Reported here is the correlation between the transfection efficiency of cationic liposome/DNA complexes (lipoplexes)
and the structural evolution that they undergo when interacting with anionic membrane lipids. Multicomponent lipoplexes,
incorporating from three to six lipid species simultaneously, presented a much higher transfection efficiency than
binary lipoplexes, which are more commonly used for gene-delivery purposes. The discovery that a high transfection
efficiency can be achieved by employing multicomponent complexes at a lower-than-ever-before membrane charge
density of lipoplexes was of primary significance. Synchrotron small-angle X-ray diffraction (SAXD) experiments
showed that anionic liposomes made of dioleoylphosphatidylglycerol (DOPG) disintegrated the lamellar phase of
lipoplexes. DNA unbinding was measured by electrophoresis on agarose gels. Most importantly, structural changes
induced by anionic lipids strictly depended on the lipid composition of lipoplexes. We found evidence of the existence
of three different regimes of stability related to the interaction between complexes and anionic membranes. Both
unstable (with low membrane charge density, !M) andhighly stable lipoplexes (withhigh !M) exhibited lowtransfection
efficiency whereas highly efficient multicomponent lipoplexes exhibited an “optimal stability”. This intermediate
regime reflects a compromise between two opposing constraints: protection of DNA in the cytosol and endosomal
escape. Here we advance the concept that structural stability, upon interaction with cellular anionic lipids, is a key
factor governing the transfection efficiency of lipoplexes. Possible molecular mechanisms underlying experimental
observations are also discussed