Self-assembly of biomembranes results from the intricate interactions between water and the lipids’ hydrophilic head groups. Therefore, the lipid–water interplay strongly contributes to modulating membrane architecture, lipid diffusion, and chemical activity. Here, we introduce a new method of obtaining dehydrated, phase-separated, supported lipid bilayers (SLBs) solely by controlling the decrease of their environment’s relative humidity. This facilitates the study of the structure and dynamics of SLBs over a wide range of hydration states. We show that the lipid domain structure of phase-separated SLBs is largely insensitive to the presence of the hydration layer. In stark contrast, lipid mobility is drastically affected by dehydration, showing a 6-fold decrease in lateral diffusion. At the same time, the diffusion activation energy increases approximately 2-fold for the dehydrated membrane. The obtained results, correlated with the hydration structure of a lipid molecule, revealed that about six to seven water molecules directly hydrating the phosphocholine moiety play a pivotal role in modulating lipid diffusion. These findings could provide deeper insights into the fundamental reactions where local dehydration occurs, for instance during cell–cell fusion, and help us better understand the survivability of anhydrobiotic organisms. Finally, the strong dependence of lipid mobility on the number of hydrating water molecules opens up an application potential for SLBs as very precise, nanoscale hydration sensors.

The reconstruction of accurate yet simplified mimetic models of cell membranes is a very challenging goal of synthetic biology. To date, most of the research focuses on the development of eukaryotic cell membranes, while reconstitution of their prokaryotic counterparts has not been fully addressed, and the proposed models do not reflect well the complexity of bacterial cell envelopes. Here, we describe the reconstitution of biomimetic bacterial membranes with an increasing level of complexity, developed from binary and ternary lipid mixtures. Giant unilamellar vesicles composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); PC and phosphatidylglycerol (PG); PE and PG; PE, PG and cardiolipin (CA) at varying molar ratios were successfully prepared by the electroformation method. Each of the proposed mimetic models focuses on reproducing specific membrane features such as membrane charge, curvature, leaflets asymmetry, or the presence of phase separation. GUVs were characterized in terms of size distribution, surface charge, and lateral organization. Finally, the developed models were tested against the lipopeptide antibiotic daptomycin. The obtained results showed a clear dependency of daptomycin binding efficiency on the amount of negatively charged lipid species present in the membrane. We anticipate that the models proposed here can be applied not only in antimicrobial testing but also serve as platforms for studying fundamental biological processes in bacteria as well as their interaction with physiologically relevant biomolecules.

Cholesterol-rich lipid rafts are found to facilitate membrane fusion, central to processes like viral entry, fertilization, and neurotransmitter release. While the fusion process involves local, transient membrane dehydration, the impact of reduced hydration on cholesterol's structural organization in biological membranes remains unclear. Here, we employ confocal fluorescence microscopy and atomistic molecular dynamics simulations to investigate cholesterol behavior in phase-separated lipid bilayers under controlled hydration. We unveiled that dehydration prompts cholesterol release from raft-like domains into the surrounding fluid phase. Unsaturated phospholipids undergo more significant dehydration-induced structural changes and lose more hydrogen bonds with water than sphingomyelin. The results suggest that cholesterol redistribution is driven by the equalization of biophysical properties between phases and the need to satisfy lipid hydrogen bonds. This underscores the role of cholesterol-phospholipid-water interplay in governing cholesterol affinity for a specific lipid type, providing a new perspective on the regulatory role of cell membrane heterogeneity during membrane fusion.

The present study describes a novel antimicrobial surface using anodic oxidation of titanium and biofunctional detonation nanodiamonds (ND). ND have been loaded with antibiotics (amoxicillin or ampicillin) using poly(diallyldimethylammonium chloride) (PDDA). Successful conjugation with PDDA was determined by dynamic light scattering, which showed increase in the hydrodynamic diameter of ND agglomerates and shift of zeta potential towards positive values. The surface loading of amoxicillin was determined using UV–vis spectroscopy and the maximum of 44% surface loading was obtained. Biofunctional ND were immobilized by anodic oxidation within a titanium oxide layer, which was confirmed by scanning electron microscopy. The in vitro antimicrobial properties of ND suspensions were examined using Kirby-Bauer test with E. coli. Modified titanium surfaces comprising biofunctional ND were evaluated with E. coli inoculum by live/dead assay staining. Both biofunctional ND suspensions and modified titanium surfaces presented inhibition of bacteria growth and increase in bacteria lethality.

For over 40 years, Laurdan has been widely used as a universal fluorescent probe for the study of lipid membranes. However, recent molecular dynamics simulations have uncovered previously unknown properties of Laurdan, revealing that it can adopt distinct conformations within the lipid bilayer, thereby influencing its molecular orientation. Despite these findings, experimental and quantitative validation has been lacking. Here, we present the first experimental study of the orientation of Laurdan in a phase-separated supported lipid bilayer, directly linking its spatial orientation to its emission spectra in liquid-ordered (L_o) and liquid-disordered (L_d) phases. Using azimuthally and radially polarized excitation beams, we show that in the L_o phase, Laurdan molecules align more parallel to the membrane normal, whereas in the L_d phase, they adopt a more planar orientation within the membrane. Interestingly, the emission spectra for both excitation modes converge at shorter wavelengths, but show deviations at longer wavelengths, particularly in the L_d phase. By refining our understanding of the behavior of Laurdan in lipid membranes, this study underlines the critical role of the molecular orientation of the dye in fluorescence-based membrane studies and highlights the need for orientation-sensitive analysis in biophysical investigations.