Study On The Preparation Of Protamine Coating PLAG Nanoparticles As Vector Of The Hepatis B Vaccine

Polylactic-glycolic acid(PLGA), a kind of polymer materials with good biodegradability and biocompatibility, has been widely used in the drug delivery system.PLGA nanoparticles are promising in the study of protein vaccine carrier. Current studies have found that antigen particles may have a stronger ability to induce the immune response compared with soluble antigen. Additionally, the positively charged modification on the surface of nanoparticles can be more effective in promoting the intracellular uptake of antigen-presenting cells(APC). In this paper, Protamine(PS) that is rich in arginine and positive charged, was adsorbed on the surface of PLGA nanoparticles loading hepatitis B virus surface antigen(HBsAg). We investigated effects of the nanoparticles on the phagocytic of DC2.4, the phenotype of mouse bone marrow-derived dendritic cells(BMDC), secretion of cytokine of BMDC, and the immune responses for mice. The research was mainly concerned with the following aspects:(1) HBsAg loaded PLGA nanoparticles(HBsAg/PLGA) and PS adsorbed on the surface of HBsAg/PLGA nanoparticles(HBsAg/PLGA/PS) were prepared by double emulsion solvent evaporation, and were characterized by the Laser particle size analyzer.HBsAg/PLGA nanoparticles had the size of 293.4 ± 6.9 nm, PDI of 0.109 ± 0.070, zeta potential of-21.8 ± 1.6mV, and the encapsulation efficiency of 45.23 ± 6.19 %.HBsAg/PLGA/PS nanoparticles had the size of 330.5 ± 14.1 nm, PDI of 0.210±0.019,zeta potential of 9.6±1.3 mV, and the encapsulation efficiency of 47.27±5.56 %. The zeta potential changed from negative to positive because of the coating of PS on the surface of PLGA nanoparticles. The TEM observations of HBs Ag/PLGA and HBsAg/PLGA/PS nanoparticles found that nanoparticles were spherical and has a typical structure of core-shell structure. In addition, the release in vitro and stability situation were also investigated.(2) FITC-OVA / PLGA nanoparticles and FITC-OVA/PLGA/PS nanoparticles were prepared. The effects of FITC-OVA/PLGA nanoparticles and FITC-OVA/PLGA/PS nanoparticles on the uptake efficiency of DC2.4 were investigated by flow cytometry. Theresults showed PS coating PLGA nanoparticles significantly enhanced the uptake of DC2.4. BMDC were isolated from mouse bone marrow cells. The influences of HBsAg/PLGA and HBsAg/PLGA/PS nanoparticles on the expression of MHC I, MHC II,CD80, CD83 and CD86 on the surface of BMDC were investigated. It was found that HBsAg/PLGA/PS nanoparticles significantly enhanced the expression of MHC I, MHC II,CD83 and CD86, while HBsAg/PLGA nanoparticles significantly enhanced the expression of MHC II and CD86. In addition, the influences of HBsAg/PLGA and HBsAg/PLGA/PS nanoparticles on the secretion of IL-4 and IL-12p70 of BMDC were investigated. It was found that HBsAg/PLGA and HBsAg/PLGA/PS nanoparticles significantly promote the secretion of IL-4 and IL-12p70. Compared with HBsAg/PLGA nanoparticles, the promoting effects of HBsAg/PLGA/PS nanoparticles were better.(3) The ability of HBsAg/PLGA and HBsAg/PLGA/PS nanoparticles to induce humoral and cellular immunity in mice were evaluated. These nanoparticles showed the ability to induce IgG in serum and Th1-type immunity induction. The results evaluated suggested that the modification of PS on the surface of PLGA nanoparticles had enhanced effect in the immunized mouses.In summary, HBsAg/PLGA and HBsAg/PLGA/PS nanoparticles were effective vaccine vectors that could enhance the immune response of HBsAg. Compared with HBsAg/PLGA nanoparticles, the promoting effects of HBsAg/PLGA/PS nanoparticles were better. Therefore, the HBsAg/PLGA/PS nanoparticles have the potential as a novel HBsAg vaccine carrier.