Mixed Lipid Phase Research Articles

Protein-driven lipid domain nucleation in biological membranes.

Lipid rafts are heterogeneous dynamic lipid domains of the cell membranes that are involved in several biological processes, such as protein and lipid specific transport and signaling. Our understanding of lipid raft formation is still limited due to the transient and elusive nature of these domains in vivo, in contrast with the stable phase-separated domains observed in artificial membranes. Inspired by experimental findings highlighting the relevance of transmembrane proteins for lipid rafts, we investigate lipid domain nucleation by coarse-grained molecular dynamics and Ising-model simulations. We find that the presence of a transmembrane protein can trigger lipid domain nucleation in a flat membrane from an otherwise mixed lipid phase. Furthermore, we study the role of the lipid domain in the diffusion of the protein showing that its mobility is hindered by the presence of the raft. The results of our coarse-grained molecular-dynamics and Ising-model simulations thus coherently support the important role played by transmembrane proteins in lipid domain formation and stability.

A Self-Microemulsifying Drug Delivery System to Overcome Intestinal Resveratrol Toxicity and Presystemic Metabolism

A mixed lipid-mixed surfactant self-microemulsifying drug delivery system (SMEDDS) was developed to exploit the health benefits of resveratrol, a Biopharmaceutical Classification System Class 2 natural polyphenol, subject to extensive intestinal presystemic metabolism. SMEDDS with a mixed lipid phase (castor oil/Capmul MCM 1:1) and a mixed surfactant phase (Kolliphor EL/Kolliphor RH 40 1:1) was developed and evaluated for its self-emulsifying properties and in vitro dispersion. The impact of SMEDDS on the permeability properties of resveratrol and its metabolite fluxes through the rat intestine and Caco-2 cells was monitored. The inhibitory effect of selected SMEDDS components on the efflux transporters multidrug resistance-associated protein and P-gp as well as cytotoxicity was assessed on Caco-2 cells. The formulation allowed for high resveratrol loading (122.5 mg/g SMEDDS), excellent self-emulsifying properties, and very rapid release. When formulated in SMEDDS, resveratrol metabolite efflux significantly declined. The formulation (SMEDDS without incorporated resveratrol) and its individual components did not compromise in vitro cell vitality and integrity. Mixed lipid-mixed surfactant SMEDDS is a prospective formulation to improve resveratrol biopharmaceutical, pharmacokinetic, and toxicological properties, leading the way to resveratrol use not only as a supplement but also as a pharmacological drug.

Mixed lipid phase SMEDDS as an innovative approach to enhance resveratrol solubility

Context: Despite its promising therapeutic activities, clinical use of resveratrol (RSV) is compromised with unfavorable biopharmaceutical properties, namely low water solubility.Objective: This work deals with improving RSV solubility and release rate through its incorporation in innovative mixed lipid phase self-microemulsifying drug delivery systems (SMEDDS).Methods: (Pseudo)ternary diagrams were constructed for different oils and surfactant mixtures. Selected systems were further evaluated for RSV solubility, self-emulsification ability, accelerated stability, dynamic viscosity, compatibility with hard gelatin capsules and in vitro dissolution of RSV.Results: Lipid phase composed of diverse lipid species, castor oil (long-chained triglyceride) and Capmul MCM (mixture of medium chain mono and diglycerides) allowed formulation of mixed lipid SMEDDS with lower surfactants content (60% Cremophor EL/RH 40/RH 60). Mixed lipid phase SMEDDS showed best self-emulsifying ability with regard to self-emulsifying time as well as droplet size and monodispersity of microemulsions obtained upon SMEDDS dilution with aqueous phase. Overall, incorporation of RSV in SMEDDS resulted in improved solubility (over 23-fold) and dissolution rate compared to crystalline RSV. All SMEDDS formulations were adequately viscous for filling into hard gelatin capsules (>150 mPaċs for empty SMEDDS; >400 mPaċs for RSV-loaded SMEDDS) and no leaking was observed during three months of storage.Conclusion: The presented work indicates the promising potential of mixed lipid SMEDDS formulations for future development of SMEDDS with lower surfactant content and no added cosolvents for incorporation of RSV and other poorly soluble drugs.

