Palmitoyloleoyl Phosphatidylcholine Research Articles

Phospholipids as emulsifiers for micro/nano droplets suitable for biotechnological systems integration

We studied the emulsifying properties of palmitoyl oleoyl phosphatidylcholine (POPC) using the biocompatible compounds water and squalene as immiscible fluid phases. We tested the solubility limit of POPC in squalene and its equilibrium distribution between bulk phases. POPC is dissolvable in squalene up to 0.3% (w/v) with an ultrasonication procedure. Above this limit, aggregates of >1 μm are formed which are resistant to prolonged ultrasonication in size and quantity. Emulsifying properties of POPC were elaborated by measuring the droplet size ranges of emulsions. Nanofluidics was studied by pressure driven transport of nanometer sized emulsion droplets through defined nanochannels whereby droplet sizes 500 nm can be produced. The mechanical properties of the emulsifying phospholipid monolayer at the water/squalene interface were studied by profile analysis tensiometry (PAT). The dynamic interfacial tension was measured and the adsorption isotherms were established from long-time approximations of the diffusion-controlled adsorption. With PAT a critical aggregation concentration was determined in the same range as the solubility limit, which was measured by dynamic light scattering. The minimum interfacial tension for POPC as emulsifier was found to be below 1 mN/m. Thus, it can be concluded that phospholipids are suitable emulsifiers for microfluidics and produce adsorbed layers of remarkably small interfacial tension.

Cholesterol modulates the fusogenic activity of a membranotropic domain of the FIV glycoprotein gp36

Lipid composition of viral envelopes is usually rich in sphingolipids and cholesterol (CHOL). These components have a stiffening effect on the membrane, thus enhancing the energetic barrier to be overcome for its fusion with the T-cell plasma membrane, a fundamental step of the infection process. In this work, we demonstrate that the octapeptide (C8) corresponding to the Trp770–Ile777 sequence of the Feline Immunodeficiency Virus gp36 is highly effective in inducing the fusion of palmitoyl oleoyl phosphatidylcholine (POPC)/sphingomyelin (SM)/CHOL membranes. We analyze the molecular mechanism of the C8–membrane interactions combining Neutron Reflectivity (NR) and Electron Spin Resonance (ESR) experiments, and molecular dynamics simulations. A strict interplay among the different lipids in the peptide-induced fusion mechanism is highlighted. Since CHOL preferentially locates close to SM, POPC molecules remain relatively free to interact with the peptide, driving its positioning at the membrane interface. Here, C8 comes in contact with CHOL-interacting SM molecules, causing a strong perturbation of acyl chain ordering, which is a necessary condition for membrane fusion. Our findings suggest that CHOL rules, by an indirect mechanism, the activity of viral fusion glycoproteins.

Effect of N-Propyl Gallate on Lipid Peroxidation in Heterogenous Model Membranes

ABSTRACTThe antioxidant n-propyl gallate (nPG) is widely used in the food industry, pharmaceutics and cosmetics. Therefore, we should be well acquainted with its exact mechanism of action and its effects on the human organism, especially at the molecular level. Because of its slight solubility in water, it could be expected that, in cells, nPG would enter the membrane structures, thus altering their properties. Up to date little is known about its interaction with heterogenous lipid bilayers. That is why we focused our present study on the influence of nPG on the phase behavior of phosphatidylcholine/sphingomyelin/cholesterol (PC/SM/CHOL) ternary mixtures. Fluorescence microscopy of giant unilamellar vesicles (GUVs) was used as an experimental approach. Two phosphatidylcholine species were compared: palmitoyl-oleoyl phosphatidylcholine (POPC) and palmitoyl-docosahexaenoyl (ω-3) phosphatidylcholine (PDPC) differing in the number of double bonds at the sn-2 position (1 for POPC and 6 for PDPC). Fluorescence microscopy observations showed that the presence of docosahexaenoic acid induced the formation of micron-scale liquid-ordered (Lo) domains (model of cellular “rafts”) at physiological and higher temperatures compared to the monounsaturated oleic acid. nPG decreased the liquid-ordered (Lo)/liquid-disordered (Ld) miscibility transition temperature, Tm, for both types of vesicles in a concentration- dependent manner. Its effect was more pronounced in GUVs composed of PDPC/SM/CHOL.

