Advantages of statistical analysis of giant vesicle flickering for bending elasticity measurements.

We show how to greatly improve precision when determining bending elasticity of giant unilamellar vesicles. Taking advantage of the well-known quasi-spherical model of liposome flickering, we analyze the full probability distributions of the configurational fluctuations instead of limiting the analysis to the second moment measurements only as usually done in previously published works. This leads to objective criteria to reject vesicles that do not behave according to the model. As a result, the confidence in the bending elasticity determination of individual vesicles that fit the model is improved and, consequently, the reproducibility of this measurement for a given membrane system. This approach uncovers new possibilities for bending elasticity studies like detection of minute influences by solutes in the buffer or into the membrane. In the same way, we are now able to detect the inhomogeneous behavior of giant vesicle systems such as the hazardous production of peroxide in bilayers containing fluorescent dyes.

Conception and realization of a non-cationic non-viral DNA vector.

Cationic non-viral DNA vectors are very successful in in vitro transfections but less efficient in in vivo tests. This seems mainly due to the cationic nature of the molecules used to complex DNA. In this article, we describe the design and the route towards the realization of a non-viral non-cationic vector. The strategy follows three steps: first, the incorporation of DNA to a lamellar phase; second, the making of multilamellar vesicles containing a high loading of DNA by shearing the lamellar phase and, finally, the grafting of peptides onto the surface of the vesicles to target a specific receptor on the cells. Throughout this process, we had to overcome many obstacles; this review describes the present state of our work and summarizes the remaining steps.

The lipid charge density at the bilayer surface modulates the effects of melittin on membranes.

The influence of melittin on two DMPA membrane systems at pH 4.2 and 8.2 has been investigated by solid-state 31P and 2H NMR, as a function of temperature and peptide concentration. Melittin promotes greater morphological changes for both systems in the fluid phase, the effect being larger at pH 4.2. Close inspection of fatty acyl chain dynamics suggests that some parallels can be drawn between the DMPA/melittin at pH 8.2 and PC/melittin systems. In addition, at pH 8.2 a direct neutralization at the interface of one of the lipid negative charges by a positive charge of the peptide occurs, as can be monitored by 31P NMR at the molecular level. For the system at pH 4.2 and at high temperature, a lipid-to-peptide molar ratio of 30 is sufficient to transform the whole system into an isotropic phase, proposed to be inverted micelles. When the system is cooled down towards the gel phase one observes an intermediate hexagonal phase in a narrow range of temperature.

Tryptophan-anchored transmembrane peptides promote formation of nonlamellar phases in phosphatidylethanolamine model membranes in a mismatch-dependent manner.

To better understand the mutual interactions between lipids and membrane-spanning peptides, we investigated the effects of tryptophan-anchored hydrophobic peptides of various lengths on the phase behavior of 1,2-dielaidoylphosphatidylethanolamine (DEPE) dispersions, using (31)P nuclear magnetic resonance and small-angle X-ray diffraction. Designed alpha-helical transmembrane peptides (WALPn peptides, with n being the total number of amino acids) with a hydrophobic sequence of leucine and alanine of varying length, bordered at both ends by two tryptophan membrane anchors, were used as model peptides and were effective at low concentrations in DEPE. Incorporation of 2 mol % of relatively short peptides (WALP14-17) lowered the inverted hexagonal phase transition temperature (T(H)) of DEPE, with an efficiency that seemed to be independent of the extent of hydrophobic mismatch. However, the tube diameter of the H(II) phase induced by the peptides was clearly dependent on mismatch and decreased with shorter peptide length. Longer peptides (WALP19-27) induced a cubic phase, both below and above T(H). Incorporation of WALP27, which is significantly longer than the DEPE bilayer thickness, did not stabilize the bilayer. The longest peptide used, WALP31, hardly affected the lipid's phase behavior, and appeared not to incorporate into the bilayer. The consequences of hydrophobic mismatch between peptides and lipids are therefore more dramatic with shorter peptides. The data allow us to suggest a detailed molecular model of the mechanism by which these transmembrane peptides can affect lipid phase behavior.