Miscibility of lactylated monoacylesters of propylene glycol with charged bilayer-forming colipids in aqueous solution

Lactylated monoacylesters of propylene glycol (LMP) are nonionic synthetic surfactants that form crystalline phases, when dispersed in aqueous solution. In this study the possibilities of controlled generation of lipid mesophases from LMP and two synthetic ionic colipids, dihexadecyldimethylammonium bromide (DHDAB) and sodium dihexadecylphosphate (DHP), were investigated applying differential scanning calorimetry (DSC) and small-angle X-ray diffraction (SAXD). We found that the bilayer forming, positively charged DHDAB is capable of forming mixed lipid phases with LMP. However, DSC data also revealed the formation of DHDAB-rich and LMP-rich domains. In mixtures of LMP with the negatively charged colipid, DHP, the DSC scans suggested the immiscibility of lipids at all mixing ratios. Since the hydrocarbon chain length is the same for both colipids, the observed differences in the thermotropic phase behaviour and the mixing behaviour were ascribed to the different steric features of the headgroups. The formation of unilamellar vesicles in the LMP/DHDAB mixture (60 : 40 w/w) was evidenced by the SAXD data. The ability to form mixed lipid phases and to self-assemble into vesicles upon hydration with an appropriate amount of colipid makes this non-phospholipid (LMP) a potential candidate for a phospholipid substitute in practical applications.

Interaction between Artificial Membranes and Enflurane, a General Volatile Anesthetic: DPPC-Enflurane Interaction

The structural modifications of the dipalmitoylphosphatidylcholine (DPPC) organization induced by increasing concentration of the volatile anesthetic enflurane have been studied by differential scanning calorimetry, small-angle, and wide-angle x-ray scattering. The interaction of enflurane with DPPC depends on at least two factors: the enflurane-to-lipid concentration ratio and the initial organization of the lipids. At 25 degrees C (gel state), the penetration of enflurane within the lipids induces the apparition of two different mixed lipid phases. At low anesthetic-to-lipid molar ratio, the smectic distance increases whereas the direction of the chain tilt changes from a tilt toward next-neighbors to a tilt between next-neighbors creating a new gel phase called L(beta')(2NNN). At high ratio, the smectic distance is much smaller than for the pure L(beta') DPPC phase, i.e., 50 A compared to 65 A, the aliphatic chains are perpendicular to the membrane and the fusion temperature of the phase is 33 degrees C. The electron profile of this phase that has been called L(beta)(i), indicates that the lipids are fully interdigitated. At 45 degrees C (fluid state), a new melted phase, called L(alpha)(2), was found, in which the smectic distance decreased compared to the initial pure L(alpha)(1) DPPC phase. The thermotropic behavior of the mixed phases has also been characterized by simultaneous x-ray scattering and differential scanning calorimetry measurements using the Microcalix calorimeter of our own. Finally, titration curves of enflurane effect in the mixed lipidic phase has been obtained by using the fluorescent lipid probe Laurdan. Measurements as a function of temperature or at constant temperature, i.e., 25 degrees C and 45 degrees C give, for the maximal effect, an enflurane-to-lipid ratio (M/M), within the membrane, of 1 and 2 for the L(alpha)(2) and the L(beta)(i) lamellar phase respectively. All the results taken together allowed to draw a pseudo-binary phase diagram of enflurane-dipalmitoylphosphatidylcholine in excess water.

Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes.

We report on the successful application of fluorescence correlation spectroscopy (FCS) to the analysis of single fluorescently labeled lipid analogue molecules diffusing laterally in lipid bilayers, as exemplified by time traces of fluorescence bursts of individual molecules entering and leaving the excitation area. FCS measurements performed on lipid probes in rat basophilic leukemia cell membranes showed deviations from two-dimensional Brownian motion with a single uniform diffusion constant. Giant unilamellar vesicles were employed as model systems to characterize diffusion of fluorescent lipid analogues in both homogeneous and mixed lipid phases with diffusion heterogeneity. Comparing the results of cell membrane diffusion with the findings on the model systems suggests possible explanations for the observations: (a) anomalous subdiffusion in which evanescent attractive interactions with disparate mobile molecules modifies the diffusion statistics; (b) alternatively, probe molecules are localized in microdomains of submicroscopic size, possibly in heterogeneous membrane phases.

