Building, Characterization And Evaluation Of In Vitro Transfection Efficiency Of Polycationic Non-viral Gene Vector Modified By Pluronic

Gene therapy is a technique to delivery gene to a specific tissue cells (target cells) and expression within the proper way to treat the disease. Currently, gene therapy vectors are divided into viral vectors and non-viral vectors. Non-viral vectors have significant clinical advantages for its targeting, low immunogenicity, low-cost, easy to scale. And other viral vectors do not have these advantages. In gene carriers, cationic polymers showing significant advantages for the transfer of gene. Through modification to cationic non-viral vectors, we can effectively shield the positively charged cations on the surface and reduce the toxicity of compounds to achieve the long circulation purposes.At present, the hydrophilic material are widely used to modify the carriers, the most commonly used modifier is polyethylene glycol (PEG).and Pluronic, as a surfactant, is rich of oil hydrophilic groups, can reduce toxicity and improve the compound's affinity for cells. Currently, there are few studies on the modification of cationic polymer carriers, but not systematically evaluated. In this project, two common carriers PEI and PAMAM are modified by Pluronic to study their cytotoxicity and transfection efficiency, and compared with the PEG system. We hope to get one or several Pluronic modification materials superior to PEG-modified carriers, and provide support to released gene delivery system.In the first part, succinimide ester was used to activation the terminal hydroxyl of Pluronic, nonionic wetting agent, to get a product with high activation efficiency and activity series. The activation compound was modified with PEI or PAMAM in the different proportion. After the purification through polydextran gel, the product was ultrafiltrated to get rid of salt and then cryodesiccated. According to the above process, we successfully got Graft-PEI or Graft-PAMAM compound with a series of different degree of modification per one PEI or PAMAM macromolecule at 1, 2, 5 and 10 Pluronic. We also used PEG which was modified with PEI or PAMAM as a comparison. The result of 1H-NMR showed the cationic polymerization synthesized by this method had a high graft ratio and PEI or PAMAM can be modified quantitatively by controlling administration dosage molar ratio of which had a satisfactory linear relationship with graft ratio. All that demonstrated that that synthetic method was stability, safety, controllability and had a high yield. There is no other impunity peak, so the purity of this cationic polymerization was high.In the second part, the related property of cationic graft polymer was investigated. By analysis blockage of electrophoresis, the N/P ratios need to completely block DNA were increased with the increment of modified degree of PEI or PAMAM, and decreased with the diminished molecular of reactor. The diameter of polymer/DNA and graft-polymer/DNA compound prepared were between 60-300nm. As the N/P ratios increased, the diameter of compound was decreased. The diameter of compound was obviously increased if it modified by macromolecular PEI or PAMAM at high modified degree. The zeta potential of compound with the same N/P ratios decreased with the increase of grafting amount of surfactant. If the modified degree and N/P ratio are same, zeta potential of compound increased a litter with the decrease of molecular of surfactant. According to the result of MTT method to test cytotoxicity, using surfactant to modify PEI or PAMAM can decrease the cytotoxicity in some degree. In the low concentration, the cytotoxicity can be decreased obviously if every PEI or PAMAM was grafted a surfactant, while in the high concentration, the cytotoxicity won't decrease unless every PEI or PAMAM was grafted more than five surfactant.In the third part, cationic graft polymer to DNA compound was evaluated for transfection in vitro. Enhanced green fluorescent protein plasmid (pEGFP) and Luciferase plasmid was selected as the report genes, respectively. In vitro transfection experiment of cationic graft polymer/DNA compound on HepG2 cell showed the PEI or PAMAM modified by P123 and PEG did not significantly decrease the transfection efficiency, and the best transfection effect of PEI or PAMAM modified by P123 is similar with that of PEI unmodified. As the increasing molecular of surfactant, grafting ratio rise and the decrease of the best transfection efficiency was followed. And at the same grafting ratio, the best transfection effect of PEI or PAMAM modified by P123 is better than that of PEI or PAMAM modified by PEG.