1-palmitoyl-2-oleoylphosphatidylcholine and 1-2-distearoyllecithin

Fluoxetine (FLX), used in the clinic to treat depression, is a well-known cationic amphiphilic antidepressant. To get a deeper insight into the effect of FLX on Langmuir monolayers, in this study the stability and relaxation of 1,2-dioctadecanoyl-sn-glycero-3-phophocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol (DSPC/POPC/CHOL) monolayers without and with FLX at different pH values were studied. The experiments involved surface pressure-area (π-A) measurements, mean molecular area-time (A-t) measurements, and atomic force microscope (AFM) analysis. It was found that intermolecular interactions decreased after the addition of FLX in the subphase but increased with increasing pH values. The relaxation of the ternary lipid monolayers with FLX was dominated by dissolution steps, and the dissolution rates decreased with increasing pH values. These findings can be easily confirmed by the analysis of thermodynamic parameters calculated for the investigated films. The data obtained in this study help to understand the effect of drugs on the ternary lipid monolayers from the molecular point of view.

Topics: Cholesterol; Fluoxetine; Hydrogen-Ion Concentration; Lipids; Microscopy, Atomic Force; Phosphatidylcholines; Thermodynamics

The plasma membrane (PM) is asymmetric in lipid composition. The distinct and characteristic lipid compositions of the exoplasmic and cytoplasmic leaflets lead to different lipid-lipid interactions and physical-chemical properties in each leaflet. The exoplasmic leaflet possesses an intrinsic ability to form coexisting ordered and disordered fluid domains, whereas the cytoplasmic leaflet seems to form a single fluid phase. To better understand the interleaflet interactions that influence domains, we compared asymmetric model membranes that capture salient properties of the PM with simpler symmetric membranes. Using asymmetric giant unilamellar vesicles (aGUVs) prepared by hemifusion with a supported lipid bilayer, we investigate the domain line tension that characterizes the behavior of coexisting ordered + disordered domains. The line tension can be related to the contact perimeter of the different phases. Compared to macroscopic phase separation, the appearance of modulated phases was found to be a robust indicator of a decrease in domain line tension. Symmetric GUVs of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)/cholesterol (chol) were formed into aGUVs by replacing the GUV outer leaflet with DOPC/chol = 0.8/0.2 in order to create a cytoplasmic leaflet model. These aGUVs revealed lower line tension for the ordered + disordered domains of the exoplasmic model leaflet.

Topics: Cholesterol; Phosphatidylcholines; Surface Tension; Unilamellar Liposomes

Carotenoids have been found to be important in improving the integrity of biomembranes in eukaryotes. However, the molecular details of how carotenoids modulate the physical properties of biomembranes are unknown. To this end, we have conducted a series of molecular dynamics simulations of different biologically-relevant membranes in the presence of carotenoids. The carotenoid effect on the membrane was found to be specific to the identity of the carotenoid and the composition of the membrane itself. Therefore, different classes of carotenoids produce a different effect on the membrane, and different membrane phases are affected differently by carotenoids. It is apparent from our data that carotenoids do trigger the bilayer to become thinner. The mechanism by which this occurs depends on two competing factors, the ability of the lipid tails of opposing monolayers to either (1) compress or (2) interdigitate as the bilayer condenses. Indeed, carotenoids directly influence the physical properties via these two mechanisms, thus compacting the bilayer. However, the degree to which these competing mechanisms are utilized depends on the bilayer phase and the carotenoid identity.

Topics: beta Carotene; Carotenoids; Cholesterol; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Zeaxanthins

