Preparation And Preliminary Functional Study Of ApoE Modified Cationic Lipoplex

Objective:1. Prepare common cationic liposomes.2. Construct a novel liposome coupled with apo E, namely apo E modified lipoplex,which had specific transfection ability on hepatocyte and hepatoma carcinoma cell.3. Detect the transfection specificity and efficiency of the apo E modified lipoplex on the Hep G2, Hu H-7 and Li-7 hepatoma carcinoma cells and the HL-7702 hepatocyte cells.4. Explore the transfection efficiency of the apo E modified lipoplexes containing different doses of apo E on the Hep G2, Hu H-7 and Li-7 cells. Explore whether the storage time can influence the transfection efficiency of the apo E modified lipoplexes.5. Evaluate the toxicity of common cationic liposomes and apo E modified lipoplexes on Hep G2 cells.Methods:1. A common cationic liposome was produced using the modified ethanol injection method and extrusion method.2. Common cationic liposome was coupled with apolipoprotein E to produce apo E modified lipoplex.3. Common cationic liposome and apo E modified lipoplex were mixed with plasmid p Genesil-1 respectively, to explore the best mixing ratio for transfection.4. The Hep G2, Hu H-7 and Li-7 cells were transfected with common cationic liposome and apo E modified lipoplex respectively, the expression of EGFP report gene was observed under a fluorescence microscope to detect the transfection specificity of the apo E modified lipoplex.5. Different doses of apo E was added to the common cationic liposomes, which were used to transfect Hep G2 and Hu H-7 cells, transfection efficiency was detected by flow cytometry and fluorescence microscope, thus to determine the best mixing ratio of apo E and liposome.6. Apo E modified lipoplex of different storage times was used to transfect the Hep G2 cells, the transfection efficiency was detected by flow cytometry and fluorescence microscope to analyze whether the storage time can influence the transfection efficiency of apo E modified lipoplex.7. Agarose electrophoresis was used to analyze the stability of apo E modified lipoplex-DNA complex and common cationic liposome- DNA complex in DNase?solution.8. MTT assay was used to analyze the cytotoxicity of common cationic liposome and apo E modified lipoplex on Hep G2 cells.Result:1. The best mixing ratio for transfection of the common cationic liposome and plasmid p Genesil-1 is 0.5 ?l : 2.5 ?g.2. The best mixing ratio for transfection of the apo E modified lipoplex and plasmid p Genesil-1 is 2 ?l : 2.5 ?g.3. Three types of hepatoma carcinoma cells, hepatocyte and other kinds of tumor cells were transfected with this two kinds of liposomes, to verify the apo E modified lipoplex had specificity on three types of hepatoma carcinoma cells and hepatocyte by fluorescence microscope.4. The best mixing ratio of apo E modified lipoplex and common cationic liposome can be determined through flow cytometry, when the content of apo E was 6.6 ug/ml, the transfection rate was the highest, the difference is statistically significant(P < 0.05).5. Flow cytometry proved storage time can influence the transfection efficiency of apo E modified lipoplex, the difference is statistically significant(P < 0.05).6. MTT test proved that apo E modified lipoplex and common cationic liposome had no significant cytotoxicity on Hep G2 cell(P > 0.05).Conclusions:1. The apo E modified lipoplex had higher transfection efficiency than the common cationic liposome when they were used to transfect hepatocyte and hepatoma carcinoma cells. The content of apo E in apo E modified lipoplexes can influence the transfection efficiency.2. The transfection efficiency of apo E modified lipoplexes decreased with the storage time increasing.3. No significant cytotoxicity was observed of apo E modified lipoplex and common cationic liposome on Hep G2 cell.