Binary Lipid Mixtures Research Articles

Permeability-driven selection in a semi-empirical protocell model: the roots of prebiotic systems evolution

The origin-of-life problem has been traditionally conceived as the chemical challenge to find the type of molecule and free-solution reaction dynamics that could have started Darwinian evolution. Different autocatalytic and ‘self-replicative’ molecular species have been extensively investigated, together with plausible synthetic pathways that might have led, abiotically, to such a minimalist scenario. However, in addition to molecular kinetics or molecular evolutionary dynamics, other physical and chemical constraints (like compartmentalization, differential diffusion, selective transport, osmotic forces, energetic couplings) could have been crucial for the cohesion, functional integration, and intrinsic stability/robustness of intermediate systems between chemistry and biology. These less acknowledged mechanisms of interaction and molecular control might have made the initial pathways to prebiotic systems evolution more intricate, but were surely essential for sustaining far-from-equilibrium chemical dynamics, given their functional relevance in all modern cells. Here we explore a protocellular scenario in which some of those additional constraints/mechanisms are addressed, demonstrating their ‘system-level’ implications. In particular, an experimental study on the permeability of prebiotic vesicle membranes composed of binary lipid mixtures allows us to construct a semi-empirical model where protocells are able to reproduce and undergo an evolutionary process based on their coupling with an internal chemistry that supports lipid synthesis.

Geodesic curvature driven surface microdomain formation

Lipid bilayer membranes are not uniform and clusters of lipids in a more ordered state exist within the generally disorder lipid milieu of the membrane. These clusters of ordered lipids microdomains are now referred to as lipid rafts. Recent reports attribute the formation of these microdomains to the geometrical and molecular mechanical mismatch of lipids of different species on the boundary. Here we introduce the geodesic curvature to characterize the geometry of the domain boundary, and develop a geodesic curvature energy model to describe the formation of these microdomains as a result of energy minimization. Our model accepts the intrinsic geodesic curvature of any binary lipid mixture as an input, and will produce microdomains of the given geodesic curvature as demonstrated by three sets of numerical simulations. Our results are in contrast to the surface phase separation predicted by the classical surface Cahn–Hilliard equation, which tends to generate large domains as a result of the minimizing line tension. Our model provides a direct and quantified description of the structure inhomogeneity of lipid bilayer membrane, and can be coupled to the investigations of biological processes on membranes for which such inhomogeneity plays essential roles.

Self-assembling complexes between binary mixtures of lipids with different linkers and nucleic acids promote universal mRNA, DNA and siRNA delivery

Protein expression and RNA interference require efficient delivery of DNA or mRNA and small double stranded RNA into cells, respectively. Although cationic lipids are the most commonly used synthetic delivery vectors, a clear need still exists for a better delivery of various types of nucleic acids molecules to improve their biological activity. To optimize the transfection efficiency, a molecular approach consisting in modifying the chemical structure of a given cationic lipid is usually performed, but an alternative strategy could rely on modulating the supramolecular assembly of lipidic lamellar phases sandwiching the nucleic acids molecules. To validate this new concept, we synthesized on one hand two paromomycin-based cationic lipids, with either an amide or a phosphoramide linker, and on the other hand two imidazole-based neutral lipids, having as well either an amide or a phosphoramide function as linker. Combinations of cationic and helper lipids containing the same amide or phosphoramide linkers led to the formation of homogeneous lamellar phases, while hybrid lamellar phases were obtained when the linkers on the cationic and helper lipids were different. Cryo-transmission electron microscopy and fluorescence experiments showed that liposomes/nucleic acids complexes resulting from the association of nucleic acids with hybrid lamellar phases led to complexes that were more stable in the extracellular compartment compared to those obtained with homogeneous systems. In addition, we observed that the most active supramolecular assemblies for the delivery of DNA, mRNA and siRNA were obtained when the cationic and helper lipids possess linkers of different natures. The results clearly show that this supramolecular strategy modulating the property of the lipidic lamellar phase constitutes a new approach for increasing the delivery of various types of nucleic acid molecules.

