Development And Research A Kind Of Nonvirus Carrier Used In Gene Transmission

Objective: To prepare DNA-Chitosan nanoparticle, a kind of nonvirus carrier used in gene transmission and investigate its physico-chemical properities and transfection effect in vitro to evaluate the possibility of its application in gene delivery. Methods: Chitosan nanoparticles acting as gene vectors were prepared by polymerid dispersion method. PEGFP-C1-Chitosan nanoparticles were prepared by electrostatic adsorption, the former acting as reporter gene. The appearances of the nanoparticles were surveyed by the scanning electron microscope and the atomic force microscope. The grain distribution and zeta potentials of the nanoparticles were determined with the laser grain analyzer. The cytotoxities of the Chitosan nanoparticles and lipofectamine to Hep G2 and L-02 cell lines were examined by the MTT assay. DNase I assay was used to study the gene protection effect of the DNA-Chitosan nanoparticles. The transfection activities of the chitosan-plasmid nanoparticles were qualitatively assessed by an in vitro gene transfection test . The transfection effect was determined by inversion fluorescence microscope and flow cytometry. We test different dose of gene which is transferred to determine the best chance of the transfection. We test the change of the transfection activity after the particles were modified with MSS-PEG. Results: Chitosan nanoparticles with positive charges can protect the DNA from degradation by DNase I. The DNA - chitosan nanoparticles without MSS-PEG modification can transfect COS7 cells, and the gene can express in these cells, but its transfection effect is very low, which is 11.6 .The better dose of DNA which can be transferred is 1.6μg. After the particles were modified with MSS-PEG, their transfection activity was still remained. The transfection effect is 22.6%, and the better dose of DNA which can be transferred is 2.4μg . It is important to find that in Cos7 cell lines the transfection activity of PEGlate nanoparticles is improved. Conclusion: The chitosan nanoparticles can delivery the gene into cells and the gene can express, so the chitosan nanoparticles are worth studying as gene medicine carriers.