centigrade for survival and maturation [1,7, 9]. Nature has provided the female reproductive system with a more acid pH range of 3.8 to 4.5, which acts as an antiseptic and protects it from bacterial vaginosis and other diseases. [43] In order to develop a pH responsive polymer that can be activated at pH values less than 4.5, it will be necessary to crosslink the cationic liposome (pH 3 5) with carboxylic acid (pH 4 - 6) at various concentrations which can then entrap any neutral charged porcine zona pellucida (PZP) antigens. Surface modification and surface treatments will ensure that for every menstrual cycle or whenever the pH sensitive polymer is in contact with sperm, then the pH level will drop from the normal value which will cause the pH sensitive polymer to release PZP. [1, 7, 9, 12, 13]. Biopolymers as discussed elsewhere [5] are an innovative way of interacting elastomeric protein chains with polymers to achieve elastomeric-like properties. Since these polymers act in a more dynamic way, and in the proposed study elasticity is being explored to achieve its intended purpose, there are real possibilities for converting the surrounding energy to mechanical energy so as to enhance the reaction rate. It is only by further experimentation that we will be able to find how these polymers will behave. Carboxylic acid and cationic liposomes can act as the core for micelles. As long as the pH is high enough, PZP will not induce cytotoxicity nor interactions with other systems under these conditions. In a similar manner drug delivery systems can be developed using surface activation by corona discharge of N-isopropyl acrylamide and anionic liposomes to achieve activation in the pH range of 6.0 - 7.2. At pH values less than 7.2 the entrapped indazole-3-carboxylic acid is an anti spermatozoa agent, is toxic to sertoli cells and affects normal spermatogenesis. When indazole-3-carboxylic acid is at a pH range of 6.0 - 6.5, it will start killing cells. First it will start apoptosis of granulose cells in the female reproductive system at normal pH levels which will effect the female reproductive system. However, it will only work over a specific pH range for the male reproductive system. [3] It is important to understand that drug release for the female reproductive system needs to be at high concentrations over an extended period of time since the length of the menstrual cycle in various breeds of female cats and dogs is comparatively short but cyclic. Recent studies by Dr Joseph Tash and Dr, Kathy Roby [3, 46] have been undertaken on the derivatives of the ionidamine cancer treatment drug. Alterations to the indazole-3-carboxylic acid structure have provided them with KU-AS-272 a filed patented product that has shown success in mature pubertal rats. Further studies are being conducted to see their effectiveness in pre-pubertal and post-pubertal rats, dogs and cats. The drug KU-AS-272 targets cells in the ovaries and testes, and same agents are currently being utilized to kill cancer cells. It should be noted that the controlled release of the same drug in both male and female reproductive systems has not until now been found effective. This needs to be explored further so that the respective levels of toxicity and their responses need to be identified precisely. The percentage concentration of the delivered drug also needs to be defined, given that it will vary depending upon the size to weight ratio of the animal under investigation.

Another study of a similar product on the market called Adjuvin uses a lonidamine derivative of indazole-3carboxylic acid, which has been grafted with FSH hormone, to induce a false attraction. This causes a decrease in total sperm count for the male reproductive system over time, and has been utilized to provide reversible contraception in the male reproductive system [39]. While PZP has been utilized for female contraception of wildlife animals, such as cats, dogs, elephants and fishes, as early as the 1980s, these early studies show that PZP contraception is reversible [20]. A study at Duke University [45], evaluated temperature and pH sensitive hydrogels for the prevention of sexually transmitted diseases through use of a microbiocidal vehicle which investigated the options using such biomaterial polymers which has served as one of the sources of inspiration behind this approach. There was no specific mechanism found that utilized a combination of PZP and indazole-3-carboxylic acid derivatives to deliver drugs in a controlled manner for the male and female reproductive systems at the same time using pH responsive polymers. Studies on using PZP with resorbable PLA/PLGA carrier materials have been attempted, but there is no product on the market that utilizes responsive polymers to deliver the same drug to both reproductive systems. That is why the novel approach of this submission is to propose the use of dual responsive polymers, i.e. both pH responsive and temperature responsive polymers, to induce controlled drug delivery in both males and females.[20] A new reversible contraceptive drug for humans that is already in Stage III of clinical trials in India and has been successful for almost 7 years now on experimental in vivo animal assays, is the drug RISUG.[40,51] This synthetic polymer has already been successfully used to induce reversible contraception in Indian males. It uses styrene maleic anhydride (SMA) monomer which, when dissolved and polymerized in dimethyl sulphoxide, blocks the production and flow of semen. In the event that contraception needs to be reversed, a reversal injection of sodium bicarbonate solution is delivered to dissolve the polymer. The effectiveness and reversibility of such RISUG polymers can be studied in terms of sperm blockage and subsequent growth in dogs and cats. Preliminary Data: The results of rat animal trials for KU-AS-272, a derivative of indazole-3-carbohydrazide, studied by Dr. Joseph Tash and Dr. Kathy Roby, can be observed in Figure 1. Note how the sperm count has decreased significantly by Week 4. Additional in vivo data for indazole-3-carbohyrazide shows considerable sperm reduction in Figure 2.[3, 49] Figure 1: Source: Tash-Roby KU-AS-272 final.ppt