The Interplay of Structure and Dynamics in Mixed Lipid Bilayers

The implication of lipids in membrane mediated processes has motivated significant theoretical and experimental research interest to understand the functional significance of lipid chemical structure variations on the biomembrane structure and dynamics. In particular, hydrophobic mismatch between lipids with different acyl chain lengths is expected to play an important role in determining the local membrane structure and collective membrane dynamics. Herein we investigated these phenomena in large unilamellar vesicles composed of binary mixtures of lipids with a 4 carbon mismatch in tail length, dimyristoylphosphatidylcholine (DMPC, 14:0 C) and distearoylphosphatidycholine (DSPC, 18:0 C), using neutron scattering techniques. Structural studies using small angle neutron scattering (SANS) revealed that the mixed lipid bilayers were thinner than expected for a simple composition-weighted average of the pure component bilayers. The structural differences were accompanied by dramatic differences in the mixed lipid membrane dynamics. Neutron spin echo spectroscopy (NSE) experiments showed that the mixed lipid bilayers were more dynamic than their single component analogs, with the mixed bilayers having a lower bending modulus and increased thickness fluctuation amplitude than the pure DMPC or DSPC bilayers. Both of these enhanced dynamics were consistent with a decrease in the area expansion modulus of the mixed systems. Together our results demonstrate the influence of lipid composition on the bilayer biophysical properties and highlight the complex interplay between structure and dynamics in lipid membranes.

Lateral Heterogeneity of Cholesterol on Binary Lipid Mixtures of POPC/CHOL: a Molecular Dynamics Study

Phase diagrams on ternary and quaternary lipid mixtures showing the existence of liquid-disordered (ld), liquid-ordered (lo) and a mixed phase (ld+lo) are now well established. Although the ld+lo is generally accepted for binary mixtures of cholesterol and phospholipids with saturated acyl chains, the existence of a phase separation in mixtures of cholesterol with phospholipids that have one saturated and one unsaturated acyl chain is not universally accepted. There are reports of a phase diagram on the mixtures POPC/Chol and POPC/Erg and nystatin activity co-related to this purported phase diagram; with a maximal activity appearing in the mixed phase.

Lateral Heterogeneity of Cholesterol on Binary Lipid Mixtures of POPC/Chol Imaged with AFM

Phase diagrams on ternary and quaternary lipid mixtures showing the existence of liquid-ordered (lo), liquid-disordered (ld) and a mixed phase (lo+ld) are now well established. However the existence of such a phase diagram on binary mixtures is still in debate despite reports of a phase diagram on the mixtures POPC/Chol and POPC/Erg and nystatin activity co-related to this purported phase diagram; with a maximal activity appearing in the mixed phase. Recent Molecular Dynamics simulations of POPC/Chol at different temperatures and cholesterol concentrations along this phase diagram, suggest the existence of cholesterol enriched nano-sites in this mixed phase, indicating the presence of cholesterol lateral heterogeneity. Here we present atomic force microscopy images of supported lipid bilayers made up of POPC and different cholesterol concentrations along the phase diagram. We observe structures that are consistent with the Molecular Dynamics predictions of order parameters and membrane thickness, supporting the idea of cholesterol lateral heterogeneity in binary mixtures, which can enrich the discussion on the existence of lipid rafts and domains in biological membranes. Funding: DGAPA-PAPIIT-RG100416.

Hysteresis and the Cholesterol Dependent Phase Transition in Binary Lipid Mixtures with the Martini Model.

Extensive Martini simulation data, totaling 5 ms, is presented for binary mixtures of dipalmitoylphosphatidylcholine (DPPC) and cholesterol. Using simulation initiated from both gel (so) and liquid-disordered (Ld) phases, significant and strongly cholesterol-dependent hysteresis in the enthalpy as a function of temperature is observed for cholesterol mole fractions from 0 to 20 mol %. Although the precise phase transition temperature cannot be determined due to the hysteresis, the data are consistent with a first order gel to fluid transition, which increases in temperature with cholesterol. At 30 mol % cholesterol, no hysteresis is observed, and there is no evidence for a continuous transition, in either structural parameters like the area per lipid or in the heat capacity as a function of temperature. The results are consistent with a single uniform phase above a critical cholesterol composition between 20 and 30 mol % in Martini, while highlighting the importance and difficulty of obtaining the equilibrium averages to locate phase boundaries precisely in computational models of lipid bilayers.