Molecular sorting of lipids by bacteriorhodopsin in dilauroylphosphatidylcholine/distearoylphosphatidylcholine lipid bilayers

A combined experimental and theoretical study is performed on binary dilauroylphosphatidylcholine/distearoylphosphatidylcholine (DLPC/DSPC) lipid bilayer membranes incorporating bacteriorhodopsin (BR). The system is designed to investigate the possibility that BR, via a hydrophobic matching principle related to the difference in lipid bilayer hydrophobic thickness and protein hydrophobic length, can perform molecular sorting of the lipids at the lipid-protein interface, leading to lipid specificity/selectivity that is controlled solely by physical factors. The study takes advantage of the strongly nonideal mixing behavior of the DLPC/DSPC mixture and the fact that the average lipid acyl-chain length is strongly dependent on temperature, particularly in the main phase transition region. The experiments are based on fluorescence energy transfer techniques using specifically designed lipid analogs that can probe the lipid-protein interface. The theoretical calculations exploit a microscopic molecular interaction model that embodies the hydrophobic matching as a key parameter. At low temperatures, in the gel-gel coexistence region, experimental and theoretical data consistently indicate that BR is associated with the short-chain lipid DLPC. At moderate temperatures, in the fluid-gel coexistence region, BR remains in the fluid phase, which is mainly composed of short-chain lipid DLPC, but is enriched at the interface between the fluid and gel domains. At high temperatures, in the fluid phase, BR stays in the mixed lipid phase, and the theoretical data suggest a preference of the protein for the long-chain DSPC molecules at the expense of the short-chain DLPC molecules. The combined results of the experiments and the calculations provide evidence that a molecular sorting principle is active because of hydrophobic matching and that BR exhibits physical lipid selectivity. The results are discussed in the general context of membrane organization and compartmentalization and in terms of nanometer-scale lipid-domain formation.

Effects of random copolymers of lysine on the thermotropic behaviour of dipalmitoylphosphatidylglycerol vesicles. A fluorescence anisotropy study.

The effects of some random copolymers of lysine on the thermotropic behaviour of phospholipid vesicles, mainly dipalmitoylphosphatidylglycerol (DPPG), were studied by monitoring the steady-state fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe. A characteristic effect of the polylysine-tyrosine 4:1 copolymer was observed: DPPG vesicles are strongly stabilized, the gel-to-fluid transition going from 40 degrees C in the absence to 52 degrees C with an excess of copolymer (corresponding to a lipid/amino-acid ratio R = 0.5). For R greater than 0.5, the gel-to-fluid transition shows a three-step profile. This triphasic transition, which appears with a much better resolution than with polylysine, demonstrates the formation of stable distinct domains of reasonable size, coexisting from 41 to 51 degrees C. Such stability, over a temperature range of 10 degrees C, is quite unusual and unexpected for a phase separation of mixed lipid phases. The peculiarity of this copolymer was proved by investigating the interactions of DPPG vesicles with polylysine-tryptophan 4:1, polyornithine-lysine 4:1 and polylysine-tyrosine 1:1. We hypothesize that the observed effects are correlated with the ability of these copolymers to adopt/not adopt an alpha-helical or beta-sheet conformation upon interacting with anionic vesicles.

A quantitative dynamic concept of the interphase partition of lipids: Application to bile salt—lecithin—cholesterol mixed micelles

A system is proposed for a quantitative classification of lipids, based on interphase partition coefficients. This system enables calculation of exchanges of lipid molecules between phases. The mass/volume chemical unit mol X cm-3, strictly derived from the CGS system, is used, thus simplifying mathematical relations. Applied to bile salt-lecithin-cholesterol mixed micelles, this dynamic concept gives new insight into the variations of physico-chemical parameters. Experimental results obtained with the glycodesoxycholate and the taurocholate show a striking difference in partition coefficients between aqueous and mixed bile salt-lecithin interfacial phases. A new model applying triangular co-ordinates to a bile salt-lecithin-cholesterol mixed lipid phase is described.

Thermotropic behavior of binary mixtures of diplamitoylphosphatidylcholine and glycosphingolipids in aqueous dispersions

The thermotropic behavior of mixtures of dipalmitoylphosphatidylcholine (DPPC) with natural glycosphingolipids (galactosylceramide, phrenosine, kerasine, glucosylceramide, lactosylceramide, asialo-G M1, sulfatide, G M3, G M1, G D1a, G T1b) in dilute aqueous dispersions were studied by high sensitivity differential scanning calorimetry over the entire composition range. The pretransition of DPPC is abolished and the cooperativity of the main transition decreases sharply at mole fractions of glycosphingolipids below 0.2. All systems exhibit non-ideal temperature-composition phase diagrams. The mono- and di-hexosylceramides are easily miscible with DPPC when the proportion of glycosphingolipids in the system is high. A limited quantity (1–6 molecules of DPPC per molecule of glycosphingolipid (GSL) can be incorporated into a homogeneously mixed lipid phase. Domains of DPPC, immiscible with the rest of a mixed GSL-DPPC phase that shows no cooperative phase transition, are established as DPPC exceeds a certain proportion in the system. One negative charge (sulfatide) or four neutral carbohydrate residues (asialo-G M1) in the oligosaccharide chain of the glycosphingolipids results in phase diagrams exhibiting coexistence of gel and liquid phases over a broad temperature-composition range. Systems containing gangliosides show complex phase diagrams, with more than one phase transition. However, no evidence for phase-separated domains of pure ganglioside species is found. The thermotropic behavior of systems containing DPPC and glycosphingolipids correlates well with their interactions in mixed monolayers at the air/water interface.

