Preparation of chitosan nanocomposite gene vector

Objective To prepare chitosan nanocomposite gene vector and investigate its physico-chemical property,cytotoxicity,stability and in vitro transfection efficiency.Methods Chitosan nanoparticles entrapping complexes of polyethylenimine(PEI)/DNA were prepared by a complex coacervation method,and measured for size and Zeta potential by nanoparticle size analyser,observed for morphology by transmission electron microscopy,and determined for cytotoxicity by MTT method.The prepared chitosan nanocomposite gene vector was stored in PBS(pH 7.4)and RPMI1640 containing 10% calf serum at 37 ℃ for 0,1,3 and 5 d separately,and evaluated for stability by 1% agarose gel electrophoresis.CNE cells were transfected with the prepared vector in vitro,and the transfection efficiency was evaluated.Results Stable chitosan nanoparticles were formed when the N(amino group of PEI)/ P(phosphate of DNA)ratio was not less than 6.The resultant chitosan nanocomposite gene vector were in even and intact spherical shape,at mean diameter of about 300 nm,which showed a surface potential of about 30 mV and low cytotoxicity.The result of 1% agarpse gel electrophoresis showed that DNA is completely wrapped in nano-vector without free DNA released in 5 d.The transfection activity in vitro of thte nanoparticles was 1 000 times higher than that of chitosan/DNA complexes,on which no interference of serum was observed.Conclusion The prepared chitosan nanocomposite gene vector is a high effective non-viral vector with low toxicity,which may be a potential vector for gene therapy in vivo.