Figure 2: Sperm count reduction in male rats can be observed on treatment with indazole-3-carbohydrazide

Figure 3: Results showing ZP digestion time for porcine oocytes.

These data support our belief that PZP provides a tougher and more durable glycoprotein layer for sperm. [50] The above experimental data gives a better idea of the effectiveness of the drug on rats and other wild life species.

Proposed Experimental Methods: Initially we will prepare samples of the drug / liposome complexes so that they are stable, activated and resorbable over a range of different pH values at 37.4 degrees Celsius. It will be necessary to characterize their chemical, solubility, thermal and physical properties prior to planning a detailed in vivo protocol in rats. In particular we need to assess their solubility and stability in different aqueous solutions such as distilled water, saline and phosphate buffered saline. The in vivo rat trials (Study 1) will commence with mature, post-pubertal rats and then continue with prepubertal rats. Both male and female contraceptive studies will be undertaken initially over the short term (4-6 weeks). The drug / liposome complex will be administered by intramuscular injection, and the initial dosage concentrations of less than 2% by weight will be used and studied to ensure drug dosage remains within toxicological limits, yet sterilization is maintained throughout the trial. The same number of male and female rats will be included for study with an immediate focus on acute toxicity and sensitization tests prior to assessing the long term evaluation over 6 months. Histological staining of the drug molecules will be performed to observe the acute effects on internal organs. After successfully performing all these tests and trials on rats in Study 1 in a proper controlled environment, the next step will involve designing a longer term in vivo animal protocol with 4 male and female cats and dogs in which the drug effectiveness will be studied on the following animals (Study 2). a. Male and female adult dogs (beagles) b. Pre-pubertal male and female dogs (beagles) c. Male and female adult cats (short hair) d. Pre-pubertal male and female cats (short hair) Variations in results with respect to sterility and cytotoxicity will occur based on the drug concentration and the animal size, which can be studied by the incidence of pregnancy and histological staining of the gonads. [3] After studying drug effectiveness in these two preliminary studies and assuming that our conclusions indicate that our approach is effective and reliable in ensuring sterility over the 6 month period, it will be time to propose a longer term in vivo study to evaluate the long-term effectiveness of the drug for at least 2 years for a range of different breeds of animals.

Statistical Analysis: Computing the data and presenting results is an important task from studying the success rate of administering sterility drugs to rats, dogs and cats in the two trials proposed above. Statistical methods will be used to compare the four populations for cats and dogs using their central values and population variances. Samples for dogs and cats will need to be assessed independently as well as in pairs since their data points will be same through out the study. The variables such as size of assay, drug dosage volume and concentration will be studied and compared using various methods such as multiple comparison studies using Scheffes S method. Variations assessed while sampling will be calculated in terms of p value to ensure appropriate reliability and confidence intervals.