A comparative study of the action of melittin on sphingomyelin and phosphatidylcholine bilayers.

To investigate whether lipid solubilization is of relevance in describing the interaction between melittin and biological membranes, we studied melittin-induced polymorphism using model membranes composed of the biological lipid sphingomyelin (bovine brain). The behavior of the system was monitored by solid state 31P-NMR and turbidity measurements and compared to the peptides well-characterized action on the synthetic lipid dipalmitoylphosphatidylcholine. It was found that melittin-induced macroscopic changes of sphingomyelin membranes are qualitatively the same as in the case of dipalmitoyl-phosphatidylcholine bilayers. The sphingomyelin/melittin system is thus proposed to show a reversible vesicle-to-disc transition (fluid-to-gel phase) through an intermediate fusion or aggregation event centered at the main transition temperature, Tm, as reported in the case of saturated phosphatidylcholine. In the case of spontaneous disc formation at 37 degrees C, the lipid-to-peptide molar ratio in the discoidal objects was determined to be approximately 20 for dipalmitoylphosphatidylcholine and about 12 in the case of natural sphingomyelin. Melittin partition coefficients between membranes and the aqueous medium at 37 degrees C were found to be 6.1 +/- 0.8 mM-1 and 3.7 +/- 0.4 mM-1 for sphingomyelin and dipalmitoylphosphatidylcholine, respectively. For very high peptide quantities (lipid-to-peptide molar ratio, Ri < or = 5) mixed micelles are formed over the entire temperature range (20 degrees to 60 degrees C) for both kinds of lipids.

Bending elasticities of model membranes: influences of temperature and sterol content.

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.

Effects of pH and cholesterol on DMPA membranes: a solid state 2H- and 31P-NMR study.

The effect of pH and cholesterol on the dimyristoylphosphatidic acid (DMPA) model membrane system has been investigated by solid state 2H- and 31P-NMR. It has been shown that each of the three protonation states of the DMPA molecule corresponds to a 31P-NMR powder pattern with characteristic delta sigma values; this implies additionally that the proton exchange on the membrane surface is slow on the NMR time scale (millisecond range). Under these conditions, the 2H-labeled lipid chains sense only one magnetic environment, indicating that the three spectra detected by 31P-NMR are related to charge-dependent local dynamics or orientations of the phosphate headgroup or both. Chain ordering in the fluid phase is also found to depend weakly on the charge at the interface. In addition, it has also been found that the first pK of the DMPA membrane is modified by changes in the lipid lateral packing (gel or fluid phases or in the presence of cholesterol) in contrast to the second pK. The incorporation of 30 mol% cholesterol affects the phosphatidic acid bilayer in a way similar to what has been reported for phosphatidylcholine/cholesterol membranes, but to an extent comparable to 10-20 mol % sterol in phosphatidylcholines. However, the orientation and molecular order parameter of cholesterol in DMPA are similar to those found in dimyristoylphosphatidylcholine.

Action of melittin on the DPPC-cholesterol liquid-ordered phase: a solid state 2H-and 31P-NMR study.

Solid-state deuterium and phosphorus-31 nuclear magnetic resonance studies of deuterium-labeled beta--[2,2',3,4,4',6-2H6]-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine have been undertaken to monitor the action of melittin on model membranes containing 30 mol% cholesterol, both at the molecular and macroscopic level. Cholesterol totally inhibits the toxin-triggered formation of large unilamellar vesicles and strongly restricts the appearance of small discs. The latter remain stable over a wide temperature range (20-60 degrees C) because of an increase in their cholesterol content as the temperature increases. This process is related to a constant disc hydrophobic thickness of approximately 29 A. The system, when not in the form of discs, appears to be composed of very large vesicles on which melittin promotes magnetically induced ellipsoidal deformation. This deformation is the greatest when the maximum of discs is observed. A model to describe both the disc formation and stability is proposed.