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We discovered a cascade vesicle division system driven by osmotic inflation. Binary giant unilamellar vesicles (GUVs) composed of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and cholesterol (Chol) were subjected to an osmotic pressure difference by encapsulating membrane-impermeable osmolytes (typically sucrose) in an external aqueous solution containing membrane-permeable osmolytes (typically fructose). This simple setup enabled the mother GUVs to repeatedly form small membrane buds and subsequently undergo divisions over several hundred seconds, resulting in the production of approximately 30-300 daughter GUVs from a single mother GUV. The observed morphological change of GUVs is well described by the mechanical balance between membrane bending, membrane tension, and osmotic pressure difference based on the spontaneous curvature model. This "osmotic spawning" behavior of GUVs does not rely on chemical reactions or functional macromolecules. Therefore, this cascade division system is compatible with various chemical systems and has the potential to implement proliferation ability in artificial cells, drug delivery systems, and protocells simply by modifying their membrane compartments and osmolytes.

Lipid compositional asymmetry across the leaflets of the plasma membrane is an ubiquitous feature in eukaryotic cells. How this asymmetry is maintained is thought to be primarily controlled by active transport of lipids between leaflets. This strategy is facilitated by the fact that long-tail phospholipids and sphingolipids diffuse through the lipid bilayer slowly-taking many hours or days. However, a lipid like cholesterol-which is the most abundant lipid in the plasma membrane of animal cells-has been harder to pinpoint in terms of its favored side. In this work we show that, when a saturated lipid is added to a mix of the unsaturated lipid palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesterol, both cholesterol and the long-tail phospholipids organize asymmetrically across the membrane's leaflets naturally. In these extruded unilamellar vesicles, most cholesterol as well as the saturated lipid-dipalmitoylphosphatidylcholine or sphingomyelin-segregated to the inner leaflet while POPC preferentially localized in the outer leaflet. This asymmetric arrangement generated a slight phospholipid number imbalance favoring the outer leaflet and thus opposite to where cholesterol and the saturated lipids preferentially partitioned. These results were obtained using magic-angle spinning nuclear magnetic resonance (MAS NMR) in combination with small-angle neutron scattering (SANS) using isotope labeling to differentiate lipid species. We suggest that sidedness in membranes can be driven by thermodynamic processes. In addition, our MAS NMR results show that the lower bound for cholesterol's flip-flop half-time at 45°C is 10 ms, which is at least two orders of magnitude slower than current MD simulations predict. This result stands in stark contrast to previous work that suggested that cholesterol's flip-flop half-time at 37°C has an upper bound of 10 ms., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The unidirectional movement of nascent secretory proteins in the cell is primarily assisted by the signal recognition particles (SRP). However, this does not completely justify the importance of the signal peptide (SP) which gets eliminated after the protein translocation. We have earlier demonstrated that a negatively charged lipid such as POPG plays an important role in the higher binding affinity and cholesterol-discriminating ability of the apolipoprotein E (ApoE) SP. In this present work, we aimed to understand the role of sphingomyelin, an important constituent of ER, on the membrane binding of ApoE SP. Our results demonstrate that sphingomyelin promotes membrane binding but cannot discriminate cholesterol. However, sphingomyelin shows a synergistic effect with POPG toward the membrane binding of the ApoE SP. We have further shown that the membrane domains do not have any impact on the binding of ApoE SP. Based on our results we propose that the lipid composition of the endoplasmic reticulum (ER) where ApoE translocates, enhances the binding of the ApoE signal peptide to the ER membrane., (© 2024 Wiley-VCH GmbH.)

Phytosterols are structurally similar to cholesterol but they are much less absorbed (<2%) than cholesterol (>50%) in the intestine. We hypothesize that phytosterols are poor substrates of intestinal acyl-CoA: cholesterol acyltransferase 2 (ACAT2), and thus minimal phytosterol esters are formed and packed into chylomicrons, leading to their low absorption. Two isotope tracing models, including a radioactive hamster microsomal ACAT2 reaction model and a differentiated Caco-2 cell model, were established to examine the specificity of ACAT2 to various sterols, including cholesterol, sitosterol, stigmasterol, and campesterol. Both models consistently demonstrated that only cholesterol but not phytosterols could be efficiently esterified by ACAT2 in a time- and dose-dependent manner. Molecular docking further suggested that unfavorable interactions existed between ACAT2 and phytosterols. In conclusion, phytosterols are poor substrates of ACAT2 and thus minimally absorbed. This work provides a theoretical basis for the use of phytosterol-based supplements in treating dyslipidemia and preventing heart diseases., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Signal transduction across biological membranes enables cells to detect and respond to diverse chemical or physical signals, and replicating these complex biological processes through synthetic methods is of significant interest in synthetic biology. Here we present an artificial signal transduction system using oriented cholesterol-tagged triplex DNA (TD) as synthetic receptors to transmit and amplify signals across lipid bilayer membranes through H + -mediated TD conformational transitions from duplex to triplex. An auxiliary sequence, complementary to the third strand of the TD, ensures a controlled and preferred outward orientation of cholesterol-tagged TD on membranes. Upon external H + stimuli, the conformational change triggers the translocation of the third strand from the outer to the inner membrane leaflet, resulting in effective transmembrane signal transduction. This mechanism enables fluorescence resonance energy transfer (FRET), selective photocleavage, catalytic signal amplification, and logic gate modulation within vesicles. Our findings demonstrate that these TD-based receptors mimic the functional dynamics of natural G protein-coupled receptors (GPCRs), providing a foundation for advanced applications in biosensing, cell signaling modulation, and targeted drug delivery systems., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The challenge of enhancing wound healing and skin regeneration, particularly in conditions like burns and diabetic wounds, necessitates innovative solutions. Cholesterol, often associated with cardiovascular diseases, plays vital roles in cellular functions, maintaining skin integrity and preserving the skin barrier. Here, we explore cholesterol's significance, its influence on keratinocytes, and its potential application in skin regeneration. The study utilizes electrospun polyimide (PI) fibers as a cholesterol carrier model and investigates its impact on HaCaT keratinocytes, marking the first time tracked cholesterol delivery from the scaffold into cells. We demonstrate that an optimal concentration of 0.7 mM cholesterol in the medium enhances cell proliferation, while higher concentrations have negative effects. Cholesterol-enriched scaffolds significantly increase cell proliferation and replicative activity, especially in a 3D culture environment. Moreover, cholesterol influences keratinocyte differentiation, promoting early differentiation while inhibiting late differentiation. These findings suggest that cholesterol-loaded scaffolds can have applications in wound healing by promoting cell growth, regulating differentiation, and potentially accelerating wound closure. Further research in this area will lead to innovative wound management and tissue regeneration strategies.

Liposomal drug delivery is a promising approach for delivering therapeutics effectively. While most liposomes are designed to be nanometer-sized for efficient cellular uptake, micron-sized liposomes are gaining interest due to their larger drug-loading capacity. When combined with macroscale structures, such as implants and hydrogels, they offer prolonged therapeutic delivery. This study investigates how solvents affect the production of micron-sized liposomes, with or without cholesterol, using the reverse-phase evaporation (RPE) method. Although the RPE method is established for producing micron-sized liposomes, the influence of solvents on liposome size and uniformity is not well understood. The study explores whether controlling the size of inverse micelles, an intermediate product, through the use of different organic solvents affects the final liposome size. Three solvents─diethyl ether, methanol, and acetone─were tested for their effect on the formation of inverse micelles and liposomes and their sizes. Results showed that without cholesterol, diethyl ether produced uniform inverse micelles, leading to mostly nanosized liposomes. Methanol and acetone resulted in phase separation, preventing uniform liposome formation, although some micron-sized liposomes appeared. The acetone sample yielded mostly oil droplets. The results showed that forming inverse micelles lead to nanosized liposomes. With cholesterol, phase separation was dominant in methanol, but micron-sized liposomes still formed. Across all cases, cholesterol reduced the liposome size. The findings reveal that inverse micelles are not always reliable predictors of the final liposome size and that the RPE method is highly sensitive to solvent polarity and lipid-solvent interactions. Overall, the findings of this study provide valuable insights into how the choice of solvent and lipid composition can influence the production of liposomes via the RPE method. These insights are critical for optimizing liposome production and influencing future designs of liposomal drug delivery systems.

