We assessed in this work how a chemical structure difference could influence a supramolecular organization and then its biological properties. In our case study, we considered two amphiphilic lipidic gene vectors. The chemical difference was situated on their hydrophilic part which was either a pure neutral thiourea head or a mixture of three thiourea function derivatives, thiourea, iminothiol, and charged iminothiol. This small difference was obtained thanks to the last chemical deprotection conditions of the polar head hydroxyl groups. Light, neutron, and X-ray scattering techniques have been used to investigate the spatial structure of the liposomes and lipoplexes formed by the lipids. The chemical structure difference impacts the supramolecular assemblies of the lipids and with DNA as shown by fluorescence correlation spectroscopy (FCS), X-ray, and neutron scattering. Hence the structures formed were found to be highly different in terms of liposomes to DNA ratio and size and polydispersity of the aggregates. Finally, the transfection and internalization results proved that the differences in the structure of the lipid aggregates fully affect the biological properties of the lipopolythiourea compounds. The lipid containing three functions is a better gene transfection agent than the lipid which only contains one thiourea moiety. As a conclusion, we showed that the conditions of the last chemical step can influence the lipidic supramolecular structure which in turn strongly impacts their biological properties.