| Plague is a kind of severe infectious disease from which both the human being and animals suffer, caused by Yersinia pestis. Hundreds of millions of people have been killed by plague in history. Immunization is one of the important measures to prevent the plague, but the current plague vaccines have their own problems:for inactivated vaccine, poor production security, inefficient protection, short period, high rate of side effects and ineffective protection against pneumonic plague; for rat poisoning epidemic live vaccine, subcutaneous injection of effective protection against bubonic plague, but more side effects, potential toxicity, can not fully protect against pneumonic plague, the immune effect of uncertainty; recombinant subunit vaccine can overcome the many short comings of traditional vaccines, do not contain infectious components, non-pathogenic, and has some immune protection to both bubonic plague and pneumonic plague. But simply effective subunit vaccine is still not ideal. Biodegradable material from the microspheres is a good vaccine carrier, and also is an adjuvant. But in the microspheres preparation, the vaccine should undergo high mechanical shear strength and organic solvents tested usually, easy to lose activity. Therefore, how to keep the activity of vaccines in the process of microspheres preparation has become the key issue. This paper aims to study a new plague subunit vaccine delivery system which can avoid defects and retain the advantages of traditional microspheres. With porous microspheres as a carrier, this delivery system has adjuvant effect and improve the effect of mucosal immunity after administration.First of all, bio-degradable material of polylactic acid-glycolic acid (PLGA) as carrier, respectively, sodium chloride, sodium bicarbonate, sodium carbonate as the porogen, we prepare porous microspheres using emulsion solvent evaporation method and study of morphological characteristics of microspheres and particle size using scanning electron microscopy. The results show that the sodium carbonate pore the best, followed by sodium bicarbonate, sodium chloride lowest in three kinds of porogen. Secondly, the selection of sodium bicarbonate and sodium carbonate as a porogen, we design multi-level single-factor experiment studied a variety of prescription and technological factors on the system of porous microspheres porosity, the results show that:with the increase of porogen in the inner water phase, the porosity of microspheres increases; more amount of hydrochloric acid by adding in curing process, the lower the porosity; with processing of the time of adding hydrochloric acid, the porosity of microspheres increased; the influence on porous microspheres by different formulation and process factors on the bicarbonate sodium and sodium carbonate by the system as a porogen were similar, but the porous microspheres of sodium carbonate as a porogen had better uniformity and greater porosity. On this basis, we determined the sodium carbonate as porogen and bovine serum albumin (BSA) as a model drug, using L18 (37) orthogonal experimental design, which influence the microspheres size, entrapment efficiency and drug loading prescription process factors. Statistical analysis showed that microspheres size distributed uniformly and had high porosity, but low adsorption drug loading. Which significantly affect the particle diameter of the prescription process factors:the concentration of porogen in internal water phase and the amount of hydrochloric acid added on curing process. The concentration of porogen was inceased and the microspheres size increased; the amount of hydrochloric acid added on curing process increased and the microspheres size decreases. Significant effect factors on microspheres porosity and the amount of absorption of microspheres is the concentration of porogen, the amount of hydrochloric acid added on curing process and the time of adding hydrochloric acid:the concentration of porogen was increased and the porosity and adsorption of microspheres increased; the amount of hydrochloric acid added on curing process was increased and the porosity and adsorption of microspheres decreased; with processing of the time of adding hydrochloric acid, the porosity and drug loading of microspheres inaeased.In order to verify the effect of the mucosal immune the porous microspheres containing vaccine, the plague F1 antigen and V antigen vaccine as targeted drugs, we used the optimal conditions of formulation and preparation of porous miaospheres to prepare the porous microspheres delivery system of plague subunit vaccine by incubation method subunit plague vaccine adsorbed on the porous microspheres. The drug loading of microspheres of F1 antigen and V antigen plague vaccine were 115μg/mg and 74μg/mg. Female BALB/c mi ce as the ani mal model, porous microspheres contai ni ng plague vaccine as the experimental group, pure antigen and simple microspheres as comparison group, we investigated protective immunity effect of different experimental groups by intramuscular injection method:detecting antibody titers in mice By ELISA and survival in mice by attacking drug. The results showed that:The administration of intramuscular form, containing the plague F1 antigen of porous microspheres and containing plague V antigen had shown a good immune protection. The antibody titers two experimental groups higher than antigen alone or simple microspheres group was significantly higher (P<0.01). Attack drug test show mice survival rate of experimental groups was significantly higher than those of pure antigen or simple microspheres group (P<0.01). Finally, we had the porous microspheres containing plague subunit vaccine for intranasal administration immunological evaluation to inspect the plague vaccine immunology effect of intranasal administration and intramuscular injection with design a different route of administration and administration dose group. Preliminary results indicated that immune efficacy of current porous microspheres of plague by nasal is lower than that of the intramuscular injection. This may because the microspheres were cleared fast in the mouse nasal, leading to the vaccine was cleared before it played role, and therefore it can not cause sufficient immune response.In summary, porous microspheres were prepared by emulsion solvent evaporation method, including adding sodium carbonate as the porogen in the internal water phase with biodegradable PLGA as carrier material. The porous microspheres can absorb plague F1 antigen and V antigen subunit vaccine by incubating. The porous microspheres which had absorbed plague Fl antigen and V antigen subunit vaccine were administered by injection and got better immune protection than that of simple antigen or microspheres. The intranasal administration of immune protection is evaluating. These results suggest that, the porous microspheres as a carrier, containing plague subunit vaccine in this study can enhance the immune effect of the vaccine itself and could become new drug delivery systems of vaccine to prevent plague. |
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