Caveolins are lipid-binding proteins that can organize membrane remodeling and oligomerize into the 8S complex. The CAV1-8S complex comprises a disk-like structure, about 15 nm in diameter, with a central beta barrel. Further oligomerization of 8S complexes remodels the membrane into caveolae vessels, with a dependence on cholesterol concentration. However, the molecular mechanisms behind membrane remodeling and cholesterol filtering are still not understood. Performing atomistic molecular dynamics simulations in combination with advanced sampling techniques, we describe how the CAV1-8S complex bends the membrane and accumulates cholesterol. Here, our simulations show an enhancing effect by the palmitoylations of CAV1, and we predict that the CAV1-8S complex can extract cholesterol molecules from the lipid bilayer and accommodate them in its beta barrel. Through backmapping to the all-atom level, we also conclude that the Martini v.2 coarse-grained force field overestimates membrane bending, as the atomistic simulations exhibit only very localized bending., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Berberine (BBR) is a natural molecule with noteworthy pharmacological properties, including the prevention of antibiotic resistance in Gram-negative bacteria. However, its oral bioavailability is poor, thus resulting in an impaired absorption and efficacy in humans. In combination with other drugs, liposomes have been shown to enhance the availability of the drug, representing a smart delivery system to target tissues and reduce negative side effects. To date, there is a lack of studies on BBR and liposomes that enable the rationalization and molecular-based design of such formulations for future use in humans. In this work, the encapsulation of BBR into liposomes is proposed to overcome current limitations using a combination of experimental and computational assays to rationalize the membrane composition of liposomes that maximizes BBR encapsulation. First, the encapsulation efficiency was measured for several membrane compositions, revealing that it is enhanced by cholesteryl hemisuccinate and, to a lesser extent, by cholesterol. The physical basis of the BBR encapsulation efficiency and permeability was clarified using molecular dynamics simulation: using the lipid composition, one can tune the capability of membranes to attract, i.e., to adsorb, the molecules onto their surface. Overall, these findings suggest a rational strategy to maximize the encapsulation efficiency of liposomes by using negatively charged lipids, thus representing the basis for designing delivery systems for BBR, useful to treat, e.g., antibiotic resistance.

The stratum corneum (SC) plays the most important role in the absorption of topical and transdermal drugs. In this study, we developed a multi-layered SC model using coarse-grained molecular dynamics (CGMD) simulations of ceramides, cholesterol, and fatty acids in equimolar proportions, starting from two different initial configurations. In the first approach, all ceramide molecules were initially in the hairpin conformation, and the membrane bilayers were pre-formed. In the second approach, ceramide molecules were introduced in either the hairpin or splayed conformation, with the lipid molecules randomly oriented at the start of the simulation. The aim was to evaluate the effects of lipid chain length on the structural and dynamic properties of SC. By incorporating ceramides and fatty acids of different chain lengths, we simulated the SC membrane in healthy and diseased states. We calculated key structural properties including the thickness, normalized lipid area, lipid tail order parameters, and spatial ordering of the lipids from each system. The results showed that systems with higher ordering and structural integrity contained an equimolar ratio of ceramides (chain length of 24 carbon atoms), fatty acids with chain lengths ≥ of 20 carbon atoms, and cholesterol. In these systems, strong apolar interactions between the ceramide and fatty acid long acyl chains restricted the mobility of the lipid molecules, thereby maintaining a compact lipid headgroup region and high order in the lipid tail region. The simulations also revealed distinct flip-flop mechanisms for cholesterol and fatty acid within the multi-layered membrane. Cholesterol is mostly diffused through the tail-tail interface region of the membrane and could flip-flop in the same bilayer. In contrast, fatty acids flip-flopped between adjacent leaflets of two bilayers in which the tails crossed the thinner headgroup region of the membrane. To conclude, our SC model provides mechanistic insights into lipid mobility and is flexible in its design and composition of different lipids, enabling studies of varying skin conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cholesterol is a fundamental lipid prevalent in eukaryotic cell membranes and circulating in the bloodstream bound to lipoproteins. It serves as a precursor to steroid hormones and is regarded as a biomarker for cardiovascular disease and other metabolic disorders. Numerous cholesterol detection methods predominantly rely on enzymes, which suffer from instability, leading to non-cost-effective biosensors with low sensitivity and poor reusability. Therefore, monitoring cholesterol levels with a feasible, rapid, and stable biosensor is critical for diagnosing and treating various disorders. This study aimed to develop a non-enzymatic cholesterol biosensor based on a selected cholesterol recognition peptide as the detection element. Screen-printed carbon electrodes (SPEs) modified with biocompatible poly-L-lactic acid (PLLA) porous nanomembranes (NMs) were utilized as support for the covalent immobilization of the peptide. Data obtained from electrochemical impedance spectroscopy (EIS) demonstrated the peptide's effective binding affinity towards cholesterol, paving the way for its implementation. The determination of cholesterol with the proposed biosensor exhibited a low limit of detection of 6.31 μM with linear responses ranging from 2-15 μM and 20-40 μM. These findings present an alternative method for cholesterol sensing by integrating novel peptides as biorecognition motifs with biocompatible polymeric materials, potentially useful as biocompatible and future point-of-care sensors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The potency of anesthetics with various structures increases exponentially with lipophilicity, which is the Meyer-Overton (MO) correlation discovered over 120 years ago. The MO correlation was also observed with various biological effects and chemicals, including alcohols; thus, the correlation represents a fundamental relationship between chemicals and organisms. The MO correlation was explained by the lipid and protein theories, although the principle remains unknown because these are still debating., Methods: The gentle hydration method was used to form giant unilamellar vesicles (GUVs) consisting of high- and low-melting phospholipids and cholesterol in the presence of n-alcohols (C 2 -C 12 ). Confocal fluorescence microscopy was used to determine the percentage of GUVs with domains in relation to the n-alcohol concentrations., Results: n-Alcohols inhibited the domain formation of GUVs, and the half inhibitory concentration (IC 50 ) in the aqueous phase (C w ) decreased exponentially with increasing chain length (lipophilicity). In contrast, the membrane concentrations (C m ) of alcohols for the inhibition, which is a product of the membrane-water partition coefficient and the IC 50 values, remained constant irrespective of the chain length., Conclusions: The MO correlation is established in GUVs, which supports the lipid theory. When alcohols reach the same critical concentration in the membrane, similar biological effects appear irrespective of the chain length, which is the principle underlying the MO correlation., General Significance: The protein theory states that a highly lipophilic compound targets minor membrane proteins due to the low C w . However, our lipid theory states that the compound targets various membrane proteins due to the high C m ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Milk fat globule membrane (MFGM) promotes the lateral phase separation of milk lipids and stabilizes the fat globules in milk. The composition and structures of lipids have a significant impact on physicochemical properties of MFGM, which in turn influences the digestion and absorption of milk lipids. Phospholipids (PL), sphingolipids, and cholesterol are the major lipid constituents of MFGM. While the effects of the head-group and structure of the fatty acids (FAs) on membrane properties are commonly studied, little is known on the impact of PL regioisomerism. The present study investigated the impact of phosphatidylcholine (PC) regioisomerism on lateral segregation of milk-sphingomyelin (milk-SM) as well as the influence on the interaction of milk-SM with ceramide and cholesterol in simulated membrane systems. The regioisomer pairs of four molecular species PC 16:0/18:1n-9, PC 16:0/18:2n-6, PC 16:0/18:3n-3, and PC 16:0/20:4n-6 were included in this study. The lateral segregation was determined using lifetime analysis of trans-parinaric acid (tPA) fluorescence. Thermostability of the domains was detected using steady-state anisotropy of tPA. Our results demonstrated a clear impact of PC regioisomerism on membrane properties. PC regioisomers having the unsaturated FAs at the sn-2 position enhanced the lateral segregation of milk-SM with and without the presence of ceramide and cholesterol compared to the regioiosmers having 16:0 at the sn-2 position. Furthermore, the characteristics i. e. the acyl chain length and degree of unsaturation of sn-2 FA of the PCs had a major impact on the milk-SM gel phase and the intermolecular forces between milk-SM and ceramide/cholesterol. This work is the first investigation showing the effect of PL regioisomerism on milk-SM domains, which might have significant influence on functional properties of MFGM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The discovery of a novel sphingolipid subclass, the (1-deoxy)sphingolipids, which lack the 1-hydroxy group, attracted considerable attention in the last decade, mainly due to their involvement in disease. They differed in their physico-chemical properties from the canonical (or 1-hydroxy) sphingolipids and they were more toxic when accumulated in cells, inducing neurodegeneration and other dysfunctions. (1-Deoxy)ceramides, (1-deoxy)dihydroceramides, and (1- deoxymethyl)dihydroceramides, the latter two containing a saturated sphingoid chain, have been studied in this work using differential scanning calorimetry, confocal fluorescence and atomic force microscopy, to evaluate their behavior in bilayers composed of mixtures of three or four lipids. When compared to canonical ceramides (Cer), a C16:0 (1-deoxy)Cer shows a lower miscibility in mixtures of the kind C16:0 sphingomyelin/cholesterol/XCer, where XCer is any (1-deoxy)ceramide, giving rise to the coexistence of a liquid-ordered phase and a gel phase. The latter resembles, in terms of thermotropic behavior and nanomechanical resistance, the gel phase of the C16:0 sphingomyelin/cholesterol/C16:0 Cer mixture [Busto et al., Biophys. J. 2014, 106, 621-630]. Differences are seen between the various C16:0 XCer under study in terms of nanomechanical resistance, bilayer thickness and bilayer topography. When examined in a more fluid environment (bilayers based on C24:1 SM), segregated gel phases are still present. Probably related to such lateral separation, XCer preserve the capacity for membrane permeation, but their effects are significantly lower than those of canonical ceramides. Moreover, C24:1 XCer show significantly lower membrane permeation capacity than their C16:0 counterparts. The above data may be relevant in the pathogenesis of certain sphingolipid-related diseases, including certain neuropathies, diabetes, and glycogen storage diseases., Competing Interests: Declaration of Competing Interest WAS was an employee of Avanti Polar Lipids (Alabaster, AL). The rest of authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

