Studies On Arginine-Modified Chitosan Non-viral Vector For Gene Delivery

Chitosan is a naturally occurring cationic polysaccharide with good biocompatibility, biodegradation, low immunogenicity and non-cytotoxicity. Chitosan is considered to be a good candidate for non-viral vector, since cationically charged chitosan can efficiently condense negatively charged DNA to form polyelectrolyte complexes via electrostatic interaction, facilitating the transport of therapeutic genes across the cell membrane. In this thesis, a novel arginine-modified chitosan(Arg-CS) non-viral vector was developed. The work mainly focus on the following two parts: 1. Synthesis of Arg-CS and characteristics of Arg-CS/DNA polyelectrolyte complexes. Chitosan was modified with arginine by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) and N-hydroxysuccinimide(NHS) as coupling agents. Arg-CS/DNA complexes were prepared by coacervation process. The formation of Arg-CS and the properties of Arg-CS/DNA complexes were investigated. FTIR and 13C NMR spectra results showed that arginine was chemically coupled to chitosan to form arginine-modified chitosan conjugates. CD spectra indicated that the interaction of DNA with chitosan merely caused a slight perturbation of DNA bonds and DNA still remained B-conformation within the complexes. TEM, AFM and DLS results revealed that the complexes were nearly spheres with mean diameter about 80-150nm, which is very suitable for gene delivery. 2. Cell culture of vascular smooth muscle cell(VSMC) by cell line and tissue culture. Taking VSMC as host cell, the preliminary cell transfection experiments were carried out to explore the possibility of Arg-CS-mediated delivery of pEGFP which encoded green fluorescent protein(GFP) into vascular smooth muscle cells. It was observed that pEGFP was successively delivered into VSMC and the GFP was expressed to a measurable degree.