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Cationic lipids are widely used for gene delivery, and inclusion of dioleoylphosphatidylethanolamine (DOPE) as a helper lipid in cationic lipid–DNA formulations often promotes transfection efficacy. To investigate the significance of DOPE's preference to adopt a hexagonal phase in the mechanism of transfection, the properties and transfection efficiencies of SAINT-2/DOPE lipoplexes were compared to those of lipoplexes containing lamellar-phase-forming dipalmitoylphosphatidylethanolamine (DPPE). After interaction with anionic vesicles, to simulate lipoplex–endosomal membrane interaction, SAINT-2/DOPE lipoplexes show a perfect hexagonal phase, whereas SAINT-2/DPPE lipoplexes form a mixed lamellar–hexagonal phase. The transition to the hexagonal phase is crucial for dissociation of DNA or oligonucleotides (ODN) from the lipoplexes. However, while the efficiencies of nucleic acid release from either …