Lipoproteins play a key role in the onset and development of atherosclerosis, the formation of lipid plaques at blood vessel walls. The plaque formation, as well as subsequent calcification, involves not only endothelial cells but also connective tissue, and is closely related to a wide range of cardiovascular syndromes, that together constitute the number one cause of death in the Western World. High (HDL) and low (LDL) density lipoproteins are of particular interest in relation to atherosclerosis, due to their protective and harmful effects, respectively. In an effort to elucidate the molecular mechanisms underlying this, and to identify factors determining lipid deposition and exchange at lipid membranes, we here employ neutron reflection (NR) and quartz crystal microbalance with dissipation (QCM-D) to study the effect of membrane charge on lipoprotein deposition and lipid exchange. Dimyristoylphosphatidylcholine (DMPC) bilayers containing varying amounts of negatively charged dimyristoylphosphatidylserine (DMPS) were used to vary membrane charge. It was found that the amount of hydrogenous material deposited from either HDL or LDL to the bilayer depends only weakly on membrane charge density. In contrast, increasing membrane charge resulted in an increase in the amount of lipids removed from the supported lipid bilayer, an effect particularly pronounced for LDL. The latter effects are in line with previously reported observations on atherosclerotic plaque prone regions of long-term hyperlipidaemia and type 2 diabetic patients, and may also provide some molecular clues into the relation between oxidative stress and atherosclerosis. (C) 2018 Elsevier B.V. All rights reserved.

High and low density lipoproteins (HDL and LDL) are thought to play vital roles in the onset and development of atherosclerosis; the biggest killer in the western world. Key issues of initial lipoprotein (LP) interactions at cellular membranes need to be addressed including LP deposition and lipid exchange. Here we present a protocol for monitoring the in situ kinetics of lipoprotein deposition and lipid exchange/removal at model cellular membranes using the non-invasive, surface sensitive methods of neutron reflection and quartz crystal microbalance with dissipation. For neutron reflection, lipid exchange and lipid removal can be distinguished thanks to the combined use of hydrogenated and tail-deuterated lipids. Both HDL and LDL remove lipids from the bilayer and deposit hydrogenated material into the lipid bilayer, however, the extent of removal and exchange depends on LP type. These results support the notion of HDL acting as the ‘good’ cholesterol, removing lipid material from lipid-loaded cells, whereas LDL acts as the ‘bad’ cholesterol, depositing lipid material into the vascular wall.

The demand for highly efficient macromolecular drugs, used in the treatment of many severe diseases, is continuously increasing. However, the hydrophilic character and large molecular size of these drugs significantly limit their ability to permeate across cellular membranes and thus impede the drugs in reaching their target sites in the body. Cell-penetrating peptides (CPP) have gained attention as promising drug excipients, since they can facilitate drug permeation across cell membranes constituting a major biological barrier. Fluorophores are frequently covalently conjugated to CPPs to improve detection, however, the ensuing change in physico-chemical properties of the CPPs may alter their biological properties. With complementary biophysical techniques, we show that the mode of biomembrane interaction may change considerably upon labeling of the CPP penetratin (PEN) with a fluorophore. Fluorophore-PEN conjugates display altered modes of membrane interaction with increased insertion into the core of model cell membranes thereby exerting membrane-thinning effects. This is in contrast to PEN, which localizes along the head groups of the lipid bilayer, without affecting the thickness of the lipid tails. Particularly high membrane disturbance is observed for the two most hydrophobic PEN conjugates; rhodamine B or 1-pyrene butyric acid, as compared to the four other tested fluorophore-PEN conjugates.

