A Gemini Cationic Lipid with Histidine Residues as a Novel Lipid-Based Gene Nanocarrier: A Biophysical and Biochemical Study

Abstract

This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C3(C16His)2). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C3(C16His)2/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120–290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies.

A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid

“…These results indicate that both the cationic lipid and plasmid show net positive and negative charges, respectively, that are quite different from their nominal ones: LYCl exhibits only~45% of its positive nominal charge (+1), while pDNA displays on average only~12% of its negative nominal charge (−2/bp). This behavior has also been reported for other lipoplexes [46,52], revealing that the supercoiled conformation adopted by the plasmid under physiological conditions retains a significant percentage of Na + counterions associated to the phosphate groups of pDNA upon lipoplex formation, while the rest are expelled to the bulk contributing to a clear entropy gain. This factor, together with strong electrostatic interactions, is considered the main driving force toward lipoplex formation.…”

“…Therefore, the starting point of all lipofection studies should be the determination of the effective charges of both members of the lipoplex (lipid vector and plasmid), which do not necessarily have to be equal to the nominal ones, as demonstrated by us in previous works [50,62]. For that purpose, we have reported a protocol (fully described elsewhere [46,49,63] and summarized in the SM) based on a wide electrochemical study that uses both zeta potential and agarose gel electrophoresis experiments to determine the electroneutrality conditions of lipoplexes (m L /m DNA ) φ and, subsequently, the effective charges of both the cationic lipid (q + eff, LYCl ) and pDNA (q − eff, pDNA ). Figure 2a shows both studies (the zeta potential curves in the main figure and agarose gel electrophoresis in the inset) for the LYCl/DOPE-pDNA lipoplexes studied in this work at different LYCl molar compositions.…”

“…In previous studies, we have also demonstrated the advantages of using gene carriers based on amino acid components within a gemini-type lipid [46,47]. Generally speaking, those works have demonstrated that the use of this kind of molecular platforms is a safe option for biological studies.…”

Lipoaminoacids Enzyme-Based Production and Application as Gene Delivery Vectors

“…Histidine-mediated membrane fusion at acidic pH via protonation of the imidazole side chain (pK a 6.04) and disruption of the endosomes contributes to the high transfection efficiency usually associated with histidine-based LAAs [16,98,99], a trend also shown by non-LAA surfactants that contain imidazole as headgroup [8]. Cationic gemini histidine-based surfactants with very low CMC showed excellent DNA binding ability in the EB exclusion assay [99].…”

“…The cationic gemini LAAs with the longer alkyl chains (C 12 , C 14 , and C 16 ) completely displaced EB at cationic surfactant/DNA charge ratio of 1.75-2 [99]. The cationic histidine gemini LAA, C 3 (C 16 His) 2 (Figure 1), efficiently compacted plasmid DNA (pDNA) when mixed with helper lipid DOPE, which formed nanoaggregates with sizes of 120-290 nm that protected pDNA from enzymatic degradation [98]. The nanoaggregates showed high transfection efficiency in COS-7 and HeLa cells and low cytotoxicity in the alamar Blue assay, exceeding 80% cell viability, when considering the threshold for cell safety [98].…”