colfosceril-palmitate and 1-2-oleoylphosphatidylcholine

Growth of plastic waste in the natural environment, and in particular in the oceans, has raised the accumulation of polystyrene and other polymeric species in eukyarotic cells to the level of a credible and systemic threat. Oligomers, the smallest products of polymer degradation or incomplete polymerization reactions, are the first species to leach out of macroscopic or nanoscopic plastic materials. However, the fundamental mechanisms of interaction between oligomers and polymers with the different cell components are yet to be elucidated. Simulations performed on lipid bilayers showed changes in membrane mechanical properties induced by polystyrene, but experimental results performed on cell membranes or on cell membrane models are still missing. We focus here on understanding how embedded styrene oligomers affect the phase behavior of model membranes using a combination of scattering, fluorescence, and calorimetric techniques. Our results show that styrene oligomers disrupt the phase behavior of lipid membranes, modifying the thermodynamics of the transition through a spatial modulation of lipid composition.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Deuterium; Humans; Kinetics; Lipid Bilayers; Liposomes; Phase Transition; Phosphatidylcholines; Polystyrenes; Seawater; Temperature; Thermodynamics; Water Pollution

We have studied the impact of cholesterol and/or melatonin on the static and dynamical properties of bilayers made of DPPC or DOPC utilizing neutron scattering techniques, Raman spectroscopy and molecular dynamics simulations. While differing in the amplitude of the effect due to cholesterol or melatonin when comparing their interactions with the two lipids, their addition ensued recognizable changes to both types of bilayers. As expected, based on the two-component systems of lipid/cholesterol or lipid/melatonin studied previously, we show the impact of cholesterol and melatonin being opposite and competitive in the case of three-component systems of lipid/cholesterol/melatonin. The effect of cholesterol appears to prevail over that of melatonin in the case of structural properties of DPPC-based bilayers, which can be explained by its interactions targeting primarily the saturated lipid chains. The dynamics of hydrocarbon chains represented by the ratio of trans/gauche conformers reveals the competitive effect of cholesterol and melatonin being somewhat more balanced. The additive yet opposing effects of cholesterol and melatonin have been observed also in the case of structural properties of DOPC-based bilayers. We report that cholesterol induced an increase in bilayer thickness, while melatonin induced a decrease in bilayer thickness in the three-component systems of DOPC/cholesterol/melatonin. Commensurately, by evaluating the projected area of DOPC, we demonstrate a lipid area decrease with an increasing concentration of cholesterol, and a lipid area increase with an increasing concentration of melatonin. The demonstrated condensing effect of cholesterol and the fluidizing effect of melatonin appear in an additive manner upon their mutual presence.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Melatonin; Molecular Dynamics Simulation; Neutron Diffraction; Phosphatidylcholines; Scattering, Small Angle

Cholesterol is the most abundant molecule in the plasma membrane of mammals. Its distribution across the two membrane leaflets is critical for understanding how cells work. Cholesterol trans-bilayer motion (flip-flop) is a key process influencing its distribution in membranes. Despite extensive investigations, the rate of cholesterol flip-flop and its dependence on the lateral heterogeneity of membranes remain uncertain. In this work, we used atomistic molecular dynamics simulations to sample spontaneous cholesterol flip-flop events in a DPPC:DOPC:cholesterol mixture with heterogeneous lateral distribution of lipids. In addition to an overall flip-flop rate at the time scale of sub-milliseconds, we identified a significant impact of local environment on flip-flop rate. We discuss the atomistic details of the flip-flop events observed in our simulations.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Kinetics; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Thermodynamics