Analysis of the Composition of Mixed Lipid Phases by the Moments of 2H NMR Spectra

The use of (2)H NMR spectral moments to determine the composition of biphasic lipid mixtures is outlined. The analysis has been applied to phosphatidylethanolamine-cholesterol (1:1), potassium palmitate, 30% (wt/wt) water and phosphatidylcholine-cholesterol (4:1) systems, as well as to membrances of Escherichia coli during phase transitions. The advantages and disadvantages of the use of spectral moments to determine fractions of coexistent phases are discussed.

Quantitative studies on lysolecithin mediated hemolysis. Benzylated lysolecithin as a probe to study effects of temperature and red cell species on the hemolytic reaction

The slow reacting hemolytic lysolecithin analog l-octadecyl-2-benzyl-glycero-3-phosphorylcholine has been employed for a detailed study of the process of lysolecithin induced hemolysis. Using a radiolabelled analog we found that the different sensitivities of red cells from different species (chicken, man, cattle) are not paralleled by the binding affinities of lysolecithin. Moreover, lysophosphatide binding to the cells is reduced at low temperatures while its hemolytic activity increases. In contrast to continuous changes of lytic activity and binding between 0 and 37°C, the velocity of the hemolytic reaction with human erythrocytes is extraordinarily fast at 10°C. Experiments in sucrose containing buffer indicated principally different lysis mechanisms below and above 15°C. We have further shown that cells loaded sublytically with the lysolecithin at 37°C undergo spontaneous lysis upon cooling to 0°C. The degree of lysis under these conditions, however, is diminished with increasing amounts of cell-bound lysolipid. Determinations of membrane microviscosities by means of fluorescence polarization revealed some qualitative relations between membrane fluidity and sensitivity to lysolecithin. The data are discussed on the basis of recent reports indicating that lysolecithin-distribution in mixed lipid phases may be heterogeneous depending on lipid composition and temperature.

Randomization of membrane lipids in relation to transport system assembly in Escherichia coli.

The distribution of newly synthesized lipid molecules in the pre-existing lipid phase of the membrane was studied in whole cells of the fatty acid requiring Escheria coli strain K1062. The fluorescence probe N-phenyl-1-naphthylamine revealed reversible lipid phase transitions in cells supplemented with cis-delta9-octadecenoate (transition temperature Tt = 14 degrees C; width of the transition deltaT = 13 degrees C) or trans-delta9-hexadecenoate (Tt = 27 degrees C; deltaT = 7 degrees C). Cells were first grown in the presence of cis-delta9-octadecenoate at 37 degrees C and subsequently for various periods in the presence of trans-delta9-hexadecenoate at 37 or 22 degrees C, i.e. above or below the transition of the newly formed lipids. Reproducible phase transitions with single, well-defined Tt values between 14 and 27 degrees C were observed under both conditions. Beta-Galactoside transport induced in a similar experiment before or during a change in the fatty acid composition showed a single change in activation energy at a temperature close to the lipid transition temperature, Tt. Starvation of cis-delta9-octadecenoate-supplemented cells for this fatty acid led to a gradual rise in the transition temperature, due to an increase in the percentage of saturated acyl chains in the membrane lipids. It is concluded that under all conditions investigated a mixed lipid phase composed of newly synthesized and pre-existing lipid molecules is formed in the membrane. Since conserved domains of newly synthesized lipids surrounding simultaneously formed transport proteins could not be demonstrated, the results do not support a membrane assembly mechanism proposed by N. Tsukagoshi and C. F. Fox [(1973), Biochemistry 12, 2822-2829]. It rather appears that newly formed lipid molecules are continuously released from their sites of synthesis into the lipid matrix by a rapid diffusion-controlled process.