Chitosan-DNA microparticles as mucosal delivery system: synthesis, characterization and transfection

Objective:The aims of this study are to encapsulate anti-carries DNA vaccine (pGJA-P/VAX) in chitosan microparticles and to investigate in vitro transfection characteristics and cytotocity of chitosan. Furthermore, the important parameters for the microparticles synthesis were studied. Methods: Plasmid-loaded chitosan microparticles were prepared by complex coacervation. A series of chitosan microparticles with various parameters were prepared, effect of N/P ratio, concentration of DNA and sodium sulfate on the characteristics of microparticles was studed according to zhengjiaTest. And then, the physicochemical structure of microparticles prepared under optimum condition was investigated by scanning electron microscopy(SEM) and confocal laser scanning microscopy(CLSM). At last,. Expression of protein in NIH3T3 cell transfected by chitosan/DNA microparticles was measured by histrochemistry, and the cytotocity of chitosan was determined by MTT assay. Results: It showed that the microparticles prepared under the optimal conditions ( N/P ratio of 2,the finally plasmids concentration of 0.1ug/ul and sodium sulphate of 50 mmol ) were about 1-2 um in size and spherical in shape. The encapsulation efficiency of DNA was 99%,almost all the DNA were captured and distributed throughout these particles. The number of positive staining cells of chitosan/pGJA-P transfected cell were more than that of naked plasmid transfect cells, but less than that of Lipofect-DNA(positive contral) complex group. Chitosan was found to be less cytotoxic compared with lipofectin. Cells grew well even at the concentration as high as 350 mg/ml. Conclusions:These results showed that chitosan/DNA microparticles may become a potential mucosal DNA vaccine delivery system .