In this work, we tested the hypothesis that the incorporation of amphiphilic drugs into lipid membranes may be regulated by their rheological properties. For this purpose, two members of the l-ascorbic acid alkyl esters family (ASCn) were selected, ASC16 and ASC14, which have different rheological properties when organized at the air/water interface. They are lipophilic forms of vitamin C used in topical pharmacological preparations. The effect of the phase state of the host lipid membranes on ASCn incorporation was explored using Langmuir monolayers. Films of pure lipids with known phase states have been selected, showing liquid-expanded, liquid-condensed, and solid phases as well as pure cholesterol films in liquid-ordered state. We also tested ternary and quaternary mixed films that mimic the properties of cholesterol containing membranes and of the stratum corneum. The compressibility and shear properties of those monolayers were assessed in order to define its phase character. We found that the length of the acyl chain of the ASCn compounds induces differential changes in the rheological properties of the host membrane and subtly regulates the kinetics and extent of the penetration process. The capacity for ASCn uptake was found to depend on the phase state of the host film. The increase in surface pressure resultant after amphiphile incorporation appears to be a function of the capacity of the host membrane to incorporate such amphiphile as well as the rheological response of the film. Hence, monolayers that show a solid phase state responded with a larger surface pressure increase to the incorporation of a comparable amount of amphiphile than liquid-expanded ones. The cholesterol-containing films, including the mixture that mimics stratum corneum, allowed a very scarce ASCn uptake independently of the membrane diffusional properties. This suggests an important contribution of Cho on the maintenance of the barrier function of stratum corneum.

Topics: Alkylation; Ascorbic Acid; Biological Transport; Biomimetic Materials; Cholesterol; Epidermis; Esters; Humans; Kinetics; Lipid Bilayers; Permeability; Phase Transition; Phosphatidylcholines; Pressure; Rheology; Surface Properties; Water

Spatial organization within lipid bilayers is an important feature for a range of biological processes. Leaflet compositional asymmetry and lateral lipid organization are just two of the ways in which membrane structure appears to be more complex than initially postulated by the fluid mosaic model. This raises the question of how the phase behavior in one bilayer leaflet may affect the apposing leaflet and how one begins to construct asymmetric model systems to investigate these interleaflet interactions. Here we report on all-atom molecular dynamics simulations (a total of 4.1 μs) of symmetric and asymmetric bilayer systems composed of liquid-ordered (Lo) or liquid-disordered (Ld) leaflets, based on the nanodomain-forming POPC/DSPC/cholesterol system. We begin by analyzing an asymmetric bilayer with leaflets derived from simulations of symmetric Lo and Ld bilayers. In this system, we observe that the properties of the Lo and Ld leaflets are similar to those of the Lo and Ld leaflets in corresponding symmetric systems. However, it is not obvious that mixing the equilibrium structures of their symmetric counterparts is the most appropriate way to construct asymmetric bilayers nor that these structures will manifest interleaflet couplings that lead to domain registry/antiregistry. We therefore constructed and simulated four additional asymmetric bilayer systems by systematically adding or removing lipids in the Ld leaflet to mimic potential density fluctuations. We find that the number of lipids in the Ld leaflet affects its own properties, as well as those of the apposing Lo leaflet. Collectively, the simulations reveal the presence of weak acyl chain interdigitation across bilayer leaflets, suggesting that interdigitation alone does not contribute significantly to the interleaflet coupling in nonphase-separated bilayers of this chemical composition. However, the properties of both leaflets appear to be sensitive to changes in in-plane lipid packing, possibly providing a mechanism for interleaflet coupling by modulating local density and/or curvature fluctuations.

Topics: Cholesterol; Diffusion; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Pressure; Surface Tension

In the present chapter we discuss the complex mixing behaviour of plasma membrane lipids. To do so, we first introduce the plasma membrane and membrane mixtures often used to model its complexity. We then discuss the nature of lipid phase behaviour in bilayers and the distinction between these phases and other manifestations of non-random mixing found in one-phase mixtures, such as clusters, micelles and microemulsions. Finally, we demonstrate the applicability of Gibbs phase diagrams to the study of increasingly complex model membrane systems, with a focus on phase coexistence, morphology and their implications for the cell plasma membrane.

