Structural characteristics of supramolecular assemblies formed by guanidinium-cholesterol reagents for gene transfection

We have recently discovered that cationic cholesterol derivatives character ized by guanidinium polar headgroups are very efficient for gene transfecti on in vitro and in vivo. In spite of being based on some rationale at the m olecular level, the development of these new synthetic vectors was neverthe less empirical. Indeed, the factors and processes underlying cationic lipid -mediated gene transfer are still poorly understood. Thus, to get a better insight into the mechanisms involved, we have examined the supramolecular s tructure of lipid/DNA aggregates obtained when using reagent bis(guanidiniu m)-tren-cholesterol (BGTC), either alone or as a liposomal formulation with the neutral phospholipid dioleoyl phosphatidylethanolamine (DOPE), We here report the results of cryotransmission electron microscopy studies and sma ll-angle x-rag scattering experiments, indicating the presence of multilame llar domains with a regular spacing of 70 Angstrom and 68 Angstrom in BGTC/ DOPE-DNA and BGTC-DNA aggregates, respectively. In addition, DNA lipoplexes with similar lamellar patterns were detected inside transfected HeLa cells by conventional transmission electron microscopy, These results suggest th at DNA condensation by multivalent guanidinium-cholesterol cationic lipids involves the formation of highly ordered multilamellar domains, the DNA mol ecules being intercalated between the lipid bilayers, These results also in vite further investigation of the intracellular fate of the internalized li pid/DNA structures during their trafficking toward the cell nucleus. The id entification of the basic features of active complexes should indeed help i n the design of improved guanidinium-based vectors.