Despite the central role of lipids in many biophysical functions, the molecular mechanisms that dictate macroscopic lipid behavior remain elusive to both experimental and computational approaches. As such, there has been much interest in the development of low-resolution, implicit-solvent coarse-grained (CG) models to dynamically simulate biologically relevant spatiotemporal scales with molecular fidelity. However, in the absence of solvent, a key challenge for CG models is to faithfully emulate solvent-mediated forces, which include both hydrophilic and hydrophobic interactions that drive lipid aggregation and self-assembly. In this work, we provide a new methodological framework to incorporate semiexplicit solvent effects through the use of virtual CG particles, which represent structural features of the solvent-lipid interface. To do so, we leverage two systematic coarse-graining approaches, multiscale coarse-graining (MS-CG) and relative entropy minimization (REM), in a hybrid fashion to construct our virtual-site CG (VCG) models. As a proof-of-concept, we focus our efforts on two lipid species, 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), which adopt a liquid-disordered and gel phase, respectively, at room temperature. Through our analysis, we also present, to our knowledge, the first direct comparison between the MS-CG and REM methods for a complex biomolecule and highlight each of their strengths and weaknesses. We further demonstrate that VCG models recapitulate the rich biophysics of lipids, which enable self-assembly, morphological diversity, and multiple phases. Our findings suggest that the VCG framework is a powerful approach for investigation into macromolecular biophysics.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Entropy; Lipid Bilayers; Molecular Dynamics Simulation; Phase Transition; Phosphatidylcholines; Solvents; Temperature

Preparation of lipid-based drug delivery systems by microfluidics has been increasingly popular, due to the reproducible, continuous and scalable nature of the microfluidic process. Despite exciting development in the field, versatility and superiority of microfluidics over conventional methods still need further evidence, since preparing clinically-relevant sterically stabilised liposomes has been lacking. The present study describes the optimisation of PEGylated liposomal formulations of various rigidity using staggered herringbone micromixer (SHM). The effect of both processing parameters (total flow rate (TFR) and aqueous-to-ethanol flow rate ratio (FRR)) and formulation parameters (lipid components and composition, initial lipid concentration and aqueous media) was investigated and discussed. Liposomal formulations consist of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), with cholesterol and PEGylated lipid (DSPE-PEG

Topics: 1,2-Dipalmitoylphosphatidylcholine; Doxorubicin; Microfluidics; Particle Size; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols

Non-fluidic bio-layer interferometry (BLI) has rapidly become a standard tool for monitoring almost all biomolecular interactions in a label-free, real-time and high-throughput manner. High-efficiency screening methods which measure the kinetics of liposomes with a variety of compounds require the immobilization of liposomes. In this work, a method is described for immobilizing liposomes for interaction studies, based on the biophysical principles of this biosensor platform. The immobilization approach includes the loading of DSPE-PEG

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adsorption; Biosensing Techniques; Biotin; Cardiolipins; Cytochromes c; Drosophila Proteins; Fluoresceins; Fluorescent Dyes; High-Throughput Screening Assays; Hydrophobic and Hydrophilic Interactions; Interferometry; Kinetics; Liposomes; Micelles; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Polyethylene Glycols; Protein Phosphatase 1; Reproducibility of Results

Long-range ordering of DNA crossover tiles with blunt ends on lipid bilayers is investigated using atomic force microscopy. "Blunt-ended" tiles do not have single-stranded complementary ends, and thus instead of assembling via base-pairing, they can interact by π-stacking of their duplex ends. This work demonstrates that the balance of base π-stacking interactions between the ends of DNA duplexes, cholesterol-mediated DNA anchoring, and electrostatic DNA binding to supported lipid bilayers (SLBs) presents an opportunity to build dynamic materials with long-range order on a soft support. The tiles are shown to organize into novel tunable surface packing morphologies on the micrometer scale. This work focuses on three-point star (3PS) tiles that are either unmodified or modified with a cholesterol unit and investigates their interactions on supported lipid bilayers. On fluid bilayers, the cholesterol tiles form extended hexagonal arrays with few defects, while the unmodified tiles do not bind. In contrast, both modified and unmodified tiles bind to gel-phase bilayers and produce arrays of new organized morphologies. With increasing tile concentration, we observe a range of motifs, that progressively favor tile-tile packing over duplex-end π-stacking. These structures can selectively pattern domains of phase-separated lipid bilayers, and the patterning is also observed for four-arm cross-tiles. Dynamic blunt end contacts promote error correction and network reconfiguration to maximize favorable interactions with the substrate and are required for the observed tile organization. These results suggest that small blunt-ended tiles can be used as a platform to organize oligonucleotides, nanoparticles, and proteins into extensive networks at the interface with biologically relevant membrane systems or other soft surface materials for applications in cellular recognition, plasmonics, light harvesting, model systems for membrane protein assemblies, or analytical devices.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; DNA; Lipid Bilayers; Microscopy, Atomic Force; Nanostructures; Nanotechnology; Nucleic Acid Conformation; Phosphatidylcholines; Static Electricity