Proteins are key components in a multitude of biological processes, of which the functions carried out by transmembrane (membrane-spanning) proteins are especially demanding for investigations. This is because this class of protein needs to be incorporated into a lipid bilayer representing its native environment, and in addition, many experimental conditions also require a solid support for stabilization and analytical purposes. The solid support substrate may, however, limit the protein functionality due to protein–material interactions and a lack of physical space. We have in this work tailored the pore size and pore ordering of a mesoporous silica thin film to match the native cell-membrane arrangement of the transmembrane protein human aquaporin 4 (hAQP4). Using neutron reflectivity (NR), we provide evidence of how substrate pores host the bulky water-soluble domain of hAQP4, which is shown to extend 7.2 nm into the pores of the substrate. Complementary surface analytical tools, including quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy, revealed successful protein-containing supported lipid bilayer (pSLB) formation on mesoporous silica substrates, whereas pSLB formation was hampered on nonporous silica. Additionally, electron microscopy (TEM and SEM), light scattering (DLS and stopped-flow), and small-angle X-ray scattering (SAXS) were employed to provide a comprehensive characterization of this novel hybrid organic–inorganic interface, the tailoring of which is likely to be generally applicable to improve the function and stability of a broad range of membrane proteins containing water-soluble domains.

In this review, the authors discuss the challenges of studying supported lipid bilayers (SLBs) deposited by vesicle fusion in terms of (1) evaluating SLB formation and quality using quartz crystal microbalance with dissipation and (2) analyzing the composition and asymmetry of SLBs composed by lipid mixtures using complementary surface sensitive techniques. An overview of the literature is presented and the inconsistencies on this topic are discussed with the objective to expand beyond simple lipid compositions and set the basis for forming and analyzing SLBs of complex natural lipid extracts formed via the vesicle fusion method. The authors conclude by providing some guidelines to successfully form SLBs of complex lipid mixtures including natural extracts.

As a result of the synthesis protocol polyoxyethylene sorbitan monooleate (polysorbate 80, PS80) is a highly complex mixture of compounds. PS80 was therefore separated into its main constituents, e.g. polyoxyethylene isosorbide esters and polyoxyethylene esters, as well as mono- di- and polyesters using preparative high-performance liquid chromatography. In this comprehensive study the individual components and their ethoxylation level were verified by matrix assisted laser desorption/ionization time-of-flight and their thermotropic behavior was analyzed using differential scanning calorimetry and X-ray diffraction. A distinct correlation was found between the average length of the ethylene oxide (EO) chains in the headgroup and the individual compounds' ability to crystallize. Importantly, a critical number of EO units required for crystallization of the headgroup was determined (6 EO units per chain or 24 per molecule). The investigation also revealed that the hydrocarbon tails only crystallize for polyoxyethylene sorbitan esters if saturated. PS80 is synthesized by reacting with approximately 20 mol of EO per mole of sorbitol, however, the number of EO units in the sorbitan ester in commercial PS80 products is higher than the expected 20 (5 EO units per chain). The complex behavior of all tested compounds revealed that if the amount of several of the linear by-products is reduced, the number of EO units in the chains will stay below the critical number and the product will not be able to crystallize by the EO chains.

Antimicrobial drug resistance is a major human health threat. Among the many attempts to tackle this problem, the synthesis of antimicrobial compounds that mimic natural antimicrobial peptides appears as a promising approach. Peptide-based dendrimers can be designed to have higher potency than natural antimicrobial peptides and at the same time they can evade the bacterial defense system. Novel dendrimers with similar chemical structure but varying potency in terms of minimum inhibitory concentration were designed. The dependency between dendrimer structure and antibacterial activity as well as their capacity to attack model cell membranes was studied. The data suggests that supramolecular structure in terms of charge distribution and amphiphilicity, rather than net charge, is the main driver for disruption of cellular membranes and this correlates well with dendrimer hemolytic activity.

We have investigated the effect of deposition temperature on supported lipid bilayer formation via vesicle fusion. By using several complementary surface-sensitive techniques, we demonstrate that despite contradicting literature on the subject, high-quality bilayers can be formed below the main phase-transition temperature of the lipid. We have carefully studied the formation mechanism of supported DPPC bilayers below and above the lipid melting temperature (Tm) by quartz crystal microbalance and atomic force microscopy under continuous flow conditions. We also measured the structure of lipid bilayers formed below or above Tm by neutron reflection and investigated the effect of subsequent cooling to below the Tm. Our results clearly show that a continuous supported bilayer can be formed with high surface coverage below the lipid Tm. We also demonstrate that the high dissipation responses observed during the deposition process by QCM-D correspond to vesicles absorbed on top of a continuous bilayer and not to a surface-supported vesicular layer as previously reported.

