Date of Award

5-1-2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Harlan P. Jones

Abstract

Targeted Immunotherapy represents a potential and innovative means to combat cancer. Cancer vaccines designed against a specific tumor antigen have been efficiently utilized to trigger immune responses against tumor cells. Despite the preliminary evidence in animal models, low immunogenicity is one of the major hurdles in the development of vaccines in humans. Several approaches including the use of an “ideal” tumor antigen, appropriate delivery techniques, immune boosting strategies with co-stimulatory molecules are being explored to surmount this obstacle. The goal of this dissertation project was to utilize polymeric nanoparticles (NPs) as a vehicle to deliver Tumor Associated Antigen (TAA) that would elicit a strong antitumor immune response. In the present study, we successfully formulated CpG surface functionalized Tag encapsulating PLGA nanoparticles (CpG-NP-Tag) and tested their efficacy using in vivo and ex vivo experimental models. Specifically, we developed and characterized NPs for physicochemical properties including particle size, surface charge, surface morphology, Polydispersity index (PDI), encapsulation efficiency and CpG ligand binding efficiency. CpGNP-Tag NPs were found be of desired size (220-230 nm) and surface charge (negative zeta potential). Particles were non agglomerated, spherical in shape and uniform in size with PDI in the range of 0.03-0.1. Due to the hydrophobic nature of the encapsulated entity (Tag), the encapsulation efficiency was limited to 30-40%. CpG ligand conjugation on the surface of NPs was confirmed using Fluorescence Correlation Spectroscopy (FCS). CpG ligand binding efficiency was found to be around 10-14%. We also found that CpG-NP-Tag NP formulation had desired properties (size, charge and morphology) for efficient uptake by phagocytic antigen presenting cells (APCs) such as dendritic cells (DCs). The major aim of our studies was to test the antitumor efficacy of NPs. Using a prophylactic syngenic Balb/c mice model, we demonstrated that CpG-NP-Tag can serve as an efficient tool to bolster antitumor immunity and thus could be used as a platform for the development of NP based immunotherapeutic interventions in future. We found that CpG-NPTag NP immunization attenuated tumor growth, proliferation, angiogenesis and induced apoptotic tumor cell death. These NPs indicated immunostimulatory potential by enhancing tumor CD4+ and CD8+ T cell infiltration as well as local IFN-γ production. Overall, from these in vivo studies we concluded that CpG-NP-Tag promotes IFN-γ secretion which possibly mediates the inhibited tumor growth, angiogenesis and enhanced T cell mediated immunity which facilitates tumor cell death via apoptosis. To understand the mechanism by which CpG-NP-Tag imparts antitumor effects we used ex vivo model of APCs. Studies were conducting using Bone Marrow Derived Dendritic Cells (BMDCs) isolated from female Balb/c mice. We demonstrated enhanced NP uptake, preferential Endosomal localization, and increased population of CD80/86 expressing BMDCs in case of CpG-NP-Tag pulsed BMDCs indicating these NPs could serve as candidates for DC based vaccines in future. In summary, both ex vivo and in vivo studies conducted with CpG-NP-Tag NPs provide insight in the development of particulate vaccines in cancer immunotherapy.

Comments

Kokate Rutika A., Formulation, characterization and validation of CpG functionalized PLGA bacteriomimetic nanoparticles for breast cancer immunotherapy. Doctor of Philosophy (Biomedical Sciences), April, 2015, 147 pp., 6 tables, 32 figures, bibliography, 143 titles.

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