Study On Microspheres To Improve The Effectiveness Of Vaccine By Oral Administration Based On Natural Cationic Polymer-chitosan

The subject is committed to obtain cationic microspheres to improve the effectiveness of vaccine by oral administration and expand the scope of application of the vaccine. The physical and chemical properties of microspheres such as particle size, hydrophobicity and electric charge have a direct impact on targeting, transfection efficiency and immune effect in particulate drug delivery system. Based on the three main factors affecting the uptake of microspheres by M cell, hydrophilic materials (sodium alginate and chitosan) and hydrophobic material (PLGA) are used as carrier separately in this study, bovine serum albumin (BSA) is used as a model drug to preare cationic microspheres coated by chitosan. Influences of different types of carrier materials on microsphere morphology, particle size, encapsulation efficiency, drug loading, surface charge and release behavior are investigated. In the first chapter, BSA–loaded alginate/chitosan microspheres are prepared by an emulsion method and further incubated in chitosan. Microspheres are obtained with more than 80% less than 10μm, positively charged on the surface, the encapsulation efficiency is (81.4±1.5)%, loading rate is (6.4±0.1)%. The initial burst release is obvious (more than 60% in half an hour) both in 0.1mol?L-1 HCl (pH 1.2) and PBS (pH 7.4). The release rate of drug is fast in PBS (pH 7.4). Drug released entirely in 2 h.In the second chapter, hydrophobic PLGA and hydrophilic chitosan are used together as carrier, BSA–loaded PLGA/chitosan microspheres are prepared by W/O/W double emulsion solvent evaporation method with more than 90% than 10μm, encapsulation efficiency is (80.4±6.3)%, loading rate is (8.4±0.3)%. With the concentration of chitosan increasing in the external water phase, negative microspheres change into positive microspheres. Compared with PLGA microsphere, the addition of chitosan significantly reduce the burst release and sustain the release rate. The molecular weight of chitosan has a significant impact on the release rate of drug.In the third chapter, using Newcastle vaccine as a model drug, a novel S/O/W emulsion-solvent evaporation method is used to prepare Newcastle vaccine PLGA/chitosan cationic microspheres. Hemagglutination reaction (HA) is used to determine the titer of Newcastle vaccine, which can be used to investigate the influences of different factors on activity of the vaccine in the process. Hemagglutination inhibition reaction (HI) is used to detect antibody levels in serum, and make a preliminary assessment of the protective effect of microspheres by oral immunization.The results show that the loss of vaccine activity mainly come from the organic solvents and stirring under room temperature for a long time. Compared with conventional W/O method, S/O method can significantly protect the activity of the vaccine. When the dose of PLGA/chitosan microspheres by oral administration is 10 times to the dose of intranasal immunization, considerable immune effect can be generated, superior to the group of PLGA microspheres by oral administration.In the fourth chapter, PLGA/chitosan cationic microspheres are prepared with hepatitis B vaccine as a model drug. The effect of their humoral immune is assessed preliminarily by ELISA method. The average particle size of the PLGA/chitosan microspheres is 3.86μm, and more than 95% is less than 10μm. Encapsulation efficiency is (70.7±3.2)%, and loading rate is (9.45±0.11)%. The antibody titer of subcutaneous group is much higher than oral groups. The immune effect of PLGA/chitosan microsphere groups by oral administration is better than PLGA microsphere group.