• Development and Characterization of a Novel Immunotherapy for Treatment of Breast Cancer by Combining Particulate Cancer Vaccines with Immune-Modulators

      Mulla, Nihal S; College of Pharmacy
      In the last half of the century, advances in the field of cancer therapy including chemotherapy, hormonal therapy and targeted therapy have been responsible for improvements in breast cancer related mortality. Although such advances have benefited all cancer types, there are considerable challenges faced by researchers striving to realize the goal of complete tumor remission. Immunotherapy is a great alternative as it has minimum side effects and several advantages over traditional cancer therapies. The aim of this project is to develop a novel immunotherapy for treatment of cancer by combining cancer vaccines with various immune-modulators. We take advantage of micron-sized particles to deliver vaccine along with other immune modifiers to target immune cells and to initiate immune response against breast cancer antigens. These particles were evaluated for their size, charge, surface morphology, release profiles, cyto-toxicity and particle uptake by various in vitro studies. The efficacy of breast cancer vaccine microparticles was tested in a murine breast cancer model. The immunized animals showed significantly lower tumor growth compared to the naive animals that did not receive any treatment. The delay in tumor growth in vaccinated animals was due to a strong immune response generated against tumor- associated antigens encapsulated within the microparticles. We observed a significant increase in the CD4+ T cell population. The suppression mechanism employed by regulatory T cells are thought to contribute significantly to the failure of current therapies that rely on potentiation of anti-tumor responses. We evaluated the therapeutic efficacy of vaccine microparticles after depleting the immunosuppressive regulatory T cells. We observed a significant improvement in the efficacy of vaccine microparticles by depleting regulatory T cells. The tumor inhibitory effect of vaccine microparticles was due to depletion of regulatory T cells by cyclophosphamide. We observed a significant increase in the CD8+ T cell population. The final aim of my research project was to enhance the immunogenicity of cancer vaccines using adjuvants. Based on our findings we conclude that Alum, Addavax (like MF59) R848 and CpG significantly enhanced the immunogenicity of breast cancer associated antigens.
    • Development and Evaluation of Micro/Nanoparticulate Vaccines for Human Papillomavirus and Influenza

      Vo, Trinh; College of Pharmacy
      Influenza and Human papillomavirus (HPV) are among the most common infectious deseases prevalent globally. The influenza viruses cause approximately 200,000 hospitalizations each year, resulting in a significant financial burden to the healthcare industry. Similarly, HPVs cause virulent infections that have multiple serotypes capable of producing genital warts and cervical cancer. In both cases the infection is spread through viral agents, The influenza virus is transmitted by aerosolized mist of coughs and sneezes with HPV is a sexually transmitted disease. Prevention of influenza and HPV is possible with the help of vaccines, and currently, there are several vaccines for both infectious diseases. However, in the case of influenza, development of a vaccine is challenging as the virus undergoes rapid mutations and hence, the vaccine needs to be modified every season. Along with the constant need for vaccination rates. In the case of HPV, the need for a novel vaccine delivery system is more crucial. HPV is directly linked to cervical cancer and developing a novel strategy to improve vaccination globally is a major priority. Commercial vaccines are not only expensive but in developing countries the financial cost of maintaining cold storage and vaccine administration is significantly challenging. The developing non-parenteral vaccines is essential to improve patient compliance without affecting safety and efficacy of the vaccine. This project has two main sections focusing on development of an oral microparticulate vaccine for influenza and HPV. In the first part of this project, we developed an oral microparticulate vaccine for influenza. The influenza vaccine consists of a M2e protein which is a viral protein common to all influenza viruses encapsulated into a microparticle. Adjuvants such as Alum, MPL. R848, MF59, Flagellin, CpG and P4 were encapsulated in microparticles separately. Microphages werestimulated with the vaccine-adjuvants combinations and subsequently., splenocytes were incubated with activated macrophages to investigate the T-cell activity. We observed significant enhancement of immune markers such as nitric oxide, CD80, CD86, and MHC II with several adjuvants. Furthermore, CD4 and CD8 T cell expression was also significantly enhanced in groups treated with particulate adjuvants. In the second part of this project, we formulated a novel oral and transdermal vaccine delivery system for HPV. Development of potent vaccines