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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.)

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.

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.)

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.)

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.

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.)

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.)

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.)

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.)

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.)

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.)

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.)

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.

The remarkable success of two FDA-approved mRNA-encapsulating vaccines (Comirnaty® and Spikevax®) indicated the importance of lipid nanoparticles (LNPs) delivery systems in clinical use. Currently, mRNA-encapsulating LNPs (mRNA-LNPs) vaccines are stored as frozen liquid at low or ultralow temperatures. We designed lyophilized LNPs utilizing FDA-approved lipids to expedite the clinical application of our developed lyophilized mRNA-LNPs in the future. The key parameters of sucrose concentration and the selection and molar ratio of the four lipids in these vaccines were optimized for long-term stability with high transfection efficiency after lyophilization. We demonstrated that 8.7% sucrose is the optimal cryoprotectant concentration to maintain the transfection efficiency of lyophilized mRNA-LNPs. Optimal lipid formulations with high transfection efficiency both before and after lyophilization were screened using an orthogonal experimental design. The ratios of distearoylphosphatidylcholine (DSPC)/cholesterol and the selection of the ionizable and PEGylated lipids are the main factors influencing the long-term stability of mRNA-LNPs. Comparative mouse transfection experiments showed that the optimal lyophilized mRNA-LNPs maintained high mRNA expression after lyophilization, predominantly in the spleen or liver, with no expression in the kidneys or eyes. Our studies demonstrated the importance of the sucrose concentration and of the selection and molar ratio of the four lipids composing LNPs for maintaining mRNA-LNP stability under lyophilization and for long-term storage under mild 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 Elsevier B.V. All rights reserved.)

Liposomes constitute a widespread drug delivery platform, gaining more and more attention from the pharmaceutical industry and process development scientists. Their large-scale production as medicinal products for human use is all but trivial, especially when parenteral administration is required. In this study an off-the-shelf microfluidic system and a methodological approach are presented for the optimization, validation and scale-up of highly monodisperse liposomes manufacturing. Starting from a Doxil®-like formulation (HSPC, MPEG-DSPE and cholesterol), a rational approach (Design of Experiments, DoE) was applied for the screening of the process parameters affecting the quality attributes of the product (mainly size and polydispersity). Additional DoEs were conducted to determine the effect of critical process parameters "CPPs" (cholesterol concentration, total flow rate "TFR" and flow rate ratio "FRR"), thus assessing the formulation and process robustness. A scale-up was then successfully accomplished. The procedure was applied to a Marqibo®-like formulation as well (sphingomyelin and cholesterol) to show the generality of the proposed formulation, process development and scale-up approach. The application of the system and method herein presented enables the large-scale manufacturing of liposomes, in compliance with the internationally recognized regulatory standards for pharmaceutical development (Quality by Design)., 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.)

Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts., Competing Interests: PC, JO, Bd, CA, TW No competing interests declared, (© 2023, Chiu, Orjuela et al.)

The study utilized transition metal chalcogenide, molybdenum diselenide (MoSe 2 ), for application in the field of bioelectrochemical sensing. The MoSe 2 was combined with carbon nanotubes (CNTs) by chemical vapor deposition to enhance the specific surface area and improve the detection sensitivity. To further increase the contact area between the electrolyte and the electrode, photolithography techniques were employed to fabricate hive-shaped CNTs, thereby enhancing the specific surface area. Next, cholesterol oxidase (ChO x ) was coated onto the electrode material, creating a cholesterol biosensor. Cyclic voltammetry was utilized to detect the concentration of cholesterol. The experiment involved segmented testing for cholesterol concentrations ranging from 0 μ M to 10 mM. Excellent sensitivity, low detection limits, and high accuracy were achieved. In the cholesterol concentration range of 0 μ M-100 μ M, the experiment achieved the highest sensitivity of 4.44 μ A μ M⋅cm -2 . Consequently, all data indicated that ChO x /MoSe 2 /CNTs functioned as an excellent cholesterol sensor in the study., (Creative Commons Attribution license.)

The development of an inhalation powder (IP) for cancer therapy is desired to improve the therapeutic response and patient compliance. The latest studies highlighted that statins, a class of drugs used in hypercholesterolemia, can have anticancer and antiinflammatory properties. Therefore, the aim of the study was to develop an IP containing liposomes loaded with simvastatin using spray drying technology, as well as to investigate the influence of formulation factors on the quality attributes of the IP by means of experimental design. Results highlighted that the composition of liposomes, namely type of phospholipid and cholesterol concentration, highly influences the quality attributes of IP, and the use of optimal concentrations of excipients, i.e. D-mannitol and L-leucine, is essential to preserve the characteristics of liposomes throughout the spray drying process. The in vitro characterization of the optimal IP formulation revealed that the total percentage of released drug is higher from the IP formulation compared to the powder of active substance (53.38 vs. 42.76%) over a period of six hours, and 39.67% of dry particles have a size less than 5 µm, making them suitable for inhalation. As a conclusion, spray drying technology can be effectively used in the development and preparation of IP containing liposomes.

