The Use Of Chitosan As A Condensing Agent To Enhance Cationic Liposome-mediated Gene Transfer

Cationic liposome has been extensively used as a non-viral vector for the delivery of reporter and therapeutic genes in vitro and in vivo. Chitosan is a natural cationic polysaccharide, which can condense DNA and protect it against nuclease degradation. Based on these facts, chitosan can be used as a condensing agent to enhance the transfection efficiency of cationic liposome. In the present study, we investigated the association and dissociation kinetics of DNA with the two kinds of nonviral gene delivery systems: DOTAP/DOPE cationic liposome and chitosan, prepared and characterized the liposome/chitosan/DNA complex (LCD), and evaluated the transfection efficiency of this LCD complex. The main results are as follows:1.Stopped-flow fluorescent spectrophotometry was used to investigate the association and dissociation kinetics of DNA with two kinds of nonviral gene delivery systerms (cationic liposome formed by 1,2-dioleoyl-3- trimethy- lammonium- propane (chloride salt)(DOTAP) and 1,2- Dioleyl-s–Glycero-3- Phosphoeth anolamine (DOPE) and chitosan). The results indicated that DNA condensation was the rate-determining step during the association process, while DNA extending and rearrangement was the rate-determining step during the DNA dissociation process. At N/P ratios before reaching the thermodynamically most stable state, the complexes of DNA and DOTAP /DOPE or DNA and chitosan were all incompact and could dissociate to some extent. The binding capacity of chitosan with plasmid DNA was more powerful than that of DOTAP/DOPE cationic liposome.2.DNA was firstly mixed with chitosan, and then DOTAP/DOPE cationic liposome was added to form ternary complexes (LCD). The formation of LCD complex was then characterized by Agarose gel electrophoresis, [Ru(phen)2dppz]2+ displacement, particle size and zeta potential measurements. The results indicated that the LCD complex was spherical shape with a relatively homogenous size, which was reduced after the condensation of DNA by chitosan compared with lipoplex. With the increasing of the N/P ratio, the zeta potential of LCD became more positive and the binding capacity of vectors with plasmid DNA was enhanced.3.The cytotoxicity and transfection efficiency of LCD were examined. It was found that LCD was devoid of any cytotoxicity. Besides, the cell viability was a little improved in the presence of chitosan. And in the presence of serum did, LCD delivered into the cells more efficiently than lipoplex and naked DNA. The in vivo experiment also obtained the same results.Based on the above experiment, these two kinds of vectors have the ability to associate with DNA through electrostatic interactions to form complexes which can dissociate to some extent. And the LCD has a higher efficient transfection capacity compared with the cationic lipoplex. It suggests that the use of chitosan can enhance transfection efficiency in vitro and extend gene transfer in vivo.