Construction And Evaluation Of A Novel Cationic Polymers Gene Delivery System

Gene therapy has been used in the treatment of tumors, cancer and various genetic diseases, and it has great exciting progress and will achieve a promising future. The key to efficient gene therapy is to design suitable and low toxicity gene delivery systems. Gene delivery systems are classified as viral vectors and nonviral vectors, synthetic nonviral vectors such as cationic polymers are being sought as alternatives, which have several advantages over viral counterparts, including ease of production, low immune response, low toxocitity, and so on.Chitosan as a natural polysaccharide, nontoxic, biodegradable, it has been reported that chitosan contains a large number of amino groups capable of forming complexes with DNA. But its water solubility and transfection efficiencyare very low, these disadvantages limit its application.Therefore, in order to improve its transfection efficiency, it's important to find a suitable way to get novel cationic chitosan derivatives as gene carrier, in the first part of this study, a series of novel water soluble chitosan derivatives were synthesized from N-maleated chitosan via Michael addition.The resulted polymers were characterized by 1H NMR spectra. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilizing agarose gel electrophoresis, particle size analysis, zeta potential and scanning electron microscopy. Furthermore, cytotoxicity of chitosan derivatives and transfection efficiency of polyplexes were evaluated in vitro. The results showed that the novel chitosan derivatives have lower cytotoxicity, good DNA condensation, and higher transfection efficiencies comparable to chitosan in both 293 T and HeLa cell lines. It indicated that these chitosan derivatives had a promising candidate as non-viral gene vectors.Covalently bonded disul?des can be formed spontaneously by autoxidation of sulfhydryls, primarily via oxidation upon exposure to air, which can reversibly be cleaved in the presence of reducing agents such as glutathione(GSH, 2-10 mM) in the cells. It will be very promising in gene delivery to combine the feature of disul?de bonds and cationic polymers. In the second part of this study, hyaluronic acid was used as skeleton and a disulfide bond was introduced, and tetraethylenepentamine was grafted to prepare a hyaluronic acid-based catonic polymer. Proton nuclear magnetic resonance spectroscopy(1HNMR) was used to characterize the carrier materials to determine its structure. As nonviral cationic vector, the performance of the carrier materials was evaluated by utilizing electrophoresis of agarose gel to evaluate the condension with plasmid DNA, and inspect the release of DNA under the high concentration of GSH; particle size analysis, zeta potential and scanning electron microscope. The result showed that the newly synthesized hyaluronic acid derivative have better DNA condensation, and can stimulate the release of DNA under the high concentration of GSH, it's a kind of potential gene carrier.Therefore, it's significant to design and develop a new type of cationic gene carrier system. For achieve the goal of improving transfection efficiency, it's availiable to modificate polysaccharide, such as hyaluronic acid and chitosan, to achieve their derivatives.Therefore, this study showed that both novel chitosan derivatives and hyaluronic acid derivatives can be used as potential non-viral gene vectors.