Topics: Cholesterol; Emulsions; Kinetics; Lipid Bilayers; Membrane Microdomains; Membrane Proteins; Micelles; Models, Chemical; Monte Carlo Method; Phase Transition; Phosphatidylcholines; Thermodynamics

The study of the ability of drug molecules to enter cells through the membrane is of vital importance in the field of drug delivery. In cases where the transport of the drug molecules through the membrane is not easily accomplishable, other carrier molecules are used. Spherical fullerene molecules have been postulated as potential carriers of highly hydrophilic drugs across the plasma membrane. Here, we report the coarse-grain molecular dynamics study of the translocation of C60 fullerene and its derivatives across a cell membrane modeled as a 1,2-distearoyl-sn-glycero-3-phosphocholine bilayer. Simulation results indicate that pristine fullerene molecules enter the bilayer quickly and reside within it. The addition of polar functionalized groups makes the fullerenes less likely to reside within the bilayer but increases their residence time in bulk water. Addition of polar functional groups to one half of the fullerene surface, in effect creating a Janus particle, offers the most promise in developing fullerene models that can achieve complete translocation through the membrane bilayer.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cell Membrane; Fullerenes; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Temperature

Cholesterol is an important component of all biological membranes as well as drug delivery liposomes. We show here that increasing the level of cholesterol in a phospholipid membrane decreases surface charge in the physiological environment. Through molecular dynamics simulation we have shown that increasing the level of cholesterol decreases Na+ ion binding. Complementary experimental ζ--potential measurements have shown a decreased ζ--potential with increasing cholesterol content, indicative of reduced surface charge. Both experiments and simulations have been carried out on both saturated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. This result is particularly important because membrane surface charge plays an important role in the interactions of biomembranes with peripheral membrane proteins and drug delivery liposomes with the immune system.

Topics: Cell Membrane; Cholesterol; Drug Delivery Systems; Humans; Lipid Bilayers; Liposomes; Membrane Lipids; Molecular Dynamics Simulation; Phosphatidylcholines; Sodium Chloride

We report the first 4-component phase diagram for the lipid bilayer mixture, DSPC/DOPC/POPC/chol (distearoylphosphatidylcholine/dioleoylphosphatidylcholine/1-palmitoyl, 2-oleoylphosphatidylcholine/cholesterol). This phase diagram, which has macroscopic Ld+Lo phase domains, clearly shows that all phase boundaries determined for the 3-component mixture containing DOPC transition smoothly into the boundaries for the 3-component mixture containing POPC, which has nanoscopic phase domains of Ld+Lo. Our studies start from two published ternary phase diagrams, and show how these can be combined into a quaternary phase diagram by study of a few hundred samples of intermediate compositions.

Topics: Cholesterol; Fluorescence Resonance Energy Transfer; Phase Transition; Phosphatidylcholines

Imaging ellipsometry (IE) has been applied to generate laterally resolved thickness maps of spin-coated membranes in both the dry and fully hydrated state. Spin-coating offers a convenient preparation method for stacked supported membranes, and in-depth thickness maps for such films can be measured by IE, thereby going beyond topography measurements of the top surface. We find that dry lipid films of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) have a highly ordered multilamellar structure which allows counting of the number of individual bilayers in a thick film from the progression in a concentration series. The average film thickness is approximately proportional to the coating concentration with a constant of proportionality of 5.2 nm/mM and 6.2 nm/mM for POPC and DSPC (1,2-distearoyl-sn- glycero-3-phosphocholine), respectively. The root-mean-square roughness of the dry films is also approximately proportional to concentration with constants of 3.7 nm/mM (DSPC) and 0.87 nm/mM (POPC). Fully hydrated POPC membranes with several stacked bilayers show decreasing thickness for increasing temperature. An apparent excess in thickness by 1.2 nm for the proximal membrane can possibly be linked to the presence of a structured water film next to the solid support. This is supported by modeling of spectroscopic data. Thickness maps of double supported ternary membranes show resolvable liquid-ordered domains in the second membrane while domains are below the resolution limit in the proximal membrane. A thickness difference of 1.69 and 1.89 nm between the liquid-ordered (lo) and liquid-disordered (ld) phases is found for two different ternary membrane compositions. This is approximately twice the height difference measured by AFM on domains, thus indicating that the relative excess thickness of the lo phase is symmetrically distributed.

