Stebelska K, Dubielecka P M, Sikorski A F
Laboratory of Cytobiochemistry, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Przybyszewskiego 63/77, Wroclaw, 51-148, Poland.
J Membr Biol. 2005 Aug;206(3):203-14. doi: 10.1007/s00232-005-0793-0.
Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca(2+), indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.
含有阳离子脂质的超分子聚集体因其能够与带负电荷的DNA分子和生物膜相互作用，已被广泛用作转染介质。导致转染过程的第一步部分是静电作用，部分是脂质体/脂质复合物与细胞和/或内体膜的疏水相互作用。生物膜带负电荷的化合物，即糖脂、糖蛋白和磷脂酰丝氨酸（PS），是与阳离子脂质体/脂质复合物接触时天然膜发生吸附、半融合、融合、成孔和不稳定等事件的原因。本通讯描述了阳离子脂质体与天然膜和人工膜相互作用对靶膜负电荷的依赖性，在大多数情况下，这些电荷是通过改变PS含量或其暴露程度产生的。靶膜的模型是PS或PG（磷脂酰甘油）含量可变的脂质体以及红细胞膜，其中PS和其他阴离子化合物的含量/暴露程度通过几种方式进行了改变。阴离子磷脂含量增加的膜与DOTAP（1,2 - 二油酰基 - 3 - 三甲基铵丙烷）脂质体的融合增加，而部分去除糖萼（尤其是其中的唾液酸）的红细胞膜显示融合能力下降。红细胞膜外翻小泡容易与阳离子脂质体融合这一事实也支持了阴离子成分的作用。特别是在正常和不对称性破坏的红细胞血影上获得的数据，尤其是在Ca(2+)存在下获得的数据，表明了由翻转酶催化的脂质翻转运动的作用。红细胞的ATP耗竭也导致与DOTAP脂质体相互作用时血红蛋白泄漏的敏感性增加。与DOTAP脂质体一起孵育的阴离子脂质体的钙黄绿素泄漏也取决于靶膜的表面电荷。在所有使用不对称膜的实验中，随着温度升高，融合水平显著增加，这支持了膜脂质流动性的作用。通过结合质粒DNA使正电荷减少以及离子强度增加会降低DOTAP脂质体/脂质复合物与红细胞血影融合的能力。较低的pH促进红细胞血影与DOTAP脂质体和脂质复合物之间的融合。所得结果表明，静电相互作用以及膜脂质流动性增加和形成负曲率结构的易感性在DOTAP脂质体与天然膜和人工膜的融合中起主要作用。