The purpose of this study is to obtain a comprehensive relationship between membrane microdomain structures of liposomes and their cellular uptake efficiency. Model liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol (Ch) were prepared with various lipid compositions. To detect distinct membrane microdomains in the liposomes, fluorescence-quenching assays were performed at temperatures ranging from 25 to 60 °C using 1,6-diphenyl-1,3,5-hexatriene-labeled liposomes and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl. From the data analysis using the response surface method, we gained a better understanding of the conditions for forming distinct domains (Lo, Ld, and gel phase membranes) as a function of lipid composition. We further performed self-organizing maps (SOM) clustering to simplify the complicated behavior of the domain formation to obtain its essence. As a result, DPPC/DOPC/Ch liposomes in any lipid composition were integrated into five distinct clusters in terms of similarity of the domain structure. In addition, the findings from synchrotron small-angle X-ray scattering analysis offered further insight into the domain structures. As a last phase of this study, an in vitro cellular uptake study using HeLa cells was conducted using SOM clusters' liposomes with/without PEGylation. As a consequence of this study, higher cellular uptake was observed from liposomes having Ch-rich ordered domains.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; HeLa Cells; Humans; Lipid Bilayers; Liposomes; Membrane Microdomains; Phosphatidylcholines; Temperature

Auxins are successfully used to improve phytoextraction efficiency of metal ions from the contaminated environment, however, the mechanism of their activity in this field is not explained. Auxins are known to exert various biochemical alterations in the plant membranes and cells, but their activity involves also direct interactions with lipids leading to changes in membrane organization. Following the suggestion that the auxins-induced modifications in membrane properties alleviate toxic effect of metal ions in this paper we have undertaken the comparative studies on the effect of metal ions and metal ions/auxins mixtures on model membrane systems. The experiments were done on lipid monolayers differing in their composition spread on water subphase and on Pb(2+), Indole-3-acetic acid (IAA), 1-Naphthaleneacetic acid (NAA) and Pb(2+)/IAA and Pb(2+)/NAA water solutions. The analysis of the collected data suggests that metal ions and auxins can change fluidity of the lipid systems and weaken the interactions between monolayer components. This manifested in the increase of the mean area per molecule and the excess area per molecule values for the films on Pb(2+), auxins as well as Pb(2+)/auxin solutions as compared to the values on pure water subphase. However, the presence of auxin in the mixture with lead(II) ions makes the alterations induced by sole metal ions weaker. This effect was more pronounced for the membranes of a higher packing. Thus it was proposed that auxins may enhance phytoextraction of metal ions by weakening their destabilizing effect on membrane.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Brassica; Cations, Divalent; Cell Membrane; Indoleacetic Acids; Lead; Liquid Phase Microextraction; Naphthaleneacetic Acids; Phosphatidylcholines; Phosphatidylglycerols; Plant Cells; Sitosterols; Soil Pollutants; Unilamellar Liposomes

