Synthesis Of Osmotic Gene Carrier FA-PEI-GDM And Its Application

In gene therapy,the gene carrier carry the gene material into the targeting cell or tissue,through replacing the disease gene or inhibiting the expression of abnormal gene to treat disease.Viral carriers and non-viral carriers are currently the most widely used in gene therapy.The non-viral carriers exceed the viral carriers in many aspects,such as the low cost,simple preparation,easy batch production,large carrying capacity,optimal security.cationic polymer carriers were widely reached in non-viral carriers applications.PEI,which act as a menber of cationic polymer carriers,has wide varieties and high charge density.Under the physiologic pH conditions,PEI has strongly buffer ability,then escape easily from the endosome and enter into the nucleus.Cationic polymer/DNA complexes(Polyplexes)and other large particulate matter enter into cells mainly by clathrin-dependent endocytosis or caveolae-mediated endocytosis.Polyplexes could be affected by the internal acid environment of lysosome and enzyme released when polyplexes enter into cells by clathrin-development endocytosis.Due to the internal environment of the caveosome is neutral,polyplexes entering into cells could sussceedly avoid the internal acid environment of lysosome and enzyme released by clathrin-dependent endocytosis,so the gene drugs could be effectively protected and delivered to the cell nucleus.Studies had shown that PEI carrier with osmotic ligand could not only inhibit the clathrin-development endocytosis but also effectibely promote the caveolae-mediated endocytosis.Because osmotic ligand connection could select stimulation caveolae-mediated endocytosis connection with induction of caveolin-1 phosphorylation through activation of Src-kinase.In this study,an osmotic gene carrier FA-PEI-GDM(FPG)was synthosized by low molecular weight PEI and glycerol dimethacrylate through Micheal addition.The physicochemical properties of FPG were tested by 1H NMR,particles size,Zete potential and TEM,results show that the characteristic peaks of different compositions in FPG was at the correct position,andthe mole ration of three PEI(600,1200,1800 Da)in FPG:45.27mol%,34.91mol%,42.24mol%,respectively.The condensation ability and protection ability of FPG for plasmid DNA were tested by agarose gel electrophoresis,results show that FPG could condense DNA and protect DNA escaping the DNase I released at low N/P ratio.And FPG-PEI1.2K could condense siRNA and protect siRNA escaping the RNase A released.FPG could compress plasmid DNA to form stabilize and spherical complexes,and particle size ranged from 85 nm to 180 nm,and Zeta potential ranged from 11 mV to 28 mV.Different concentration of FPG was added in HeLa,293T,NCI-H446 cell lines,the cell viability was greater than 80%by MTT.The transfection efficiency and gene silence efficiency of FPG were used plasmids DNA(luciiferase and pEGFP-N2),results show that the transfection efficiency and gene silence efficiency of FPG exceed the PEI 25K at above N/P of 20.Above all,FPG with plasmid DNA could select caveolae-mediated endocytosis into cells,ensuring the high transfection efficiency.