Thermo-responsive Chitosan-g-NIPAAm/VL Copolymer Transgene Vector

In this thesis, the preparation of thermo-responsive chitosan -graft-NIPAAm/VL copolymer (PNVLCS), the characteristics of PNVLCS/DNA polyelectrolyte complexes, and gene transfection mediated by this nonviral vector were investigated. The main work includes three parts as follows: 1. A thermoresponsive PNVLCS copolymer was prepared by grafting copolymerization of NIPAAm and vinyl laurate with chitosan. Several methods were employed to examine the physicochemical characteristics of PNVLCS. The results of FTIR and 1H NMR confirmed that chitosan had been coupled with NIPAAm/VL copolymer, and GPC was used to determine the molecular weight of PNVLCS. The thermal analysis of copolymer indicated that the aqueous solution of PNVLCS displayed an obvious thermal transition, and the lower critical solution temperature(LCST) was about 26℃. 2. The properties of thermo-sensitive vector PNVLCS/plasmid DNA complexes were investigated by a variety of methods. The results of 31P NMR and circular dichroism (CD) indicated that the interaction of DNA with PNVLCS caused a slight perturbation of DNA bonds in the complex, which induced B→C type transformation; PNVLCS tended to be bound to the minor grooves of DNA. Temperature tunable CD and UV results proved that PNVLCS formed tight complexes with DNA because of vector phase transition when the temperature was increased above LCST; TEM images and DLS data showed that the size of complex particles varied with charge ratios. The morphology of PNVLCS/DNA complexes took on uniform globules and smaller nanoparticles were formed at higher charge ratios. The analytical results of electrophoresis demonstrated that PNVLCS was capable of condensing DNA, and comparatively the complexes formed at 37℃ were of higher stability and showed greater retardation. 3. CS and PNVLCS were used to transfer plasmid-encoding β-galactosidase into C2C12 cells. The transfection efficiency was evaluated by determining the β-galactosidase activity, and the influence of temperature on transfection efficiency was elucidated as well. The results revealed that PNVLCS could transfer pTracerTM-CMV/Bsd/lacZ Vector into C₂C₁₂ cell lines. The transfection efficiency of chitosan vector was 4-5 times higher than that of naked DNA; while the transfection level of PNVLCS could be further increased upon lowering temperature during transfection.