How Does the Spacer Length of Cationic Gemini Lipids Influence the Lipoplex Formation with Plasmid DNA? Physicochemical and Biochemical Characterizations and their Relevance in Gene Therapy

Abstract

Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) alkanes family referred to as C16CnC16, where n=2, 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, qpDNA−, a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of qDNA−=−2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, α, of the lipid mixture, and the effective charge ratio of the lipoplex, ρeff, the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEMand SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipid bilayers of lamellar structures within a monolayer of ∼2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out with HEK293T, HeLa, CHO, U343, and H460 cells. The α and ρeff values for each lipid mixture were optimized on HEK293T cells for transfection, and using these values, the remaining cells were also transfected in absence (-FBS-FBS) and presence (-FBS+FBS) of serum. The transfection efficiency was higher with the CLs of shorter gemini spacers (n=2 or 3). Each formulation expressed GFP on pDNA transfection and confocal fluorescence microscopy corroborated the results. C16C2C16/DOPE mixtures were the most efficient toward transfection among all the lipid mixtures and, in presence of serum, even better than the Lipofectamine2000, a commercial transfecting agent. Each lipid combination was safe and did not show any significant levels of toxicity. Probably, the presence of two coexisting lamellar structures in lipoplexes synergizes the transfection efficiency of the lipid mixtures which are plentiful in the lipoplexes formed by CLs with short spacer (n=2, 3) than those with the long spacer (n=5, 12).

Structural, biocomplexation and gene delivery properties of hydroxyethylated gemini surfactants with varied spacer length

“…Superior results were obtained for ethanediyl-and propanediyl versus the longer (-CH 2 -) 5 and (-CH 2 -) 12 polymethylene spacers in ref. [30 24], while the gemini with pentanediyl spacer appeared the most effective in work [31].…”

Stimuli responsive polymorphism of C12NO/DOPE/DNA complexes: Effect of pH, temperature and composition

“…For example, the MIC of C 12 NO, the most effective of the C n NOs, n = 6-18 homologous series, detected at Escherichia coli and Staphylococcus aureus was found 3-340 times higher [32,39] in comparison to gemini surfactant pentane-1,5-diylbis(dodecyldimethylammonium bromide) used in the liposomal DNA delivery vector in transfection experiments [40,41]. In absolute unit scale, Warisnoicharoen et al [38] found 0.08 mg/ml (~350 μM) as the C 12 NO concentration that caused 50% cell death (i.e.…”

Tunable pDNA/DODAB:MO lipoplexes: The effect of incubation temperature on pDNA/DODAB:MO lipoplexes structure and transfection efficiency

“…In a first approach, this optimization has been done by the empirical testing of newly synthesized lipids that had never been used before for gene therapy applications, and that led to the appearance of some promising lipoid molecules such as the gemini (dimeric) surfactants [3,4], the PEGylated (stealth) lipids [5,6], the pH-sensitive amphiphiles [7,8], the amino acidderived surfactants [9,10], or the neutral (helper) lipids [11,12]. Simultaneously, the increasing understanding of the transfection mechanism has led the researchers to explore the highly tunable physicochemical properties of lipoplexes (namely size [13,14], surface charge [15,16], curvature [17,18] and membrane fluidity [13]) as a means of enhancing transfection efficiency.…”