It was the aim of this study to design charge converting lipid nanoparticles (LNP) via a microfluidic mixing technique used for the preparation and coating of LNP. LNP consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, N-(carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG-2000-DSPE), and various cationic surfactants were prepared at diverging flow rate ratios (FRR) via microfluidic mixing. Utilizing a second chip in the microfluidic set-up, LNP were coated with polyoxyethylene (9) nonylphenol monophosphate ester (PNPP). LNP were examined for their stability in different physiologically relevant media as well as for hemolytic and cytotoxic effects. Finally, phosphate release and charge conversion of PNPP-coated LNP were evaluated after incubation with alkaline phosphatase and on Caco2-cells. LNP produced at an FRR of 5:1 exhibited a size between 80 and 150 nm and a positive zeta potential. Coating with PNPP within the second chip led to LNP exhibiting a negative zeta potential. After incubation with 1 U/ml alkaline phosphatase for 4 h, zeta potential of the LNP containing 1,2-dioleoyloxy-3-trimethylammonium-propane chloride (DOTAP) as cationic component shifted from - 35 mV to approximately + 5 mV. LNP prepared with other cationic surfactants remained slightly negative after enzymatic phosphate cleavage. Manufacturing of LNP containing PNPP and DOTAP via connection of two chips in a microfluidic instrument proves to show efficient change in zeta potential from negative to positive after incubation with alkaline phosphatase., (© 2024. The Author(s).)

Double-headed warheads focusing on the pharmacological aspects as well as membrane permeability can contribute a lot to medicinal chemistry. Over the past few decades, a lot of research has been conducted on steroid-heterocycle conjugates as possible therapeutic agents against a variety of disorders. In the second half of the 20th century, successful research was conducted on cholesterol-based heterocyclic moieties. Keeping in view the biological significance of various triazoles, research on fusion with cholesterol has emerged. This review has been designed to explore the chemistry of cholesterol-based triazoles for the duration from 2010 to 2023 and their significance in medicinal chemistry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Cationic liposomes represent a promising non-viral carrier platform for gene delivery. The successful intracellular delivery of genes to the target cell is highly influenced by lipid compositions in the liposomal formulation. In the present study, a Box-Behnken design was applied to investigate the optimal lipid composition for the liposome-based transfection agent., Methods: The concentrations of DOTAP, DSPE-PEG, and cholesterol were set as independent factors. A total of 15 lipid compositions were generated and tested for specific responses, including particle size, encapsulation efficiency, cell viability, and cell transfection. The data were then analyzed to predict the optimal composition using response surface methodology (RSM)., Results: The results for particle size, encapsulation efficiency, cell viability and fluorescence intensity ranged from 158.7 to 2064 nm, 48.19-95.72%, 81.50-122.67%, and 0.0-9.08, respectively. Compositions of liposome-based transfection agent without DOTAP, those without cholesterol, and those containing DSPE-PEG2000 with a molar ratio equal to or greater than that of cholesterol tended to exhibit low encapsulation efficiency. The ability of the liposome to complex DNA, as determined through electrophoresis gel retardation assay, showed that the composition without DOTAP produced DNA bands, indicating that the prepared liposomes had a less ability to complex DNA. The cytotoxicity test results indicated that all lipid compositions were considered non-toxic, as they exhibited >80% cell viability. The cell transfection assay demonstrated that the lipid composition containing a combination of DOTAP and cholesterol was able to transfect DNA into cells. According to response analysis, RSM predicted that the optimal lipid composition consisted of 2.75 μmol DOTAP and 0.91 μmol cholesterol, with a desirability value of 0.85., Conclusions: Although the equation model is still acceptable for predicting the optimal lipid composition, further study is needed to obtain a model with higher desirability, such as by using more lipid compositions, increased replications, and different variable responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Liposomes (LIP) loaded with natural active ingredients have significant potential in the food industry. However, their low loading efficiency (LE) hampers the advancement of liposomal products. To improve the loading capacity of functional compounds, bionic oleosomes (BOLE) with a monolayer of phospholipid membranes and a glyceryl tricaprylate/caprate (GTCC) oil core have first been engineered by high-pressure homogenization. TEM revealed that the core of BOLE consists of GTCC instead of water, thereby extending the hydrophobic space. Steady-state fluorescence and active loading experiments confirmed that cholesterol (CH) detached from the phospholipid membrane and entered the oil core, where it repelled cannabidiol (CBD). Based on the extending hydrophobic space, CBD-BOLE was prepared and its LE was 3.13 times higher than CBD-LIP. The CBD-phospholipid ratio (CPR) of CBD-BOLE significantly improved at least 7.8 times. Meanwhile, the free radical scavenging activity of CBD was increased and cytotoxicity was reduced., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Sphingomyelin (SM) and cholesterol are the major lipids in the signaling platforms of cell membranes, known as lipid rafts. In particular, SM with a stearoyl chain (C18-SM) is abundant in specific tissues such as the brain, the most cholesterol-rich organ, whereas the distribution of palmitoyl (C16)-SM is ubiquitous. Here, we reveal the differences between palmitoyl- and stearoyl-SM in lipid-lipid interactions based on the tie lines obtained from the 2 H solid-state NMR spectra of bilayer systems composed of SM/dioleoylphosphatidylcholine/cholesterol 33:33:33 and 40:40:20. Lipid probes carrying position-selective deuterations, 10',10'- d 2 -SM, 24- d 1 -cholesterol, and 6″,6″- d 2 -dioleoyl-phosphatidylcholine, were incorporated into the membranes. 2 H NMR peaks from these probes in the membranes directly provide the lipid compositions of the liquid-ordered (Lo) and liquid-disordered (Ld) regions. Without using bulky fluorescent groups, these probes allow us to obtain the end points of the tie lines in a ternary phase diagram based on the lever rule. Consequently, the tie lines of the stearoyl-SM membranes were steeper than those of the palmitoyl-SM membranes, indicating that cholesterol content was higher in the Lo domains of stearoyl-SM, regardless of the total concentration of unsaturated phospholipids. When comparing the content of unsaturated lipids in the Lo domain, the stearoyl-SM membranes had a higher content than palmitoyl-SM membranes. These results revealed that stearoyl-SM is suitable for stabilizing biologically functional microdomains in cholesterol-rich organs, whereas palmitoyl-SM may be better suited for stabilizing domains in tissue membranes with normal cholesterol content. The small but significant differences in the lipid interactions between stearoyl-SM and palmitoyl-SM may be related to the spatiotemporal formation of functional domains in biological environments.

