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Novel Biodegradable Amphiphilic Nanoadjuvants with Immunomodulatory Capabilities

Maria P. Torres1, Jennifer H. Wilson2, Michael Wannemuehler2, Balaji Narasimhan1, and Surya K. Mallapragada1. (1) Chemical & Biological Engineering, Iowa State University, 2114 Sweeney Hall, Ames, IA 50011, (2) Veterinary Microbiology & Preventive Medicine, Iowa State University, 2180 Vet Med, Ames, IA 50011

Our overall objectives are to design novel vaccine adjuvants based on biodegradable polymers and to understand the cellular and molecular mechanisms that establish immunologic memory. The novel amphiphilic polyanhydride system based on the anhydride monomers 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) provides a favorable and biocompatible environment for protein stabilization and release. Our hypothesis is that the encapsulation of polypeptides into polyanhydride nanospheres will provide superior adjuvanticity and provide insights for a broad spectrum of applications in vaccine-based therapies. Since it is well known that particle size plays a major role for efficient antigen presentation, nanospheres of different CPTEG:CPH compositions have been fabricated by three non-aqueous methods: solid/oil/oil double emulsion, cryogenic atomization, and nanoprecipitation. The ability of these polyanyhydride nanospheres to stabilize model antigens (i.e., ovalbumin) was investigated. Ovalbumin was incubated in aqueous solutions saturated with the individual diacids or combinations of diacids to determine protein structure and function. Next, the stability of both the encapsulated as well as the released Ova was checked using gel electrophoresis, circular dichroism, and fluorescence spectroscopy. It was found that the CPTEG:CPH system preserves the structural hierarchy of the encapsulated protein and retains the activity of the released protein. The biocompatibility of the CPTEG:CPH system was evaluated using the XTT assay and it was found that in the dosage range of 50-1600 ug/mL, cell viability was not affected by the nanospheres. Since the induction of an immune response require that antigen presenting cells interact with and phagocytose the antigen-loaded nanospheres, the efficient uptake of ovalbumin-loaded CPTEG:CPH nanospheres by bone marrow-derived dendritic cells and the respective cytokine profiles were evaluated and chemistries that induce Th1-inducing cytokines were identified. Altogether this integrated approach will provide novel insights into the activity of polymer adjuvants as it relates to the relationships between adjuvant chemistry and microenvironment. Because of their importance in the initiation of an immune response, the stability, uptake, and presentation of the antigen by APCs (i.e., macrophages and dendritic cells) will be assessed. Development and application of this technology will facilitate the rational design of vaccines and the ability to appropriately redirect the immune response to develop protective immunity.