Design Of Gene Delivery Systems Using Chitosan Derivatives And Preliminary Studies On Their Biosafety

Chitosan has been considered to be a good candidate for gene delivery system, since it is already known as a biocompatible, biodegradable, and low toxic material with high cationic potential. However, low specificity and low transfection efficiency of chitosan need to be overcome prior to clinical trial. It is necessary to develop a new gene delivery system using chitosan derivatives to gain high transfection efficiency. Moreover, an evaluation of biosafety must be carried out prior to clinical trial.In this dissertation, the work mainly focus on the following points:A novel method to synthesize chitosan-L-arginine was developed. Fmoc-Arg(Pbf)-OH was grafted to chitosan by using coupling agents EDC and NHS. Then Fmoc group and Pbf group were removed one after the other by deprotection reaction. Thus the final product, chitosan-L-arginine, was yielded. The formation of this product was confirmed by the ¹H NMR and FT-IR spectra.Chitosan, N-alkyl chitosan, chitosan-L-arginine was used as gene vector to deliver plasmid DNA into vascular smooth muscle cell and K562 cell. Green fluorescence observed indicated that the plasmid DNA was successfully delivered into cell. The derivatives/DNA complex particles were labeled by ⁹⁹Tc^m to investigate their efficiency of entering cell. The transfection efficiency was figured out by calculating the percentage of cells expressing green fluorescent protein.A preliminary study on biosafety of these three kinds of gene vectors was carried out. An evaluation of cytotoxicity was performed by MTT assay. Cytotoxicity caused by chitosan derivatives was observed. The cytotoxicity of Chitosan-L-arginine with different molecular weights was also tested. The results show that there is a correlation between molecular weight and cytotoxicity.