In-situ single pass intestinal permeability and pharmacokinetic study of developed Lumefantrine loaded solid lipid nanoparticles

The present investigation aims to develop lumefantrine loaded binary solid lipid nanoparticles (LF-SLNs) to improve its poor and variable oral bioavailability. The oral bioavailability of LF is poor and variable due to its limited aqueous solubility and P-gp mediated efflux occurring in small intestine. LF-SLNs were prepared using binary lipid mixture of stearic acid and caprylic acid stabilized with TPGS (D-alpha tocopheryl polyethylene glycol 1000 succinate) and Poloxamer 188. Developed LF-SLNs were characterized for particle size distribution, zeta potential, entrapment efficiency, solid state properties and biopharmaceutical properties including in situ intestinal permeability and oral bioavailability. The particle size distribution, zeta potential and entrapment efficiency of optimized batch (LF-SLN7) was found to be 357.7±43.27nm, 25.29±1.15mV and 97.35±0.30%, respectively. DSC thermographs showed loss of crystalline nature of lumefantrine in LF-SLNs. In situ single pass intestinal permeability study (SPIP) study indicated significant enhancement in the effective intestinal permeability of LF from LF-SLN7 as compared to that of control. Pharmacokinetic study also showed significant increase in Cmax and area under curve (AUC0-∞) from LF-SLN7 (3860±521ng/mL and 43181±2557h×ng/mL, respectively) as compared to that of LF-control suspension (1425±563ng/mL and 19586±1537h×ng/mL, respectively). Thus, developed LF-SLNs can be promising to overcome P-gp efflux pump and enhance the oral bioavailability of lumefantrine.

Mixed DPPC/POPC Monolayers: All-atom Molecular Dynamics Simulations and Langmuir Monolayer Experiments

To elucidate the consequences of the saturated-unsaturated nature of lipid surface films, monolayers formed by an equimolar mixture of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipids are investigated in a wide range of surface pressures. As such mixtures share some features with naturally-occurring surfactants, for example the lung surfactant, the systems are studied at the temperature relevant for human body. All-atom molecular dynamics simulations and Langmuir trough experiments are employed. The binary lipid mixture is compared with the corresponding one-component systems. Atomistic-level alterations of monolayer molecular properties upon lateral compression are scrutinized. These involve elevation of lateral ordering of lipid chains, modulation of chain and headgroup orientation, and reduction of lipid hydration. The presence of the unsaturated POPC in the DPPC/POPC mixture reduces the liquid expanded-liquid condensed coexistence region and moderates the phase transition. Simulations predict that nanoscale lipid de-mixing occurs with small transient DPPC clusters emerging due to local fluctuations of the lateral lipid arrangement. A vertical sorting of lipids induced by lateral compression is also observed, with DPPC transferred toward the water phase. Both the conformational lipid alterations due to monolayer compression as well as the existence of lateral dynamic inhomogeneities of the lipid film are potentially pertain to dynamic and non-homogeneous lipid interfacial systems.

Lipid Spontaneous Curvatures Estimated from Temperature-Dependent Changes in Inverse Hexagonal Phase Lattice Parameters: Effects of Metal Cations.

Recently we reported a method for estimating the spontaneous curvatures of lipids from temperature-dependent changes in the lattice parameter of inverse hexagonal liquid crystal phases of binary lipid mixtures. This method makes use of 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) as a host lipid, which preferentially forms an inverse hexagonal phase to which a guest lipid of unknown spontaneous curvature is added. The lattice parameters of these binary lipid mixtures are determined by small-angle X-ray diffraction at a range of temperatures and the spontaneous curvature of the guest lipid is determined from these data. Here we report the use of this method on a wide range of lipids under different ionic conditions. We demonstrate that our method provides spontaneous curvature values for DOPE, cholesterol, and monoolein that are within the range of values reported in the literature. Anionic lipids 1,2-dioleoyl-sn-glycerol-3-phosphatidic acid (DOPA) and 1,2-dioleoyl-sn-glycerol-3-phosphoserine (DOPS) were found to exhibit spontaneous curvatures that depend on the concentration of divalent cations present in the mixtures. We show that the range of curvatures estimated experimentally for DOPA and DOPS can be explained by a series of equilibria arising from lipid-cation exchange reactions. Our data indicate a universal relationship between the spontaneous curvature of a lipid and the extent to which it affects the lattice parameter of the hexagonal phase of DOPE when it is part of a binary mixture. This universal relationship affords a rapid way of estimating the spontaneous curvatures of lipids that are expensive, only available in small amounts, or are of limited chemical stability.