Harnessing a Physiologic Mechanism for siRNA Delivery With Mimetic Lipoprotein Particles

Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice. We further demonstrated enhancement of silencing by administration of chol-siRNA pre-associated with isolated high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In this study, we investigated mimetic lipoprotein particle prepared from recombinant apolipoprotein A1 (apoA) and apolipoprotein E3 (apoE) as a delivery vehicle for chol-siRNAs. We show that apoE-containing particle (E-lip) is highly effective in functional delivery of chol-siRNA to mouse liver. E-lip delivery was found to be considerably more potent than apoA-containing particle (A-lip). Furthermore, E-lip–mediated delivery was not significantly affected by high endogenous levels of plasma LDL. These results demonstrate that E-lip has substantial potential as delivery vehicles for lipophilic conjugates of siRNAs.

Nonintercalating nanosubstrates create asymmetry between bilayer leaflets.

The physical properties of lipid bilayers can be remodeled by a variety of environmental factors. Here we investigate using molecular dynamics simulations the specific effects of nanoscopic substrates or external contact points on lipid membranes. We expose palmitoyl-oleoyl phosphatidylcholine bilayers unilaterally and separately to various model nanosized substrates differing in surface hydroxyl densities. We find that a surface hydroxyl density as low as 10% is sufficient to keep the bilayer juxtaposed to the substrate. The bilayer interacts with the substrate indirectly through multiple layers of water molecules; however, despite such buffered interaction, the bilayers exhibit certain properties different from unsupported bilayers. The substrates modify transverse lipid fluctuations, charge density profiles, and lipid diffusion rates, although differently in the two leaflets, which creates an asymmetry between bilayer leaflets. Other properties that include lipid cross-sectional areas, component volumes, and order parameters are minimally affected. The extent of asymmetry that we observe between bilayer leaflets is well beyond what has been reported for bilayers adsorbed on infinite solid supports. This is perhaps because the bilayers are much closer to our nanosized finite supports than to infinite solid supports, resulting in a stronger support-bilayer electrostatic coupling. The exposure of membranes to nanoscopic contact points, therefore, cannot be considered as a simple linear interpolation between unsupported membranes and membranes supported on infinite supports. In the biological context, this suggests that the exposure of membranes to nonintercalating proteins, such as those belonging to the cytoskeleton, should not always be considered as passive nonconsequential interactions.

Interactions between Ionizable Amino Acid Side Chains at a Lipid Bilayer–Water Interface

Potentials of mean force (PMF) between ionizable amino acid side chains (Arg, Lys, His, Glu) in the headgroup area of a palmitoyl oleoyl phosphatidylcholine lipid bilayer were obtained from all-atom molecular dynamics simulations and the adaptive biasing force method. Simulations in bulk water were also performed for comparison. Side chains were constrained in collinear, stacking, and orthogonal (T-shaped) orientations. The most structured and attractive PMFs were observed for hydrogen-bonded side chains. Contact minima occurred at a distance of 2.6-3.1 Å between selected atoms or centers of mass with the most attractive interaction (-9.6 kcal/mol) observed between Arg(+) and Glu(-). Hydrogen bonds play a significant role in stabilizing these interactions. Interactions between like charged side chains can also be very attractive if the charges are screened by surrounding molecules or groups (e.g., the PMF value at the contact minimum for Arg(+)···Arg(+) is -7.6 kcal/mol). Like charged side chains can have contact minima as close as 3.6 Å. The PMFs depend strongly on the relative orientation of the side chains. In agreement with experimental studies and other simulations, we found the stacking arrangement of like charged side chains to be the most favorable orientation. Interaction energies and Lennard-Jones energies between side chains, headgroups, and water molecules were analyzed in order to rationalize the observed PMFs and their dependence on orientation. In general, the results cannot be explained by simple dielectric arguments.

