Chitosan-Derivated Non-viral Vectors 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, two novel non-viral vectors, arginine-modified chitosan (Arg-CS) and N-methylene phosphonic chitosan (NMPCS), were developed. The physicochemical properties of the vectors,the interactions between the vectors and plasmid DNA and gene tranfection activities were investigated. The parameters of labeling chitosan-DNA nanoparticles with fluorescein isothiocyanate were optimized. The potential application of Arg-CS/DNA nanoprticles (ACDNPs) coated stent in endovascular gene delivery was studied. The work mainly focus on the following parts:1. Synthesis of Arg-CS and interactions between Arg-CS and DNA.. 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. Gene transfection mediated by Arg-CS. Luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan. MTT assay indicated that Arg-CS was a safe non viral vector.3. Synthesis of NMPCS and interactions between NMPCS and DNA. FTIR and 13C NMR spectra were used to study the structure of NMPCS. NMPCS/DNA complexes were obtained using a complex coacervation process, characterized by agarose gel electrophoresis retardation assay for their stabilities, atomic force microscopy (AFM), transmission electron microscopy (TEM) and laser size analysizer observation for morphology and size, results showed that at charge ratio 2:1 or above, DNA could be completely entrapped and spherical complexes with mean size of 80~210nm were formed.4. Gene transfection mediated by NMPCS. The results indicated that the transfection efficiencies were strongly dependent on the charge ratio and pH value of culture medium, for HeLa cells, the highest efficiency obtained at ratio of 4:1 and pH 6.2 was greatly higher than that from CS/DNA complexes or naked DNA and approximate to that from PEI/DNA complexes. Polymer-inorganic salt type NMPCS-Ca vector showed some similarities with traditional Ca-P, giving high efficiencies probably owe to positive role of Ca2+ ion channel.5. Optimization of the assay for labeling chitosan-DNA nanoparticles with fluorescein isothiocyanate. To achieve the appropriate nanoparticle size and cell transfection efficacy, the FCNP should be prepared with the FITC-chitosan labeled by incubating the FITC and chitosan in a pH 7.5 solution at 25℃for 4 hours with the molar ratio of chitosan to FITC being 25:1.6. Study on Arg-CS/DNA nanoprticles (ACDNPs) coated stent in endovascular gene delivery. In A10 cell culture, the ACDNP-stents with plasmid pIRES-EGFP induced high level of GFP expression in cells grown on the stent surface and along the adjacent area. In animal studies, luciferase activity was observed only in the region of the artery in contact with the ACDNP-stents(high expression) but not in adjacent arterial segments or at the distal organs(2 liver samples showed low luciferase activity). Arg-CS/DNA nanoprticles (ACDNPs) coated stents demonstrate great potential as a novel and effective endovascular gene delivery system.