Synthesis And Transfection Of Stimuli-responsive Cationic Gene Vectors

Gene therapy refers to the use of DNA recombination technology to introduce functionalized nucleic acid molecules(DNA or RNA)into target cells to correct or compensate for disease genes,and it has become a new biomedical technology allowing cells to completely(or partially)return to normal physiological metabolic states for therapeutic purposes.The advantage of gene therapy lies in its ability to treat diseases at a fundamental(genetic level).The main technical problem facing gene therapy at present is the lack of safe and efficient gene delivery vectors.Commonly used gene vectors mainly include two types of viral vectors and non-viral vectors.Although viral vectors have high transfection efficiency,yet they have drawbacks such as safety risks,immunogenicity,gene transfer size limitations,and high prices.As a commonly used non-viral gene carrier,cationic polymers and cationic liposomes have the potential to be excellent gene carriers due to their diverse structure,easy modification and functionalization,good biocompatibility,low toxicity and degradability.In recent years,it has attracted the attention of more and more researchers.The first chapter of this paper introduces gene therapy and gene vectors,and reviews thestimulus-responsive cationic gene vectors,gene transfection processes,the problems and the solutions.In the second chapter,we designed and synthesized four ROS-responsive cationic polymers by ring-opening polymerization of diepoxide containing a stimulus-responsive thioacetal group with low Molecular weight polyethyleneimine(PEI).Thro?gh a series of experiments,we found that these cationic polymers have good DNA binding ability,and the formed polyplexes have suitable sizes(180~300 nm)and zeta-potentials(+35~50 mV),which is stable to serum but decomposed under ROS conditions.In vitro cell experiments showed that these cationic polymers have lower cytotoxicity and the transfection efficiency is higher than the commercial reagents Lipofectamine 2000 and 25 KDa PEI.In the presence of 10% serum,the transfection efficiency of the polymer is even 10 times higher than 25 KDa PEI and 9 times higher than Lipofectamine 2000,with the ability to act as a potential transfection reagent.In the third chapter,we designed and synthesized four reduction-sensitive amino acid cationic lipids 3-4a,3-4b,3-4c and 3-6a with fluorescent dye naphthalene as the parent and octylamine as the hydrophobic group.Their DNA binding ability and transfection activity were studied by gel electrophoresis,particle size,zeta potential,cytotoxicity and in vitro transfection.The results showed that 3-4b and 3-4c have better DNA binding capacity and transfection efficiency than 3-4a and 3-6a.The cytotoxicity of the four fluorescent lipids is comparable to the commercial reagents Lipofectamine 2000 and 25 KDa PEI,but their transfection efficiency is lower than that of commercial reagents.We speculate that the hydrophobic chain length may be too short.Therefore,in order to further improve the transfection efficiency,we have further modified the hydrophobic group.Based on the results of previous experiments,lipids with high charge density of arginine(such as 3-4b)as hydrophilic head have better DNA binding ability and higher transfection efficiency.Therefore,based on the arginine hydrophilic head,we designed and synthesized the reducing-responsive cationic lipids 3-4d,3-4e and 3-4f with hydrophobic tails of octadecylamine,oleylamine and alpha-tocopherol.The relationship between the structure and activity of this series of cationic lipids was investigated.