Timeline: S No Event 1 Grant Application Submission and Approval

Duration June - August 2012

Preparation & characterization of liposome drug complex, effectiveness study on male and female rats (Study 1). Data Analysis & Conclusions

August December 2012

Justification Application process time ends June 1st, approval within 4 to 6 weeks Experimentation and data gathering

January February 2013 February 2013 July 2013

In Vivo Trial (Study 2) Development of protocol and performing experimental work for intended 4 groups of dogs and cats Data compilation and analysis

Understanding of data and results of Study 1 Experimentation and data gathering

August 2013 October 2013

Results and analysis of Study 2

Once the preliminary studies (Study 1 and 2) are completed, if their outcomes are successful, then we will propose conducting further studies using a number of different breeds of animals for at least 2- year in which the number of testable factors will be expanded. Animal Use Justification: We will be starting our study with rats, and later continuing with cats and dogs. All the animals used in the experimental studies will be spayed and neutered once the experimentation has been performed. IACUC approvals and regulations will be required at each stage of the studies. The protocol used throughout the trials will include the requirements as defined by the standard National Research Councils Guide for the care and use of Laboratory Animals guideline (2008) in accordance with IACUC regulations. IACUC inspectors approval will be required, and a visit will be made by the inspectors to the animal holding facilities at the College of Veterinary Medicine, NC State University, before the start of the project as required. An initial proposal and justification on the use of laboratory animals will be submitted to the local

IACUC Committee so that prior approval can be obtained in all matters requiring the testing of rats, cats and dogs before the studies start. If there is a need for any change in the study protocols, details of the proposed changes will be submitted to the local IACUC Committee for prior approval. Proper food, diet, exercise, temperature and housing for animals will be provided at all times. Budget: Project timeline: 3 year, 1st Year Budget: $80,000, 2nd Year Budget: $60,000 and 3rd Year Budget: $60,000. Overall Budget: $266,000. The estimated cost is proposed in a realistic manner to cover the costs of Studies 1 and 2. Justification for Proposed Budget for 2012 - 2015 Personnel: There will be only one Principal Investigator but a co-investigator will be added, such as Dr. Kyle Mathews, DVM, Professor of Surgery at the College of Veterinary Medicine, NC State University, once the project is funded and the animal studies will start. Professor Dr. Martin W. King, will be the principal investigator and will lead the research. Two full-time graduate student research assistants (RAs) will be required to prepare and characterize the drug / liposome complex biomaterials, and to undertake the in vivo animal trials and complete the statistical analysis. The annual cost of a graduate student assistantship including out-of-state tuition and health insurance coverage is approximately $60,000. As defined by NCSU regulations, faculty fringe benefits of 19.1% of salary will be added to the first years budget. Equipment: All equipment purchases will be made within the policy and guidelines of NC State University. Travel: The graduate student and/or faculty member will be expected to travel and attending national and international conferences to present the results of this work. The costs for travel, registration and accommodation while attending such a conference will be covered by the budget. (Total: $6,000) Other Direct Costs: Materials, Animals and Supplies: For the purchase maintenance, care, food and housing of the animals the estimated cost will be approximately $110,000 per year. Maintenance Cost: Maintenance and calibration costs of the equipment and facilities are charged each month at 2.5% and are taken care by NCSU administration except for purchased items, such as rats, dogs and cats. (Total: $20,000) Graduate Student tuition: All research assistantships at NCSU have their in-state tuition paid and supported by federal grant, teaching grants and fellowship grants. The grant amount may wary starting from $9,000 to $16,000 for every academic year. (Total $40,000) Facilities & Administrative Costs: All equipments and facilities utilized by graduate students for experimentation are charged and billed each month to the Principal Investigator. (Total: $30,000)

Rahim Jindani Home Address: 701 Centenial Parkway, Apt 307 Mission Valley Apt, Raleigh, NC, 27606 (248) 821-0923