Interaction of Actin with Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1) Receptor in Liposomes Is Ca2+- and Phospholipid-dependent

The regulation of binding of G-actin to cytoplasmic domains of cell surface receptors is a common mechanism to control diverse biological processes. To model the regulation of G-actin binding to a cell surface receptor we used the cell-cell adhesion molecule carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-S) in which G-actin binds to its short cytoplasmic domain (12 amino acids; Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749-5760). A liposome model system demonstrates that G-actin binds to the cytosolic domain peptide of CEACAM1-S in the presence of negatively charged palmitoyl-oleoyl phosphatidylserine (POPS) liposomes and Ca(2+). In contrast, no binding of G-actin was observed in palmitoyl-oleoyl phosphatidylcholine (POPC) liposomes or when a key residue in the peptide, Phe-454, is replaced with Ala. Molecular Dynamics simulations on CEACAM1-S in an asymmetric phospholipid bilayer show migration of Ca(2+) ions to the lipid leaflet containing POPS and reveal two conformations for Phe-454 explaining the reversible availability of this residue for G-actin binding. NMR transverse relaxation optimized spectroscopic analysis of (13)C-labeled Phe-454 CEACAM1-S peptide in liposomes plus actin further confirmed the existence of two peptide conformers and the Ca(2+) dependence of actin binding. These findings explain how a receptor with a short cytoplasmic domain can recruit a cytosolic protein in a phospholipid and Ca(2+)-specific manner. In addition, this model system provides a powerful approach that can be applied to study other membrane protein interactions with their cytosolic targets.

Stable and Unstable Lipid Domains in Ceramide-Containing Membranes

We applied x-ray diffraction, calorimetry, and infrared spectroscopy to lipid mixtures of palmitoyl-oleoyl phosphatidylcholine, sphingomyelin, and ceramide. This combination of experimental techniques allowed us to probe the stability and structural properties of coexisting lipid domains without resorting to any molecular probes. In particular, we found unstable microscopic domains (compositional/phase fluctuations) in the absence of ceramide, and macroscopically separated fluid and gel phases upon addition of ceramide. We also observed phase fluctuations in the presence of ceramide within the broad phase transition regions. We compare our results with fluorescence spectroscopy data and complement the previously reported phase diagram. We also obtained electron paramagnetic resonance data to assess the possible limitations of techniques employing a single label. Our study demonstrates the necessity of applying a combination of experimental techniques to probe local/global structural and fast/slow motional properties in complex lipid mixtures.

Controlled Release from Cleavable Polymerized Liposomes upon Redox and pH Stimulation

A gallate derivative with three propargyl groups was coupled to palmitoyl oleoyl phosphoethanolamine (POPE). The resulting anionic lipid was formulated with common lipids such as palmitoyl oleoyl phosphatidyl choline (POPC) to form large unilamellar vesicles (LUVs). Polymerization of the LUVs was accomplished by the Cu(I)-catalyzed click reaction between the propargyl groups and the azide groups in the cross-linker. When the cross-linker contained a disulfide or ketal group, the resulting polymerized liposomes depolymerized and released entrapped contents upon the addition of a reducing thiol or under weakly acidic conditions. The click reaction allowed simultaneous multivalent surface functionalization during cross-linking, making these cleavable polymerized liposomes (CPLs) potentially very useful in the delivery and controlled release of pharmaceutical agents.

Observing the Solubilization of Lipid Bilayers by Detergents with Optical Microscopy of GUVs

The solubilization of lipid bilayers by detergents was studied with optical microscopy of giant unilamellar vesicles (GUVs) composed of palmitoyl oleoyl phosphatidylcholine (POPC). A solution of the detergents Triton X-100 (TX-100) and sodium dodecyl sulfate (SDS) was injected with a micropipette close to single GUVs. The solubilization process was observed with phase contrast and fluorescence microscopy and found to be dependent on the detergent nature. In the presence of TX-100, GUVs initially showed an increase in their surface area, due to insertion of TX-100 with rapid equilibration between the two leaflets of the bilayer. Then, above a solubility threshold, several holes opened, rendering the bilayer a lace fabric appearance, and the bilayer gradually vanished. On the other hand, injection of SDS caused initially an increase in the membrane spontaneous curvature, which is mainly associated with incorporation of SDS in the outer layer only. This created a stress in the membrane, which caused either opening of transient macropores with substantial decrease in vesicle size or complete vesicle bursting. In another experimental setup, the extent of solubilization/destruction of a collection of GUVs was measured as a function of either TX-100 or SDS concentration.