The present work investigates the in vitro cholesterol reduction bioactivity of epigallocatechin gallate (EGCG) prior to and after nano-encapsulation using potato starch nanoparticle (SNP) as wall material. EGCG encapsulation in potato SNPs was achieved through a green inclusion complexation method. The encapsulated EGCG was characterized for its morphology, thermal, and crystalline properties using FESEM, DSC, XRD, and Fourier transform infrared (FTIR) studies. The bioactivity of EGCG to reduce gut cholesterol was studied using in vitro micellar cholesterol solubility study. The encapsulated EGCG exhibited enhanced thermal and crystalline properties. The FESEM results indicated successful nano-encapsulation of EGCG at 20-120 nm diameter. The melting point enhanced from 225.7°C in EGCG to 282.9°C in encapsulated EGCG. The crystallinity also enhanced and could be observed through the increased intensity in the encapsulated EGCG. The FTIR results affirmed a shifting of peaks at 3675, 2927, 1730, and 1646 cm -1 , which corresponds to formation of new H bonds and confirms successful encapsulation of EGCG in SNPs. Further, EGCG had significantly reduced the cholesterol concentration by 91.63% as observed through the in vitro micellar inhibition study. The encapsulated EGCG was not able to reduce cholesterol as observed in the in vitro micellar cholesterol solubility study. This effect occurred due to the unavailability of EGCG after it formed a complex with SNPs. PRACTICAL APPLICATION: This study first investigates the utilization of newly synthesized potato starch nanoparticles as a coating material for nano-encapsulation of EGCG. The enhanced thermal and crystalline properties of these nanoparticles contribute to improved attributes in the nano-encapsulated EGCG. Such properties hold promise for applications in functional food matrices subjected to high-temperature processing, including functional cookies, bread, and cakes. Furthermore, this research explores the bioactivity of EGCG concerning its capacity to reduce gut cholesterol levels. It also examines the potential application of nano-encapsulated EGCG in lowering gut cholesterol through a micellar solubility study., (© 2024 Institute of Food Technologists.)

Cholesterol-rich nanoemulsion (LDE) can carry chemotherapeutic agents in the circulation and can concentrate those agents in the neoplastic and inflammatory tissues. This method improves the biodistribution of the drug and reduces toxicity. However, the structural stability of LDE particles, without or with associated drugs, has not been extensively investigated. The aim of the present study is to investigate the structural stability of LDE and LDE associated to paclitaxel, etoposide or methotrexate in aqueous solution over time by small-angle X-ray scattering (SAXS and Ultra SAXS) and dynamic light scattering (DLS). The results show that LDE and LDE associated with those chemotherapeutic agents had reproducible and stable particle diameter, physical structure, and aggregation behavior over 3-month observation period. As estimated from both DLS and Ultra-SAXS methods, performed at pre-established intervals, the average particle diameter of LDE alone was approx. 32 nm, of LDE-paclitaxel was 31 nm, of LDE-methotrexate was 35 nm and of LDE-etoposide was 36 nm. Ultra-SAXS analysis showed that LDE nanoparticles were quasi-spherical, and SAXS showed that drug molecules inside the particles showed a layered-like organization. Formulations of LDE with associated PTX, ETO or MTX were successfully tested in animal experiments and in patients with cancer or with cardiovascular disease, showing markedly low toxicity, good tolerability and possible superior pharmacological action. Our results may be useful for ensuing clinical trials of this novel Nanomedicine tool, by strengthening the knowledge of the structural aspects of those LDE formulations., 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 article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cholesterol is one of the essential component of lipid in membrane. We present a polarizable atomic multipole force field (FF) for the molecular dynamic simulation of cholesterol. The FF building process follows the computational framework as the atomic multipole optimized energetics for biomolecular applications (AMOEBA) model. In this framework, the electronics parameters, including atomic monopole moments, dipole moments, and quadrupole moments calculated from ab initio calculations in the gas phase, are applied to represent the charge distribution. Furthermore, the many-body polarization is modeled by following the same pattern of distributed atomic polarizabilities. Then, the bilayers composed of two typical phospholipid molecules, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in a range of different cholesterol concentrations are built and implemented by molecular dynamics (MD) simulations based on the proposed polarizable FF. The simulation results are statistically analyzed to validate the feasibility of the proposed FF. The structural properties of the bilayers are calculated to compare with the related experimental values. The MD values show the same trend of experimental values changes.Communicated by Ramaswamy H. Sarma.