against HPV can prevent serious complications such as genital warts and cervical cancer. Therefore a novel non-parenteral vaccine against HPV may significantly reduce incidence of HPV worldwide. In this study, three different microparticualte vaccine formulations have been prepared and characterized: Bovine Albumin Serum (BSA) cross0linked with glutaradehyde; b-cyclodextrin, and cellulose acetate succinate matrices. We systematically studied activation of the immune markers such as nitirc oxide. CD80, CD86, CD40 and MHC-II. In vivo studies demonstrated elevated humoral and cell-mediated immune response in murine models. We observed elevated IgG titers in mice vaccinated with microparticulate HPV16 VLP vaccine, Furthermore, flow cytometry analysis revealed expansion of specific T-cell populations in vivo. This study proves the efficacy of the HPV-16 oral microparticulate vaccine as a promising alternative to conventional vaccines, In the alter part of the HPV vaccine project we investigated the potential of transdermal HPV vaccine as a novel strategy to improve vaccinations globally. In order to combat HPV infection, vaccines must be affordable, self-administered and efficacious. Transdermal vaccines can be self-administered. are almost painless and tap the immune cells present under the skin. Transdermal vaccines have gained immense attention due to their efficacy. In this project, our main aim was to develop a microparticulate HPV16 VLP vaccine for transdermal vaccination. HPV-16 VLP was encapsulated in microparticles and characterized for size, zeta potential, encapsulation efficiency and microparticle yield. Scanning electron microscope images confirmed the particles were irregular with surface indentations. Western blot analysis was performed on HPV16 VLP extracted from microparticles, which confirmed the retention of antigenicity of the epitope following microparticles, which confirmed the retention of antigenicity of the epitope following microparticle fromulation. Following in vitro characterization studies, we performed extensive in vivo studies to investigate the potential of the microparticulate vaccine to initiate a robust and sustained immune response. IgC titers for mice vaccinated with microparticulate vaccine were significantly elevated as compared to antigen solutions adminstered by the transdermal route. Flow cytometry was performed on vaccinated and control groups to investigate the expansion of specific T-cell populations. Vaccinated mice demonstrated significant increase in CD4, CD45R, CD27 and CD62L cell populations. Expansion of these specific T-cell subsets demonstrates the efficacy of the microparticulate vaccine when administered transdermally. Thus, our study proves the efficacy of the microparticulate vaccine over vaccine solutions and the novel approach of administering the vaccine through a minimally invasive, pain-free route that will help to improve acceptability of vaccination against HPV.
    • Development of Microencapsulated Formulations for Prostate Cancer Vaccine and Live Cells

      Akalkotkar, Archana; College of Pharmacy
      In the current trend, novel particle based platform technology has paved its way to various therapeutic benefits in our lives. In this study, we explored the way of harnessing this technology in order to formulate effective vaccines in the quest for mass immunization in a cost-effective manner. Here we propose a custom formulation of vaccines, which will render effects of sustained release, site specificity and cost-effective delivery of various antigens to enhance the immunity. We used the novel nano-micro technology based of Spray drying as a platform to deliver vaccines against Prostate cancer and encapsulated live cells. In this study, we formulated a whole cell lysate vaccine against prostate cancer via oral and transdermal route of administration. The formulation intended for oral adminstration was optimized for M-cell targeting, The formulation increased the immunogenicity of the vaccine by incorporating the antigen into an albumin matrix having a size of around 0.35-1.2 um that acted as a synthetic adjuvant. The animals were vaccinated with 1 prime and 4 booster doses administered every 14 days over 10 weeks duration, followed by challenge with live tumor cells which showed protection after oral vaccination. Unlike subcutaneous injections, administration-using microneedles is painless and in general can increase the permeability of many compounds ranging in size from small molecules to proteins and microparticles that do not normally penetrate the skin. The mice vaccinated via transdermal route showed promising results in delaying the tumor growth compared to the controls. The study was extrapolated in a therapeutic approach. This would mimic the real clinical scenario where the patient who was diagnosed with prostate cancer would come in the clinic for immunotherapy. The microparticle based delivery system showed protection in vaccinated group compared to the controls. This shows the microparticle based delivery system can be an useful tool for delivery as well as live cells in vivo.