Phase Separation on Bicontinuous Cubic Membranes: Symmetry Breaking, Reentrant, and Domain Faceting.

We study the phase separation of binary lipid mixtures that form bicontinuous cubic phases. The competition between the nonuniform Gaussian membrane curvature and line tension leads to a very rich phase diagram, where we observe symmetry breaking of the membrane morphologies and reentrant phenomena due to the formation of bridges between segregated domains. Upon increasing the line tension contribution, we also find faceting of lipid domains that we explain using a simple argument based on the symmetry of the underlying surface and topology.

Cholesterol-dependent thermotropic behavior and organization of neuronal membranes

The composition of neuronal membranes is unique with diverse lipid composition due to evolutionary requirement. The organization and dynamics of neuronal membranes are crucial for efficient functioning of neuronal receptors. We have previously established hippocampal membranes as a convenient natural source for exploring lipid-protein interactions, and organization of neuronal receptors. Keeping in mind the pathophysiological role of neuronal cholesterol, in this work, we used differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) to explore thermotropic phase behavior and organization (thickness) of hippocampal membranes under conditions of varying cholesterol content. Our results show that the apparent phase transition temperature of hippocampal membranes displays characteristic linear dependence on membrane cholesterol content. These results are in contrast to earlier results with binary lipid mixtures containing cholesterol where phase transition temperature was found to be not significantly dependent on cholesterol concentration. Interestingly, SAXS data showed that hippocampal membrane thickness remained more or less invariant, irrespective of cholesterol content. We believe that these results constitute one of the early reports on the thermotropic phase behavior and organizational characterization of hippocampal membranes under varying cholesterol content. These results could have implications in the functioning of neuronal receptors in healthy and diseased states.

Phase Diagrams and Ordering in Charged Membranes: Binary Mixtures of Charged and Neutral Lipids.

We propose a model describing the phase behavior of two-component membranes consisting of binary mixtures of electrically charged and neutral lipids. We take into account the structural phase transition (main-transition) of the hydrocarbon chains, and investigate the interplay between this phase transition and the lateral phase separation. The presence of charged lipids significantly affects the phase behavior of the multicomponent membrane. Due to the conservation of lipid molecular volume, the main-transition temperature of charged lipids is lower than that of neutral ones. Furthermore, as compared with binary mixtures of neutral lipids, the membrane phase separation in binary mixtures of charged lipids is suppressed, in accord with recent experiments. We distinguish between two types of charged membranes: mixtures of charged saturated lipid/neutral unsaturated lipid and a second case of mixtures of neutral saturated lipid/charged unsaturated lipid. The corresponding phase behavior is calculated and shown to be very different. Finally, we discuss the effect of added salt on the phase separation and the temperature dependence of the lipid molecular area.

Intermolecular Headgroup Interaction and Hydration as Driving Forces for Lipid Transmembrane Asymmetry.

Variations between the inner and outer leaflets of cell membranes are crucial for cell functioning and signaling, drug-membrane interactions, and the formation of lipid domains. Transmembrane asymmetry can in principle be comprised of an asymmetric charge distribution, differences in hydration, specific headgroup/H-bonding interactions, or a difference in the number of lipids per leaflet. Here, we characterize the transmembrane asymmetry of small unilamellar liposomes consisting of zwitterionic and charged lipids in aqueous solution using vibrational sum frequency scattering and second harmonic scattering, label-free methods, specifically sensitive to lipid and water asymmetries. For single component liposomes, transmembrane asymmetry is present for the charge distribution and lipid hydration, but the leaflets are not detectably asymmetric in terms of the number of lipids per leaflet, even though geometrical packing arguments would predict so. Such a lipid transmembrane asymmetry can, however, be induced in binary lipid mixtures under conditions that enable H-bonding interactions between phosphate and amine groups. In this case, the measured asymmetry consists of a different number of lipids in the outer and inner leaflet, a difference in transmembrane headgroup hydration, and a different headgroup orientation for the interacting phosphate groups.

