185. Poly-lactide-co-glycolide Acid (PLGA) Nanoparticles for DNA Vaccination

Abstract

Immunization with pcDNA encoding antigenic proteins is emerging as a novel vaccination strategy. Advantages of DNA vaccination include: Eliciting of both humoral and cellular response, ease of production and stability of pcDNA. Among the various gene carriers, viral vectors such as retrovirus and adenovirus are still the most widely investigated because of their transfection efficiency. However, drawbacks like immunogenecity, integration with host genome and limitation of the amount of genomic information that can be introduced by the vectors led researchers to look for non-viral methods. We offer to use a biodegradable nanopolymeric system made of Poly-lactide-co-glycolide acid (PLGA) as a carrier for DNA vaccination. PLGA-nanoparticles (NPs) encapsulating pcDNA could serve as an efficient sustained release DNA delivery system, which can be easily taken by antigen presenting cells. Therefore, biodegradable PLGA nanoparticles containing pcDNA were prepared by the double emulsion (water/oil/water) solvent evaporation technique using a sonicator. The nanoparticles were characterized by investigating their morphology, size, loading efficiency, surface potential and release kinetics as a function of different formulation parameters. PLGA with a narrow size distribution and DNA loading efficiency of 85% were formulated. PLGA was found to protect pcDNA from shear stresses during sonication steps as was demonstrated by gel electrophoresis. Viability of several cell lines was examined post incubation with a suspension of PLGA particles. PLGA particles were found to be non-toxic. Uptake and distribution of the nanoparticles in cells of the immune system were studied [figure 1] using fluorescent dyes including 6-coumarin, Lysotracker Red and DRAQ5. The effect of temperature and size of particles on uptake by cells was studied. Using confocal microscopy the uptake and localization of nanoparticles in cells were studied. Nanoparticles were detected mainly in the cytoplasm and inside the endo-lysosome compartments. pcDNA that was released from the nanoparticles as well as pcDNA extracted from the nanoparticles retained their biological activity. Using these DNA extracts cells were successfully transfcted with a commercial transfecting reagent. In addition, transfection of cells with the PLGA-NPs system was studied and was found to be 200-fold higher compared to control. Plasmid expression was maintained for 3 weeks [figure 2]. Our results suggest that PLGA-NPs can serve as a non-viral DNA delivery system for DNA vaccination.