Oberle V, Bakowsky U, Zuhorn I S, Hoekstra D
Department of Physiological Chemistry, University of Groningen, Faculty of Medical Sciences, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Biophys J. 2000 Sep;79(3):1447-54. doi: 10.1016/S0006-3495(00)76396-X.
Cellular transfection can be accomplished by the use of synthetic amphiphiles as gene carrier system. To understand the mechanism and hence to improve the efficiency of transfection, insight into the assembly and properties of the amphiphile/gene complex is crucial. Here, we have studied the interaction between a plasmid and cationic amphiphiles, using a monolayer technique, and have examined complex assembly by atomic force microscopy. The data reveal a three-step mechanism for complex formation. In a first step, the plasmids, interacting with the monolayer, display a strong tendency of orientational ordering. Subsequently, individual plasmids enwrap themselves with amphiphile molecules in a multilamellar fashion. The size of the complex formed is determined by the supercoiled size of the plasmid, and calculations reveal that the plasmid can be surrounded by 3 to 5 bilayers of the amphiphile. The eventual size of the transfecting complex is finally governed by fusion events between individually wrapped amphiphile/DNA complexes. In bulk phase, where complex assembly is triggered by mixing amphiphilic vesicles and plasmids, a similar wrapping process is observed. However, in this case, imperfections in this process may give rise to a partial exposure of plasmids, i.e., part of the plasmid is not covered with a layer of amphiphile. We suggest that these exposed sites may act as nucleation sites for massive lipoplex clustering, which in turn may affect transfection efficiency.
细胞转染可以通过使用合成两亲物作为基因载体系统来实现。为了理解其机制并进而提高转染效率，深入了解两亲物/基因复合物的组装和性质至关重要。在此，我们使用单层技术研究了质粒与阳离子两亲物之间的相互作用，并通过原子力显微镜检查了复合物的组装情况。数据揭示了复合物形成的三步机制。第一步，与单层相互作用的质粒表现出强烈的取向有序倾向。随后，单个质粒以多层方式被两亲物分子包裹。形成的复合物的大小由质粒的超螺旋大小决定，计算表明质粒可以被3到5层两亲物双层包围。最终转染复合物的大小最终由个体包裹的两亲物/DNA复合物之间的融合事件决定。在本体相中，通过混合两亲性囊泡和质粒触发复合物组装时，观察到类似的包裹过程。然而，在这种情况下，该过程中的缺陷可能导致质粒部分暴露，即部分质粒没有被一层两亲物覆盖。我们认为这些暴露位点可能作为大量脂质体聚集体的成核位点，进而可能影响转染效率。