An evolutionary conserved motif is responsible for Immunoglobulin heavy chain packing in the B cell membrane

All species of vertebrates synthesize immunoglobulin molecules, which differ in an number of aspects but also share a few common features responsible for their function, such as the presence of a transmembrane domain in the membrane bound form of the immunoglobulin heavy chain (IgTMD) that ensures communication with the signal transducing Igα-Igβ peptides. We have analyzed the gene sequence encoding the IgTMD of different heavy chain isotypes of very distant species, from shark to mammals. The IgTMD sequences show a high degree of sequence identity and their encoding nucleotide sequences were shown to be subject to purifying selection at most sites. We have built molecular models of seven IgTMDs from different vertebrate species and have investigated the formation of homodimer in a palmitoyl oleoyl phosphatidylcholine (POPC) lipid bilayer by molecular dynamics simulations. We found that the conserved FXXXFXXS/TXXXS motif, never observed to date in protein transmembrane chains, is responsible for the two heavy chains association through two pairs of Phe–Phe hydrophobic interactions and two pairs of Ser/Thr–Ser/Ser hydrogen bonds. This interaction pattern, which stabilizes the dimer conformation in the lipid bilayer, was unique, being different from any other pattern identified in transmembrane helices to date.

Self-organization and phase structure of ternary lipid membranes: A spin label EPR study

The phase structure of membranes formed by ternary lipid mixtures, palmitoyl sphingomyelin (PSM)/palmitoyl oleoyl phosphatidylcholine (POPC)/Cholesterol (Chol), the composition of which simulates that of cell membranes, was studied by EPR using spin-labeled phospholipids. It is shown that, in the two-component POPC/Chol membranes and ternary membranes with a low (less than 24%) fraction of PSM, no phase boundaries are observed and the transition from the liquid disordered (ld) phase to the liquid ordered (lo) phase occurs continuously. In contrast, two-component EPR spectra and tie lines are detected in the other two-component systems, PSM/POPC and PSM/Chol, as well as in the ternary systems with a low content of Chol or POPC, respectively. These features are indicative of the coexistence of the gel phase (Lβ) and two liquid phases, ld or lo, respectively. The tie lines are approximately parallel to the Gibbs triangle sides Chol = 0 and POPC = 0, respectively. In the central part of the Gibbs triangle, where a sufficiently fast rotational diffusion of the spin labels indicates the membrane liquid state, we also observed two-component EPR spectra with quite different magnetic anisotropy and tie lines, indicating the coexistence of ld and lo phases. The boundaries are found for all the two-phase coexistence regions. The localization of the three-phase (Lβ, ld, and lo) coexistence region between the two-phase regions (Lβ-ld, Lβ-lo, and ld-lo) was proposed on the basis of an EPR-line shape analysis.

Molecular dynamics simulation of the transmembrane subunit of BtuCD in the lipid bilayer

Based on the crystal structure of the vitamin B(12) transporter protein of Escherichia coli (BtuCD) a system consisting of the BtuCD transmembrane domain (BtuC) and the palmitoyloleoyl phosphatidylcholine (POPC) lipid bilayer was constructed in silica, and a more-than-57-nanosecond molecular dynamics (MD) simulation was performed on it to reveal the intrinsic functional motions of BtuC. The results showed that a stable protein-lipid bilayer was obtained and the POPC lipid bilayer was able to adjust its thickness to match the embedded BtuC which underwent relatively complicated motions. These results may help to understand the mechanism of transmembrane substrate transport at the atomic level.

Revealing the Lytic Mechanism of the Antimicrobial Peptide Gomesin by Observing Giant Unilamellar Vesicles

