Preparation And Evaluation Of Lipopolyplexes(LP-PAE) As A Gene Vector

Objective:To prepare a lipopolyplexes gene vector with low toxicity and high transfection efficiency,and to evaluate lipopolyplexes(LP-PAE)in vitro and study its endocytosis mechanism.Methods:1.Preparation and evaluation of cationic liposome(LP)Cationic liposomes were prepared by ethanol dilution method.Use of these method that the DNA block experiment,cytotoxicity experiments and transfection efficiency in vitro to screen liposomal prescriptions.The Particle size and Zeta potential of LP/DNA were measured by nanoBrook 90 plus zeta.The morphology of the liposomes was observed under the Transmission Electron Microscope(TEM).The mechanism of transfection was studied by using endocytosis inhibitors.In addition,the programmed cell death 4 was used as a model drug to test the expression of LP/pdcd4 in SPC-A1,and its effect on the transmission of therapeutic genes was evaluated.2.Preparation and evaluation of lipopolyplexes(LP-PAE)LP-PAE/DNA was prepared by mixed the LP(50:25:25),PAE and DNA directly.The optimal prescription of LP-PAE/DNA were screened by these method which were gel retardation,cytotoxicity experiments and the transfection efficiency that was test by Luciferase reporter assay.The Particle size and Zeta potential of LP-PAE/DNA were measured by nanoBrook 90 plus zeta.The morphology of the liposomes was observed under the TEM.The mechanism of transfection was studied by using endocytosis inhibitors.In addition,the programmed cell death 4 was used as a model drug to test the expression of LP/pdcd4 in SPC-A1,and its effect on the transmission of therapeutic genes was evaluated.Result:1.Preparation and evaluation of cationic liposome(LP)Particle size and Zeta potential of LP(50:25:25)/DNA were about 200 nm and 30 mV,respectively.The morphology was spherical under TEM.Results of cytotoxicity experiments showed that cell survival rates were higher than 80%when the weight ratios of LP(50:25:25)/DNA and LP(40:30:30)/DNA were more than 30;Transfection experiments showed that the group of LP(50:25:25)/pGL3-N2 and LP(40:30:30)/pGL3-N2 had a higher transfection efficiency,especially that the transfection efficiency of LP(50:25:25)was similar to lipofectamin2000.The result of endocytosis showed that LP/pGL3-N2 mainly entered into cells through clathrin-mediated method.At the same time,a small part was taken up through caveolin-mediated endocytosis.The result of western blot showed that the expression of PDCD4,BAD and BAX proteins in the LP(50:25:25)/DNA(PDCD4)group was significantly increased in vitro.Furthermore,the result showed that the LP could effectively deliver the therapeutic gene pdcd4.2?Preparation and evaluation of lipopolyplexes(LP-PAE)The migration of DNA was completely retarted when the LP-PAE/DNA at any weight ratio.The cell viability of LP-PAE/pGL3-N2 was more than 80%except that the mass of PAE was 2 ?g in Hela,A549 and SPC-A1 cells.The results of cell transfection showed that the transfection efficiency of LPPAE/pGL3-N2 was significantly higher than that of positive control lipofectamine 2000/pGL3-N2 in Hela,A549 and SPC-A1 cells.The cellular uptake pathway experiments showed that after the clathrin-mediated endocytosis inhibitor chlorpromazine was used,the transfection efficiency of the LP-PAE/pGL3-N2 group decreased by 90%,while LP/DNA was 88%.Significantly,experiments using methyl-?-cyclodextrin which was caveolin-mediated endocytosis pathway inhibitor showed that the transfection efficiency of the LP-PAE/pGL3-N2 group decreased by approximately 65%and the LP/pGL3-N2 group decreased by approximately 45%.The results of macrophage drinking inhibitor-drugmannin-treated cells showed that the LP-PAE/pGL3-N2 group decreased by about 4%,and LP/pGL3-N2 had no significant effect.The result of western blot showed that the protein expression of PDCD4,BAD and BAX proteins in LP-PAE/pdcd4 was higher than LP/pdcd4.Furthermore,the result showed that the LP could effectively deliver the therapeutic gene pdcd4.Conclusion:In this experiment,liposomes(LP/DNA)and lipopolyplexes LP-PAE/DNA with low toxicity and high transfection efficiency were successfully prepared.The results showed that the transfection of lipopolyplexes(LP-PAE/DNA)was higher than cationic liposomes LP/DNA.The LP and PAE had synergistic effects as gene carriers.