Thermosensitive N-isopropylacrylamide-allylamine Copolymers And Their Guanidino Derivatives Based Non-Viral Vectors

In this work, thermosensitive Poly(N-isopropylacrylamide-co-allylamine) random copolymers (PNI-AAM) were synthesized by free radical copolymerization and their guanidinylated derivatives PNI-AAM-G were prepared through guanidinylation reaction. The main work includes three parts as follows:(1) The structures of PNI-AAM and PNI-AAM-G were confirmed by 1H NMR and 13C NMR. Element Analysis proved the high purity of resultant products. The lower critical solution temperatures (LCSTs) of copolymer solutions were determined by cloud point method. The result indicated that the LCSTs were increased along with the increment in the content of AAM and guanidino moieties, and the lowest LCST of copolymer solutions was about 38℃.(2) The interaction between copolymer vectors and plasmid DNA was investigated, and some influencing factors like copolymer structure and temperature were examined. It could be deduced from the results of temperature tunable agarose electrophoresis and UV that all copolymers were able to condense DNA effectively to form vector/DNA complexes, the high temperature exerted a more evident effect on the complexes with less AAM contents, and the general DNA condensing capability was: PNI-AAM 1-3 > PNI-AAM-G 1-3> PNI-AAM 1-1 >PNI-AAM-G 1-1. The size and morphology of complex particles were measured through Particle Size Analyzer and TEM at different weight ratios. It was shown that the complexes presented relatively uniform nanospheres, and smaller particles were found in the complexes containing the vectors with stronger DNA condensing capability and at higher weight ratios.(3) All copolymer vectors were used to transfer pGL3 plasmid encoding luciferase gene reporter into C2C12 cells. The data of luciferase activity showed that PNI-AAM-G 1-3 vector could successfully transfer pGL3 plasmid into C2C12 cell lines. The transfection efficiency was 100~400 times higher than that of naked DNA, 1~5 folds PEI and two orders of magnitude lower relative to Lipofectamine 2000. Meanwhile, this type of copolymer vectors exhibited a good serum resistance. It is expected that the transfection efficiency of PNI-AAM-G 1-3 could be further improved by changing its LCST below the body temperature.