An intracellular lamellar–nonlamellar phase transition rationalizes the superior performance of some cationic lipid transfection agents

Abstract

Two cationic phospholipid derivatives with asymmetric hydrocarbon chains were synthesized: ethyl esters of oleoyldecanoylethylphosphatidylcholine (C18:1͞C10-EPC) and stearoyldecanoylethylphosphatidylcholine (C18:0͞C10-EPC). The former was 50 times more effective as a DNA transfection agent (human umbilical artery endothelial cells) than the latter, despite their similar chemical structure and virtually identical lipoplex organization. A likely reason for the superior effectiveness of C18:1͞C10-EPC relative to C18:0͞C10-EPC (and to many other cationic lipoids) was suggested by the phases that evolved when these lipoids were mixed with negatively charged membrane lipid formulations. The saturated C18:0͞C10-EPC remained lamellar in mixtures with biomembranemimicking lipid formulations [e.g., dioleoyl-phosphatidylcholine͞ dioleoyl-phosphatidylethanolamine͞dioleoyl-phosphatidylserine͞ cholesterol at 45:20:20:15 (wt͞wt)]; in contrast, the unsaturated C18:1͞C10-EPC exhibited a lamellar-nonlamellar phase transition in such mixtures, which took place at physiological temperatures, Ϸ37°C. As is well known, lipid vehicles exhibit maximum leakiness and contents release in the vicinity of phase transitions, especially those involving nonlamellar phase formation. Moreover, nonlamellar phase-forming compositions are frequently highly fusogenic. Indeed, FRET experiments showed that C18:1͞C10-EPC exhibits lipid mixing with negatively charged membranes that is several times more extensive than that of C18:0͞C10-EPC. Thus, C18:1͞C10-EPC lipoplexes are likely to easily fuse with membranes, and, as a result of lipid mixing, the resultant aggregates should exhibit extensive phase coexistence and heterogeneity, thereby facilitating DNA release and leading to superior transfection efficiency. These results highlight the phase properties of the carrier lipid͞cellular lipid mixtures as a decisive factor for transfection success and suggest a strategy for the rational design of superior cationic lipid carriers.lipofection ͉ lipoplex ͉ mesophase I mportant therapeutic procedures, such as gene transfection and gene silencing, require efficient delivery of genetic material to cells. Synthetic cationic lipoids, which form complexes (lipoplexes) with polyanionic DNA, are promising gene carriers (1). Understanding the mechanism of lipid-mediated DNA delivery (lipofection) is essential for the successful application and rational design and synthesis of novel cationic lipoid compounds for enhanced gene delivery. Although considerable improvement in the transfection properties of cationic lipoids has come from the synthesis of new kinds of cationic amphiphiles or from the inclusion of noncationic helper lipids, an effective alternative strategy was recently described: The combination of two cationic lipid derivatives having the same headgroup but different hydrocarbon chains can synergistically enhance transfection (2). For example, the optimal combination of the long chain͞medium chain lipoids, dioleoyl-and dilauroyl-ethylphosphatidylcholines, de...

Development of Dioctadecyldimethylammonium Bromide/Monoolein Liposomes for Gene Delivery

“…MO-based aggregates induce a relatively low cytotoxicity level, which further reinforces its use as a new helper lipid in this type of non-viral systems. In vivo evaluation of MO-based lipoplexes shall confirm the potential for this neutral surfactant to replace classic helpers in lipofection formulations, although some promising results have already been obtained for other cationic lipid formulations that also form inverted bicontinuous cubic structures [86][87][88]. Ethylphosphatidylcholines are a family of positively charged membrane lipid derivatives that promote the formation of QII G and QII P structures, having been linked to high levels of transfection efficiency with low cytotoxicity in several animal cell lines [89,90], consubstantiating MO's potential role in gene delivery.…”

Effect of spacer attachment sites and pH-sensitive headgroup expansion on cationic lipid-mediated gene delivery of three novel myristoyl derivatives

“…This is accomplished by efficient compaction and condensation of DNA by the vector for protection against enzymatic degradation, binding of the cationic liposome-DNA complex (lipoplex) to the cellular surface, internalization of the lipoplex within an endosome, and lipoplex fusion with the endosomal membrane for DNA release into the cytosol where the exogenous genetic material can be translocated to the nucleus for expression of the target gene [8], as illustrated in Figure 1. Membrane fusion has garnered particular attention recently through lipid mixing studies highlighting the fusogenic properties of cationic vesicles as critical for successful transfection [9][10][11].…”

Polyamine – Lipid Conjugates as Effective Gene Carriers: Chemical Structure, Morphology, and Gene Transfer Activity

“…With increasing N/P ratio, the morphology of the lipoplex transforms from large bead-like structure into smaller particles, wherein DNA molecules are condensed more tightly ( Figures 6C and D). The size of the lipoplexes, whose ζ-potential is positive, is 150-400 nm, is more favorable for cellular uptake via endocytosis (Koynova, Wang & MacDonald, 2006). Given that the lamellar assembly in the lipoplex is responsible for its effectiveness as a gene carrier (Koltover et al, 1998;Koynova, Wang & MacDonald, 2006), the population of active species for gene transfer would increase with increasing in the N/P.…”

“…For cationic lipids/DNA complexes (lipoplexes), it has been proposed that a morphological change from lamellar to inverted hexagonal phase in the acidic endosomal environment facilitates the endosomal release and escape of DNA (Bell et al, 2003;Xu & Szoka, 1996). In addition to investigation of intracellular trafficking of polycation-DNA complexes (polyplexes and lipoplexes), observation of morphology and metamorphosis of the complexes is very important to shed light on the mechanism of gene transfer and provide information for development of novel synthetic carriers (Koynova, Wang & MacDonald, 2006;Wan et al, 2008;Tarahovsky, Koynova & MacDonald, 2004). We have reported that polycationic liposomes (PCL) containing cetylated polyethylenimine (cetyl-PEI) possess high gene transfer activity (Yamazaki et al, 2000;Oku et al, 2001;Matsuura et al, 2003).…”