    • Development of Spray Dried Microparticulate Delivery Systems for Vaccines and Oligonucleotides

      Ubale, Ruhi V; College of Pharmacy
      In recent years, research related to the development of biotherapeutics has gained tremendous interest in industry as well as academia. There are a number of biologics available in the market and many more currently being evaluated in clinical trials. Their development has gained momentum due to the range of advantages they offer over traditional small molecule drugs. However, there still remain challenges with their delivery mainly due to the poor stability of these molecules in harsh physiological conditions. A number of strategies are being devised for the delivery of biomolecules by non-invasive routes to make delivery easier and more patient compliant. Microparticles are one of the options being explored as they offer protection, sustained release and enhanced bioactivity to the biomolecule. In this project, we have studied the use of microparticles for the development of formulations for a meningococcal polysaccharide vaccine and an antisense oligonucleotide to NF-kB. Meningitis occurs majorly in children and is caused by Neisseria meninigitidis. Infection by this bacterium spreads rapidly through the body resulting in loss of limbs, hearing and can also be fatal. Hence, vaccination is considered as the only means prevention for meninigitis. Marketed vaccines use protein conjugated bacterial capsular polysaccharides as the antigen. Polysacchatides are considered inherently weak antigens with a T- independent immune response. With the development of a microparticulate vaccine using a protien matrix, we expected to stimulate the T-dependent immune pathway for these antigens avoiding the use of a conjugated Diphtheria toxoid. We have studied the enhancement of innate immune responses by the microparticulate vaccine. The antigen used N. meningitidis polysaccharide A, aslo an adjuvant- kdtA(unglecosylated lipid A)- was studied for its response. The microparticulate vaccine was characterized for its physicochemical characteristics like size, charge and toxicity. Also it was studied for the innate immune responses the microparticles can elicit after incubation with murine and human macrophages. Release of innate immune markers like TNF-a, IL-1B, IL-8, nitric oxide and reactive oxygen species from macrophages was studied and the microparticulate vaccine was observed to elicit a desirable innate immune response. Antigen presentation by macropaphages stimulated with the microparticulate vaccine was also studied by visualizing autophagy. Lung inflammation occurs doe to an infection or a damaging agent that irritates the lung lining or pleura. NF-kB is a nuclear transcription factor activated by stimuli like endotoxin and bacteria that initiates the synthesis of pro inflammatory cytokines such as TNF-a, IL-1, IL-6, etc. Antisense are single stranded RNA complementary to a chosen sequence that offer great potential to inhibit the synthesis of individual proteins by interfering with protein translation. As intracellular penetration of antisence compounds has proven to be a limiting factor in their effectiveness, we have proposed to develop a microparticulate formulation for pulmonary delivery of antisense to NF-kB and evaluate their efficacy. The microparticulate formulation of antisense to NF-kB was characterized for its physiochemical characteristics like size, charge, antisense content and release. Since enhancing uptake into cells was the crux of the study, we also visualized and quantified uptake of microparticles by microphages. We looked at the period of lung resistance and the biodistribution of the microparticles using a near infrared bioimager. Serum levels of proinflammatory cytokines were studied after induction of lung inflammation in a rat model. Animals that were delivered antisense microparticles to the lung showed reduced levels of TNF-a and IL-1b.