rjindan@ncsu.edu Work Address: 1267 College of Textiles Box 8301, Raleigh, NC

Objective: My objective is to work at NCSU in a challenging environment and utilize my skills with advisors to solve problems that are affecting the world around us by coming up with innovative solutions for highlighted problems at large in developing countries. Education: August2011 Dec2014 North Carolina State University M.S in Textile Technology GPA: 3.58/4.0 Concentrations in medical textiles and nonwovens August 2006 May 2010 Textile Institute of Pakistan (TIP), Karachi BSc in Textile Sciences GPA: 3.02 /4.0 Thesis: Enhancing surface properties of textile materials using organic solvents. Advisor: Faiza Saeed, ISBN: 978- 3-8433-8615-9, Published by LAP Experience: March 2011 July 2011 United Registrar of Systems (URS), Pakistan Worked on Global Reporting Indicators (GRi3), with Engro Pakistan and AkzoNobel Pakistan and verified their sustainability initiatives taken in year 2010 Certified Lead Auditor for ISO 9001:2008, Quality Management System, audited various conglomerates in the capacity of Auditor. Studied Courses from Chartered Management Institute of U.K. on financial Control, Resource Management and Marketing and Planning. June 2010 Dec 2010 Management Trainee, Artistic Milliners (AM), Pakistan Assisted in Product Development for GAP, (Gap Mens, Kids, Women) Counter Development and Quality Control for proto-samplings before Premier Vision (France) Show Fall 2010. Studied Better Cotton (BT) initiatives taken by H&M. Co-curricular Activates: Headed Rotract TIP-Karsaz as Vice President Worked with UNICEF, WHO on Polio eradication Working Currently with The Citizen Foundation to ensure better education for all young students, Worked as TCF mentor, planner in Pakistan, currently member of TCF-USA team for awareness and promotion of TCF work internationally. Working with Ray of Hope in Africa on HIV Aids project proposal.

References Cited and Readings: 1. Horacio B. Croxatto*, Mechanism that explain the contraceptive action of progestin implants for women Contraception 65 (2002) 21-27 2. Aristide Floridi, Marco Giorgio Paggi, Stenfania D`Atri, et al. Effect of Lonidamine on the Energy Metabolism of Ehrlich Ascites Tumor Cells Cancer Res 1981:41:4661-4666 3. KU-AS-272, as a Potential Single-dose Non-hormonal Male and Female Sterilant for Cats and Dogs, Dr. Joseph Tash, Dr. Kathy Roby http://michelson.foundanimals.org/resources 4.ReproductiveBiology101Dr.MichelleKutzlerhttp://michelson.foundanimals.org/resource 5. Marica Martino, Tiziana Perri, Antonio M. Tamburro*, Biopolymers and Biomaterials Based on Elastomeric Proteins Macromolecular Bioscience 2002, 2, 319-328 *] 7. http://www.drmalpani.com/azoospermia.htm 8. http://women.webmd.com/vaginal-wet-mount 9. Muzaffer Tas, Mithat Evecen, Ozen Banu Ozdas, Umut Cirit, kamber Demir, Sema Birler, Serhat Pabuccuglu, Effect of Transport and storage temperature of ovaries on in vitro maturation of bitch oocytes. Animal Reproduction Science 96(2006) 30-34 11. M.R Barber, S.M Lee, W.L Steffens, M.Ard and R.A Fayer-Hosken, Immunolocalization of Zona Pellucida Antigens in the ovarian follicle of dogs, cats, horses and elephants, Department of Large Animal Medicine and Physiology and Pharmacology, Department of Pathology, College of Veterinary Medicine, University of Georgia Athens, Georgia December 13, 2000 12. , Elizabeth R. Gillies and Jean M. J. Frechet Development of Acid-Sensitive Co-polymer micelles for drug delivery, Pure Appl. Chem., Vol. 76, Nos. 7-8, pp. 1295-1307,2004 2004 IUPAC 13.Arnaud E. Felber, Marie-Helene Dufresne, Jean-Christophe Leroux, pH-Sensitive Vesicles, Polymeric micelles, and Nano spheres prepared with polycarboxylates Institute of Pharmaceutical Sciences, ADR12189;No of Pages 14 14. Edward C. Yurewicz, Anthony G. Sacco, and Marappa G. Subramania, Structural Characterization of the Mr = 55,000 Antigen (ZP3) of Porcine Oocyte Zona Pellucida, Journal of Biological Chemistry 1987, Vol. 262, No: 2, Issue of January15, pp, 556-571 15. M.L.Papale, A.Grillo, E.Leonardi, G.Giuffrida, M.Palumbo and G. Palumbo, Assessment of the relevance of Zona Pellucida antibodies in tollicular fluid of in-vitro fertilization (IVF) patients, Human reproduction Vol.9 no.10 pp.1827-1831, 1994. 16. C.R. Brown and W.K.T Cheng, Changes in composition of the porcine zona pellucida during development of the oocyte to the 2- to 4-cell embryo.exp. Morph, 92, 183-191 (1986) 18. J.Guo*, L.Li, Y.Ti, J. Zhu, Synthesis and Properties of Novel pH Sensitive Poly (N-vinyl-pyrrolidone-cosulfadiazine) hydrogel, eXPRESS Polymer Letters Vol.1, No.3 (2007) 166-172 19. PATENT TITLE: Molecules presenting a multitude of active moieties, Application No. AU 199871069 B2 (10) Patent No. 743028 20. Satish K. Gupta, Villuppanoor A.Srinivasan, Pankaj Suman, Sriraman Rajan, Singanallur B. Nagendrakumar, Neha Gupta, Abhinav Shrestha, Pooja Joshi, Amulya K. Panda, Contraceptive Vaccines Based on the Zona Pellucida Glycoproteins for Dogs and Other Wildlife Population Management, National Institute of Immunology, Newdelhi-110 067. 21. M. Constantin, I. Oanea, V. Harabagiu, P. Ascenzi, G. Fundueanu, DNA Complexation by Cationic Pullulan Possessing Thermo-sensitive Units, Digest Journal of Nanomaterials and Biostructures Vol.6, No 2, April June 2011, p. 849-861

