Basic Study On Application Of Gene Carrier In Gene Therapeutic Drugs

According to the independent intellectual property patent process route,a branched and 30KD cationic polymer was synthesized,which the sofast gene carrier was constructed based on.The sofast gene carrier can be applied as big molecular nucleic acids carrier both in vitro and in vivo.In this paper we investigated its transfection mechanism as a gene carrier through formation of sofast/DNA complex,particle size,zeta potential analysis,anti-nuclease assays,buffering capacity assays,DNA combining assayes and effect of diINF-7 and salt concentration of working solutions.The in vivtro asssays results showed that the sofast gene carrier had high transfection efficiency,can be applied to various cell lines transfection.It also had the property of low cytotoxicity,anti-serum capacity,long-time stability(4℃for 3 years) and simple operation.In in vivo assayes, acute toxicity test,sbuacute and chronic toxicity test,LD50 assays,allergic assays and pyrogen assays were taken.The results indicated the sofast gene carrier had low in vivo cytotoxicity.The in vivo transfection assays results showed that its tansfection efficiency was higher than cationic-liposome Lipofectamine 2000 which prevailed in the market. So the sofast gene carrier is an new kinds of easy handling,widely-fitting with the property of high efficiency,low toxicity,long stability,lipid-cationic polymer gene carrier.Based on the construction of sofast gene carrier,we synthesized 50KD brassidic acid branched lipid-cationic polymer,then constructed the lipid-cationic polymer siRNA gene carrier to mediated siRNA transfection both in vitro and in vivo.In this paper we investigated its transfection mechanism as a gene carrier through formation of sofast/DNA complex,particle size,zeta potential analysis,anti-nuclease assays,buffering capacity assays,DNA combining assayes and effect of diINF-7 and proton pump inhibitor. It showed that the lipid cationic polymer gene carrier is composed of the cationic polymer backbone and the branched brassidic acid.So it bore the characteristics of cationic polymer and cationic liposome.It's transfection efficiency is higher than the Lipofectamine 2000.Further specificity test,cytotoxicity test,bio-compatibility test, degradation test,stability test and suitable cell line test indicated that the lipid-cationic polymer gene carrier mediated siRNA transfection is safe,efficient,specific and stable. The siRNA induced silence after the transcriptiom of cox-2 showed the effectiveness of gene carrier in mRNA level.The western blotting analysis and immunohistochemistry analysis using self-made COX-2 polyclonal and monoclonal antibodies showed the effectiveness of the gene carrier mediated gene silence in protein expression level.So the lipid-cationic polymer gene carrier can be applied in areas of embryo development,virus infection,carcinogenesis,drug screening,gene function and genetic therapy etc.And it is hoped that the lipid-cationic polymer gene carrier can break the bottle neck in genetic therapy the gene carrier in gene therapeutic drugs.