Phospholipase A2 (PLA2) is a peripheral membrane protein that can hydrolyze phospholipids to produce lysolipids and fatty acids. It has been found to play crucial roles in various cellular processes and is thought as a potential candidate for triggering drug release from liposomes for medical treatment. Here, we directly observed that PLA2 hydrolysis reaction can induce the formation of PLA2-binding domains at lipid bilayer interface and found that the formation was significantly influenced by the fluidity of the lipid bilayer. We prepared supported lipid bilayers (SLBs) with various molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to adjust the reactivity and fluidity of the lipid bilayers. A significant amount of the PLA2-induced domains was observed in mixtures of DPPC and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) but not in either pure DPPC or pure DOPC bilayer, which might be the reason that previous studies rarely observed these domains in lipid bilayer systems. The fluorescently labeled PLA2 experiment showed that newly formed domains acted as binding templates for PLA2. The AFM result showed that the induced domain has stepwise plateau structure, suggesting that PLA2 hydrolysis products may align as bilayers and accumulate layer by layer on the support, and the hydrophobic acyl chains at the side of the layer structure may be exposed to the outside aqueous environment. The introduced hydrophobic region could have hydrophobic interactions with proteins and therefore can attract the binding of not only PLA2 but also other types of proteins such as proteoglycans and streptavidin. The results suggest that the formation of PLA2-induced domains may convert part of a zwitterionic nonsticky lipid membrane to a site where biomolecules can nonspecifically bind.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Bee Venoms; Bees; Insect Proteins; Lipid Bilayers; Membrane Microdomains; Phosphatidylcholines; Phospholipases A2

The adsorption behaviour of amyloid beta (Aβ), thought to be a key peptide for understanding Alzheimer's disease, was investigated by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS). Aβ aggregates depending on the lipid membrane condition though it has not been fully understood yet. In this study, Aβ samples on different lipid membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were observed with ToF-SIMS and the complex ToF-SIMS data of the Aβ samples was interpreted using data analysis techniques such as principal component analysis (PCA), gentle-SIMS (G-SIMS) and g-ogram. DOPC and DMPC are liquid crystal at room temperature, while DPPC is gel at room temperature. As primary ion beams, Bi3(+) and Ar cluster ion beams were used and the effect of an Ar cluster ion for evaluating biomolecules was also studied. The secondary ion images of the peptide fragment ions indicated by G-SIMS and g-ogram were consistent with the PCA results. It is suggested that Aβ is adsorbed homogeneously on the liquid-crystalline-phase lipid membranes, while it aggregates along the lipid on the gel-phase lipid membrane. Moreover, in the results using the Ar cluster, the influence of contamination was reduced.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Amino Acid Sequence; Amyloid beta-Peptides; Dimyristoylphosphatidylcholine; Glycerylphosphorylcholine; Image Processing, Computer-Assisted; Membrane Lipids; Molecular Sequence Data; Phosphatidylcholines; Principal Component Analysis; Spectrometry, Mass, Secondary Ion

The response of cell membranes to the local physical environment significantly determines many biological processes and the practical applications of biomaterials. A better understanding of the dynamic assembly and environmental response of lipid membranes can help understand these processes and design novel nanomaterials for biomedical applications. The present work demonstrates the directed assembly of lipid monolayers, in both liquid and gel phases, on the surface of a monolayered reduced graphene oxide (rGO). The results from atomic force microscopy indicate that the hydrophobic aromatic plane and the defect holes due to reduction of GO sheets, along with the phase state and planar surface pressure of lipids, corporately determine the morphology and lateral structure of the assembled lipid monolayers. The DOPC molecules, in liquid phase, probably spread over the rGO surface with their tails associating closely with the hydrophobic aromatic plane, and accumulate to form circles of high area surrounding the defect holes on rGO sheets. However, the DPPC molecules, in gel phase, prefer to form a layer of continuous membrane covering the whole rGO sheet including defect holes. The strong association between rGO sheets and lipid tails further influences the melting behavior of lipids. This work reveals a dramatic effect of the local structure and surface property of rGO sheets on the substrate-directed assembly and subsequent phase behavior of the supported lipid membranes.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Gels; Graphite; Hydrophobic and Hydrophilic Interactions; Membranes, Artificial; Microscopy, Atomic Force; Molecular Structure; Oxidation-Reduction; Oxides; Phase Transition; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Pressure; Rhodamines; Surface Properties

Hybrids composed of amphiphilic block copolymers and lipids constitute a new generation of biological membrane-inspired materials. Hybrid membranes resulting from self-assembly of lipids and polymers represent adjustable models for interactions between artificial and natural membranes, which are of key importance, e.g., when developing systems for drug delivery. By combining poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) amphiphilic copolymers (PDMS-b-PMOXA) with various phospholipids, we obtained hybrid films with modulated properties and topology, based on phase separation, and the formation of distinct domains. By understanding the factors driving the phase separation in these hybrid lipid-polymer films, we were able to use them as platforms for directed insertion of membrane proteins. Tuning the composition of the polymer-lipids mixtures favored successful insertion of membrane proteins with desired topological distributions (in polymer or/and lipid regions). Controlled insertion and location of membrane proteins in hybrid films make these hybrids ideal candidates for numerous applications where specific spatial functionality is required.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Dimethylpolysiloxanes; Membrane Proteins; Membranes, Artificial; Models, Biological; Phosphatidylcholines; Phosphatidylethanolamines; Polyamines; Polymerization; Thermodynamics

Biological membranes are supposed to have heterogeneous structure containing lipid rafts-lateral micro- and nanodomains enriched in cholesterol (chol) and sphingolipids. In this work, lipid bilayers containing a small amount of the spin-labeled chol analogue 3β-doxyl-5α-cholestane (chlstn) were studied using electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR). Bilayers were prepared from an equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with chol added at different concentrations. The ESE decays recorded at 77 K become faster with increase of chlstn concentration. The chlstn-dependent contribution to ESE decay is remarkably nonexponential; however, the logarithm of this contribution can be rescaled for different chlstn concentrations to a universal function with the rescaling factor approximately proportional to concentration. This result shows that the chlstn-dependent contribution to the ESE decay can be employed to estimate the local (at the nanometer scale of distances) chlstn concentration. Analogous rescaling behavior is also observed for the bilayers with different chol concentrations, with the rescaling factor increasing with increase of the chol concentration. This result is evidence that chlstn molecules are distributed heterogeneously in the chol-containing bilayer and form clusters with enhanced chlstn (and probably chol) local concentration. The local concentration of chlstn molecules for large chol content (∼30 mol %) was enhanced by at least ∼70% versus chol-free bilayers. The suggested approach appears to be useful for exploring heterogeneities in lipid composition of biological membranes of different types.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Electron Spin Resonance Spectroscopy; Glycerylphosphorylcholine; Lipid Bilayers; Models, Molecular; Phosphatidylcholines; Spin Labels

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

Dimethyl sulfoxide (DMSO) has been broadly used in biology as a cosolvent, a cryoprotectant, and an enhancer of membrane permeability, leading to the general assumption that DMSO-induced structural changes in cell membranes and their hydration water play important functional roles. Although the effects of DMSO on the membrane structure and the headgroup dehydration have been extensively studied, the mechanism by which DMSO invokes its effect on lipid membranes and the direct role of water in this process are unresolved. By directly probing the translational water diffusivity near unconfined lipid vesicle surfaces, the lipid headgroup mobility, and the repeat distances in multilamellar vesicles, we found that DMSO exclusively weakens the surface water network near the lipid membrane at a bulk DMSO mole fraction (XDMSO) of <0.1, regardless of the lipid composition and the lipid phase. Specifically, DMSO was found to effectively destabilize the hydration water structure at the lipid membrane surface at XDMSO <0.1, lower the energetic barrier to dehydrate this surface water, whose displacement otherwise requires a higher activation energy, consequently yielding compressed interbilayer distances in multilamellar vesicles at equilibrium with unaltered bilayer thicknesses. At XDMSO >0.1, DMSO enters the lipid interface and restricts the lipid headgroup motion. We postulate that DMSO acts as an efficient cryoprotectant even at low concentrations by exclusively disrupting the water network near the lipid membrane surface, weakening the cohesion between water and adhesion of water to the lipid headgroups, and so mitigating the stress induced by the volume change of water during freeze-thaw.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Diffusion; Dimethyl Sulfoxide; Fatty Acids, Monounsaturated; Magnetic Resonance Spectroscopy; Membranes, Artificial; Phosphatidylcholines; Phosphatidylglycerols; Quaternary Ammonium Compounds; Scattering, Small Angle; Water; X-Ray Diffraction