    • Enhancement Technologies for Delivery of Small Molecules Into and Across Skin

      Kale, Madhura; College of Pharmacy
      Transdermal drug delivery is a non-invasive route of drug administration through the skin. It offers several benefits, such as bypassing the first-pass metabolism, avoiding fluctuations in plasma concentrations, and easy termination of therapy. Furthermore, local and systemic delivery via this non-invasive route makes it patient-friendly, and this route avoids compliance issues, especially for pediatric and geriatric populations. However, although unionized drugs, having molecular weight <500 Da and moderate lipophilicity (log P 1-3) can passively permeate through the skin, larger molecules and hydrophilic drugs cannot pass through the stratum corneum, the tightly packed lipophilic layer which acts as a barrier to drug delivery. Thus, to overcome this barrier, physical enhancement techniques are applied to achieve permeation into and across the skin. Physical enhancement techniques like iontophoresis, skin microporation using microneedles or an ablative laser, and microdermabrasion have been explored for their drug delivery potential. In this study, we evaluated the topical delivery of nordihydroguaretic acid (NDGA), a compound having the potential to attenuate arsenic toxicity. Studies showed that this compound has the potential to get retained in the skin, which is the target site of delivery. Also, skin exposed to UV radiation too showed similar delivery with that of normal skin. Thus, topical delivery of NDGA is feasible for reducing arsenic toxicity. We also evaluated the topical delivery of kinetin, an antiaging phytohormone that inhibits senescence in plants and helps increase catalase activity. We enhanced the topical delivery using two approaches: increasing the drug loading in the formulation and using microdermabrasion. Our studies showed that increasing the drug loading as well as abrading the skin led to enhancement in the topical delivery of kinetin. We also evaluated the transdermal delivery of donepezil, a cholinesterase inhibitor that is used for managing Alzheimer’s. Physical enhancement were used alone and in combination to control the delivery of donepezil. We observed that the flux profiles could be tailored successfully using a combination of skin microporation and iontophoresis, and therapeutic levels of donepezil can be delivered. In this project, we also developed an iontophoretic patch for the transdermal delivery of zanamivir. A conducting crosslinked chitosan membrane was formulated that, upon hydration, can conduct current. Prophylactic levels of zanamivir were delivered transdermally with this iontophoretic patch. Thus, we successfully delivered pharmaceutical actives across the skin using passive and physical enhancement techniques that have the potential to manage diseases and help patients lead better lives.
    • Facilitated Topical and Transdermal Delivery of Small Molecules

      Yeh, Jihee; College of Pharmacy
      The skin is the largest organ of the body that is easily accessible and can be utilized as a route of administration to deliver drugs locally and systemically to achieve targeted therapeutic effects. It provides several advantages over conventional routes of administration (e.g. oral and parenteral) such as bypassing first-pass metabolism, reduction of adverse effects and enhanced patient compliance. However, drug delivery through skin can be challenging due to protective barrier of the skin, especially stratum corneum. Stratum corneum serves as a rate-limiting layer and only allows the permeation of drug molecules with certain physicochemical properties. Chemical enhancers can be used to reversibly alter the structure of stratum corneum to be more permeable without causing long-term compromise of the skin. Also, physical enhancement technique such as iontophoresis utilize an additional energy as a driving force to actively disrupt the barrier nature of stratum corneum to enhance the drug delivery.In the present study, different enhancement strategies such as addition of permeation enhancers to the drug formulation and/or electrically assisted technique such as anodal and cathodal iontophoresis were investigated to enhance the delivery of therapeutic (N-acetylcysteine and minoxidil) and dermatological (adapalene) drugs into and across the skin. Also, iontophoresis was explored for enhancement of topical and transfollicular drug delivery. In summary, chemical enhancers and physical enhancement technique were shown to significantly enhance the skin permeation of different drugs into and across the skin, as compared to their respective passive permeation controls. Furthermore, studies of iontophoresis with different durations of current application revealed that lower duration was adequate to achieve significant amount of minoxidil in hair follicles with reduced amount of drug penetration across the skin, thereby potentially minimizing systemic exposure.
    • Formulation and Evaluation of Microparticulate System for the Development of Pneumonia and Influenza Vaccines

      Nagaraja Shastri, Prathap; College of Pharmacy
      In recent years vaccine research has gained a tremendous interest from both industries as well from the academic sectors. There are number of vaccines available in the market and still there is a scope of improvement in most of the marketed vaccines. The antigens used in vaccination are in general large molecules, either protein or polysaccharide based. These antigens can lead to specific antibodies that will protect our body from the infection. Some of the antigens are stable, however majority of them are instable and sensitive resulting in problems during formulation, storage. Formulation of protein or polysaccharide has always been a challenge for scientists due to several characteristics of the antigen and the dosage from itself. Microparticle is on the the dosage forms that have shown promising results in several vaccine studies in the past. In this study we have evaluated microparticle formulations for two infectious diseases namely, Pneumonia and Influenza. Both these are respiratory infections and the vaccinations against these are highly recommended by the Center for Disease Control. In particularly for Influenza the vaccination is recommended every year. In this research we have used two novel approaches to formulate these antigens using microparticles. The pneumococcal polysaccharide antigens are usually less immunogenic in nature and hence to potentiate their immune response the antigens were formulated in a cross linked albumin matrix. In case of Influenza vaccines, we have attempted to vaccinate via oral route of administration after formulating inactivated form of influenza virus in an enteric coated microparticle formulation. Upon formulation both theses vaccines were characterized for their physical properties such as particle size, zeta potential and also the bioactivity of these antigens in microparticles were measured using antigen specific bio assays, Further invivo studies were carried out in mice to evaluate the adaptive immune responce elicited by microparticle based vaccines. The results have been promising with increase in antibody titers for vaccine formulations and also better protection was observed in case of Influenza vaccine, Overall these promising results further emphasize the use of microparticles as a tool to deliver vaccine antigens effectively.
