The concentration of cholesterol in cell membranes affects membrane fluidity and thickness, and might regulate different processes such as the formation of lipid rafts. Since interpreting experimental data from biological membranes is rather intricate, investigations on simple models with biological relevance are necessary to understand the natural systems. We study the effect of cholesterol on the molecular structure of multi-lamellar vesicles (MLVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a phospholipid ubiquitous in cell membranes, with compositions in the range 0–60 mol% cholesterol. Order parameters, |S_CH|, are experimentally determined by using ¹H–¹³C solid-state nuclear magnetic resonance (NMR) spectroscopy with segmental detail for all parts of both the cholesterol and POPC molecules, namely the ring system and alkyl chain of the sterol, as well as the glycerol backbone, choline headgroup and the sn-1 and sn-2 acyl chains of POPC. With increasing cholesterol concentration the acyl chains gradually adopt a more extended conformation while the orientation and dynamics of the polar groups are rather unaffected. Additionally, we perform classical molecular dynamics simulations on virtual bilayers mimicking the POPC–cholesterol MLVs investigated by NMR. Good agreement between experiments and simulations is found for the cholesterol alignment in the bilayer and for the |S_CH| profiles of acyl chains below 15 mol% cholesterol. Deviations occur for the choline headgroup and glycerol backbone parts of POPC, as well as for the phospholipid and cholesterol alkyl chains at higher cholesterol concentrations. The unprecedented detail of the NMR data enables a more complete comparison between simulations and experiments on POPC–cholesterol bilayers and may aid in developing more realistic model descriptions of biological membranes.

Although it is well described in model membranes, little is known about phase separation in biological membranes. Here, we provide evidence for a coexistence of at least two different lipid bilayer phases in the apical plasma membrane of epithelial cells. Phase connectivity was assessed by measuring long-range diffusion of several membrane proteins by fluorescence recovery after photobleaching in two polarized epithelial cell lines and one fibroblast cell line. In contrast to the fibroblast plasma membrane, in which all of the proteins diffused with similar characteristics, in the apical membrane of epithelial cells the proteins could be divided into two groups according to their diffusion characteristics. At room temperature (≈25°C), one group showed fast diffusion and complete recovery. The other diffused three to four times slower and, more importantly, displayed only partial recovery. Only the first group comprises proteins that are believed to be associated with lipid rafts. The partial recovery is not caused by topological constraints (microvilli, etc.), cytoskeletal constraints, or protein-protein interactions, because all proteins show 100% recovery in fluorescence recovery after photobleaching experiments at 37°C. In addition, the raft-associated proteins cannot be coclustered by antibodies on the apical membrane at 12°C. The interpretation that best fits these data is that the apical membrane of epithelial cells is a phase-separated system with a continuous (percolating) raft phase <25°C in which isolated domains of the nonraft phase are dispersed, whereas at 37°C the nonraft phase becomes the continuous phase with isolated domains of the raft phase dispersed in it.

Small extracellular vesicles (sEVs), through their natural ability to interact with biological membranes and exploit endogenous processing pathways to convey biological information, are quintessential for the delivery of therapeutically relevant compounds, such as microRNAs (miRNAs) and proteins. Here, we used a fluorescently‐labelled miRNA to quantify the efficiency of different methods to modulate the cargo of sEVs. Our results showed that, compared with electroporation, heat shock, permeation by a detergent‐based compound (saponin) or cholesterol‐modification of the miRNA, Exo‐Fect was the most efficient method with > 50% transfection efficiency. Furthermore, qRT‐PCR data showed that, compared with native sEVs, Exo‐Fect modulation led to a > 1000‐fold upregulation of the miRNA of interest. Importantly, this upregulation was observed for sEVs isolated from multiple sources. The modulated sEVs were able to delivery miR‐155‐5p into a reporter cell line, confirming the successful delivery of the miRNA to the target cell and, more importantly, its functionality. Finally, we showed that the membrane of Exo‐Fect‐loaded sEVs was altered compared with native sEVs and that enhanced the internalization of Exo‐Fect‐loaded sEVs within the target cells and decreased the interaction of those modulated sEVs with lysosomes.

Broad-spectrum antimicrobial activity of quaternary ammonium surfactants (QAS) makes them attractive and cheap topical prophylactic options for sexually transmitted infections and perinatal vertically transmitted urogenital infections. Although attributed to their high affinity for biological membranes, the mechanisms behind QAS microbicidal activity are not fully understood. We evaluated how QAS structure affects antimicrobial activity and whether this can be exploited for use in prophylaxis of bacterial infections. Acute toxicity of QAS to in vitro models of human epithelial cells and bacteria were compared to identify selective and potent bactericidal agents. Bacterial cell viability, membrane integrity, cell cycle and metabolism were evaluated to establish the mechanisms involved in selective toxicity of QAS. QAS toxicity normalized relative to surfactant critical micelle concentration showed n-dodecylpyridinium bromide (C₁₂PB) to be the most effective, with a therapeutic index of ∼10 for an MDR strain of Escherichia coli and >20 for Neisseria gonorrhoeae after 1 h of exposure. Three modes of QAS antibacterial action were identified: impairment of bacterial energetics and cell division at low concentrations; membrane permeabilization and electron transport inhibition at intermediate doses; and disruption of bacterial membranes and cell lysis at concentrations close to the critical micelle concentration. In contrast, toxicity to mammalian cells occurs at higher concentrations and, as we previously reported, results primarily from mitochondrial dysfunction and apoptotic cell death. Our data show that short chain (C₁₂) n-alkyl pyridinium bromides have a sufficiently large therapeutic window to be good microbicide candidates.

Several classes of polysaccharides have been described to have hypocholesterolemic potential, namely cholesterol bioaccessibility and bioavailability. This review will highlight the main mechanisms by which polysaccharides are known to affect cholesterol homeostasis at the intestine, namely the effect (i) of polysaccharide viscosity and its influence on cholesterol bioaccessibility; (ii) on bile salt sequestration and its dependence on the structural diversity of polysaccharides; (iii) of bio-transformations of polysaccharides and bile salts by the gut microbiota. Different quantitative structure–hypocholesterolemic activity relationships have been explored depending on the mechanism involved, and these were based on polysaccharide physicochemical properties, such as sugar composition and ramification degree, linkage type, size/molecular weight, and charge. The information gathered will support the rationalization of polysaccharides’ effect on cholesterol homeostasis and highlight predictive rules towards the development of customized hypocholesterolemic functional food.