Preparation Of Chitosan Derivate As Intelligent Polymer And Application Of Modified Chitosan Derivate As Gene Carrier In Gene Delivery

Chitosan is a well-known abundant natural polymer. Due to its biodegradability, biocompatibility and non-toxicity, it has been found applications in many areas such as biomedical and agriculture areas. Nevertheless, the crystallinity and poor solubility limit its further applications. Thus, chemical modification has been used to improve the properties of this rigid aminopolysaccharide and enlarge their application fields. Because the amino group at C-2 has a higher reactivity than the hydroxyl groups at C-6 and C-3 on the chitosan backbone, most chemical modifications deal with the former. However, the amino groups in chitosan impart its various advanced functions, including biological activity and cationic polymer properties. Considering these excellent properties of chitosan, it is better for chemical modification of chitosan without the loss of amino groups.On the other hand, stimuli-sensitive system, which alter their volume and shape reversibly according to the various external physiochemical factors have received much attention in recent years. It is expected that graft copolymers based on chitosan become stimuli-sensitive polymers with free amino groups. To achieve the goal, we first prepared O-maleoyl-N-phthaloyl-chitosan (MPCS) as an intermediate which had double bond for the subsequent graft copolymerization of vinyl monomers. Novel stimuli-sensitive graft copolymer was prepared by grafting monomer onto MPCS and the deprotection of the phthaloyl groups yielded copolymers with free amino groups, such as chitosan-g-PAA and chitosan-g-PNIPAAm. In addition, chitosan-g-phenyloboric acid was synthesized via a new protection-graft-deprotection procedure, the side chain of which was glucose-responsive at alkaline aqueous solutions.Chitosan, compared with synthetic polycations, has been emerged as a biocompatible alternative, suitable for in vivo gene delivery to mucosal tissues. But the low transfection efficiency of chitosan-DNA complex limits its applications in gene delivery. It had been proved that modification of chitosan could improve the transfection efficiency of chitosan-DNA complex. In this dissertation, thiolated N-Alkylated Chitosan (HWCS) were prepared first, and then HWCS-DNA nanoparticles were obtained using a similar complex coacervation approach. The particle size and zeta potential of the nanoparticles were characterized by dynamic light scatter (DLS) and transfection efficiency for nanoparticles in HEK-293 cell was evaluated.1. A novel temperature-sensitive graft copolymer was prepared by grafting N-isopropylacrylamide (NIPAAm) onto a chitosan derivate whose amino groups were protected by phthaloyl groups. The deprotection of the phthaloyl groups yields chitosan-g-PNIPAAm copolymers with free amino groups. The graft copolymers exhibits good solubility in aqueous solutions with a broader pH range and it exhibits a thermally sensitivity in aqueous solution.2. The graft copolymerization of HEMA onto chitosan was carried out with MPCS as intermediate in homogeneous system and initiated by azo-bis-isobutyronitrile (AIBN).3. The pH-sensitive graft copolymer chitosan-g-PAA was achieved by grafting acrylic acid (AA) onto chitosan backbone with MPCS as intermediate. The effect of various experimental conditions on the grafting degree was investigated. The result of turbidity showed that spontaneous turbidity appeared when aqueous solution of chitosan-g-PAA was acidified. It was complained that polyelectrolyte complex nanoparticles was self-assembled by electrostatic interactions.4. Poly (acrylic acid)-modified chitosan amphiphilic gels were prepared via O-maleoyl-N-phthaloyl-chitosan as an intermediate by grafting with poly (acrylic acid). The reaction was carried out in a homogeneous system byγ-ray irradiation. The swelling behavior of chitosan samples with different grafting degree was studied in different buffers as a function of pH. It could be found that all the samples swollen slightly at low pH but very well at high pH. Moreover, the swelling behavior of chitosan samples in DMF was evaluated.5. A novel thermo-sensitive switching membrane has been prepared by radiation-induced simultaneous grafting N-isopropylacrylamide (NIPAAm) onto chitosan membrane. Pure water flux measurements showed that water flux of the grafted membrane decreased with the increase of temperature, while that of chitosan membrane was constant. It was proved that grafted membrane was sensitive to temperature.6. HWCS-DNA nanoparticles were prepared using a complex coacervation process. Results showed that HWCS could effectively bind DNA in sodium acetate buffer and protect DNA from nuclease degradation. Transfection efficiency in HEK-293 for HWCS-DNA nanoparticles was much higher than chitsoan-DNA nanoparticles. It could be explained that the introduction of alkyl and thiol groups for chitsoan were favorable for cell entry and the unpacking of genes.7. Chitosan-g-phenyloboric acid was synthesized via a new protection-graft-deprotection procedure. The chemical structure of the copolymers was characterized by FT-IR and ~1H-NMR spectroscopy.