Aspirin Interacts with Cholesterol-Containing Membranes in a pH-Dependent Manner.

Aspirin has been used for broad therapeutic treatment, including secondary prevention of cardiovascular disease associated with increased cholesterol levels. Aspirin and other nonsteroidal anti-inflammatory drugs have been shown to interact with lipid membranes and change their biophysical properties. In this study, mixed lipid model bilayers made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) comprising varying concentrations of cholesterol (10:1, 4:1, and 1:1 mole ratio of lipid:chol), prepared by the droplet interface bilayer method, were used to examine the effects of aspirin at various pH on transbilayer water permeability. The presence of aspirin increases the water permeability of POPC bilayers in a concentration-dependent manner, with a greater magnitude of increase at pH 3 compared to pH 7. In the presence of cholesterol, aspirin is similarly shown to increase water permeability; however, the extent of the increase depends on both the concentration of cholesterol and the pH, with the least pronounced enhancement in water permeability at high cholesterol levels at pH 7. A fusion of data from differential scanning calorimetry, confocal Raman microspectrophotometry, and interfacial tensiometric measurements demonstrates that aspirin can promote significant thermal, structural, and interfacial property perturbations in the mixed-lipid POPC or DOPC membranes containing cholesterol, indicating a disordering effect on the lipid membranes. Our findings suggest that aspirin fluidizes phosphocholine membranes in both cholesterol-free and cholesterol-enriched states and that the overall effect is greater when aspirin is in a neutral state. These results confer a deeper comprehension of the divergent effects of aspirin on biological membranes having heterogeneous compositions, under varying physiological pH and different cholesterol compositions, with implications for a better understanding of the gastrointestinal toxicity induced by the long term use of this important nonsteroidal anti-inflammatory molecule.

Trans-Resveratrol Decreases Membrane Water Permeability: A Study of Cholesterol-Dependent Interactions.

Resveratrol (RSV), a biologically active plant phenol, has been extensively investigated for cancer prevention and treatment due to its ability to regulate intracellular targets and signaling pathways which affect cell growth and metastasis. The non-specific interactions between RSV and cell membranes can modulate physical properties of membranes, which in turn can affect the conformation of proteins and perturb membrane-hosted biological functions. This study examines non-specific interactions of RSV with model membranes having varying concentrations of cholesterol (Chol), mimicking normal and cancerous cells. The perturbation of the model membrane by RSV is sensed by changes in water permeability parameters, using Droplet Interface Bilayer (DIB) models, thermotropic properties from Differential Scanning Calorimetry, and structural properties from confocal Raman spectroscopy, all of which are techniques not complicated by the use of probes which may themselves perturb the membrane. The nature and extent of interactions greatly depend on the presence and absence of Chol as well as the concentration of RSV. Our results indicate that the presence of RSV decreases water permeability of lipid membranes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), indicating a capability for RSV in stiffening fluidic membranes. When Chol is present, however, (at 4:1 and 2:1 mol ratio DOPC to cholesterol), the addition of RSV has no significant effect upon the water permeability. DSC thermograms show that RSV interacts with DOPC and DOPC/Chol bilayers and influences their thermotropic phase behavior in a concentration-dependent manner, by decreasing the main phase transition temperature and enthalpy, with a phase separation shown at the higher concentrations of RSV. Raman spectroscopic studies indicate an ordering effect of RSV on DOPC supported bilayer, with a lesser extent of ordering in the presence of Chol. Combined results from these investigations highlight a differential effect of RSV on Chol-free and Chol-enriched membranes, respectively, which results constitute a bellwether for increased understanding and effective use of resveratrol in disease therapy including cancer.

Ibuprofen and the Phosphatidylcholine Bilayer: Membrane Water Permeability in the Presence and Absence of Cholesterol.

The interactions between drugs and cell membranes can modulate the structural and physical properties of membranes. The resultant perturbations of the membrane integrity may affect the conformation of the proteins inserted within the membrane, disturbing the membrane-hosted biological functions. In this study, the droplet interface bilayer (DIB), a model cell membrane, is used to examine the effects of ibuprofen, a nonsteroidal anti-inflammatory drug (NSAID), on transbilayer water permeability, which is a fundamental membrane biophysical property. Our results indicate that the presence of neutral ibuprofen (pH 3) increases the water permeability of the lipid membranes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). When cholesterol is present with the DOPC, however, the water permeability is not influenced by addition of ibuprofen, regardless of the cholesterol content in DOPC. Given the fact that cholesterol is generally considered to impact packing in the hydrocarbon chain regions, our findings suggest that a potential competition between opposing effects of ibuprofen molecules and cholesterol on the hydrocarbon core environment of the phospholipid assembly may influence the overall water transport phenomena. Results from confocal Raman microspectroscopy and interfacial tensiometry show that ibuprofen molecules induce substantial structural and dynamic changes in the DOPC lipid bilayer. These results, demonstrating that the presence of ibuprofen increases the water permeability of pure DOPC but not that of DOPC-cholesterol mixtures, provide insight into the differential effect of a representative NSAID on heterogeneous biological membranes, depending upon the local composition and structure, results which will signal increased understanding of the gastrointestinal damage and toxicity induced by these molecules.

Structural determination of model phospholipid membranes by Raman spectroscopy: Laboratory experiment.

In an upper-division interdisciplinary laboratory experiment, students use Raman spectroscopy to highlight how the overall structure and conformational order of lipid bilayers can be influenced by their individual phospholipid composition. Students prepare a supported lipid bilayer, as a model cell membrane, by spreading liposomes made of various phospholipids on a solid support. The characterization of phospholipid bilayers, a major component of cellular membranes, can advance our fundamental understanding of important biological phenomena, with significant implications in various fields including drug delivery and development. We use Raman spectroscopy as an analytical tool to investigate the structural and packing properties of model cell membranes. The spectral frequency, intensity, and line-width of lipid Raman bands are extremely sensitive to structural alterations. This experimental module effectively exposes students to the fundamentals of Raman spectroscopy and teaches students the importance of interdisciplinary education as they integrate concepts from chemical structure, molecular interactions, and analytical spectroscopic techniques to gain a more holistic understanding of biological membrane properties.