Construction And Evaluation Of Multifunctional Liposomes As Gene Vectors

More and more attentions are paid to the multifunctional liposomes for gene vectors in gene therapy and a broad application prospects has been showed. In this paper, the multifunctional liposomes are prepared with folate-polyethylene glycol-cholesteryl hemisuccinate (F-PEG-CHEMS) and stearyl octaarginine(R8) as the functional moieties and then use it as ASODN and plasmid DNA vectors, investigate its physicochemical properties, behavior of the cellular uptake and transfection efficiency, and explore its viability as the generic drugs intracellular delivery system.First of all, in this paper the synthetic routes of cholesteryl hemisuccinate (CHEMS), Polyethylene glycol monomethyl ether-cholesteryl hemisuccinate (MPEG-CHEMS) and F-PEG-CHEMS are designed based on the research literatures. CHEMS is synthesized using cholesterol and Succinic anhydride as raw material via esterification reactivity. MPEG-CHEMS is synthesized using polyethylene glycol (MPEG) and CHEMS as raw material via esterification reactivity. The synthesis of F-PEG-CHEMS is divided into three steps:firstly, F-PEG-NH2is synthesized by reacting polyethylene glycol bisamine (NH2-PEG-NH2) with folate; secondly, CHEMS-NHS is synthesized by reacting cholesteryl hemisuccinate(CHEMS) and N-hydroxysuccinimide(NHS); finally, F-PEG-CHEMS is synthesized by reacting Folate-PEG-NH2with NHS-CHEMS. The products of synthesis and purification are confirmed as the target products with TLC, IR, MS and1H-HMR. As liposome membrane material, MPEG-CHEMS and F-PEG-CHESM of synthesis and purification laid the foundation of preparation of multifunctional liposomes.Secondly, conventional liposome are prepared with the method of thin film dispersion, and R8modified liposome(R8-Lip) are preparation with the method of R8aqueous solution incubated with normal liposome in45℃for1hour, and both of them are evaluated on physicochemical properties and cell level. The experimental results show that after conventional liposome is modified by R8, particle size and distribution are nearly unchanged, while zeta potential is turned from-10.24mV to+38.69mV. When the ratio of+/-in lipolexes is more than4:1, R8-Lip/ASODN has been combined into lipolexes with positive charge completely. When the R8concentration is less than10μg/mL, the cell survival rate is more than75%. The efficiency of celluar uptake is dependend on the concentration of the R8. When the concentration of the R8increase from0.3μg/mL to9.6μg/mL (+/-from1:2to16:1), intracellular fluorescence intensity increases gradually. When the+/-ratio of R8-Lip/ASODN lipolexes is16:1, the efficient of celluar uptake is better than the commercially available reagents Lipofectamine2000.After the R8-Lip surface modified with PEG, promotion the efficiency of celluar uptake is very poor. In order to further improve the efficiency of cell uptake, folate ligands are grafted to the end of the PEG, prepare the multifunctional liposome R8-Lip-F, and it is evaluated on physicochemical properties and celluar level also. The experimental results show that the liposomes have prefect morphology, and almost all of them are spherical. When the+/-ratio of R8-Lip-F/ASODN lipolexes is16:1, the lipolexes was completely stuck in the loading hole. The celluar uptake of R8-Lip-F/ASODN lipolexes is increased during the six hours. The R8-Lip-F/ASODN lipolexes enters into cell by endocytic pathways, and it is dependent on energy. The mechanism into the cell is mainly clathrin mediated endocytosis and macropinocytosis pathway. Efficiency of celluar uptake will decrease due to the competitive inhibition of free folate. After ASODN enter into the cell, most of them are distributed in the cytoplasm, nearly none in the nucleus. Compared with the commercially available transfection reagent Lipofectamine2000, the R8-Lip-F can better promote celluar uptake efficiency of ASODN, but nearly has no effect on the transfection of plasmid DNA.Finally, multifunctional cationic liposomes R8-DOTAP-F are prepared with F-PEG-CHEMS and R8on the basis of liposome DOTAP, and then evaluated on physicochemical properties and cell level. The experimental results show that the R8-DOTAP-F liposomes have prefect morphology, and almost all of them are spherical. The mass ratio of DOTAP/pDNA or ASODN is30(+/-ratio or N/P is about13:1) in the lipolexes, pDNA or ASODN are combined completely. The celluar uptake of R8-DOTAP-F/ASODN lipolexes is increased during the six hours. To modify liposome with folate ligands can significantly improve the cellular uptake and transfection effect of liposome lipolexes and the liposome modified with R8can further improve the effect too. The R8-DOTAP-F/ASODN lipolexes can enter into cell by endocytic pathways, and it is dependent on energy. The mechanism into the cell is mainly clathrin mediated endocytosis and macropinocytosis pathway. Efficiency of celluar uptake will decrease due to the competitive inhibition of free folate. After ASODN enter into the cell, most of them are distributed in the nucleus, almost none in the lysosomes. Compared with the commercially available transfection reagent Lipofectamine2000, the R8-DOTAP-F can better promote celluar uptake efficiency of ASODN, but has less effect on the transfection of plasmid DNA.