Supported lipid bilayers (SLBs) are simple and robust biomimics with controlled lipid composition that are widely used as models of both mammalian and bacterial membranes. However, the lipids typically used for SLB formation poorly resemble those of bacterial cell membranes due to the lack of available protocols to form SLBs using mixtures of lipids relevant for bacteria such as phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Although a few reports have been published recently on the formation of SLBs from Escherichia coli lipid extracts, a detailed understanding of these systems is challenging due to the complexity of the lipid composition in such natural extracts. Here, we present for the first time a simple and reliable protocol optimized to form high-quality SLBs using mixtures of PE and PG at compositions relevant for Gram-negative membranes. We show using neutron reflection and quartz microbalance not only that Ca2+ ions and temperature are key parameters for successful bilayer deposition but also that mass transfer to the surface is a limiting factor. Continuous flow of the lipid suspension is thus crucial for obtaining full SLB coverage. We furthermore characterize the resulting bilayers and report structural parameters, for the first time for PE and PG mixtures, which are in good agreement with those reported earlier for pure POPE vesicles. With this protocol in place, more suitable and reproducible studies can be conducted to understand biomolecular processes occurring at cell membranes, for example, for testing specificities and to unravel the mechanism of interaction of antimicrobial peptides.

Supported lipid bilayers are widely used for sensing and deciphering biomolecular interactions with model cell membranes. In this paper, we present a method to form supported lipid bilayers from total lipid extracts of Escherichia coli by vesicle fusion. We show the validity of this method for different types of extracts including those from deuterated biomass using a combination of complementary surface sensitive techniques; quartz crystal microbalance, neutron reflection and atomic force microscopy. We find that the head group composition of the deuterated and the hydrogenated lipid extracts is similar (approximately 75% phosphatidylethanolamine, 13% phosphatidylglycerol and 12% cardiolipin) and that both samples can be used to reconstitute high-coverage supported lipid bilayers with a total thickness of 41 ± 3 Å, common for fluid membranes. The formation of supported lipid bilayers composed of natural extracts of Escherichia coli allow for following biomolecular interactions, thus advancing the field towards bacterial-specific membrane biomimics.

The interaction mechanism of a novel amphiphilic antimicrobial peptide dendrimer, BALY, with model lipid bilayers was explored through a combination of neutron reflection and molecular dynamics simulations. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phos-phocholine (DPPC) lipid bilayers were examined at room temperature to extract information on the interaction of BALY with fluid and gel phases, respectively. Furthermore, a 1:4 mixture of POPC and DPPC was used as a model of a phase-separated membrane. Upon interaction with fluid membranes, BALY inserted in the distal leaflet and caused thinning and disordering of the headgroups. Membrane thinning and expansion of the lipid cross-sectional area were observed for gel phase membranes, also with limited insertion to the distal leaflet. However, dendrimer insertion through the entire lipid tail region was observed upon crossing the lipid phase transition temperature of DPPC and in phase separated membranes. The results show clear differences in the interaction mechanism of the dendrimer depending on the lipid membrane fluidity, and suggest a role for lipid phase separation in promoting its antimicrobial activity.

Phosphoinositide (PIP) lipids are anionic phospholipids playing a fundamental role for the activity of several transmembrane and soluble proteins. Among all, phosphoinositol-3',4',5'-trisphosphate (PIP3) is a secondary signaling messenger that regulates the function of proteins involved in cell growth and gene transcription. The present study aims to reveal the structure of PIP-containing lipid membranes, which so far has been little explored. For this purpose, supported lipid bilayers (SLBs) containing 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',4',5'-trisphosphate (DOPIP3) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were used as mimics of biomembranes. Surface sensitive techniques, i.e. Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Atomic Force Microscopy (AFM) and Neutron Reflectometry (NR), provided detailed information on the formation of the SLB and the location of DOPIP3 in the lipid membrane. Specifically, QCM-D and AFM were used to identify the best condition for lipid deposition and to estimate the total bilayer thickness. On the other hand, NR was used to collect experimental structural data on the DOPIP3 location and orientation within the lipid membrane. The two bilayer leaflets showed the same DOPIP3 concentration, thus suggesting the formation of a symmetric bilayer. The headgroup layer thicknesses of the pure POPC and the mixed POPC/DOPIP3 bilayer suggest that the DOPIP3-headgroups have a preferred orientation, which is not perpendicular to the membrane surface, but instead it is close to the surrounding lipid headgroups. These results support the proposed PIP3 tendency to interact with the other lipid headgroups as PC, so far exclusively suggested by MD simulations.