22. P Sipila, J Jalkanen, IT Huhtaniemi and M Poutanen, Novel Epididymal Proteins as Targets for the Development of Post-Testicular Male Contraception Reproduction Advance Publication REP-08-01-0132. 23. S B Meroni, M F Riera, E H Pellizzari and S B Cigorraga, Regulation of Rat Sertoli Cell function by FSH: possible role of phosphatidylinositol 3-kinase/protein kinase B pathway Hournal of endocrinology (2002) 174, 195-204 24. Yongjin Liu, Bin Zhang and Bing Yan, Enabling Anticancer Therapeutics by Nanoparticle Carriers: The Delivery of Paclitaxel, International Journal of Molecular Sciences ISSN 1422-0067. 25. Darrel J. Kesler, Remotely delivered Contraception with Needle-less Norgestomet Implants. 26. Donatella Del Bufalo*, Annamaria Biroccio*, Silvia Soddu, Nina Laudonio, Carmen D Angelo, Ada Sacchi, and Gabriella Zupi, Lonidamine Induces Apoptosis in drug-resistant cells independently of the p53 Gene, C.R.S., 00158 Rome Italy 27. H. Ian Robins, Walter L. Longo, Rhonda K. Lagoni, e al. Phase I Trial of Lonidamine with Whole Body Hyperthermia in Advanced Cancer, Cancer res 1988;48;6587-6592 27. E. Manias, M. Rackaitis, Temperature-Responsive polymers for Biological Applications, Pennsylvania State University, University Park, PA 28. Liliana Verestiuc, Oana Nastasecu, Eugen Barbu, Indrajeetsinh Sarvaiya, Keith L. Green, John Tsibouklis, Functionalized Chitosan/NIPAM (HEMA) hybrid polymer networks as inserts for ocular drug delivery: Synthesis, in vitro assessment, and in vivo evaluation., Pharmacy and Biomedical Sciences 29. Man Fai Leung, Junmin Zhu, Frank W. Harris, Pei Li*, Novel Synthesis and Properties of Smart CoreShell Microgels,Macromol. Symp. 2005,226, 177-185. 30. Marica Martino, Tiziana Perri, Antonio M. Tamburro*, Biopolymers and Biomaterials Based on Elastomeric Proteins, Macromol. Biosci 2002, 2, 319-328 31. Pier L. san Bhagio, Francesco Madonia, The Overlap of Elastomeric Polypeptide coils in solution required for single phase initiation of elastogensis, Chemical Physics Letter Volume 145, number 6, 22 April 1988. 32. Dana L. Nettles, Ashutosh chilkoti, Lori A. Setton, Applications of elastin-like polypeptides in tissue engineering, Advanced Drug Delivery Reviews 62 (2010) 1479-1485 33. J. carlos Rodriguez-Cabello*, Javier Reguera, Alessandra Girotti, Matilde Alonso, Ana M. Testera, Developing functionality in elastin-like polymers by increasing their molecular complexity: the power of the genetic engineering approach, Prog. Polym. Sci. 30(2005) 1119-1145. 34. Megan Cathey, DVM, and Mushtaq A. Memon, BVSc, PhD, DACT, Nonsurgical Methods of Contraception in dogs and cats: Where are we now?, PEER REVIEWED 35. Tao chen, Deirdre Macintosh, Yuehua He, Jungsoo Kim, David A. Tirrell, Peter Scerrer, David B. Fenske, Ammen P. Sandhu and Pieter R. Cullis, Alkylated derivatives of poly(ethylacrylic acid) can be inserted into preformed liposomes and trigger pH-dependent intracellular delivery of liposomal contents, Molecular Memrane Biology, 2004, 21, 385-393 36. Material Matters (Materials for biomedical Applications), Volume5, Number3.2010 by ALDRICH Material Science. 37. M.R. Aguilar*, C. Elvira, A. Gallardo, B. Vazquex, and J.S Roman, Smart Polymers and their applications as Biomaterials, Topics in Tissue Engineering. Vol 3, 2007 38. Dolores D Mruk, Ching-Hang Wong, Bruno Silverstrini & C Yan Cheng, AMale Contrceptive targeting germ cell adhesion, Nature Publishing Group 2006. 39. Katja M. Wolski, Dolores d. Mruk, And Don F. Cameron, The Sertoli-Spermatid Junctional complex adhesion Strength in affected in vitro by Adjuvin, Journal of Andrology vol.27, No. 6, November/December 2006 40. Panache BIZ Indian Pharmaceutical Association Maharashtra State Branch-Students Forum.