Gomesin (Gm) is a potent cationic antimicrobial peptide from a Brazilian spider. Here we use optical and fluorescence microscopy to study the interaction of Gm, its low active linear analogue, [Ser(2,6,11,15)]-Gm (GmL), and a fluorescent labeled analogue, Gm-Rh, with giant unilamellar vesicles (GUVs) composed of mixtures of the neutral lipid palmitoyloleoyl phosphatidylcholine (POPC) with the negatively charged lipid palmitoyloleoyl phosphatidylglycerol (POPG) or cholesterol, so as to mimic bacterial and mammalian cell membranes, respectively. We observed the effect of injecting a peptide solution with a micropipet close to GUVs. As a result of peptide-lipid interaction, GUVs burst suddenly. Stable pores, which result in leaky vesicles, were not observed. Fluorescence microscopy of Gm-Rh injected on GUVs confirmed the high peptide/lipid affinity. These facts lead us to suggest that Gm and GmL disrupt the membrane via the carpet model. In order to quantify the lytic activity of both peptides against different membrane composition, a solution of GUVs was diluted in increasing concentration of peptides and the fraction of burst GUVs was measured as a function of time. The lytic activity of both peptides was enhanced by the presence of POPG and decreased upon addition of cholesterol. GmL exhibited lower lytic activity as compared to Gm, but this difference vanished at high POPG molar fraction.

Selective Tethering of Ligands and Proteins to a Microfluidically Patterned Electroactive Fluid Lipid Bilayer Array

We report a new, quantitative methodology to pattern and present ligands from planar, supported, fluid lipid bilayers. By combining microfluidic lithography (microFL) with an electroactive, chemoselective interfacial reaction strategy, a number of ligands as well as protein concanavalin A were immobilized in lipid microarrays. Electroactive vesicles were generated after the spontaneous insertion of hydroquinone-tethered alkane (H(2)Q) into egg palmitoyl-oleoyl phosphatidylcholine (egg-POPC), followed by subsequent fusion to a siloxane-terminated self-assembled monolayer (SAM) on gold. An advantage of the H(2)Q system is that it can be electrochemically oxidized to the corresponding quinone (Q), followed by rapid chemoselective conjugation with oxyamine-functionalized (RONH(2)) ligands. The oxime product is also electroactive, and the reaction can be monitored and the amount of ligand bound can be quantified by electrochemistry. The bilayers were characterized by electrochemistry, fluorescence microscopy, and ellipsometry and were determined to be fluid by fluorescence recovery after photobleaching (FRAP). This strategy provides a synergistic method to pattern and present a number of ligands or biomolecules from the bilayer surface for the evaluation of enzyme or protein binding to biomembranes.

Interaction of DNA-PAMAM Dendrimers with a Model Biological Membrane

The systemic delivery of DNA for gene therapy requires control of DNA compaction by an agent, such a lipid, surfactant or a polymer (e.g. cationic dendrimers). The crucial step here is to make the DNA cross the membrane to gain entry into the cell. Poly (amido amine) (PAMAM) dendrimers show great promise as synthetic gene-transfection agent. We have studied the structure of the complexes formed between DNA and PAMAM dendrimers as well as their interaction with model membranes, using a range of biophysical experimental techniques and simulation. We noted that the structure of the complex formed strongly depends on the generation of the dendrimer. The results show that generation 2 (G2) and 4 (G4) PAMAM dendrimers are able to penetrate surface deposited bilayers, consisting of palmitoyl oleoyl phosphatidyl choline as shown by neutron reflectometry (NR). The ability of the dendrimers to penetrate lipid bilayers is confirmed by coarse-grained simulations. The experimental and simulation data show that the DNA-dendrimer complex has a reduced ability to penetrate the bilayer, compared to the naked dendrimers. We will discuss these results in relation to the design of efficient transfection mediators with membrane permeating ability.

Potential of Mean Force Between Ionizable Amino Acid Side Chains in Lipid Bilayer

Potentials of mean force (PMF) between ionizable amino acid side chains (Arg, Lys, His, Glu/Asp) in different protonation states in palmitoyl oleoyl phosphatidylcholine lipid bilayer were obtained from all-atom explicit solvent molecular dynamics simulations and the adaptive biasing force approach available with NAMD.1,2 Side chains (SC) were constrained in different orientations: collinear, stacked and T-shaped and placed into the bilayer interface. The most structured PMFs were observed for unlike-charged ions or pairs with neutral SCs in collinear orientation. Contact pairs (CP) occurred at a distance of 2.6-3.1 A with the strongest interaction of −9.6 kcal/mol between Arg+ and Glu- ions. Like-charged SCs in this orientation displayed less stable contact minima at greater distances or solvent separated minima. All pairs in stacking approach showed similar, well-structured PMF profiles with CPs at ∼3.8 A. The strongest interaction between like-charged pairs was observed for stacked arginines. Like-charged pairs constrained in T-shaped geometry mostly displayed slightly stable solvent separated minima. A relationship between water and phosphate coordination numbers, contact pair minima and free energy barriers was found. There is also dependence of PMF shapes on H-bonding between amino acids. Generally, interactions between ionizable SCs are more attractive and the PMFs are more structured in a lipid bilayer than in water.31 Darve, E.; Pohorille, A. J. Chem. Phys. 2001, 115, 9169.2 Darve, E.; Wilson, M.; Pohorille, A. Mol. Simul.2002, 28, 113.3 Masunov, A.; Lazaridis, T.J. Am. Chem. Soc.2003125, 1722.

