Membrane fusion plays an important role in gene transfection mediated by cationic liposomes

FEBS Letters

Nakanishi, Mamoru¹  Furuno, Tadahide¹  Kawaura, Chiyo¹  Noguchi, Ari¹ 

[1] Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467, Japan

关键词: Gene transfection;    Cationic liposome;    Membrane fusion;    Antisense oligonucleotide;    Confocal laser scanning microscopy;    Fluorescence resonance energy transfer;    Nigericin;    CLSM;    confocal laser scanning microscopy;    DIC;    differential interference contrast;    DOPE;    1;    2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine;    Rho-PE;    N-(6-tetramethylrhodamine thiocarbamoyl)-1;    2-dihexadecanoyl-sn-glycero-3-phosphoethanol amine;    NBD-PE;    N-(7-nitrobenz-2-oxa-1;    3-diazol-4-yl)-1;    2-dihexadecanoyl-sn-glycero-3-phosphoethanol amine;    MEM;    minimum essential medium;    DMEM;    Dulbecco's modified Eagle medium;    DOPG;    1;    2-dioleoyl-sn-glycero-3-phosphatidylglycerol;    DOPC;    1;    2-dioleoyl-sn-glycero-3-phosphatidylcholine;    CAT;    chloramphenicol acetyltransferase;    FBS;    fetal bovine serum;   

DOI  :  10.1016/S0014-5793(98)00837-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

By confocal laser scanning microscopy (CLSM) we have studied the membrane fusion between cationic liposomes and the endosome membranes involved in gene transfection mediated by cationic liposomes. Antisense oligonucleotides were transferred by cationic liposomes with a cationic cholesterol derivative, cholesteryl-3β-carboxyamidoethylenedimethylamine (I). Cationic liposomes were made by a mixture of the derivative I and DOPE. The intracellular distribution of fluorescein-conjugated antisense oligonucleotides (phosphorothioate) was studied by CLSM. The images showed that the antisense oligonucleotides were preferentially transferred into the nucleus of target cells (NIH3T3, COS-7 and HeLa cells) by the liposomes with derivative I. However, their transfection was completely blocked by nigericin which was able to dissipate the pH gradient across the endosome membranes, although the liposome/DNA complex was found in the cytoplasm of the target cells. This was quite in contrast with the fluorescence images of the target cells treated with wortmannin, an inhibitor of endocytosis. The results suggest that at least two steps are effective for gene transfection mediated by the cationic liposomes with cationic cholesterol derivatives. One is the endocytosis of the liposome/DNA complex into the target cells and the other is the removal of antisense oligonucleotides (plasmid DNAs) from the complex in the endosomes. The latter step was preferentially preceded by the membrane fusion between the cationic liposomes and the endosome membranes at around pH 5.0.

【 授权许可】

Unknown