Atherosclerosis (AS) poses a significant threat to human life and health. However, conventional antiatherogenic medications exhibit insufficient targeting precision and restricted therapeutic effectiveness. Moreover, during the progression of AS, macrophages undergo polarization toward the proinflammatory M1 phenotype and generate reactive oxygen species (ROS) to accelerate the occurrence of inflammatory storms, and ingest excess lipids to form foam cells by inhibiting cholesterol efflux. In our study, we developed a macrophage membrane-functionalized hollow mesoporous manganese dioxide nanomedicine (Col@HMnO 2 -MM). This nanomedicine has the ability to evade immune cell phagocytosis, enables prolonged circulation within the body, targets the inflammatory site of AS for effective drug release, and alleviates the inflammatory storm at the AS site by eliminating ROS. Furthermore, Col@HMnO 2 -MM has the ability to generate oxygen autonomously by breaking down surplus hydrogen peroxide generated at the inflammatory AS site, thereby reducing the hypoxic microenvironment of the plaque by downregulating hypoxia-inducible factor (HIF-1α), which in turn enhances cholesterol efflux to inhibit foam cell formation. In an APOE -/- mouse model, Col@HMnO 2 -MM significantly reduced inflammatory factor levels, lipid storage, and plaque formation without significant long-term toxicity. In summary, this synergistic treatment significantly improved the effectiveness of nanomedicine and may offer a novel strategy for precise AS therapy., (© 2024. The Author(s).)

Cholesterol (CHO) is an essential constituent of human cellular tissues and a crucial activity indicator for the clinical diagnosis and prevention of various diseases. Abnormal CHO levels can lead to various cardiovascular diseases, including coronary heart disease, cerebral thrombosis, and atherosclerosis. Thus, developing simple and effective methods for CHO detection is of great significance. Herein, a novel calix[6]arene-modified photonic hydrogel biosensor (PAAH@SCX6) was developed for naked-eye monitoring of CHO based on supramolecular host-guest interactions between 4-sulfocalix[6]arene (SCX6) and CHO molecules. This sensor was constructed by embedding Fe 3 O 4 colloidal nanocrystal cluster chains into a poly(acrylamide- co -acrylic acid) smart hydrogel (PAAH), followed by incorporation of plentiful SCX6 units into the PAAH via hydrogen bonding interactions. The specific recognition of SCX6 to CHO leads to the volume expansion of the hydrogel, causing a shift in the photonic band gap and a change in the hydrogel's structural color. The sensor demonstrated a linear detection range of 2.83-5.20 mM, covering the typical CHO levels in the human body. Importantly, the PAAH@SCX6 biosensor showed high selectivity and satisfactory stability, making it highly suitable for practical applications. Such a photonic hydrogel-based biosensor provides a convenient, simple, and efficient strategy for visual CHO detection.

Ratiometric probing of analytes presents a substantial advancement in molecular recognition, offering self-calibrating signals that enhance the measurement accuracy and reliability. We present a dual-emitting probe based on (6,5) chirality-enriched single-walled carbon nanotubes (SWCNTs) with oxygen defects for cholesterol (Chol) detection using ratiometric fluorescence readouts. The interaction with Chol induced significant intensity variations in the E 11 and E 11 * emission peaks of oxygen defect-induced SWCNTs, giving rise to ratiometric fluorescence changes. The sensitivity of these probes toward Chol in water and serum was 0.28 ± 0.01 and 0.72 ± 0.05 μM, respectively, which is comparable to that of common gold standards for cholesterol detection used in clinical samples. By utilizing ratiometric readouts, our approach enhanced selectivity over numerous competing analytes, including amino acids, sugars, cations, anions, proteins, steroid hormones, surfactants, and phospholipids. Mechanistic investigations revealed that Chol detection by defect-integrated SWCNTs was facilitated by Chol incorporation within micelles formed by sodium cholate, the surfactant dispersant used for the SWCNT suspension. Oxygen defects played a crucial role by directly interacting with Chol. This strategy employing defect-integrated dual-peak NIR-emitting SWCNTs as sensors for Chol in aqueous and serum environments not only enables background-free detection of biologically relevant analytes but also advances biosensing using SWCNTs through tailored surface functionalization and advanced read-out concepts.

Based on the SARS-CoV-2 fusion peptide (FP) structure determined from the NMR experiment, we created six FP models under different environmental conditions to explore the effects of salt and cholesterol on FP-membrane binding. The all-atom molecular dynamics (MD) simulation results indicated that ionic environments notably impact the FP structure as well as the stability of the helical elements within the peptide. Our findings highlighted the unpredictable influence of ions on the secondary structures and dynamics of the FP, emphasizing the complexity and sensitivity of the peptide's conformations to ionic conditions. When exploring the peptide's interaction with a cholesterol-free phospholipid bilayer membrane, we found that the helical elements of the FP remain stable irrespective of the salt type (Na + or Ca 2+ ). This result emphasizes the crucial role of phospholipid bilayer membranes in supporting the secondary structures of the FP. The MD simulation results showed that Ca 2+ ions facilitated deeper membrane penetration than Na + ions, highlighting the critical role of calcium ions in the FP-membrane binding. Our study indicates the essential role of the aromatic residues (such as Phe833 and Tyr837) in the FP-membrane binding process. Finally, we investigated the FP-membrane binding patterns in the presence of cholesterol. The MD simulation results demonstrated that the coupling of Ca 2+ ions and cholesterol would also benefit the FP-membrane binding. Furthermore, our findings reveal that while the type of ion and cholesterol content exert varied and unpredictable influences on FP-membrane binding patterns, aromatic residues like tyrosine (Tyr) and phenylalanine (Phe) play an essential role in FP-membrane binding. In particular, deep mutational scanning (DMS) experiments have confirmed that mutating phenylalanine in the FP significantly decreases viral mutational fitness, emphasizing the pivotal role of phenylalanine residues in membrane fusion. This knowledge can aid in developing more effective therapeutic strategies targeting the viral fusion peptide and its key amino acids, ultimately contributing to developing treatments and vaccines against the virus.

The majority of clinically approved nanoparticle-mediated therapeutics are lipid nanoparticles (LNPs), and most of these LNPs are liposomes containing cholesterol. LNP formulations significantly alter the drug pharmacokinetics (PK) due to the propensity of nanoparticles for uptake by macrophages. In addition to readily engulfing LNPs, the high expression of cholesterol hydroxylases and reactive oxygen species (ROS) in macrophages suggests that they will readily produce oxysterols from LNP-associated cholesterol. Oxysterols are a heterogeneous group of cholesterol oxidation products that have potent immune modulatory effects. Oxysterols are implicated in the pathogenesis of atherosclerosis and certain malignancies; they have also been found in commercial liposome preparations. Yet, the in vivo metabolic fate of LNP-associated cholesterol remains unclear. We review herein the mechanisms of cellular uptake, trafficking, metabolism, and immune modulation of endogenous nanometer-sized cholesterol particles (i.e., lipoproteins) that are also relevant for cholesterol-containing nanoparticles. We believe that it would be imperative to better understand the in vivo metabolic fate of LNP-associated cholesterol and the immune implications for LNP-therapeutics. We highlight critical knowledge gaps that we believe need to be addressed in order to develop safer and more efficacious lipid nanoparticle delivery systems.

