Preparation and Characterization of Solid-Supported Lipid Bilayers Formed by Langmuir-Blodgett Deposition: A Tutorial.

The structure, phase behavior, and properties of cellular membranes are derived from their composition, which includes phospholipids, sphingolipids, sterols, and proteins with various levels of glycosylation. Because of the intricate nature of cellular membranes, a plethora of in vitro studies have been carried out with model membrane systems that capture particular properties such as fluidity, permeability, and protein binding but vastly simplify the membrane composition in order to focus in detail on a specialized property or function. Supported lipid bilayers (SLB) are widely used as archetypes for cellular membranes, and this instructional review primarily focuses on the preparation and characterization of SLB systems formed by Langmuir deposition methods. Typical characterization methods, which take advantage of the planar orientation of SLBs, are illustrated, and references that go into more depth are included. This invited instructional review is written so that nonexperts can quickly gain in-depth knowledge regarding the preparation and characterization of SLBs. In addition, this work goes beyond traditional instructional reviews to provide expert readers with new results that cover a wider range of SLB systems than those previously reported in the literature. The quality of an SLB is frequently not well described, and details such as topological defects can influence the results and conclusions of an individual study. This article quantifies and compares the quality of SLBs fabricated from a variety of gel and fluid compositions, in correlation with preparation techniques and parameters, to generate general rules of thumb to guide the construction of designed SLB systems.

Interaction Forces between Lipid Rafts.

Cellular membranes containing sphingolipids and cholesterol have been shown to self-organize into lipid rafts-specialized domains that host integral membrane proteins and modulate the bioactivity of cells. In this work, force-distance profiles between raft membranes in the liquid-ordered phase consisting of singly unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a complex mixture of brain sphingomyelin (BSM), and cholesterol were measured using the surface force apparatus (SFA). Two distinct force profiles were detected corresponding to uniform raft membranes and raft membranes with a higher level of topological membrane defects (heterogeneous) as corroborated by atomic force microscopy (AFM) scans. In all cases a weak, long-range electrostatic repulsion was observed with some variation in the surface charge density. The variation in electrostatic repulsion was attributed to charged lipid species primarily from the constituent lipids in the BSM mixture. The adhesion between the uniform raft membranes was comparable to our previous work with pure component, liquid-ordered POPC-DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)-cholesterol membranes. Raft membranes with more topological defects adhered more strongly owing to hydrophobic attraction between exposed acyl chains. Even though the rafts were in the liquid-ordered phase and membrane defects were present in the contact region, the raft membranes were stable, and no structural rearrangement was observed throughout the measurements. Our findings demonstrate that liquid-ordered membranes are stable to mechanical loading and not particularly sensitive to compositional variation.

Interaction forces and membrane charge tunability: Oleic acid containing membranes in different pH conditions.

Oleic acid is known to interact with saturated lipid molecules and increase the fluidity of gel phase lipid membranes. In this work, the thermodynamic properties of mixed monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and oleic acid at the air-water interface were determined using Langmuir isotherms. The isotherm study revealed an attractive interaction between oleic acid and DPPC. The incorporation of oleic acid also monotonically decreased the elastic modulus of the monolayer indicative of higher fluidity with increasing oleic acid content. Using the surface force apparatus, intermembrane force-distance profiles were obtained for substrate supported DPPC membranes containing 30mol% oleic acid at pH5.8 and 7.4. Three different preparation conditions resulted in distinct force profiles. Membranes prepared in pH5.8 subphase had a low number of nanoscopic defects ≤1% and an adhesion magnitude of ~0.6mN/m. A slightly higher defect density of 1-4% was found for membranes prepared in a physiological pH7.4 subphase. The presence of the exposed hydrophobic moieties resulted in a higher adhesion magnitude of 2.9mN/m. Importantly, at pH7.4, some oleic acid deprotonates resulting in a long-range electrostatic repulsion. Even though oleic acid increased the DPPC bilayer fluidity and the number of defects, no membrane restructuring was observed indicating that the system maintained a stable configuration.

Characterization of solid-supported dipalmitoylphosphatidylcholine membranes containing cholesterol.

The incorporation of cholesterol into dipalmitoylphosphatidylcholine (DPPC) membranes, even in small amounts, has been shown to significantly alter the properties of the membrane. In this work, force-distance interaction profiles of DPPC membranes containing 8 mol % cholesterol obtained using the surface force apparatus are analyzed in the context of high-resolution structural characterization by atomic force microscopy and neutron reflectometry. The adhesion between the mixed membranes was greater than that for pure DPPC and was variable-depending on the number of defects in the outer membrane leaflets. These defects were only detectable by atomic force microscopy and had an average size of 230 ± 30 nm and 1-5% surface density in the outer leaflet. The adhesion between the membranes monotonically increased as the thickness of the membrane decreased-in direct correlation with the number of defects present (exposed hydrophobic groups) in the membrane contact region. Because of the low diffusion rate of gel-phase membranes, the interaction force profiles were stable and no membrane restructuring was observed.

Interaction forces between ternary lipid bilayers containing cholesterol.

Interaction force-distance profiles between substrate-supported membranes composed of equimolar ternary mixtures of unsaturated phosphotidylcholine (PC) lipid, saturated PC lipid, and cholesterol were determined using the surface force apparatus. Both double and single unsaturated PC lipids were studied. In all cases, the membranes were slightly negatively charged, resulting in a weak, long-range electrostatic repulsion. Corroborative atomic force microscopy, zeta potential, and fluorescence microscopy measurements were used to establish that a small level of charged lipid impurities (∼1/400 lipid molecules) were responsible for the repulsive electrostatic interaction between the membranes. At contact, the membranes were adhesive. The magnitude of the adhesion was greater than the van der Waals interaction between pure PC membranes without cholesterol. The enhanced adhesion was primarily attributed to hydrophobic attraction due to the presence of nanoscopic membrane defects which exposed the underlying membrane leaflet. The interaction force-distance profiles also demonstrated that the nanoscopic defects enabled membrane restructuring in the contact region.