    • Formulation and In Vivo Evaluation of Particulate Breast Cancer Vaccine

      Chablani, Lipika; College of Pharmacy
      Purpose: This research work concentrates on formulating two particulate breast cancer vaccines, which are further evaluated in vivo using two murine breast cancer models. As breast cancer continues to be the most fatal cancer among women throughout the world, there is an immediate need to develop a vaccine to combat it. Considering the potential of particulate delivery vehicles to impart robust systemic as well as mucosal immune response, they have been explored not only against infectious diseases but also against cancer. In this project we take advantage of these micron sized particulate delivery vehicles to target immune cells and to initiate immune response against breast cancer antigens, Also, these particles have been fabricated in such a manner that they can be administered via patient-compliant routes of administration including oral and intraepidermal delivery. Research methodology: We have explored various polymers to optimize two enteric protected particulate delivery systems with desired physical properties making them susceptible to particle uptake and there by leading to anticipated immune response. These particles have been evaluated carefully for their size, charge, surface morphology, release profiles, cyto-toxicity and particle uptake by various in vitro studies. Further the particle uptake of vaccines by the M (microfold) cells in the Peyer's patches of the small intestine when given orally is studied extensively along with characterization of microchannels created to deliver the microparticulate vaccine. Also, the vaccine efficacy was evaluated in vivo in female mice model using the two marine breast cancer cell lines which mimic the progression of breast cancer as seen in humans, The vaccine was administered via oral, intraepidermal and sub-cutaneous routes resulting in immune response against the breast cancer antigens used in the vaccine. Results and Conclusion: The vaccinated animal had sugnificantly smaller tumor volumes than the control animals, as seen after challenging the animal upon termination of treatment. Immune responses in all the animals were monitored to gauge the role of humoral and cellular immunity in generating protection by several in vitro and ex vivo studies. Promising results from these two prophylactic particulate breast cancer vaccine studies have advanced our laboratory to explore a therapeutic breast cancer vaccine. These particulate delivery systems possess the potential of entering the clinical trials and mimicking the real-time situation where patient's own tumor cells extracted after a surgery can serve as the source of antigens for an individualized particulate vaccine.
    • Formulation Design and Development of Theranostic Nanoparticles for Tumor Targeted Drug Delivery

      Kolluru, Lakshmi Prasanna; College of Pharmacy
      Cancer is among the leading cause of death in the world, accounting for one in every four deaths in United States. Researchers from academia and industry are working on discovering new drug targets, developing better drug products and enhancing efficacy of drug delivery systems. In spite of the advancements, drug delivery still remains a challenge in the management of cancer. Currently, Chemotherapy (mostly in combination with radiation) is a major therapeutic approach for the treatment of cancer. However several chemotherapeutic drugs lack the ability to differentiate between normal and tumor tissues and suffer from drawbacks such as dose limiting toxicity, low specificity and emergence of multidrug resistance. Major concerns associated with current anti-cancer agents which are gaining wide importance include rapid elimination from kidney and non-specific biodistribution. In addition, the rapid clearance of the drug from the body might require administration of larger doses which can cause toxic effects. One approach to reduce the systemic toxicity and enhance the efficacy of the drugs us to administer through selective drug delivery carriers. Polymeric nanoparticles offer promise for targeted drug delivery as they have the potential for passive targeting of drugs by Enhanced Permeable and Retention (EPR) effect, controlled/sustained release of drug, reduced clearance and ability for surface fuctionalization with tumor targeting ligands. In this studym we have successfully fabricated polymeric nanoparticles for the delivery of diagnostic and therapeutic agents using Bovine Serum Albumin (BSA) polymeric nanoparticles. Near Infrared dye, Indocyanine green and anti-cancer drug, Doxorubicin are used as model diagnostic and therapeutic agents respectively. Folic acid and cyclic RGD are used as tumor targeting ligands to target tumor microenvironment and tumor cells. This project focuses on the preformulation, formulation development, in vitro characterization and in vivo evaluation of the drug-dye loaded nanoparticles and evaluation of the active targeting potential is proposed. Spray drying and Nanoprecipitation techniques are evaluated and nanoprecipotation technique is used in final preparation. Nanoparticle suspension was then subjected to lyophilization. The formulation is further extensively characterized in vitro by Dynamic light scattering (DLS), Release Studies, Differential SCanning Calorimetry (DSC) and MTS Cytotoxicity Assey. In addition, the formulation is also evaluated in vivo for its tumor targeting potential by monitoring the biodistribution of entrapped near infrared dye using whole body non-invasive imaging technique. Results of our work demonstrated that diagnostic and therapeutic agents can be effectively delivered in a single delivery system. Our work further emphasizes that nanoparticle based system can enhance localization of diagnostic (or therapeutic agents) into the tumor, thereby contributing to reduces side effects and enhanced efficacy.