41. L.Lo Leggio, R.M. Williams and R. Jones, Some effects of Zona Pellucida glycoproteins and sulfated polymers on the autoactivation of boar sperm proacrosin and activity of Beta-acrosin, Journal of Reproduction and fertility (1994) 100, 177-185 42. Rupert konradi, Synthesis, Swelling Behavior, Complex Formation and Micropatterning, University of Hedelberg. 43. Katia sutyak Noll,The safe, Natural antimicrobial peptide Subtilosin for control of bacterial Vaginosis, Microbiology and Molecular genetics, University of New Jersey. 44. C.E. Hoesl, F.Saad, M. Poppel, J.E. Altwein, Review Reversible, Non-Barrier Male contraception: status and Prospects, European Urology 48 (2005) 712-723 45. Kavita M. Gupta, Scott R. Barnes, Rachel A. Tangaro, Meredith C. Roberts, Derek H. Owen, David F. Katz, Patrick F. Kiser, Temperature and pH Sensitive Hydrogels: An Approach Towards Smart SemenTriggered Vaginal Microbiocidal Vehicles, Department of Biomedical Engineering, Duke University DOI 10.1002/jps.20752 46. Targeted delivery of Cytotoxins for Sterilization of Cats and Dogs, 4th International symposium on Nonsurgical Contraceptive Methods of Pet Population Control 47. N. Levine and Helen Kelly, Measurement of pH in the rat epididymis in Vivo, J. Reprod. Fert. (1978) 52, 333-335 48. Maria Elsa Traina, Maria Guarino, Elisabetta Urbani, Luciano Saso, Patrizia Eleuteri, Eugenia Cordelli, Michele Rescia, Giorgio Later, Marcello Spano, Lonidamine transiently affects spermatogenesis in pubertal CD1 mice, Contraception 72 (2005) 262-267 49. C. Yan Cheng, Bruno Silvestrini, Josephine Grima, Meng-yun Mo, Li-ji Zhu, Elof Johansson, Luciano Saso, Maria-Grazia Leone, Maura Palmery and Dolores Mruk, Two New Male Contraceptives Exert Their effects by Depleting Germ Cells Prematurely from the Testis. 50. Picture Source: Pilar Coy*,Sebastin Cnovas*, Irene Mondjar*, Maria Dolores Saavedra*, Raquel Romar*, Luis Grulln*, Carmen Mats*, and Manuel Avils, Oviduct-specific glycoprotein and heparin modulate spermzona pellucida interaction during fertilization and contribute to the control of polyspermy. 51. Rakhi K Jha, Pradepp k Jha, Sujoy K Gulha, Smart RISUG: Apotential new contraceptive and its magnetic field-mediated sperm interaction. 52. Responsive Polymer/liposome Complexes: Design, Characterization and Application, Department of Chemistry, Mc Master University, 1280 Main St W, Hamilton, Ontario Canada L8S 4M1, Francoise M. Winnik and Tania Principi 53. Contraceptive Vaccines for Wildlife: A Review by Jay F. Kirkpatrick, Robin O. Lyda, Kimberly M. Frank, The Science and Conservation Center, Zoo Montana, Billing, MT, USA