α7 nicotinic acetylcholine receptors (nAChRs) are homopentameric ligand-gated ion channels with critical roles in the nervous system. Recent studies have resolved and functionally annotated closed, open, and desensitized states of these receptors, providing insight into ion permeation and lipid binding. However, the process by which α7 nAChRs transition between states remains unclear. To understand gating and lipid modulation, we generated two ensembles of molecular dynamics simulations of apo α7 nAChRs, with or without cholesterol. Using symmetry-adapted Markov state modeling, we developed a five-state gating model. Free energies recapitulated functional behavior, with the closed state dominating in absence of agonist. Open-to-nonconducting transition rates corresponded to experimental open durations. Cholesterol relatively stabilized the desensitized state, and reduced open-desensitized barriers. These results establish plausible asymmetric transition pathways between states, define lipid modulation effects on the α7 nAChR conformational cycle, and provide an ensemble of structural models applicable to rational design of lipidic pharmaceuticals., (© 2024. The Author(s).)

Membrane active peptides (MAPs) can provide novel means to trigger the release of liposome encapsulated drugs to improve the efficacy of liposomal drug delivery systems. Design of MAP-based release strategies requires possibilities to carefully tailor the interactions between the peptides and the lipid bilayer. Here we explore the influence of lipid vesicle properties on the function of conjugation-dependent MAPs, specifically focusing on two de novo designed peptides, JR2KC and CKV 4 . Utilizing liposomes with differences in size, lipid composition, and surface charge, we investigated the mechanisms and abilities of the peptides to induce controlled release of encapsulated cargo. Our findings indicate that liposome size modestly affects the structural changes and function of the peptides, with larger vesicles facilitating a minor increase in drug release efficiency due to higher peptide-to-liposome ratios. Notably, the introduction of negatively charged lipids significantly enhanced the release efficiency, predominantly through electrostatic interactions that favor peptide accumulation at the lipid bilayer interface and subsequent membrane disruption. The incorporation of cholesterol and a mix of saturated and unsaturated lipids was shown to alter the vesicle's phase behavior, thus modulating the membrane activity of the peptides. This was particularly evident in the cholesterol-enriched liposomes, where JR2KC induced lipid phase separation, markedly enhancing cargo release. Our results underscore the critical role of lipid vesicle composition in the design of MAP-based drug delivery systems, suggesting that precise tuning of lipid characteristics can significantly influence their performance.

Niosomes are key nanocarriers composed of bilayer vesicles formed by non-ionic surfactants and cholesterol, offering advantages such as high physicochemical stability, biodegradability, cost-effectiveness, and low toxicity. This review discusses their significant role in drug delivery, including applications in anticancer therapy and vaccine delivery. It also highlights the impact of non-ionic surfactants on niosome formation, drug delivery pathways, and protein corona formation-a relatively underexplored topic. Furthermore, the application of artificial intelligence in optimizing niosome design and functionality is examined. Future research directions include enhancing formulation techniques, expanding application scopes, and integrating advanced technologies. This review provides comprehensive insights and practical guidance for advancing niosome-based drug delivery systems., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Gao et al.)

Pharmacokinetics of nanomedicines can be improved by a temporal blockade of mononuclear phagocyte system (MPS) through the interaction with other biocompatible nanoparticles. Liposomes are excellent candidates as blocking agents, but the efficiency of the MPS blockade can greatly depend on the liposome properties. Here, we investigated the dependence of the efficiency of the induced MPS blockade in vitro and in vivo on the size of blocking liposomes in the 100-500 nm range. Saturation of RAW 264.7 macrophage uptake was observed for phosphatidylcholine/cholesterol liposomes larger than 200 nm in vitro . In mice, liposomes of all sizes exhibited a blocking effect on liver macrophages, prolonging the circulation of subsequently administrated magnetic nanoparticles in the bloodstream, reducing their liver uptake, and increasing accumulation in the spleen and lungs. Importantly, these effects became more pronounced with the increase of liposome size. Optimization of the size of the blocking liposomes holds the potential to enhance drug delivery and improve cancer therapy., (Creative Commons Attribution license.)

In a recent study, spectroscopic observations of modified cholesterol in both lipid-coated nanoparticles and liposomes provided evidence for a disorder-to-order orientational transition with increasing temperature. Below a critical temperature, in a membrane composed of modified cholesterol, saturated (DPPC) lipid, and anionic (DOPS) lipid, a roughly equal population of head-out and head-in conformations was observed. Surprisingly, as temperature was increased the modified cholesterol presented an abrupt transition to a population of all head-in orientations. Additionally, when saturated DPPC lipids were replaced by unsaturated DOPC the disorder-to-order transition was eliminated. To gain insight into this curious transition, we use all-atom molecular dynamics simulations to characterize the structure and fluctuations of lipid bilayers composed of saturated and unsaturated lipids, in the presence of normal and modified cholesterol. Free energy differences between head-out and head-in conformations are computed as a function of varying lipid membrane composition for normal and modified cholesterol. In bilayers primarily composed of DPPC, the orientation of modified cholesterol is observed to depend sensitively on the orientation of the surrounding normal or modified cholesterol molecules, suggesting cooperative Ising-like interactions favoring an ordered state. In bilayers primarily composed of DOPC, spontaneous flip-flop of modified cholesterol is observed, consistent with the measured small free energy barrier separating the head-in and head-out orientations. This combined experimental and computational study effectively characterizes the orientational dimorphism and provides novel insight into the fundamental nature of cholesterol interactions in membrane.