Topics: Chemistry Techniques, Analytical; Particle Size; Phosphatidylcholines; Surface Properties; Water

Though the aggregation of glycosaminoglycans (GAGs) in the presence of liposomes and divalent cations has been previously reported, the effects of different GAG species and minor changes in GAG composition on the aggregates that are formed are yet unknown. If minor changes in GAG composition produce observable changes in the liposome aggregate diameter or zeta potential, such a phenomenon may be used to detect potentially dangerous oversulfated contaminants in heparin. We studied the mechanism of the interactions between heparin and its oversulfated glycosaminoglycan contaminants with liposomes. Herein, we demonstrate that Mg(2+) acts to shield the incoming glycosaminoglycans from the negatively charged phosphate groups of the phospholipids and that changes in the aggregate diameter and zeta potential are a function of the glycosaminoglycan species and concentration as well as the liposome bilayer composition. These observations are supported by TEM studies. We have shown that the organizational states of the liposome bilayers are influenced by the presence of GAG and excess Mg(2+), resulting in a stabilizing effect that increases the T(m) value of DSPC liposomes; the magnitude of this effect is also dependent on the GAG species and concentration present. There is an inverse relationship between the percent change in aggregate diameter and the percent change in aggregate zeta potential as a function of GAG concentration in solution. Finally, we demonstrate that the diameter and zeta potential changes in POPC liposome aggregates in the presence of different oversulfated heparin contaminants at low concentrations allow for an accurate detection of oversulfated chondroitin sulfate at concentrations of as low as 1 mol %.

Topics: Calorimetry, Differential Scanning; Glycosaminoglycans; Heparin; Liposomes; Magnesium; Microscopy, Electron, Transmission; Phosphatidylcholines; Phospholipids

The purpose of this study was to elucidate the effect of trehalose distribution across the membrane on the freeze-related physical changes of liposome suspensions and their functional stability upon freeze-thawing. Cooling thermal analysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposome suspensions showed exotherm peaks of bulk (-15 °C to -25 °C) and intraliposomal (approx. -45 °C) solution freezing initiated by heterogeneous and homogeneous ice nucleation, respectively. The extent of the intraliposomal solution freezing exotherm depended on liposome size, lipid composition, cosolutes, and thermal history, suggesting that osmotic dehydration occurred due to the increasing difference in solute concentrations across the membrane. A freeze-thawing study of carboxyfluorescein-encapsulated liposomes suggested that controlling the osmotic properties to avoid the freeze-induced intraliposomal solution loss either by rapid cooling of suspensions containing trehalose in both sides of the membrane (retention of the intraliposomal supercooled solution) or by cooling of suspensions containing trehalose in the extraliposomal media prior to freezing (e.g., osmotic shrinkage) led to higher retention of the water-soluble marker. Evaluation and control of the osmotically mediated freezing behavior by optimizing the formulation and process factors should be relevant to the cryopreservation and freeze-drying of liposomes.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Calorimetry, Differential Scanning; Chemistry, Pharmaceutical; Cryopreservation; Cryoprotective Agents; Dimyristoylphosphatidylcholine; Fluoresceins; Freeze Drying; Freezing; Light; Liposomes; Osmosis; Particle Size; Phosphatidylcholines; Scattering, Radiation; Technology, Pharmaceutical; Trehalose

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Algorithms; Binding, Competitive; Cell Membrane; Chlorobenzenes; Cholesterol; Dimyristoylphosphatidylcholine; Gels; Kinetics; Lipid Bilayers; Microarray Analysis; Microscopy, Fluorescence; Molecular Structure; Phase Transition; Phosphatidylcholines; Pyrimidinones; Reproducibility of Results; Spectrometry, Fluorescence

We have found modulated phase morphology in a particular region of composition within the liquid-ordered + liquid-disordered coexistence region in the four-component lipid bilayer mixture DSPC/DOPC/POPC/Chol. By controlling lipid composition, we could see distinct types of modulated liquid-liquid phase morphologies, including linear, irregular, and angular features in giant unilamellar vesicles. We used a combination of confocal, two-photon, wide-field fluorescence, and differential interference contrast microscopies, and used stringent controls to minimize light-induced artifacts. These studies establish that both the size and morphology of membrane rafts can be controlled by the concentration and the type of low-melting lipid in mixtures with cholesterol and a high-melting lipid.