    • Formulation Develoment and Characaterization of Polycaprolacton/Pluronic F108 NanoParticles for Targeted Breast Cancer Therapy

      Chandran, Thripthy; College of Pharmacy
      Breast Cancer ranks first among cancer deaths for women after malignant lung cancer in the United States. Despite the major advancements in the treatment of breast cancer, it still poses a major challenge. While chemotherapeutic intervention remains the major treatment approach fr cancer, they suffer from several drawbacks including dose limiting toxicity, non-specific biodistribution, and emergence of resistance in solid tumors, thus posing a risk of relapse. Furthermore, the excipients used for the administration of the anticancer agents also cause several undesirable systemic effects such as nephrotoxicity, neurotoxicity, and hypersensitivity reactions when given intravenously. Biodegradable polymeric nanoparticles have emerged as promising targeted drug delivery systems for the delivery of anticancer drug owing to their size characteristics., their ability to passively accumulate by Enhanced Permeability and Retention (EPR) effect in the tumors, ability to protect the active ingredients from degradation, providing a controlled/sustained release of the active ingredients and b tunable for the attachment of active ligands according to a patient's tumor profile and thus provide a personalized therapy. In this study, we designed polymeric nanoparticles for the delivery of anticancer agent docetaxel using poly-e-caprolactone (PCL), which forms the core of the the nanopartilces. Pluronic polymer F108 is used as an emulsifying agent/stabilizer for the PCL nanoparticles, providing a hydrophilic PEG coating thus stealth properties to the nanoparticles. The passive targeting ability of the nanoparticles is evaluated using a near infrared carbocyanine dye (Dir). Trastuzmab is used as an active targeting ligand to target the HER-2 receptors present on the human breast cancer BT-474 cells. Thus, this project focuses on the formulation development, in vitro characterization of docetaxel loaded nanoparticles and finally the characterization of trastuzumab conjugated nanoparticles.
    • Oral Microparticulate Prostate Cancer Vaccine: A Promising Immunotherapeutic Approach

      Parenky, Ashwin; College of Pharmacy
      Prostate cancer is one of the leading causes of cancer-related deaths among men in the United States. Currently, there are 5 new agants approved in the United States against prostate cancer which include Sipuleucel-T, cabazitaxel, abiraterone acetate, enzalutamide and radium-223. Introduction of these agents into the clinic are important strides; however, resistance to chemotherapeutic agents is still a significant challenge, Furthermore, when patients suffer from recurrence of prostate cancer, survival is less than five months. Hence, there is an urgent need to investigate alternative approaches to treat castration resistant prostate cancer and prevent relapse. Immunotherapeutic approaches to treat cancer are under intense investigation owing to their specificity and potency to eliminate tumors. One of the most intensely studied areas in the cancer research is identification of cancer antigens that can help indicate progression of cancer, in certain scenarios these antigens may also serve as vaccines for cancer immunotherapy. These cancer antigens are important because they boost the immune system to specifically recognize and kill tumors. In our studies, we have investigated two different antigens to combat prostate cancer. Sperm protein-17 and tumor associated antigens extracted from TRAMP C2 murine prostate cancer cell line were investigated as potential therapeutic vaccines. The above mentioned antigens are proteins and protein antigens themselves have poor bioavailability and absorption thus making it difficult to initiate immune response against the cancer. Particulate delivery systems encapsulating protein antigens have been proved to improve the delivery and efficacy of vaccine responses. Thus, particulate delivery systems are crucial in improving the delivery of these potent cancer antigens for a sustained and systemic anti-cancer activity, Another important aspect of vaccine delivery is the route of vaccination owing to its patient compliance and ease of administration. However, several challenges such as harsh gastric environment and tolerance induction has hindered successful clinical effectiveness of oral vaccines. Adjuvants have always