The Study Of Efficient Carriers For Mucosal Administration Of Anti-caries DNA Vaccine

A major challenge for clinical application of genetic medicines is to develop suitable carriers to overcome their hydrophilicity, likely to be degradated. Nanotechnology promises great versatility in the field of drug delivery, especially in DNA vaccine. Liposome is the most extensively described and studied nanoparticles. In this study, DOTAP/DOPE liposome was firstly prepared, then developed to cationic lipid emulsion after adding an oil phase. The complexes were made by incubating with DNA respectively. Finally, ternary complexes were obtained by introducing a polycationic peptide namely protamine sulfate. The whole study was as follows:(1) DOTAP/DOPE cationic liposomes were prepared by the method of film dispersion, DOTAP/DOPE/Tween 80/Squalene cationic lipid emulsions were obtained by sonication. We investigated the size and zeta potential, morphology and physical stability of these plain lipid carriers. The results showed that the repeatability of the preparation technologies was well and the colloids were stabile.(2) Four kinds of complexes were prepared, namely cationic liposome/DNA, cationic liposome/protamine/DNA, cationic lipid emulsion/DNA and cationic lipid emulsion/protamine/DNA, and their physicochemical properties were characterized. The results showed that stable and positively charged complexes were formed at N/P ratios above 2. Nuclease digestion assay showed that the complexes efficiently protected DNA from enzymatic digestion. In addition, we researched the suitable methods to prepared complexes at high concentrations which required for the study in vivo.(3) MTT assay and transfection study on CHO-K1 cell showed that the cytotoxicities of the complexes were relatively slight, the optimum N/P ratio for transfection was 2, the transfection efficiency of liposome was much higher than lipid emulsion, protamine sulfate could enhance the transfection, while serum decreased it.(4) The immune efficacy of anti-caries DNA vaccine delivered by different carriers was evaluated in Balb/c mouse. The results demonstrated that protamine sulfate had positive effect on the expression of antibodies about 3-4 times, compared with the study in vitro, the difference between emulsion and liposome had been greatly decreased. In conclusion, complexes were formed between DOTAP/DOPE cationic lipid carriers and DNA through electrostatic force, ternary complexes were obtained after adding protamine sulfate. Four kinds of complexes could delivery DNA vaccine in vitro and in vivo. The results indicated emulsion be a good candidate for gene delivery, and protamine sulfate enhance the expression of gene in vitro and in vivo, deserving further research.