DESIGN AND DEVELOPMENT OF CD205 TARGETED PLGA NANOPARTICLES AND EVALUATION OF ANTIGEN SPECIFIC IMMUNE RESPONSES

Abstract

Stimulation of a patient’s immune system to fight cancer is the underlying mechanism of immunotherapy. Cancer immunotherapy manipulates the dendritic cells (DCs) to identify the non-self present in the immunosuppressive microenvironment. This vaccination strategy based on nanoparticulate drug delivery system has the potential to treat cancer through packaging of therapeutic cargoes and delivering them to target immune cells (DCs). FDA approved poly-(D, L-lactic-co-glycolide) is approved for use in human due to its widely accepted properties such as low immunogenicity, minimal toxicity, biocompatibility and biodegradability. The goal of this project is to develop an understanding of a comprehensive relationship between nanoparticle (NP) structure and activity; and address the important requirements of NP structure and chemistry to selectively target specific markers. Plain NPs were prepared by emulsification solvent evaporation method with number of preparation variables. Double emulsification solvent evaporation method was used to prepare ovalbumin (OVA) and/or adjuvant loaded NPs. The DC targeting ligand (anti-CD205 monoclonal antibody) was attached to the NPs through two methods: covalent binding in presence of spacer molecule and physical adsorption method. Infra-red (IR) spectroscopy was performed to ensure the structural modification of NPs. Formulations were evaluated in respect to particle size, polydispersity index, zeta potential, surface display, cytotoxicity assay, OVA release studies, structural integrity of OVA in formulations, DC uptake study, DC maturation study, T cell proliferation study, estimation of total IgG and cytokine secretion profile. Results indicated that different formulation groups of NPs with different viscosity grades had desirable physicochemical properties. In case of the ligand (anti-CD205 antibody) conjugated NP’s spectrum, there was presence of amide-I vibrations resulted from C=O stretching vibration near 1610 cm-1 and N-H stretching of high intensity between 3310 to 3250 cm-1. These characteristic IR peak reflects the antibody conjugation with the NPs. DC uptake study shows when ligand was adsorbed onto the surface, these NPs were better uptaken compared to covalently attached formulations. No significant correlation was observed in uptake due to change of polymer viscosity and type. Highest expression of markers CD40, CD86 and MHCII molecules were observed with adjuvant (monophosphoryl lipid A)-antigen loaded targeted NPs. Though, high viscosity grade polymers (ester or COOH terminated) had higher OVA loading, they expressed lower percentage of markers compared to low viscosity formulations. These could be attributed to the release mechanism of the respective PLGA NP formulation. In addition, sufficient secretion of T helper cell 1 (Th1) and Th2 cytokines was observed. This confirmed the maturation of DCs as well as activation of the T cells. The T cell proliferation study confirmed the proliferation of cells in-vitro for both wild type balb/c and TCR transgenic (OT1) mice. Results from OT1 mice confirmed the OVA-specific immune response. Secretion of higher level of OVA-specific IgG by the formulations confirms the antigen specific immune response. Therefore, a comprehensive evaluation of effect of formulation parameters (polymer viscosity, polymer end group, conjugation methods) was performed to modulate the antigen specific immune response. These findings would be insightful when designing a vaccine formulation for a specific type of cancer.