Atherosclerosis and its clinical consequences are the leading cause of death in the western hemisphere. While many studies throughout the last decades have aimed at understanding the disease, the clinical markers in use today still fail to accurately predict the risks. The role of the current main clinical indicator, low density lipoprotein (LDL), in depositing fat to the vessel wall is believed to be the onset of the process. However, many subfractions of the LDL, which differ both in structure and composition, are present in the blood and among different individuals. Understanding the relationship between LDL structure and composition is key to unravel the specific role of various LDL components in the development and/or prevention of atherosclerosis. Here, we describe a model for analyzing small-angle X-ray scattering data for rapid and robust structure determination for the LDL. The model not only gives the overall structure but also the particular internal layering of the fats inside the LDL core. Thus, the melting of the LDL can be followed in situ as a function of temperature for samples extracted from healthy human patients and purified using a double protocol based on ultracentrifugation and size-exclusion chromatography. The model provides information on: (i) the particle-specific melting temperature of the core lipids, (ii) the structural organization of the core fats inside the LDL, (iii) the overall shape of the particle, and (iv) the flexibility and overall conformation of the outer protein/hydrophilic layer at a given temperature as governed by the organization of the core. The advantage of this method over other techniques such as cryo-TEM is the possibility of in situ experiments under near-physiological conditions which can be performed relatively fast (minutes at home source, seconds at synchrotron). This approach now allows the monitoring of structural changes in the LDL upon different stresses from the environment, such as changes in temperature, oxidation, or external agents used or currently in development against atherosclerotic plaque build-up and which are targeting the LDL.

Atherosclerosis is the main killer in the western world. Today's clinical markers include the total level of cholesterol and high-/low-density lipoproteins, which often fails to accurately predict the disease. The relationship between the lipid exchange capacity and lipoprotein structure should explain the extent by which they release or accept lipid cargo and should relate to the risk for developing atherosclerosis. Here, small-angle neutron scattering and tailored deuteration have been used to follow the molecular lipid exchange between human lipoprotein particles and cellular membrane mimics made of natural, "neutron invisible" phosphatidylcholines. We show that lipid exchange occurs via two different processes that include lipid transfer via collision and upon direct particle tethering to the membrane, and that high-density lipoprotein excels at exchanging the human-like unsaturated phosphatidylcholine. By mapping the specific lipid content and level of glycation/oxidation, the mode of action of specific lipoproteins can now be deciphered. This information can prove important for the development of improved diagnostic tools and in the treatment of atherosclerosis.

The naturally occurring peptide indolicidin from bovine neutrophils exhibits strong biological activity against a broad spectrum of microorganisms. This is believed to arise from selective interactions with the negatively charged cytoplasmic lipid membrane found in bacteria. We have investigated the peptide interaction with supported lipid model membranes using a combination of complementary surface sensitive techniques: neutron reflectometry (NR), atomic force microscopy (AFM), and quartz crystal microbalance with dissipation monitoring (QCM-D). The data are compared with small-angle X-ray scattering (SAXS) results obtained with lipid vesicle/peptide solutions. The peptide membrane interaction is shown to be significantly concentration dependent. At low concentrations, the peptide inserts at the outer leaflet in the interface between the headgroup and tail core. Insertion of the peptide results in a slight decrease in the lipid packing order of the bilayer, although not sufficient to cause membrane thinning. By increasing the indolicidin concentration well above the physiologically relevant conditions, a deeper penetration of the peptide into the bilayer and subsequent lipid removal take place, resulting in a slight membrane thinning. The results suggest that indolicidin induces lipid removal and that mixed indolicidin-lipid patches form on top of the supported lipid bilayers. Based on the work presented using model membranes, indolicidin seems to act through the interfacial activity model rather than through the formation of stable pores.

Polysorbates (PS), commonly known as Tween (TM), are some of the most extensively used excipients and protein stabilisers in biopharmaceutical products worldwide. It is stipulated in the pharmacopoeia specifications that these ethoxylated surfactants are complex mixtures comprised of a wealth of molecular species. While little is known about the propensity of PSs to crystallise, they are used in applications ranging from food products, cosmetics, different types of drug dosage forms like creams and oral products to parenteral applications. However, in recent years a range of issues and safety concerns have appeared when using them for stabilising biopharmaceutical products including precipitation, particle formation, and adverse biological effects. Therefore, the aim of this study was to thoroughly characterise the thermotropic behaviour and mechanism of crystallisation of polysorbates with different hydrocarbon tails and their non-ethoxylated sorbitan ester equivalents for comparison. A systematic and comprehensive product characterisation was carried out, taking advantage of a combination of complementary techniques such as differential scanning calorimetry, matrix assisted laser desorption ionisation time-of-flight and small- and wide-angle X-ray diffraction. We show that polysorbate 80, having an unsaturated hydrocarbon tail, crystallises by the ethylene oxide chains in the headgroup. Polysorbate 20, 40, and 60, containing saturated hydrocarbon esters tails, crystallise not only by the ethylene oxide chains but also by their hydrocarbon tails. An analogous behaviour was observed for the PS non-ethoxylated equivalents, the sorbitan esters. Sorbitan esters with saturated hydrocarbon tails displayed a crystallisation of the tail upon cooling, whereas the sorbitan ester with unsaturated hydrocarbon tail displayed no crystallisation.

Cholesterol is an essential component of mammalian membranes and is known to induce a series of physicochemical changes in the lipid bilayer. Such changes include the formation of liquid-ordered phases with an increased thickness and a configurational order as compared to liquid-disordered phases. For saturated lipid membranes, cholesterol molecules localize close to the lipid head group-tail interface. However, the presence of polyunsaturated lipids was recently shown to promote relocation of cholesterol toward the inner interface between the two bilayer leaflets. Here, neutron reflection is used to study the location of cholesterol (both non-deuterated and per-deuterated versions are used) within supported lipid bilayers composed of a natural mixture of phosphatidylcholine (PC). The lipids were produced in a genetically modified strain of Escherichia coli and grown under specific deuterated conditions to give an overall neutron scattering length density (which depends on the level of deuteration) of the lipids matching that of D2O. The combination of solvent contrast variation method with specific deuteration shows that cholesterol is located closer to the lipid head group-tail interface in this natural PC extract rather than in the center of the core of the bilayer as seen for very thin or polyunsaturated membranes.

Lipoproteins play a central role in the development of atherosclerosis. High and low-density lipoproteins (HDL and LDL), known as 'good' and 'bad' cholesterol, respectively, remove and/or deposit lipids into the artery wall. Hence, insight into lipid exchange processes between lipoproteins and cell membranes is of particular importance in understanding the onset and development of cardiovascular disease. In order to elucidate the impact of phospholipid tail saturation and the presence of cholesterol in cell membranes on these processes, neutron reflection was employed in the present investigation to follow lipid exchange with both HDL and LDL against model membranes. Mirroring clinical risk factors for the development of atherosclerosis, lower exchange was observed in the presence of cholesterol, as well as for an unsaturated phospholipid, compared to faster exchange when using a fully saturated phospholipid. These results highlight the importance of membrane composition on the interaction with lipoproteins, chiefly the saturation level of the lipids and presence of cholesterol, and provide novel insight into factors of importance for build-up and reversibility of atherosclerotic plaque. In addition, the correlation between the results and well-established clinical risk factors suggests that the approach taken can be employed also for understanding a broader set of risk factors including, e.g., effects of triglycerides and oxidative stress, as well as local effects of drugs on atherosclerotic plaque formation.