G protein-coupled receptors (GPCRs) are lipid-regulated transmembrane proteins that play a central role in cell signaling and pharmacology. Although the role of membrane lipids in GPCR function is well established, the underlying GPCR-lipid interactions have not been thermodynamically characterized due to the complexity of these interactions. In this work, we estimate the energetics and dynamics of lipid association from coarse-grain simulations of the serotonin 1A receptor embedded in a complex membrane. We show that lipids bind to the receptor with varying energetics of 1-4 kT, and timescales of 1-10 μs. The most favorable energetics and longest residence times are observed for cholesterol, glycosphingolipid GM1, phosphatidylethanolamine (PE) and phosphatidylserine (PS) lipids. Multi-exponential fitting of the contact probability suggests distinct dynamic regimes, corresponding to ps, ns and μs timescales, that we correlate with the annular, intermediate and non-annular lipid sites. The timescales of lipid binding correspond to high barrier heights, despite their relatively weaker energetics. Our results highlight that GPCR-lipid interactions are driven by both thermodynamic interactions and the dynamical features of lipid binding., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Researchers choose different methods of making giant unilamellar vesicles to satisfy different constraints of their experimental designs. A challenge that arises when researchers use a variety of methods is that each method may produce vesicles with a different average lipid ratio, even if all experiments use lipids from a common stock mixture. Here, we use mass spectrometry to investigate ratios of lipids in vesicle solutions made by five common methods: electroformation on indium tin oxide slides, electroformation on platinum wires, gentle hydration, emulsion transfer, and extrusion. We made vesicles from either five-component or binary mixtures of lipids chosen to span a wide range of physical properties: di(18:1)PC, di(16:0)PC, di(18:1)PG, di(12:0)PE, and cholesterol. For a mixture of all five of these lipids, ITO electroformation, Pt electroformation, gentle hydration, and extrusion methods result in only minor shifts in lipid ratios (≤5 mol %) relative to a common stock solution. In contrast, emulsion transfer results in ∼80% less cholesterol than expected from the stock solution, which is counterbalanced by a surprising overabundance of saturated PC-lipid relative to all other phospholipids. Experiments using binary mixtures of saturated and unsaturated PC-lipids and cholesterol largely support results from the five-component mixture. In general, our results imply that experiments that increment lipid ratios in small steps will produce data that are highly sensitive to the technique used and to sample-to-sample variations. For example, sample-to-sample variations are ∼±2 mol % for five-component vesicles produced by a single technique. In contrast, experiments that explore larger increments in lipid ratio or that seek to explain general trends and new phenomena will be less sensitive to sample-to-sample variation and the method used., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cholesterol is a major component of plasma membranes and plays a significant role in actively regulating the functioning of several membrane proteins in humans. In this study, we focus on the role of cholesterol depletion on the voltage-gated sodium channel Na v 1.7, which is primarily expressed in the peripheral sensory neurons and linked to various chronic inherited pain syndromes. Coarse-grained molecular dynamics simulations revealed key dynamic changes of Na v 1.7 upon membrane cholesterol depletion: A loss of rigidity in the structural motifs linked to activation and fast-inactivation is observed, suggesting an easier transition of the channel between different gating states. In-vitro whole-cell patch clamp experiments on HEK293t cells expressing Na v 1.7 validated these predictions at the functional level: Hyperpolarizing shifts in the voltage-dependence of activation and fast-inactivation were observed along with an acceleration of the time to peak and onset kinetics of fast inactivation. These results underline the critical role of membrane composition, and of cholesterol in particular, in influencing Na v 1.7 gating characteristics. Furthermore, our results also point to cholesterol-driven changes of the geometry of drug-binding regions, hinting to a key role of the membrane environment in the regulation of drug effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Water permeation through phospholipid/cholesterol bilayers is the key to understanding tension-induced rupture of biological cell membranes. We performed molecular dynamics simulations of stretched phospholipid/cholesterol bilayers to investigate changes in the free energy profile of water molecules across the bilayer and the lipid structure responsible for water permeation. We modeled stretching of the bilayer by applying areal strain. In stretched phospholipid/cholesterol bilayers, the hydrophobic tail of the phospholipids became disordered and the free energy barrier to water permeation decreased. Upon exceeding the critical areal strain, a phase transition to an interdigitated gel phase occurred before rupture, and the hydrophobic tail ordering as well as the free energy barrier were restored. In pure phospholipid bilayers, we did not observe such recoveries. These transient recoveries in the phospholipid/cholesterol bilayer suppressed water permeation and membrane rupture, followed by an increase in the critical areal strain at which the bilayer ruptured. This result agrees with experimental results and provides a reasonable molecular mechanism for the toughness of phospholipid/cholesterol bilayers under tension.Communicated by Ramaswamy H. Sarma.

In this work, the B, N co-doped carbon dots (B, N-CDs) were synthesized via facile hydrothermal approach with 6-aminopyridine boronic acid as precursor. In addition to emitting intense blue luminescence when exposed to ultraviolet light, the prepared B, N-CDs displayed remarkable peroxidase-like activity, which could efficiently catalyze the oxidation of 3, 3', 5, 5' -tetramethylbenzidine (TMB) to blue ox-TMB in the presence of hydrogen peroxide (H 2 O 2 ). Furthermore, the fluorescence intensity of B, N-CDs increased gradually upon the addition of H 2 O 2 . Since cholesterol oxidase (ChOx) can catalyze the oxidation of cholesterol to form H 2 O 2 , the as-prepared B, N-CDs was then used as both colorimetric and fluorometric sensors for the detection of cholesterol with detection limit of 0.87 and 2.31 μM, respectively. Finally, the dual-mode approach based on B, N-CDs was effectively utilized for detecting cholesterol levels in serum samples, proving the potential application of B, N-CDs in the field of biological assay., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Activating the cGAS-STING pathway of circulating tumor cell clusters (CTC clusters) represents a promising strategy to mitigate metastases. To fully exploit the potential of cholesterol-regulating agents in activating CTCs' STING levels, we developed a nanoparticle (NP) composed of metal complex lipid (MCL). This design includes MCL-miriplatin to increase NP stiffness and loads lomitapide (lomi) modulating cholesterol levels, resulting in the creation of PLTs@Pt-lipid@lomi NPs. MCL-miriplatin not only enhances lomi's eliciting efficacy on STING pathway but also increases NPs' stiffness, thus a vital factor affecting the penetration into CTC clusters to further boost lomi's ability. Demonstrated by cy5 tracking experiments, PLTs@Pt-lipid@lomi NPs quickly attach to cancer cell via platelet membrane anchorage, penetrate deep into the spheres, and reach the subcellular endoplasmic reticulum where lomi regulates cholesterol. Additionally, these NPs have been shown to track CTCs in the bloodstream, a capability not demonstrated by the free drug. PLTs@Pt-lipid@lomi NPs more efficiently activate the STING pathway and reduce CTC stemness compared to free lomi. Ultimately, PLTs@Pt-lipid@lomi NPs reduce metastasis in a post-surgery animal model. While cholesterol-regulating agents are limited in efficacy when being repositioned as immunomodulatory agents, this MCL-composing NP strategy demonstrates the potential to effectively deliver these agents to target CTC clusters., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability became compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and caused a reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogues of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. Importantly, binding and membrane aggregation of natamycin was paralleled by a decrease of the dipole potential in the PM, and this effect was enhanced in the presence of myriocin. We conclude that ergosterol promotes binding and aggregation of natamycin in the yeast PM, which can be synergistically enhanced by inhibitors of sphingolipid synthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