been administered along with vaccines for decades. FDA approved vaccines such as Gardasil and Cerverix both have adjuvants to boost immunity. Thus, identifying appropriate adjuvants that will help boost anti-tumor activity is paramount. In order to increase the potency of our vaccine, several toll-like receptor (TLR) and non-TLR adjuvants were also studied. In this study, we have developed an oral microparticulate vaccine encapsulating two distinct antigens against prostate cancer. SP17 and antigens extracted from murine prostate cancer cell line were encapsulated separately in microparticles. Microparticulate vaccines were characterized for their physiochemical properties in vitro and evaluated for their antigenicity on murine dendritic cells. In order to protentiate vaccine efficacy, we also included adjuvants in microparticulate formulations and evaluated their potential to enhance the antigenicity of our vaccine formulations. SP17 administration. Several adjuvants such as R848, MPL, MF59 and alum were selected for future studies in vivo studies. The second part of this project focuses on formulation of a microparticulate vaccine encapsulating tumor associated antigens extracted from a murine prostate cancer cell line (TRAMP C2). We investigated the potential of our formulated vaccine along with two adjuvant microparticles, ALUM and MF59, to boost anti-tumor response against prostate cancer. Finally, to prove the effectiveness of our vaccine and overcome the tumor "immune escape" mechanism in cancer, we also performed a therapeutic in vivo study on a murine prostate cancer model. Encouraging results from the in vivo study demonstrate excellent anti-tumor activity of our therapeutic vaccine. We observed a significant reduction in tumor volume and sustained anti-tumor T-cell activity in vivo. Thus, we could also demonstrate , in our experiments, the importance of combination therapy which inhibits cancer "immune escape" mechanisms and improves vaccine efficacy.
    • Overian Cancer Microparticulate Vaccines: Effect of Routes of Administration

      Tawde, Suprita Ashok; College of Pharmacy
      Ovarian cancer is the most lethal gynecological cancer in the U.S. First-line treatment for advanced cancer involves surgery followed by chemotherapy. However, cancer relapses within short periods of time even after treatment. Therefore, alternative approach of immunotherapy is being investigated. We studied vaccination with microparticles containing the ovarian cancer antigens can prevent/retard ovarian cancer growth. Oral and transdermal routes are attractive modes of immunization because of their ease of administration and patient compliance. In this project we explored microparticulate system to target immune cells and to initiate response against ovarian cancer antigens via oral, subcutaneous and transdermal delivery. We selected a murine cell line which correlates closely to human cell line in terms of antigen expression and tumor formulation. We prepared the antigenic lysate and characterized it for the presence of a known antigen. We loaded micro-particulate delivery systems with the lysate. These particles were formulated with desired physical properties suitable for particle uptake and for anticipated immune response. In the present study, we demonstrate the efficacy of vaccine formulations wuth was evaluated in vivo in mouse tumor model, using the murine ovarian cancer cell line as a solid tumor model via oral, transdermal, subcutaneous and via combination of oral and transdermal routes. The tumor volumes upon challenge with live tumor cells were monitored in vaccinated animals and control animals. Humoral and cellular immune response in all the animals were monitored to determine the immune mechanism initiated by the vaccine microparticles. Encouraging results from these prophylactic particulate overian cancer studies provided the basis to design therapeutic study to mimic the real-time scenario where patients with residual tumors after a surgery are the recipients of such vaccine therapy. Further, to elucidate the role of M-cells in particle uptake once administered orally, we induced M-cells in murine models and administered microparticles loaded with a model antigen with and without immune adjuvant. The immune response generated via these vaccine particles in mice models with induced M-cells was compared to control animals, to determine whether particles followed the M-cell pathway for their uptake in order to trigger immune cells.