Topics: Cholesterol; Microscopy, Fluorescence; Nanoparticles; Phase Transition; Phosphatidylcholines; Temperature; Unilamellar Liposomes

Phase diagrams of ternary lipid mixtures containing cholesterol have provided valuable insight into cell membrane behaviors, especially by describing regions of coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. Fluorescence microscopy imaging of giant unilamellar vesicles has greatly assisted the determination of phase behavior in these systems. However, the requirement for optically resolved Ld + Lo domains can lead to the incorrect inference that in lipid-only mixtures, Ld + Lo domain coexistence generally shows macroscopic domains. Here we show this inference is incorrect for the low melting temperature phosphatidylcholines abundant in mammalian plasma membranes. By use of high compositional resolution Förster resonance energy transfer measurements, together with electron spin resonance data and spectral simulation, we find that ternary mixtures of DSPC and cholesterol together with either POPC or SOPC, do indeed have regions of Ld + Lo coexistence. However, phase domains are much smaller than the optical resolution limit, likely on the order of the Förster distance for energy transfer (R(0), ∼2-8 nm).

Topics: Cholesterol; Electron Spin Resonance Spectroscopy; Ergosterol; Fluorescence Resonance Energy Transfer; Lipid Bilayers; Membrane Microdomains; Phase Transition; Phosphatidylcholines; Porphobilinogen; Surface Properties

Zero mode waveguides (ZMWs), subwavelength optical nanostructures with dimensions ranging from 50 to 200 nm, have been used to study systems involving ligand-receptor interactions. We show that under proper conditions, lipid membranes will invaginate into the nanostructures, which confine optical excitation to subattoliter volumes. Fluorescence correlation spectroscopy (FCS) was used to characterize the diffusion of fluorescently tagged lipids in liquid-disordered phase 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and gel phase 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) membranes incubated on the nanostructured surface. In contrast to the POPC, DSPC membranes did not appear to enter the structures, suggesting that invagination is dependent on membrane rigidity. Although correlation curves obtained from POPC membranes conformed to previously derived models for diffusion in the evanescent field within the nanostructure, the diffusion constants obtained were systematically lower than expected. The validity of the one-dimensional diffusion model for membrane diffusion is discussed and it is concluded that the erroneous diffusion constants are a result of nontrivial membrane conformation within the ZMWs. Additionally, FCS was used to characterize the fraction of fluorescently labeled tetanus toxin C fragment bound to a ganglioside-populated POPC membrane within the ZMWs. This allowed the determination of the toxin's equilibrium binding constant at a concentration of 500 nM; higher than possible with diffraction-limited FCS. To our knowledge, the results presented here are the first reported for supported lipid bilayers in nanostructured devices. Furthermore, they open the possibility of studying membrane imbedded receptors and proteins at physiological concentrations with single-molecule resolution.

Topics: Gangliosides; Lipid Bilayers; Models, Biological; Nanostructures; Phosphatidylcholines; Spectrometry, Fluorescence; Tetanus Toxin

Recent work on surfactant protein A (SP-A) has shown that Ca(2+) induces an active conformation, SP-A, which binds rapidly to liposomes and mediates their aggregation. Employing sensitive real time assays, we have now studied the lipid binding characteristics of the SP-A liposome interaction. From the final equilibrium level of the resonant mirror binding signal, an apparent dissociation constant of ca. K(d)=5 microM is obtained for the complex between SP-A and dipalmitoylphosphatidylcholine (DPPC) liposomes. At nanomolar SP-A concentrations, this complex is formed with a subsecond (0.3 s) reaction time, as measured by light-scattering signals evoked by photolysis of caged Ca(2+). With palmitoyloleoylphosphatidylcholine (POPC), the complex formation proceeds at half the rate, compared to DPPC, leading to a lower final equilibrium level of SP-A lipid interaction. Distearoylphosphatidylcholine (DSPC) shows a stronger interaction than DPPC. Regarding the phospholipid headgroups, phosphatidylinositol (PI) and sphingomyelin (SM) interact comparable to DPPC, while less interaction is seen with phosphatidylethanolamine (PE) or with phosphatidylglycerol (PG). Thus both headgroup and fatty acid composition determine SP-A phospholipid interaction. However, the protein does not exhibit high specificity for either the polar or the apolar moiety of phospholipids.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Kinetics; Liposomes; Phosphatidylcholines; Phospholipids; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants