Low-melting Lipid Research Articles

Taste Masking of Dexketoprofen Trometamol Orally Disintegrating Granules by High-Shear Coating with Glyceryl Distearate.

Orally disintegrating granules (ODGs) are a pharmaceutical form commonly used for the administration of NSAIDs because of their easy assumption and fast dispersion. The development of ODGs is not easy for drugs like dexketoprofen trometamol (DXKT), which have a bitter and burning taste. In this work, high-shear coating (HSC) was used as an innovative technique for DKXT taste masking. This study focused on coating DXKT granules using the HSC technique with a low-melting lipid excipient, glyceryl distearate (GDS). The HSC technique allowed for the coating to be developed through the thermal rise resulting from the friction generated by the granules movement inside the equipment, causing the coating excipient to soften. The design of the experiment was used to find the best experimental coating conditions in order to gain effective taste masking by suitably reducing the amount of drug released in the oral cavity. The influence of the granule dimensions was also investigated. Coating effectiveness was evaluated using a simulated saliva dissolution test. It was found that low impeller speed (300 rpm) and a 20% coating excipient were effective in suitably reducing the drug dissolution rate and then in taste masking. The coated granules were characterized for their morphology and solid-state properties by SEM, BET, XRPD, DSC, and NIR analyses. A human taste panel test confirmed the masking of DXKT taste in the selected batch granules.

Pore-spanning membranes as a tool to investigate lateral lipid membrane heterogeneity.

Over the years, it has become more and more obvious that lipid membranes show a very complex behavior. This behavior arises in part from the large number of different kinds of lipids and proteins and how they dynamically interact with each other. In vitro studies using artificial membrane systems have shed light on the heterogeneity based on lipid-lipid interactions in multicomponent bilayer mixtures. Inspired by the raft hypothesis, the coexistence of liquid-disordered (ld) and liquid-ordered (lo) phases has drawn much attention. It was shown that ternary lipid mixtures containing low- and high-melting temperature lipids and cholesterol can phase separate into a lo phase enriched in the high-melting lipids and cholesterol and a ld phase enriched in the low-melting lipids. Depending on the model membrane system under investigation, different domain sizes, shapes, and mobilities have been found. Here, we describe how to generate phase-separated lo/ld phases in model membrane systems termed pore-spanning membranes (PSMs). These PSMs are prepared on porous silicon substrates with pore sizes in the micrometer regime. A proper functionalization of the top surface of the substrates is required to achieve the spreading of giant unilamellar vesicles (GUVs) to obtain PSMs. Starting with lo/ld phase-separated GUVs lead to membrane heterogeneities in the PSMs. Depending on the functionalization strategy of the top surface of the silicon substrate, different membrane heterogeneities are observed in the PSMs employing fluorescence microscopy. A quantitative analysis of the heterogeneity as well as the dynamics of the lipid domains is described.

Enzymatic Janus Liposome Micromotors

A liposome-based micromotor system that utilizes regional enzymatic conversion and gas generation to achieve directional motion in water is presented. Constituted mainly of a low-melting lipid and a high-melting lipid together with cholesterol, these liposomes maintain stable Janus configuration at room temperature as a result of lipid liquid-liquid phase separation. Local placement of enzymes such as horseradish peroxidase is realized via affinity binding between avidin and biotin, the latter as a lipid conjugate sorted specifically into one domain of these Janus liposomes as a minor component. In the presence of the substrate, hydrogen peroxide, these enzyme-decorated Janus liposomes undergo directional motion, yielding velocities exceeding thermal diffusion by three folds in some cases. Experimental details on liposome size control, motor assembly, and substrate distribution are presented; effects of key experimental factors on liposome motion, such as substrate concentration and liposome Janus ratio, are also examined. This work thus provides a viable approach to building asymmetrical lipid-assembled, enzyme-attached colloids and, in addition, stresses the importance of asymmetry in achieving particle directional motion.

Utilizing asymmetric giant unilamellar vesicles to determine how low-melting phospholipids affect interleaflet coupling strength.

Model membranes that mimic cell membrane asymmetry can be used to study coupling between lateral and transverse lipid organization. Theory predicts that, in a leaflet composed of lipids that would otherwise phase separate in a symmetric bilayer, domain formation can be suppressed by strong coupling to a uniformly mixed leaflet. Conversely, when coupled to a leaflet with a tendency to demix, an otherwise miscible leaflet can be induced to demix. Each of these outcomes has been observed in model asymmetric membranes of different composition, suggesting that cells may be able to control domain formation through changes in lipid content and/or asymmetric lipid distribution. We have explored the influence of the low-melting (low-TM) lipid in a ternary mixture on macroscopic domain formation in asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs composed of DPPC/low-TM lipid/Chol (39/39/22 mol%) with varied low-TM chain length (14:1-PC, 16:1-PC, 18:1-PC, 20:1-PC, and 22:1-PC) were investigated using confocal fluorescence microscopy; the mixtures containing 14:1-PC, 16:1-PC, and 18:1-PC showed macroscopic phase separation at room temperature, while the mixtures containing 20:1-PC and 22:1-PC did not. Cryogenic electron microscopy, small-angle neutron scattering, and Förster resonance energy transfer confirmed the presence of domains in the first three systems and revealed nanoscopic heterogeneity in the mixture containing 20:1-PC, while the 22:1-PC mixture was uniform. aGUVs were then prepared by calcium-induced hemifusion of the GUVs to a supported lipid bilayer composed of the corresponding low-TM lipid and Chol (80/20 mol%). The aGUVs showed a range of outer leaflet exchange, which revealed a correlation between phase behavior and extent of asymmetry. Domains in the 16:1-PC and 18:1-PC systems were suppressed in aGUVs with more than 50 and 60% asymmetry, respectively, indicating a relatively large free energy cost for antiregistered domains.

Investigating the influence of low-melting phospholipids on interleaflet coupling in asymmetric giant unilamellar vesicles

Compositional asymmetry is a characteristic of the plasma membrane whose biological importance is still mostly unknown. Asymmetric model membranes mimicking cell membrane composition offer a useful, highly controlled system for studying the coupling between lateral and transverse lipid organization. We have explored the influence of the low-melting (low-TM) lipid in a ternary mixture on macroscopic domain formation in asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs composed of DPPC/low-TM lipid/Chol (39/39/22 mol%) with varied low-TM chain length (14:1-PC, 16:1-PC, 18:1-PC, 20:1-PC, and 22:1-PC) were prepared by electroformation.

The Origin of Lipid Rafts.

The time-averaged lateral organization of the lipids and proteins that make up mammalian cell membranes continues to be the subject of intense interest and debate. Since the introduction of the fluid mosaic model almost 50 years ago, the "lipid raft hypothesis" has emerged as a popular concept that has captured the imagination of a large segment of the biomembrane community. In particular, the notion that lipid rafts play a pivotal role in cellular processes such as signal transduction and membrane protein trafficking is now favored by many investigators. Despite the attractiveness of lipid rafts, their composition, size, lifetime, biological function, and even the very existence remain controversial. The central tenet that underlies this hypothesis is that cholesterol and high-melting lipids have favorable interactions (i.e., they pull together), which lead to transient domains. Recent nearest-neighbor recognition (NNR) studies have expanded the lipid raft hypothesis to include the influence that low-melting lipids have on the organization of lipid membranes. Specifically, it has been found that mimics of cholesterol and high-melting lipids are repelled (i.e., pushed away) by low-melting lipids in fluid bilayers. The picture that has emerged from our NNR studies is that lipid mixing is governed by a balance of these "push and pull" forces, which maximizes the number of hydrocarbon contacts and attractive van der Waals interactions within the membrane. The power of the NNR methodology is that it allows one to probe these push/pull interaction energies that are measured in tens of calories per mole.

Bilayer compositional asymmetry influences the nanoscopic to macroscopic phase domain size transition

The eukaryotic plasma membrane (PM) exhibits lipid mixing heterogeneities known as lipid rafts. These lipid rafts, the result of liquid-liquid phase separation, can be modeled by coexisting liquid ordered (Lo) and liquid disordered (Ld) domains. Four-lipid component systems with a high-melting lipid, a nanodomain-inducing low-melting lipid, a macrodomain-inducing low-melting lipid, and cholesterol (chol) can give rise to domains of different sizes. These four-component systems have been characterized in experiments, yet there are few studies that model the asymmetric distribution of lipids actually found in the PM. We used molecular dynamics (MD) simulations to analyze the transition from nanoscopic to macroscopic domains in symmetric and in asymmetric model membranes. Using coarse-grained MD simulations, we found that asymmetry promotes macroscopic domain growth in a case where symmetric systems exhibit nanoscopic domains. Also, macroscopic domain formation in symmetric systems is highly dependent on registration of like phases in the cytoplasmic and exoplasmic leaflets. Using united-atom MD simulations, we found that symmetric Lo domains are only slightly more ordered than asymmetric Lo domains. We also found that large Lo domains in our asymmetric systems induce a slight chain ordering in the apposed cytoplasmic regions. The chol fractions of phase-separated Lo and Ld domains of the exoplasmic leaflet were unchanged whether the system was symmetric or asymmetric.

An investigation into the formations of the internal microstructures of solid dispersions prepared by hot melt extrusion

Hot melt extrusion (HME) is a widely used manufacturing process for pharmaceutical solid dispersions. The complexity of the HME formulations and the number of excipients used in the process are increasing with the advancement of the relevant knowledge. However, one of the areas that is still significantly lacking understanding is the control of internal microstructure of extrudates. Internal microstructure, consisting of voids, in hot melt extruded amorphous solid dispersions is often observed without the causes having been systemically investigated in the literature. In this study, we investigated a range of factors that demonstrated their impacts on the formation of the voids. These include the effect of the types of the materials (i.e. drug, polymer and additive) used in the formulation, the quantity of the drug and the additives used, the key extrusion processing parameters, the type of extruder, and the drying of the raw materials prior to extrusion. The results indicate that the appropriate viscosity and the presence of phase-separated particulates are essential for the formation of the voids. The particulates act as nuclei for the entrapped gas bubbles and the viscosity of the mixture during extrusion governs the collapse/escape of the bubbles. To minimise void formation, the results of this study indicate that slow screw speed, low moisture content of the raw materials, fewer particulates and the addition of lubricants, such as low melting lipid excipients, could be beneficial. This study systematically examines the mechanism of void formation in HME extrudates and generates new strategies that can be used to manage such void formations.

Transverse lipid organization dictates bending fluctuations in model plasma membranes.

Membrane undulations play a vital role in many biological processes, including the regulation of membrane protein activity. The asymmetric lipid composition of most biological membranes complicates theoretical description of these bending fluctuations, yet experimental data that would inform any such a theory is scarce. Here, we used neutron spin-echo (NSE) spectroscopy to measure the bending fluctuations of large unilamellar vesicles (LUV) having an asymmetric transbilayer distribution of high- and low-melting lipids. The asymmetric vesicles were prepared using cyclodextrin-mediated lipid exchange, and were composed of an outer leaflet enriched in egg sphingomyelin (ESM) and an inner leaflet enriched in 1-palmitoyl-2-oleoyl-phosphoethanolamine (POPE), which have main transition temperatures of 37 °C and 25 °C, respectively. The overall membrane bending rigidity was measured at three temperatures: 15 °C, where both lipids are in a gel state; 45 °C, where both lipids are in a fluid state; and 30 °C, where there is gel-fluid co-existence. Remarkably, the dynamics for the fluid asymmetric LUVs (aLUVs) at 30 °C and 45 °C do not follow trends predicted by their symmetric counterparts. At 30 °C, compositional asymmetry suppressed the bending fluctuations, with the asymmetric bilayer exhibiting a larger bending modulus than that of symmetric bilayers corresponding to either the outer or inner leaflet. We conclude that the compositional asymmetry and leaflet coupling influence the internal dissipation within the bilayer and result in membrane properties that cannot be directly predicted from corresponding symmetric bilayers.

Lipid nanoparticles with fats or oils containing β-carotene: Storage stability and in vitro digestibility kinetics.

The aim of this work was to study the formation of lipid nanoparticles (LNPs) with low (corn and olive oil) or high temperature melting lipids (cocoa butter and hydrogenated coconut oil). Moreover, their β-carotene stability and in vitro digestibility kinetics was evaluated. Submicron LNPs (d43 ≈ 570-780 nm) were stabilized with Tween 80 at a surfactant-to-oil ratio (SOR) of 0.1. It was reduced below 200 nm at an SOR of 1. The β-carotene retention at 25 °C was not related to the lipid type but rather to the particle size, being lower in samples with smaller particle sizes. Cocoa butter LNPs presented an equally complete digestion as corn oil LNPs and a high β-carotene bioaccessibility, which was related to the high degree of micellarization of monoacylglycerols. This work evidences the potential of LNPs to protect lipophilic bioactive compounds with a high digestibility and bioaccessibility.

Bending Moduli of Ternary Mixture Models of the Cell Plasma Membrane

Cells must be able to maintain and change membrane shape. This membrane curvature can be influenced by any lateral phase heterogeneity. Ternary mixtures of high-melting lipid, low-melting lipid, and cholesterol exhibit a region of liquid-ordered (Lo)+ liquid-disordered (Ld) phase coexistence analogous to raft + non-raft behavior in cells. Although curvature can induce separation and sorting in vitro, a number of highly-curved membranes in vivo have raft-like composition, which is puzzling since the raft-like compositions have greater rigidity in lipid model mixtures. We find that including transmembrane helical WALP peptides as a component of the model mixtures greatly changes their bending behavior. Measurements were made by shape analysis of giant unilamellar vesicles with both fluorescence and phase contrast microscopy. Vesicles of a single phase were made by the gentle hydration method to obtain a more narrow distribution of vesicle tensions than is obtained by electroformation. We find that a WALP peptide component rigidifies the liquid-disordered phase whereas the liquid-ordered phase is unaffected. This rigidifying effect might be related to the monolayers sliding relative to each other. Further, we have investigated the effects of peptides of different lengths and results suggest a role for hydrophobic mismatch of the peptide component relative to the bilayer thickness.

Multiscale modeling of four-component lipid mixtures: domain composition, size, alignment, and properties of the phase interface.

Simplified lipid mixtures are often used to model the complex behavior of the cell plasma membrane. Indeed, as few as four components-a high-melting lipid, a nandomain-inducing low-melting lipid, a macrodomain-inducing low-melting lipid, and cholesterol (chol)-can give rise to a wide range of domain sizes and patterns that are highly sensitive to lipid compositions. Although these systems are studied extensively with experiments, the molecular-level details governing their phase behavior are not yet known. We address this issue by using molecular dynamics simulations to analyze how phase separation evolves in a four-component system as it transitions from small domains to large domains. To do so, we fix concentrations of the high-melting lipid 16:0,16:0-phosphatidylcholine (DPPC) and chol, and incrementally replace the nanodomain-inducing low-melting lipid 16:0,18:2-PC (PUPC) by the macrodomain-inducing low-melting lipid 18:2,18:2-PC (DUPC). Coarse-grained simulations of this four-component system reveal that lipid demixing increases as the amount of DUPC increases. Additionally, we find that domain size and interleaflet alignment change sharply over a narrow range of replacement of PUPC by DUPC, indicating that intraleaflet and interleaflet behaviors are coupled. Corresponding united atom simulations show that only lipids within ∼2 nm of the phase interface are significantly perturbed regardless of domain composition or size. Thus, whereas the fraction of interface-perturbed lipids is negligible for large domains, it is significant for smaller ones. Together, these results reveal characteristic traits of bilayer thermodynamic behavior in four-component mixtures, and provide a baseline for investigation of the effects of proteins and other lipids on membrane phase properties.

Push and pull forces in lipid raft formation: the push can be as important as the pull.

Nearest-neighbor recognition measurements have been made using exchangeable mimics of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the liquid-ordered (lo) and liquid-disordered (ld) states. In the ld phase, the net interaction between these two lipids is repulsive. In the lo phase, their interactions are neither attractive nor repulsive. These results, together with previous nearest-neighbor measurements, imply that the overall driving force for lipid domain formation in bilayers composed of high-melting lipids, low-melting lipids, and cholesterol, corresponds to a strong pull (attraction) between the high-melting lipids and cholesterol, a significant push (repulsion) between the low-melting and high-melting lipids, and a significant push between the low-melting lipids and cholesterol. In a broader context, these results provide strong support for the notion that repulsive forces play a major role in the formation of lipid rafts.

The Effects of Walp Peptides on Phase Behavior in Quaternary Lipid Mixtures: A Molecular Dynamics Study

Quaternary lipid mixtures containing a high-melting lipid, a nanodomain-inducing low-melting lipid, a macrodomain-inducing low-melting lipid and cholesterol reveal behaviors not observed in ternary mixtures. Through fixing the amounts of high-melting lipid, total low-melting lipid and cholesterol and altering only the relative amounts of the two low-melting lipids, domain size in quaternary mixtures can be finely tuned from nanoscopic to macroscopic, with an intermediate patterned phase morphology. We have previously used extensive coarse grain and atomistic molecular dynamics simulations to study one such quaternary lipid mixture, containing the high-melting lipid DPPC (16:0,16:0-PC), the low-melting lipids PUPC (16:0,18:2-PC) and DUPC (18:2,18:2-PC), and cholesterol. In particular, we quantified the effect of the two low-melting lipids on domain size, alignment, lipid order and lipid tilt. Using those simulations as a control, we are currently evaluating how adding WALP peptides to the quaternary mixture affects the sizes, alignment and properties of coexisting phases using coarse grained molecular dynamics. We examine how these properties are affected by both the length of the WALP peptide as well as their concentration. A main focus of our work is analyzing the extent to which the WALPs alter the onset of large-scale phase separation and domain alignment. The addition of WALP to the quaternary systems makes these simulations some of the most complex and biologically relevant membranes studied to date with molecular dynamics.

Appearance of Modulated Bilayer Morphology for Coexisting LD and LO Phases is Correlated with Line Tension

Giant Unilamelar Vesicles (GUVs) appear uniform for the brain-SM/POPC/Chol bilayer mixture, whereas GUVs exhibit macroscopic domains of coexisting liquid-disordered + liquid-ordered phases for the brain-SM/DOPC/Chol mixture. We can travel through composition space in order to study this transition from nanodomains with POPC to macrodomains with DOPC. Using ρ defined as the ratio [DOPC]/[DOPC]+[POPC], the domain morphology undergoes a transition regime where “modulated phases” appear as a function of ρ. The formation of these different morphologies on a GUV can be understood as a competition between line tension, which favors large domains, and bending energy, which favors small domains (Amazon et al). We measured the ρ values where modulated phases appear for different 4-component mixtures, using brain-SM, or egg-SM, or palmitoyl-SM as the high-melting lipid, and DOPC/POPC or DOPC/SOPC as the low melting lipids, for a total of six different 4-component mixtures. We then measured the line tension of the macroscopic domains vs ρ, and found the same line tension at ρ values at the transition between modulated phases and macroscopic phases. This finding implies that line tension has major control over domain morphology. We are currently studying how the transmembrane peptide, GWALP-23, changes the morphology by changing the values of the competing interactions.

Membrane Bending Modulus for Ternary Mixture Models of the Cell Plasma Membrane

Ternary mixtures of high-melting lipid, low-melting lipid, and cholesterol exhibit a region of liquid-ordered + liquid-disordered phase coexistence analogous to raft + non-raft behavior in cells. These coexisting phases manifest domain sizes that range from a few nanometers to many microns, depending strongly on the nature of the low-melting lipid. When POPC, which gives rise to nanodomains, is replaced by DOPC, which yields macrodomains, an intermediate region is observed of patterned, or modulated phases. This domain morphology can be explained as a competition between line tension and bending energies with patterns occurring when the two are nearly balanced. Necessary for testing this model are measurements of line tension, and bending moduli for both phases. Here we report the bending moduli of coexisting Lo and Ld phases from mixtures that produce domains ranging in size from nanoscopic to macroscopic. Measurements were made by shape analysis of giant unilamellar vesicles with both fluorescence and phase contrast microscopy. Vesicles of a single phase were made by the gentle hydration method to obtain a more narrow distribution of vesicle tensions than is obtained by electroformation. A transmembrane helical peptide, WALP23, strongly partitions to the liquid disordered phase, and changes the size of coexisting Lo + Ld domains. We are currently investigating the effects of WALP23 on membrane mechanical properties and line tension.

High-Melting Lipid Mixtures and the Origin of Detergent-Resistant Membranes Studied with Temperature-Solubilization Diagrams

The origin of resistance to detergent solubilization in certain membranes, or membrane components, is not clearly understood. We have studied the solubilization by Triton X-100 of binary mixtures composed of egg sphingomyelin (SM) and either ceramide, diacylglycerol, or cholesterol. Solubilization has been assayed in the 4–50°C range, and the results are summarized in a novel, to our knowledge, form of plots, that we have called temperature-solubilization diagrams. Despite using a large detergent excess (lipid/detergent 1:20 mol ratio) and extended solubilization times (24–48 h) certain mixtures were not amenable to Triton X-100 solubilization at one or more temperatures. DSC of all the lipid mixtures, and of all the lipid + detergent mixtures revealed that detergent resistance was associated with the presence of gel domains at the assay temperature. Once the system melted down, solubilization could occur. In general adding high-melting lipids limited the solubilization, whereas the addition of low-melting lipids promoted it. Lipidomic analysis of Madin-Darby canine kidney cell membranes and of the corresponding detergent-resistant fraction indicated a large enrichment of the nonsolubilized components in saturated diacylglycerol and ceramide. SM-cholesterol mixtures were special in that detergent solubilization was accompanied, for certain temperatures and compositions, by an independent phenomenon of reassembly of the partially solubilized lipid bilayers. The temperature at which lysis and reassembly prevailed was ∼25°C, thus for some SM-cholesterol mixtures solubilization occurred both above and below 25°C, but not at that temperature. These observations can be at the origin of the detergent resistance effects observed with cell membranes, and they also mean that cholesterol-containing detergent-resistant membrane remnants cannot correspond to structures existing in the native membrane before detergent addition.

The Structural Role of Cholesterol in Cell Membranes: From Condensed Bilayers to Lipid Rafts

CONSPECTUS: Defining the two-dimensional structure of cell membranes represents one of the most daunting challenges currently facing chemists, biochemists, and biophysicists. In particular, the time-averaged lateral organization of the lipids and proteins that make up these natural enclosures has yet to be established. As the classic Singer-Nicolson model of cell membranes has evolved over the past 40 years, special attention has focused on the structural role played by cholesterol, a key component that represents ca. 30% of the total lipids that are present. Despite extensive studies with model membranes, two fundamental issues have remained a mystery: (i) the mechanism by which cholesterol condenses low-melting lipids by uncoiling their acyl chains and (ii) the thermodynamics of the interaction between cholesterol and high- and low-melting lipids. The latter bears directly on one of the most popular notions in modern cell biology, that is, the lipid raft hypothesis, whereby cholesterol is thought to combine with high-melting lipids to form "lipid rafts" that float in a "sea" of low-melting lipids. In this Account, we first describe a chemical approach that we have developed in our laboratories that has allowed us to quantify the interactions between exchangeable mimics of cholesterol and low- and high-melting lipids in model membranes. In essence, this "nearest-neighbor recognition" (NNR) method involves the synthesis of dimeric forms of these lipids that contain a disulfide moiety as a linker. By means of thiolate-disulfide interchange reactions, equilibrium mixtures of dimers are then formed. These exchange reactions are initiated either by adding dithiothreitol to a liposomal dispersion to generate a small amount of thiol monomer or by including a small amount of thiol monomer in the liposomes at pH 5.0 and then raising the pH to 7.4. We then show how such NNR measurements have allowed us to distinguish between two very different mechanisms that have been proposed for cholesterol's condensing effect: (i) an umbrella mechanism in which the acyl chains and cholesterol become more tightly packed as cholesterol content increases because they share limited space under phospholipid headgroups and (ii) a template mechanism whereby cholesterol functions as a planar hydrophobic template at the membrane surface, thereby maximizing hydrophobic interactions and the hydrophobic effect. Specifically, our NNR experiments rule out the umbrella mechanism and provide strong support for the template mechanism. Similar NNR measurements have also allowed us to address the question of whether the interactions between low-melting kinked phospholipids and cholesterol can play a significant role in the formation of lipid rafts. Specifically, these NNR measurements have led to our discovery of a new physical principle in the lipids and membranes area that must be operating in biological membranes, that is, a "push-pull" mechanism, whereby cholesterol is pushed away from low-melting phospholipids and pulled toward high-melting lipids. Thus, to the extent that lipid rafts play a role in the functioning of cell membranes, low-melting phospholipids must be active participants.

Multiscale Modeling of Four Component Lipid Mixtures: Coarse Grained and United Atom Simulations Reveal Trends in Phase Separation

The cell plasma membrane is often modeled using three-component mixtures containing a high melting lipid, a low melting lipid and cholesterol. These ternary mixtures exhibit either nanoscopoic or macroscopic liquid-liquid phase coexistence. An additional patterned phase morphology can exist in four component systems which combine a high melting lipid, cholesterol, a nanodomain-inducing low melting lipid and a macrodomain-inducing low melting lipid. The molecular-level details governing these different phase morphologies are not yet known. Here, we utilize molecular dynamics simulations to analyze how phase separation evolves in a four component mixture. We present data for 11 mixtures at a fixed composition of (16:0,16:0)-pc/(18:2,18:2)-pc/(16:0,18:2)-pc/Cholesterol (DPPC/DLiPC/PLiPC/Chol), where PLiPC is incrementally replaced by DLiPC from one simulation to the next. Each simulation was run to equilibrium over 25 μs using the Martini coarse grained forcefield and was then converted to united atom and run for a further 100 ns. We investigate trends in domain size, composition, interleaflet coupling and properties of the domain interface as a function of replacement of PLiPC by DLiPC.

Hybrid and Nonhybrid Lipids Exert Common Effects on Membrane Raft Size and Morphology

Cell membranes perform multiple functions that may be facilitated by the lateral organization of lipids and proteins into nanoscale compartments, termed membrane rafts. Understanding the origins of raft domains and the physicochemical mechanisms that control their finite small size is important for elucidating their functional significance and manipulating their properties. However, it has proven difficult to characterize rafts in cells due to the chemical complexity of biological membranes, and various models have been advanced based on theory and experiments of representative model membranes. In one popular model, a special role in the domains existence and properties has been postulated for chain-asymmetric or hybrid lipids having a saturated sn-1 chain and an unsaturated sn-2 chain. It was proposed that these lipids align in a preferred orientation at the boundary of ordered and disordered phases, lowering the interfacial energy and thus reducing domain size. Such a unique "line-active" role for hybrid lipids is an appealing explanation for nanoscopic rafts, as animal cell membranes contain few symmetric low-melting lipids but an abundance of hybrid lipids. We present data from small-angle neutron scattering and fluorescence techniques demonstrating the existence of nanoscopic and modulated liquid phase domains in a mixture composed entirely of nonhybrid lipids and cholesterol. Our results are indistinguishable from those obtained previously for mixtures containing hybrid lipids, conclusively showing that hybrid lipids are not required for the formation of nanoscopic liquid domains. These findings present new challenges for current theoretical descriptions of nanodomains.