Understanding the ionization behavior of lipid membranes is a key parameter for successful development of lipid-based drug delivery systems. Accurate determination of the ionization state of a titratable species incorporated in a lipid bilayer however requires special care. Herein we investigated the behavior of titratable lipids in liposomes by fluorescence spectroscopy and determined which extrinsic parameters-i.e., besides those directly related to their molecular structure-determine their ionization state. Two fluorescent dyes, TNS and R18, have been used to investigate basic and acidic titratable lipids, respectively. Our results suggest that the titration behavior of the ionizable lipid in the membrane is more sensitive to the composition of the membrane and to its physical state than to the presence of solutes in the aqueous phase. Essentially overlooked in earlier studies on ionizable lipid assemblies, the concentration of the titratable lipid in the membrane was found to have a major effect on the ionization state of the lipid polar head. This may result in a shift in the apparent pKa value which may be as large as two pKa units and cannot be satisfactorily predicted.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Drug Delivery Systems; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Lipid Bilayers; Liposomes; Molecular Structure; Naphthalenesulfonates; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Spectrometry, Fluorescence

Lipid-cholesterol interactions are responsible for different properties of biological membranes including those determining formation in the membrane of spatial inhomogeneities (lipid rafts). To get new information on these interactions, electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR), was applied to study 3β-doxyl-5α-cholestane (DCh), a spin-labeled analog of cholesterol, in phospholipid bilayer consisted of equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. DCh concentration in the bilayer was between 0.1 mol.% and 4 mol.%. For comparison, a reference system containing a spin-labeled 5-doxyl-stearic acid (5-DSA) instead of DCh was studied as well. The effects of "instantaneous diffusion" in ESE decay and in echo-detected (ED) EPR spectra were explored for both systems. The reference system showed good agreement with the theoretical prediction for the model of spin labels of randomly distributed orientations, but the DCh system demonstrated remarkably smaller effects. The results were explained by assuming that neighboring DCh molecules are oriented in a correlative way. However, this correlation does not imply the formation of clusters of cholesterol molecules, because conventional continuous wave EPR spectra did not show the typical broadening due to aggregation of spin labels and the observed ESE decay was not faster than in the reference system. So the obtained data evidence that cholesterol molecules at low concentrations in biological membranes can interact via large distances of several nanometers which results in their orientational self-ordering.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Glycerylphosphorylcholine; Lipid Bilayers; Phosphatidylcholines; Spin Labels

Nanosized ordered domains formed in 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOPC/DPPC) and DOPC/cholesterol (Ch) liposomes were characterized using a newly developed (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) quenching method. The membrane fluidity of the DOPC/DPPC liposomes, evaluated by the use of 1,6-diphenyl-1,3,5-hexatriene (DPH), increased significantly above their phase-transition temperature. The fluorescence spectra of 6-lauroyl-2-dimethylamino naphthalene (Laurdan) indicated the formation of an immiscible ordered phase in the DOPC/DPPC (50/50) liposomal membrane at 30 °C. The analysis of the membrane polarity indicated that the surface of the liquid-disordered phase was hydrated whereas that of the ordered phase was dehydrated. DOPC/DPPC and DOPC/Ch (70/30) liposomes exhibited heterogeneous membranes, indicating that nanosized ordered domains formed on the surface of the DOPC/DPPC liposomes. The size of these nanosized ordered domains was estimated using the TEMPO quenching method. Because TEMPO can quench DPH distributed in the disordered phases, the remaining fluorescence from DPH is proportional to the size of the ordered domain. The domain sizes calculated for DOPC/DPPC (50/50), DOPC/DPPC (25/75), DOPC/Ch (70/30), and DOPC/DPPC/Ch (40/40/20) were 13.9, 36.2, 13.2, and 35.5 Å, respectively.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Cyclic N-Oxides; Glycerylphosphorylcholine; Models, Molecular; Nanostructures; Particle Size; Phosphatidylcholines