We investigated the influence of archaeal lipids (C 25,25 ) isolated from thermophilic archaeon Aeropyrum pernix K1 on physicochemical properties of liposomes comprised of egg sphingomyelin (SM) and cholesterol (CH) using fluorescence emission anisotropy, calcein release studies, dynamic light scattering, transmission electron microscopy and phase analysis light scattering. The 2 mol% addition of archaeal lipids enabled formation of small unilamellar vesicles by sonication while also having significant effect on reducing mean size, polydispersity index and zeta potential of C 25,25 /SM/CH vesicles. Increasing the ratio of C 25,25 lipids in mixture of C 25,25 /SM/CH decreased lipid ordering parameter in dose dependent manner at different temperatures. We also demonstrated that adding 15 mol% C 25,25 to SM/CH mixture will cause it to notably interact with fetal bovine serum which could make them a viable alternative adjuvant to synthetic ether-linked lipids in development of advanced liposomal vaccine delivery systems. The prospect of combining the proven strengths of SM/CH mixtures with the unique properties of C 25,25 opens exciting possibilities for advancing drug delivery technologies, promising to yield formulations that are both highly effective and adaptable to a range of therapeutic applications., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The utilization of biomimetic membranes supported by advanced self-assembled monolayers is gaining attraction as a promising sensing tool. Biomimetic membranes offer exceptional biocompatibility and adsorption capacity upon degradation, transcending their role as mere research instruments to open new avenues in biosensing. This study focused on anchoring a sparsely tethered bilayer lipid membrane onto a self-assembled monolayer composed of a biodegradable polymer, functionalized with poly(ethylene glycol)-cholesterol moieties, for lipid membrane integration. Real-time monitoring via quartz crystal microbalance, coupled with characterization using surface-enhanced infrared absorption spectroscopy and electrochemical impedance spectroscopy, provided comprehensive insights into each manufacturing phase. The resulting lipid layer, along with transmembrane pores formed by gramicidin A, exhibited robust stability. Electrochemical impedance spectroscopy analysis confirmed membrane integrity, successful pore formation, and consistent channel density. Notably, gramicidin A demonstrated sustained functionality as an ion channel upon reconstitution, with its functionality being effectively blocked and inhibited in the presence of calcium ions. These findings mark significant strides in developing intricate biodegradable nanomaterials with promising applications in biomedicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Oral administration of BCS class IV anticancer agents has always remained challenging and frequently results in poor oral bioavailability. The goal of the current study was to develop hybrid nanoparticles (HNPs) employing cholesterol and poloxamer-407 to boost paclitaxel's (PTX) oral bioavailability. A series of HNPs with different cholesterol and poloxamer-407 ratios were developed utilizing a single-step nanoprecipitation technique. The PTX loaded HNPs were characterized systematically via particle size, zeta potential, polydispersity index, surface morphology, in vitro drug release, FTIR, DSC, XRD, acute oral toxicity analysis, hemolysis evaluation, accelerated stability studies, and in vivo pharmacokinetic analysis. The HNPs were found within the range of 106.6±55.60 - 244.5±88.24 nm diameter with the polydispersity index ranging from 0.20±0.03 - 0.51±0.11. SEM confirmed circular, nonporous, and smooth surfaces of HNPs. PTX loaded HNPs exhibited controlled release profile. The compatibility between the components of formulation, thermal stability, and amorphous nature of HNPs were confirmed by FTIR, DSC, and XRD, respectively. Acute oral toxicity analysis revealed that developed system have no deleterious effects on the animals' cellular structures. HNPs demonstrated notable cytotoxic effects and were hemocompatible at relatively higher concentrations. In vivo pharmacokinetic profile (AUC 0-∞ , AUMC 0-∞ , t 1/2, and MRT 0-∞ ) of the PTX loaded HNPs was improved as compared to pure PTX. It is concluded from our findings that the developed HNPs are hemocompatible, biocompatible and have significantly enhanced the oral bioavailability of PTX., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Enveloped viruses enter the host cell by fusing at the cell membrane or entering the cell via endocytosis and fusing at the endosome. Conventional inhibitors target the viral fusion protein to inactivate it for inducing fusion. These target-specific vis-à-vis virus-specific inhibitors fail to display their inhibitory efficacy against emerging and remerging viral infections. This necessitates the need to develop broad-spectrum entry inhibitors that are effective irrespective of the virus. Using a broad range of targeting techniques, the fusion inhibitors can modify the physical characteristics of the viral membrane, making it less prone to fusion. We have previously shown that two tryptophan-aspartic acid (WD)-containing hydrophobic peptides, TG-23 and GG-21, from coronin 1, a phagosomal protein, inhibit membrane fusion by modulating membrane organization and dynamics. In the present work, we designed two WD-containing hydrophilic peptides, QG-22 and AG-22, using coronin 1 as a template and evaluated their fusion inhibitory efficacies in the absence and presence of membrane cholesterol. Our results demonstrate that QG-22 and AG-22 inhibit membrane fusion irrespective of the concentration of membrane cholesterol. Our measurements of depth-dependent membrane organization and dynamics reveal that they impede fusion by enhancing the acyl chain order. Overall, our results validate the hypothesis of designing fusion inhibitors by modulating the membrane's physical properties. In addition, it demonstrates that chain hydrophobicity might not be a critical determinant for the development of peptide-based fusion inhibitors.

Cholesterol is a major component of biomembranes that impacts membrane order, permeability, and lateral organization, but the precise molecular mechanisms of cholesterol's actions are still under investigation. Density functional theory (DFT) calculations have opened the fingerprint vibration bands of large molecules to detailed spectral analysis. For cholesterol, Raman spectral interpretation for conformational structure and hydrogen bonding is now possible. Here, DFT calculations of cholesterol conformers identify 10 structure types that also have unique low-frequency Raman spectra. By fitting experimental spectra to these types, the distribution of cholesterol structures present in phospholipid (PL) membrane vesicles was measured. The distributions reveal that the cholesterol iso-octyl chain tends to align with saturated PL chains and shifts to a thermal distribution for unsaturated PL chains. The results agree with the templating effect of cholesterol on PL membranes and show that the top of the iso-octyl chain is rigid like the rings. It is also shown that the inclusion of water molecules hydrogen bonded to the cholesterol hydroxy group in the DFT calculations may improve the spectral fitting for future studies.

Cascade-enzyme reaction systems have emerged as promising tools for treating malignant tumors by efficiently converting nutrients into toxic substances. However, the challenges of poor localized retention capacity and utilization of highly active enzymes often result in extratumoral toxicity and reduced therapeutic efficacy. In this study, we introduced a cell membrane-DNA nanoanchor (DNANA) with a spatially confined cascade enzyme for in vivo tumor therapy. The DNANAs are constructed using a polyvalent cholesterol-labeled DNA triangular prism, ensuring high stability in cell membrane attachment. Glucose oxidase (GOx) and horseradish peroxidase (HRP), both modified with streptavidin, are precisely confined to biotin-labeled DNANAs. Upon intratumoral injection, DNANA enzymes efficiently colonize the tumor site through cellular membrane engineering strategies, significantly reducing off-target enzyme leakage and the associated risks of extratumoral toxicity. Furthermore, DNANA enzymes demonstrated effective cancer therapy in vitro and in vivo by depleting glucose and producing highly cytotoxic hydroxyl radicals in the vicinity of tumor cells. This membrane-engineered cascade-enzyme reaction system presents a conceptual approach to tumor treatment.

Serotonin is distinct among synaptic neurotransmitters because it is amphipathic and released from synaptic vesicles at concentrations superior to its water solubility limit (270 mM in synaptic vesicles for a solubility limit of 110 mM). Hence, serotonin is mostly aggregated in the synaptic cleft, due to extensive aromatic stacking. This important characteristic has received scant attention, as most representations of the serotonergic synapse take as warranted that serotonin molecules are present as monomers after synaptic vesicle exocytosis. Using a combination of in silico and physicochemical approaches and a new experimental device mimicking synaptic conditions, we show that serotonin aggregates are efficiently dissolved by gangliosides (especially GM1) present in postsynaptic membranes. This initial interaction, driven by electrostatic forces, attracts serotonin from insoluble aggregates and resolves micelles into monomers. Serotonin also interacts with cholesterol via a set of CH-π and van der Waals interactions. Thus, gangliosides and cholesterol act together as a functional serotonin-collecting funnel on brain cell membranes. Based on this unique mode of interaction with postsynaptic membranes, we propose a new model of serotonergic transmission that takes into account the post-exocytosis solubilizing effect of gangliosides and cholesterol on serotonin aggregates.

Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins. They are believed to underlie the experimentally observed lateral heterogeneity of eukaryotic plasma membranes and implicated in many cellular processes, such as signaling and trafficking. Ternary model membranes consisting of saturated lipids, unsaturated lipids, and cholesterol are common proxies because they exhibit phase coexistence between a liquid-ordered (lo) and liquid-disordered (ld) phase and an associated critical point. However, plasma membranes are also asymmetric in terms of lipid type, lipid abundance, leaflet tension, and corresponding cholesterol distribution, suggesting that rafts cannot be examined separately from questions about elasticity, curvature torques, and internal mechanical stresses. Unfortunately, it is challenging to capture this wide range of physical phenomenology in a single model that can access sufficiently long length- and time scales. Here we extend the highly coarse-grained Cooke model for lipids, which has been extensively characterized on the curvature-elastic front, to also represent raft-like lo/ld mixing thermodynamics. In particular, we capture the shape and tie lines of a coexistence region that narrows upon cholesterol addition, terminates at a critical point, and has coexisting phases that reflect key differences in membrane order and lipid packing. We furthermore examine elasticity and lipid diffusion for both phase separated and pure systems and how they change upon the addition of cholesterol. We anticipate that this model will enable significant insight into lo/ld phase separation and the associated question of lipid rafts for membranes that have compositionally distinct leaflets that are likely under differential stress-like the plasma membrane., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

Triacontanol is a long-chain primary alcohol derived from policosanol, known for its diverse biological activities, including functioning as a plant growth regulator and exhibiting anti-inflammatory and antitumoral effects. However, its application is limited due to its high hydrophobicity, resulting in poor absorption and reduced therapeutic effectiveness. A potential solution to this problem is the use of niosomes. Niosomes are carriers composed of non-ionic surfactants, cholesterol, charge-inducing agents, and a hydration medium. They are effective in encapsulating drugs, improving their solubility and bioavailability. The objective of this study was to optimize and synthesize nano-niosomes for the encapsulation of triacontanol. Niosomes were synthesized using a thin-film hydration method combined with ultrasonication, following a Box-Behnken design. Niosomes were characterized using various techniques including dynamic light scattering, Fourier-transform infrared spectroscopy (FTIR), confocal microscopy, high-resolution scanning electron microscopy, and transmission electron microscopy (TEM). Formulation 14 of niosomes achieved the desired size, polydispersity index (0.198 ± 0.008), and zeta potential (-31.28 ± 1.21). FTIR analysis revealed a characteristic signal in the 3400-300 cm -1 range, indicating intermolecular interactions due to a bifurcated hydrogen bond between cholesterol and S60. Confocal microscopy confirmed the presence of triacontanol through Nile Red fluorescence. TEM revealed the spherical structure of niosomes.

