Study On Protamine Coating PLGA Nanoparticles As Hepatis B Vaccine Delivery Vector

Part1:Antimicrobial peptides are small peptides with immunomodulatory and antibacterial activity to defense exogenous microbial invasion, widely existing in lower organisms and higher organisms, including human being. Due to the excellent bactericidal effect, broad spectrum antibiotic, variety, and difficulty to produce drug resistance, antimicrobial peptides are considered to be the best drugs to substitute antibiotics. The antimicrobial peptide SK84, isolated and purified from the maggot by Dr. Lu Jie in2004, has efficient antimicrobial activity. The peptide belongs to the non-cationic peptide of Gly-rich, with Gly diad, Gly hexamer, GxxxG. The Gly-rich zone existing in the N-terminal which can form a flexible structure, is a key to cleavage of the cell membrane. In order to study the relationship between antimicrobial peptides structure and function, we truncated N-terminal18amino acids to get a mutant protein.Then,we carried out the following work:(1) SK84-6His,S19K84-6His were expressed and purified from Prokaryotic induced expression.(2) The results of inhibition zone test and micro-method showed that antibacterial activity of S19K84was significantly decreased compared to SK84.(3) Circular dichroism analysis showed that the a-helix was decrease by53.8%in S19K84, while the a-helix is a fundamental structure for the antibacterial peptides.Part2:PLGA nanoparticles encapsulating antigens not only maintaine the activity of drugs, increase uptake of drugs, but also regulate drugs release rate. Studies have shown that protamine-coated PLGA nanoparticles can significantly promote coss-presentation of exogenous antigen. To explore the delivery of protamine-coated PLGA nanoparticles for hepatitis B vaccine, we prepared PLGA-OVA nanoparticles, PLGA-HBsAg nanoparticles and PLGA-HBsAg/PS nanoparticles, using double emulsion-solvent evaporation method with Ovalbumin (OVA) and Hepatitis B surface Antigen (HBsAg) as a model drug. The main results are as follows:(1) PLGA-OVA nanoparticles were prepared by double emulsion-solvent evaporation method. Their size, PDI, zeta potential were334.2±10.5nm,0.093±0.027,-9.3±1.4mv, the encapsulation efficiency were63.35±3.24%.And we investigated the release rate of the drugs in vitro, which divided into the burst release phase and sustained release phase.(2) We prepared PLGA-HBsAg nanoparticles using the same method. Preparation procedures were optimized in5aspects:the types of polyvinyl alcohol (PVA), the concentration of PVA, ultrasonic time, the external aqueous phase volume, HBsAg concentration. The average size, PDI, zeta potential were237.9±14.4nm,0.089±0.006,-13.2±2.1mv. At4℃, the nanoparticles could be stably stored20d, while the particle size and dispersibility remained basically unchanged. The zeta potential of nanoparticles transferred from negative to positive after PS coated. The particle size, PDI, zeta potential were268.4±10.6nm,0.162±0.015and9.6±1.3mv.(3) Examined by transmission electron microscope (TEM),both PLGA-HBsAg nanoparticles and PLGA-HBsAg/PS nanoparticles were spherical in core-shell shape with smooth surface.