Synthesis, Characterizations And Bioassays Of Spermine Derivatives As Polycationic Gene Vector

Whether the regeneration treatment or the anti-viral treatment based on genetic materials, one of the most critical bottlenecks is how to deliver the DNA or siRNA to target sites safely and effectively. The objective of this study is using a series of cationic Polymers formed of a natural monomer (spermine) existing in the human sperm. The Polymers can do many things such as condensing DNA, circulating in vivo, targeting to special cells, escaping from lysosome, releasing DNA and degrading to nontoxic metabolin. Compared with viral vectors,Polycationic gene carriers have no potential genotoxic, bigger Gene density, and the Chemical Structure can easily be modified.In this study, endogenous monomer spermine is employed as the building block of the Polymer, and ethylene bis-(chloroformate) and bissuccinyl chloride are used as a linkers to form the linkage structures of carbamate and amide respectively. We named these two Polymers Polycarbam-sp and Polyamide-SP. The third step, I use mPEG-SC to block the end of the Polymer, in order to reduce the positive charge. We called these Polymer PEG-Polyamide-SP and PEG-Polycarbam-SP.The two Polymers were able to condense genes when add to a gene solution. They can form a Polyplex, the particle size of which is about100-120nm in water solution and 150-200nm in saline solution. Theζpotential of the Polyplex is +10mv to +20mv. Agarose gel electrophoresis indicated that PEG-Polyamide-sp can condense DNA at the w/w ratio of 10 or above, and PEG-Polycarbam-sp can condense DNA at the w/w ratio of 5 or above. The AFM picture demonstrated that the Polyplex is spheroid. And the particle size is about 100-200nm.The transfection of luciferase plasmids to COS-7 and Hep G2 indicated that both PEG-Polycarbam-SP and PEG-Polyamide-SP exhibit competitive transfection efficiencies compared to the positive controls of PEI25KDa and Lipofectamine 2000. Cytotoxicity assays suggest that all Polymers are less toxic than PEI 25kDa with the increase of Polymer concentrations. The more important is that PEG-Polyamide-SP has even lower toxicity, lower than lipofectamine 2000. So PEG-Polyamide-SP is the best Polymer in my study.Our work suggests that small linkers could also play a significant role in shaping the biological effects of Polymers. As Polymers show higher transfection efficiencies and improved cytotoxicity, this work could also be used as the reference for the rational design of better gene carriers.