A multidisciplinary strategy, including both biochemical and biophysical studies, was proposed here to evaluate the potential of lipid nanoaggregates consisting of a mixture of a gemini-bolaamphiphilic lipid (C6C22C6) and the well-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) to transfect plasmid DNA into living cells in an efficient and safe way. For that purpose, several experimental techniques were employed, such as zeta potential (phase analysis light scattering methodology), agarose gel electrophoresis (pDNA compaction and pDNA protection assays), small-angle X-ray scattering, cryo-transmission electron microscopy, atomic force microscopy, fluorescence-assisted cell sorting, luminometry, and cytotoxicity assays. The results revealed that the cationic lipid and plasmid offer only 70 and 30% of their nominal positive (q(nom, C6C22C6)(+) = +2.0) and negative charges (q(nom, pDNA)(-) = -2/bp), respectively. Upon mixing with DOPE, they form lipoplexes that self-aggregate in typical multilamellar La lyotropic liquid-crystal nanostructures with sizes in the range of 100-200 nm and low polydispersities, very suitably fitted to remain in the bloodstream and cross the cell membrane. Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNA plasmids (pEGFP-C3, encoding the green fluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with an efficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/- charge ratio was maintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic because the viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosystem an optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experiments.