Micelles and Bicelles as Membrane Mimics for Membrane Protein Characterization

Micelles and bicelles are used to isolate membrane proteins for biophysical characterization, functional assays, and high-resolution structure determination. Utilizing these membrane mimics requires an extensive screening process to identify the appropriate amphiphilic environment to stabilize a functional membrane protein. To enable rational mimic selection, the goal of this research is to investigate the structural properties of micelles and bicelles to determine how their physical characteristics can forward membrane protein research. Small angle X-ray scattering and small angle neutron scattering were used to investigate the structure of micelles and bicelles, specifically binary mixtures of detergents and lipids. To determine the impact of membrane mimic structure on protein function, outer membrane phospholipase A1, the protease OmpT, and the lipid A palmitoyltransferase PagP were purified in detergent micelles with varying properties, and the overall protein activity was evaluated. Hypotheses based on the correlations between activity and micelle properties were tested using mixed micelles and bicelles with tunable properties. The information gained from these studies will determine the physical properties important in stabilizing membrane proteins and provide guiding principles for selecting membrane mimics.

Dynamics and Statics in Phase Separating, Adhering Lipid Membranes

When living cells adhere to one another or their environment they organize their cellular membrane components into physical structures responsible for biological functions. One striking example is the immunological synapse formed at the adhesion site of immune cells which exhibits dual heterogeneities—characterized by differences in proteins and lipids—that are at first dynamic for tens of seconds to minutes and then stable for tens of minutes to an hour (Grakoui et al., Science 285, 221-227 (1999)).We employ a minimalist biophysical approach to investigate the dynamic formation and static persistence of lipid and protein heterogeneities at the adhesion site of lipid membranes. In our experiments we adhere mixed-lipid model membrane vesicles to a supported bilayer substrate via biotin-avidin binding. In one set of experiments, our vesicle membranes are made of a lipid mixture near an Lo-Ld phase boundary but are kept above the miscibility transition temperature as measured in free floating vesicles (S. L. Veatch, S. L. Keller, Biophysical journal 85, 3074-3083 (2003)). When the membranes adhere, they form two coexisting heterogeneities at the adhesion site that are compositionally distinct from the non-adhered portion of the membrane: (1) a central region that excludes a membrane dye—which marks the Ld phase—and is devoid of binders and (2) a peripheral region that is enriched in the membrane dye and is dense with binders. In a second set of experiments, our vesicle membranes are made of binary lipid mixtures (DOPC and Cholesterol) that are reported to be miscible at all accessible temperatures. Remarkably, when these membranes adhere, they form domains that exclude the membrane dye at the adhesion site and the domains grow on seconds-to-minutes-long time scales. We suggest the domain growth in this system is a nonequilibrium phase transition.

The Mechanism of HIV Entry Inhibition by 25-Hydroxycholesterol

Among the known biological activities of 25-hydroxycholesterol (25HC) are its potential immunological functions. A recent work demonstrated that 25HC blocks HIV-1 entry, preventing the release of the viral content into the host cell, which suggests an action at the membrane level.The aim of this work is to clarify 25HC antiviral mechanism, studying its effects on membrane properties and on the viral fusion process, using lipids and HIV-fusion peptide (HIV-FP). Different biophysical studies, with small unilamellar vesicles (SUVs) and lipid monolayers, were performed to understand the differences that occur at the membrane level when cholesterol is substituted by 25HC. The effect of binary lipid mixtures, with 25HC or cholesterol, on HIV-FP structure was evaluated by Fourier transform infrared spectroscopy (FTIR).HIV-FP-induced lipid mixing assays were performed to calculate fusion efficiency. When cholesterol is substituted by 25HC, membrane fusion efficiency is reduced by 50%, demonstrating that 25HC can directly block membrane. Fusion fluorescence anisotropy, dipole potential and surface pressure assays show that the conversion of cholesterol in 25HC leads to a loss of the cholesterol modulating effect on membrane properties. On the contrary, FTIR reveals a similar HIV-FP structure in the presence of both sterols.Overall, our data show that 25HC can directly block HIV entry through the inhibition of HIV-FP-induced membrane fusion. The mechanism supporting this activity is not based on effects at the level of HIV-FP structure, but in changes of membrane biophysical properties, associated with the broad-spectrum activity against different enveloped viruses.