Effect of Ceramide on Nonraft Proteins

The currently accepted model of biological membranes involves a heterogeneous, highly dynamic organization, where certain lipids and proteins associate to form cooperative platforms ("rafts") for cellular signaling or transport processes. Ceramides, a lipid species occurring under conditions of cellular stress and apoptosis, are considered to stabilize these platforms, thus modulating cellular function. The present study focuses on a previously unrecognized effect of ceramide generation. In agreement with previous studies, we find that ceramide leads to a depletion of sphingomyelin from mixtures with palmitoyl oleoyl phosphatidylcholine bilayers, forming a ceramide-sphingomyelin-rich gel phase that coexists with a fluid phase rich in palmitoyl oleoyl phosphatidylcholine. Interestingly, however, this latter phase has an almost fourfold smaller bending rigidity compared to a sphingomyelin-palmitoyl oleoyl phosphatidylcholine mixture lacking ceramide. The significant change of membrane bulk properties can have severe consequences for conformational equilibria of membrane proteins. We discuss these effects in terms of the lateral pressure profile concept for a simple geometric model of an ion channel and find a significant inhibition of its activity.

Thermodynamic and Fluorescence Studies on the Interaction of Cholesterol with Palmitoyl-Oleoyl Phosphatidylcholine and Sphingomyelin

Studies on the interaction of cholesterol (CHOL) with palmitoyl-oleoyl phosphatidylcholine (POPC) and sphingomyelin (SPM) are considered to be important because of the occurrence of strong interactions between sphingolipids and CHOL, which lead to the formation of microdomains or rafts within biological membranes. In the present investigation, studies on the surface pressure (π)-area ( A ) measurements and fluorescence microscopic studies on monolayers of the above mentioned system have been reported. Ideality/nonideality of mixing, excess area, and free energy changes during the formation of the monolayers at different surface pressures for the mixed lipid systems were evaluated from the π– A data. Interactions were found to be repulsive at lower CHOL content, which became associative at higher CHOL content. Condensing effects of CHOL on the mixed monolayers were found. Fluorescence studies on the systems revealed similar overall results.

Influence of Mono- And Divalent Ions on the Formation of Supported Phospholipid Bilayers via Vesicle Adsorption

We have used the quartz crystal microbalance with dissipation monitoring (QCM-D) technique to investigate how mono- and divalent cations influence the formation of supported (phospho)lipid bilayers (SPB, SLB), occurring via deposition of nanosized palmitoyloleoyl phosphatidylcholine (POPC) vesicles on a SiO2 support. This process is known to proceed via initial adsorption of intact vesicles until a critical surface coverage is reached, where the combination of vesicle-surface and vesicle-vesicle interaction causes the vesicles to rupture. New vesicles then rupture and the lipid fragments fuse until a final continuous bilayer is formed. We have explored how this process and the critical coverage are influenced by different mono- and divalent ions and ion concentrations, keeping the anions the same throughout the experiments. The same qualitative kinetics is observed for all cations. However, different ions cause quite different quantitative kinetics. When compared with monovalent ions, even very small added concentrations of divalent cations cause a strong reduction of the critical coverage, where conversion of intact, adsorbed vesicles to bilayer occurs. This bilayer promoting effect increases in the order Sr2+<Ca2+<Mg2+. Monovalent cations exhibit a much weaker but similar effect in the order Li+>Na+>K+. The results are of practical value for preparation of lipid bilayers and help shed light on the role of ions and on electrostatic effects at membrane surfaces/interfaces.