Rumiana Koynova

Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: A measure for transfection efficiency

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by approximately 10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, Lalpha-->HII, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct Lalpha-->HII phase transition and promoted formation of an inverted cubic phase between the Lalpha and HII phases. In contrast, moderate and weak transfection agents retained the direct Lalpha-->HII transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.

Cationic O-ethylphosphatidylcholines and their lipoplexes: phase behavior aspects, structural organization and morphology.

Ethylphosphatidylcholines, positively charged membrane lipid derivatives in which the anionic charge of the phosphate oxygen has been eliminated by ethylation, are promising nonviral, metabolizable transfection agents. We studied in detail the phase behavior, structural organization and morphology of the ethylphosphatidylcholines and their lipoplexes. Unlike the other phospholipids, dehydration does not change the melting transition temperature of O-ethyl-dipalmitoylphosphatidylcholine (EDPPC). Neither does an isoelectric amount of DNA, when added to the EDPPC aqueous dispersion. This is ascribed to the inability of EDPPC to form hydrogen bonds because of its headgroup modification. Similarly to its parent lipid DPPC, EDPPC displays a subtransition at 15 degrees C in its differential scanning calorimetry (DSC) heating scans after prolonged low-temperature incubation. The cooling behavior of the O-ethylphosphatidylcholines is sensitive to the thermal prehistory and the ionic strength. Different aggregate morphologies in the solid and the liquid-crystalline phases-respectively lamellar sheets and vesicles, as documented by light microscopy-are considered responsible for the cooling pattern. The interconversion between these morphologies is slow or even kinetically hindered, however, increasing the ionic strength to physiological values facilitates the conversion. The interdigitated chain arrangement of EDPPC gel phase tolerates incorporation of DNA between the bilayers. The minimum observed separation between the DNA strands is approximately 30-32 A, at DNA/lipid molar ratio > or =1. Formation of lipoplexes with DNA ordered in a 1-D lattice sandwiched between interdigitated lipid bilayers is reported for the first time.

Analysis of Lipoplex Structure and Lipid Phase Changes

Efficient delivery of genetic material to cells is needed for tasks of utmost importance in the laboratory and clinic, such as gene transfection and gene silencing. Synthetic cationic lipids can be used as delivery vehicles for nucleic acids and are now considered the most promising nonviral gene carriers. They form complexes (lipoplexes) with the polyanionic nucleic acids. A critical obstacle for clinical application of the lipid-mediated DNA delivery (lipofection) is its unsatisfactory efficiency for many cell types. Understanding the mechanism of lipid-mediated DNA delivery is essential for their successful application, as well as for a rational design and synthesis of novel cationic lipoid compounds for enhanced gene delivery. A viewpoint now emerging is that the critical factor in lipid-mediated transfection is the structural evolution of lipoplexes within the cell, upon interacting and mixing with cellular lipids. In particular, recent studies showed that the phase evolution of lipoplex lipids upon interaction and mixing with membrane lipids appears to be decisive for transfection success: specifically, lamellar lipoplex formulations, which were readily susceptible to undergoing lamellar-nonlamellar phase transition upon mixing with cellular lipids and were found rather consistently associated with superior transfection potency, presumably as a result of facilitated DNA release. Thus, understanding the lipoplex structure and the phase changes upon interacting with membrane lipids is important for the successful application of the cationic lipids as gene carriers.

Bilayer structural destabilization by low amounts of chlorophyll a

The present study shows that small admixtures of one chlorophyll a (Chla) molecule per several hundred lipid molecules have strong destabilizing effect on lipid bilayers. This effect is clearly displayed in the properties of the L(alpha)-H(II) transformations and results from a Chla preference for the H(II) relative to the L(alpha) phase. Chla disfavors the lamellar liquid crystalline phase L(alpha) and induces its replacement with inverted hexagonal phase H(II), as is consistently demonstrated by DSC and X-ray diffraction measurements on phosphatidylethanolamine (PE) dispersions. Chla lowers the L(alpha)-H(II) transition temperature (42 degrees C) of the fully hydrated dipalmitoleoyl PE (DPoPE) by approximately 8 degrees C and approximately 17 degrees C at Chla/DPoPE molar ratios of 1:500 and 1:100, respectively. Similar Chla effect was recorded also for dielaidoyl PE dispersions. The lowering of the transition temperature and the accompanying significant loss of transition cooperativity reflect the Chla repartitioning and preference for the H(II) phase. The reduction of the H(II) phase lattice constant in the presence of Chla is an indication that Chla favors H(II) phase formation by decreasing the radius of spontaneous monolayer curvature, and not by filling up the interstitial spaces between the H(II) phase cylinders. The observed Chla preference for H(II) phase and the substantial bilayer destabilization in the vicinity of a bilayer-to-nonbilayer phase transformation caused by low Chla concentrations can be of interest as a potential regulatory or membrane-damaging factor.