Localization of Cholesterol within Supported Lipid Bilayers Made of a Natural Extract of Tailor-Deuterated Phosphatidylcholine.

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.

On the antimicrobial activity of various peptide-based dendrimers of similar architecture.

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.

Unraveling dendrimer translocation across cell membrane mimics.

Poly(amidoamine) (PAMAM) dendrimers are promising candidates in several applications within the medical field. However, it is still to date not fully understood whether they are able to passively translocate across lipid bilayers. Recently, we used fluorescence microscopy to show that PAMAM dendrimers induced changes in the permeability of lipid membranes but the dendrimers themselves could not translocate to be released into the vesicle lumen. Because of the lack of resolution, these experiments could not assess whether the dendrimers were able to translocate but remained attached to the membrane. Using quartz crystal microbalance with dissipation monitoring and neutron reflectivity, a structural investigation was performed to determine how dendrimers interact with zwitterionic and negatively charged lipid bilayers. We hereby show that dendrimers adsorb on top of lipid bilayers without significant dendrimer translocation, regardless of the lipid membrane surface charge. Thus, most likely dendrimers are actively transported through cell membranes by protein-mediated endocytosis in agreement with previous cell studies. Finally, the higher activity of PAMAM dendrimers for phosphoglycerol-containing membranes is in line with their high antimicrobial activity against Gram-negative bacteria.

Effects of ethylene oxide chain length on crystallization of polysorbate 80 and its related compounds.

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.

Lipoprotein ability to exchange and remove lipids from model membranes as a function of fatty acid saturation and presence of cholesterol.

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.

Towards biomimics of cell membranes: Structural effect of phosphatidylinositol triphosphate (PIP3) on a lipid bilayer.

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.

Understanding the formation of supported lipid bilayers via vesicle fusion-A case that exemplifies the need for the complementary method approach (Review).

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.