Lipoplex formation under equilibrium conditions reveals a three-step mechanism

Cellular transfection can be accomplished by the use of synthetic amphiphil es as gene carrier system. To understand the mechanism and hence to improve the efficiency of transfection, insight into the assembly and properties o f the amphiphile/gene complex is crucial. Here, we have studied the interac tion between a plasmid and cationic amphiphiles, using a monolayer techniqu e, and have examined complex assembly by atomic force microscopy. The data reveal a three-step mechanism for complex formation. In a first step, the p lasmids, interacting with the monolayer, display a strong tendency of orien tational ordering. Subsequently, individual plasmids enwrap themselves with amphiphile molecules in a multilamellar fashion. The size of the complex f ormed is determined by the supercoiled size of the plasmid, and calculation s reveal that the plasmid can be surrounded by 3 to 5 bilayers of the amphi phile. The eventual size of the transfecting complex is finally governed by fusion events between individually wrapped amphiphile/DNA complexes. In bu lk phase, where complex assembly is triggered by mixing amphiphilic vesicle s and plasmids, a similar wrapping process is observed. However, in this ca se, imperfections in this process may give rise to a partial exposure of pl asmids, 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 massi ve lipoplex clustering, which in turn may affect transfection efficiency.