Skin barrier function is localized in its outermost layer, the stratum corneum (SC), which is comprised of corneocyte cells embedded in an extracellular lipid matrix containing ceramides (CERs), cholesterol (CHOL), and free fatty acids (FFAs). The unique structure and composition of this lipid matrix are important for skin barrier function. In this study, experiments and molecular dynamics simulation were combined to investigate the structural properties and phase behavior of mixtures containing nonhydroxy sphingosine CER (CER NS), CHOL, and FFA. X-ray scattering for mixtures with varying CHOL levels revealed the presence of the 5.4 nm short periodicity phase in the presence of CHOL. Bilayers in coarse-grained multilayer simulations of the same compositions contained domains with thicknesses of approximately 5.3 and 5.8 nm that are associated with elevated levels, respectively, of CER sphingosine chains with CHOL, and CER acyl chains with FFA chains. The prevalence of the thicker domain increased with decreasing CHOL content. This might correspond to a phase with ∼5.8 nm spacing observed by x-rays (other details unknown) in mixtures with lower CHOL content. Scissoring and stretching frequencies from Fourier transform infrared spectroscopy (FTIR) also indicate interaction between FFA and CER acyl chains and little interaction between CER acyl and CER sphingosine chains, which requires CER molecules to adopt a predominantly extended conformation. In the simulated systems, neighbor preferences of extended CER chains align more closely with the FTIR observations than those of CERs with hairpin ceramide chains. Both FTIR and atomistic simulations of reverse mapped multilayer membranes detect a hexagonal to fluid phase transition between 65 and 80°C. These results demonstrate the utility of a collaborative experimental and simulation effort in gaining a more comprehensive understanding of SC lipid membranes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Photoswitchable lipids, particularly azobenzene-derivatized phosphatidylcholine (azoPC) lipids, offer a unique mechanism for reversible modification of membrane properties upon exposure to ultraviolet (UV) radiation. Through all-atom molecular dynamics simulations, we explore how UV irradiation-induced trans-to-cis photoisomerization (TCPI) of AzoPC lipid influences the structure and dynamics of a lipid membrane, composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol with similar composition to that of the DOXIL®. Structural and dynamical analyses of two states of the membrane, 'dark' state (containing cis-azoPC lipid) and 'bright' state (containing 85 % cis-azoPC and 15 % trans-azoPC lipids) reveal that the TCPI reduces membrane packing density and increases diffusivity of lipids. We have demonstrated an enhanced intercalation of doxorubicin (DOX), an anticancer drug, in the 'bright' state of the membrane compared to that in the 'dark' state. This study - elucidating the complex interplay between lipid composition, photoswitching, and lipid-drug interactions - contributes to the design of lipid-based systems for targeted drug delivery and biomedical applications., (© 2024 Wiley-VCH GmbH.)

Annexins (ANXAs), calcium-sensitive phospholipid-binding proteins, are pivotal for cellular membrane repair, which is crucial for eukaryotic cell survival under membrane stress. With their unique trimeric arrangements and crystalline arrays on the membrane surface, ANXA4 and ANXA5 induce membrane curvature and rapidly orchestrate plasma membrane resealing. However, the influence of cholesterol and anionic lipid headgroups on annexin-induced membrane curvature remains poorly understood at the molecular level. Using all-atom molecular dynamics simulations, we measured the local curvature-induced underneath ANXA4 and ANXA5 monomers and trimers when they bind to lipid bilayers of distinct lipid compositions: PSPC (20% POPS, 80% POPC), PAPC (20% POPA, 80% POPC), and PSPCCHL (14% POPS, 56% POPC, 30% cholesterol). Laser injury experiments were conducted on MCF7 cells transfected to transiently express fluorescently labeled ANXA4 or ANXA5 to facilitate the examination of protein and lipid accumulation at the damage site. Annexin trimers induce higher curvature than monomers, particularly with cholesterol present. Annexin trimers induce similar curvatures on both PAPC and PSPC membranes. Notably, among monomers, ANXA5 induces the highest curvature on PAPC, suggesting more efficient recruitment of ANXA5 compared with ANXA4 in the early stages of membrane repair near a lesion. Laser injury experiments confirm rapid coaccumulation of phosphatidic acid lipids with ANXA4 and ANXA5 at repair sites, potentially enhancing the accumulation of annexins in the early stages of membrane repair.

Liposomes are used as model membranes in many scientific fields. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures can produce liposomes at specific concentrations and lipid compositions, and researchers often assume that the concentration and composition of their liposomes are similar or identical to what would be expected if no lipid loss occurred. However, lipid loss and concomitant biasing of lipid composition can in principle occur at any preparation step due to nonideal mixing, lipid-surface interactions, etc. Here, we report a straightforward HPLC-ELSD method to quantify the lipid concentration and composition of liposomes and apply that method to study the preparation of simple cholesterol/POPC liposomes. We examine common liposome preparation steps, including vortexing during resuspension, lipid film hydration, extrusion, freeze-thaw, and sonication. We found that the resuspension step can play an outsized role in determining the lipid loss (up to ∼50% under seemingly rigorous procedures). The extrusion step yielded smaller lipid losses (∼10-20%). Freeze-thaw and sonication could both be employed to improve lipid yields. Hydration times up to 60 min and increasing cholesterol concentrations up to 50 mol % had little influence on lipid recovery. Fortunately, even conditions with large lipid loss did not substantially influence the target membrane composition, as long as the lipid mixture was below the cholesterol solubility limit. From our results, we identify best practices for producing maximum levels of lipid recovery and minimal changes to lipid composition during liposome preparation for cholesterol/POPC liposomes., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Statins are among the most widely used drugs for the inhibition of cholesterol biosynthesis, prevention of cardiovascular diseases, and treatment of hypercholesterolemia. Additionally, statins also exhibit cholesterol-independent benefits in various diseases, including neuroprotective properties in Alzheimer's disease, anti-inflammatory effects in coronary artery disease, and antiproliferative activities in cancer, which likely result from the statins' interaction and alteration of lipid bilayers. However, the membrane-modulatory effects of statins and the mechanisms by which statins alter lipid bilayers remain poorly understood. In this work, we explore the membrane-modulating effects of statins on model lipid bilayers and live cells. Through the use of fluorescence lifetime imaging microscopy (FLIM) combined with viscosity-sensitive environmental probes, we demonstrate that hydrophobic, but not hydrophilic, statins are capable of changing the microviscosity and lipid order in model and live cell membranes. Furthermore, we show that hydrophobic simvastatin is capable of forming nanoscale cholesterol-rich domains and homogenizing the cholesterol concentrations in lipid bilayers. Our results provide a mechanistic framework for understanding the bimodal effects of simvastatin on the lipid order and the lateral organization of cholesterol in lipid bilayers. Finally, we demonstrate that simvastatin temporarily decreases the microviscosity of live cell plasma membranes, making them more permeable and increasing the level of intracellular chemotherapeutic drug accumulation.