Combined effect of synthetic protein, Mini-B, and cholesterol on a model lung surfactant mixture at the air–water interface

The overall goal of this work is to study the combined effects of Mini-B, a 34 residue synthetic analog of the lung surfactant protein SP-B, and cholesterol, a neutral lipid, on a model binary lipid mixture containing dipalmitolphosphatidylcholine (DPPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG), that is often used to mimic the primary phospholipid composition of lung surfactants. Using surface pressure vs. mean molecular area isotherms, fluorescence imaging and analysis of lipid domain size distributions; we report on changes in the structure, function and stability of the model lipid-protein films in the presence and absence of varying composition of cholesterol. Our results indicate that at low cholesterol concentrations, Mini-B can prevent cholesterol's tendency to lower the line tension between lipid domain boundaries, while maintaining Mini-B's ability to cause reversible collapse resulting in the formation of surface associated reservoirs. Our results also show that lowering the line tension between domains can adversely impact monolayer folding mechanisms. We propose that small amounts of cholesterol and synthetic protein Mini-B can together achieve the seemingly opposing requirements of efficient LS: fluid enough to flow at the air–water interface, while being rigid enough to oppose irreversible collapse at ultra-low surface tensions.

Structures of malonic acid diamide/phospholipid composites and their lipoplexes.

As a continuation of previous work, the self-assembly process of cationic lipid formulations in the presence and absence of DNA was investigated with respect to binary lipid mixtures suitable as polynucleotide carrier systems. The lipid blends consist of one malonic-acid-based cationic lipid with a varying alkyl chain pattern, either N-{6-amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl}-N'-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2-hexadecylpropandiamide () or N-[6-amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N'-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2-hexadecylpropandiamide (), and one neutral co-lipid, either 1,2-di-[(9Z)-octadec-9-enoyl]-sn-glycero-3-phosphocholine (DOPE) or 1,2-di-(hexadecanoyl)-sn-glycero-3-phosphocholine (DPPC). Although the cationic lipids exhibit only slight differences in their structure, the DNA transfer efficiency varies drastically. Therefore, self-assembly was studied in 3D systems by small- and wide-angle X-ray scattering (SAXS and WAXS) and transmission electron microscopy (TEM) as well as in 2D systems by infrared reflection-absorption spectroscopy (IRRAS) on Langmuir films. The investigated lipid mixtures show quite different self-assembly in the absence of DNA, with varying structures from vesicles (/DOPE; /DOPE) and tubes (/DOPE) to discoid structures (/DPPC; /DPPC). Twisted ribbons and sheets, which were stabilized due to hydrogen-bond networks, were found in all investigated lipid mixtures in the absence of DNA. The addition of DNA leads to the formation of lamellar lipoplexes for all the investigated lipid compositions. The lipoplexes differ in crucial parameters, such as the lamellar repeat distance and the spacing between the DNA strands, indicating differences in the binding strength between DNA and the lipid composition. The formation of associates with an ideal charge density might emerge as a key parameter for efficient DNA transfer. Furthermore, the structures observed for the different lipid compositions in the absence of DNA prepare the way for other applications besides gene therapy.

Coupling between pore formation and phase separation in charged lipid membranes.

We investigated the effect of charge on the membrane morphology of giant unilamellar vesicles (GUVs) composed of various mixtures containing charged lipids. We observed the membrane morphologies by fluorescent and confocal laser microscopy in lipid mixtures consisting of a neutral unsaturated lipid [dioleoylphosphatidylcholine (DOPC)], a neutral saturated lipid [dipalmitoylphosphatidylcholine (DPPC)], a charged unsaturated lipid [dioleoylphosphatidylglycerol (DOPG((-)))], a charged saturated lipid [dipalmitoylphosphatidylglycerol (DPPG((-)))], and cholesterol (Chol). In binary mixtures of neutral DOPC-DPPC and charged DOPC-DPPG((-))), spherical vesicles were formed. On the other hand, pore formation was often observed with GUVs consisting of DOPG((-))) and DPPC. In a DPPC-DPPG((-)))-Chol ternary mixture, pore-formed vesicles were also frequently observed. The percentage of pore-formed vesicles increased with the DPPG((-))) concentration. Moreover, when the head group charges of charged lipids were screened by the addition of salt, pore-formed vesicles were suppressed in both the binary and ternary charged lipid mixtures. We discuss the mechanisms of pore formation in charged lipid mixtures and the relationship between phase separation and the membrane morphology. Finally, we reproduce the results seen in experimental systems by using coarse-grained molecular dynamics simulations.