Multidisciplinary approach to the transfection of plasmid DNA by a nonviral nanocarrier based on a Gemini-Bolaamphiphilic hybrid lipid

Abstract

A multidisciplinary strategy, including bothbiochemical and biophysical studies, was proposed here toevaluate the potential of lipid nanoaggregates consisting of amixture of a gemini−bolaamphiphilic lipid (C6C22C6) and thewell-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidy-lethanolamine (DOPE) to transfect plasmid DNA into livingcells in an efficient and safe way. For that purpose, severalexperimental techniques were employed, such as zeta potential(phase analysis light scattering methodology), agarose gelelectrophoresis (pDNA compaction and pDNA protectionassays), small-angle X-ray scattering, cryo-transmission electronmicroscopy, atomic force microscopy,fluorescence-assisted cellsorting, luminometry, and cytotoxicity assays. The resultsrevealed that the cationic lipid and plasmid offer only 70 and30% of their nominal positive (=++q2.0nom,C C C6226) and negative charges (=−−q2/bpnom,pDNA), respectively. Upon mixing withDOPE, they form lipoplexes that self-aggregate in typical multilamellar Lαlyotropic liquid-crystal nanostructures with sizes in therange of 100−200 nm and low polydispersities, very suitablyfitted 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 DNAplasmids (pEGFP-C3, encoding the greenfluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with anefficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/−charge ratio wasmaintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic becausethe viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosysteman optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experiments