Yamanashi et al., conducted a study on the absorption of cholesterol and β-sitosterol, as well as the inhibitory effect of ezetimibe (EZE). They used CaCo-2 cells to simulate the intestines and investigated how different mixed micelles, acting as carriers, were absorbed into these cells through the Niemann-Pick C1-like 1 (NPC1L1) protein. The study focused on the impact of micelle shape, size, and zeta potential on absorption and the inhibitory effect of EZE. I utilized small-angle X-ray scattering and a zeta potential measuring device to measure these characteristics. The findings revealed a two-step mechanism: NPC1L1 selectively bound micelles based on their shape and size, and once bound, the absorption was regulated by the molecular structure of the micelle components. EZE's inhibitory effect changed with micelle composition, influencing micelle size and shape. EZE initially acted on the micelle's shape and size, and then NPC1L1 selectively bound micelles based on their shape and size, allowing EZE to directly inhibit absorption by interacting with NPC1L1. This groundbreaking discovery challenges existing concepts and holds significant implications for researchers in drug development, as well as physicians and pharmacists.

Background: Vitamin E ( -tocopherol) and cholesterol are crucial components in cellular protection and physiological processes. Their uses in biological media face challenges due to their poor solubility and stability., Objective: The study investigated the complex interactions of these bioactive compounds in various encapsulation systems of cyclodextrin and liposome, as well as dispersion in PEG-6000, in an attempt to improve the viability, motility, and preservation of ovine sperm cells., Materials and Methods: The work explored the in vitro dissolution kinetics of vitamin E (d-tocopherol) and cholesterol using semi-empirical models., Results: The release profiles of VitE and Chl varied considerably, depending on the specific carrier systems. For liposome-loaded VitE and Chl, the Korsmeyer-Peppas model gave the best fit; for CD/VitE and CD/Chl, the Higuchi model provided the best fit, whereas for PEG-6000 dispersions (VitE and Chl) both the Higuchi and Korsmeyer-Peppas models demonstrated the excellent fit. All systems indicated a Fickian diffusion mechanism dictated by the concentration gradient. The delivery of VitE and Chl with CD, liposome and PEG dispersion significantly increased sperm mobility and motility. The effect on the VCL parameter was the greatest by liposome-loaded VitE and Chl, followed by CD encapsulation and PEG-6000 dispersion., Conclusion: The dynamics of vitamin E and cholesterol within innovative delivery systems offers valuable insights into the development of advanced solutions in reproductive health, particularly on improving the viability, motility of refrigerated ovine sperm cells. Doi.org/10.54680/fr24510110712.

Immunoglobulin E (IgE) plays a pivotal role in allergic responses 1,2 . The high-affinity IgE receptor, FcεRI, found on mast cells and basophils, is central to the effector functions of IgE. FcεRI is a tetrameric complex, comprising FcεRIα, FcεRIβ and a homodimer of FcRγ (originally known as FcεRIγ), with FcεRIα recognizing the Fc region of IgE (Fcε) and FcεRIβ-FcRγ facilitating signal transduction 3 . Additionally, FcRγ is a crucial component of other immunoglobulin receptors, including those for IgG (FcγRI and FcγRIIIA) and IgA (FcαRI) 4-8 . However, the molecular basis of FcεRI assembly and the structure of FcRγ have remained elusive. Here we elucidate the cryogenic electron microscopy structure of the Fcε-FcεRI complex. FcεRIα has an essential role in the receptor's assembly, interacting with FcεRIβ and both FcRγ subunits. FcεRIβ is structured as a compact four-helix bundle, similar to the B cell antigen CD20. The FcRγ dimer exhibits an asymmetric architecture, and coils with the transmembrane region of FcεRIα to form a three-helix bundle. A cholesterol-like molecule enhances the interaction between FcεRIβ and the FcεRIα-FcRγ complex. Our mutagenesis analyses further indicate similarities between the interaction of FcRγ with FcεRIα and FcγRIIIA, but differences in that with FcαRI. These findings deepen our understanding of the signalling mechanisms of FcεRI and offer insights into the functionality of other immune receptors dependent on FcRγ., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Bacterial membrane vesicles (MVs) facilitate the long-distance delivery of virulence factors crucial for pathogenicity. The entry and trafficking mechanisms of virulence factors inside host cells are recently emerging; however, whether bacterial MVs can fuse and modulate the physicochemical properties of the host lipid membrane and membrane lipid parameter for fusion remains unknown. In this study, we reconstituted the interaction of bacterial MVs with host cell lipid membranes and quantitatively showed that bacterial MV interaction increases the fluidity, dipole potential, and compressibility of a biologically relevant multicomponent host membrane upon fusion. The presence of cylindrical lipids, such as phosphatidylcholine, and a moderate acyl chain length of C16 help the MV interaction. While significant binding of bacterial MVs to the raft-like lipid membranes with phase-separated regions of the membrane was observed, however, MVs prefer binding to the liquid-disordered regions of the membrane. Furthermore, the elevated levels of cholesterol tend to hinder the interaction of bacterial MVs, as evident from the favorable excess Gibbs free energy of mixing bacterial MVs with host lipid membranes. The findings provide new insights that might have implications for the regulation of host machinery by bacterial pathogens through manipulation of the host membrane properties.

The binding of the HIV-1 Gag polyprotein to the plasma membrane is a critical step in viral replication. The association with membranes depends on the lipid composition, but its mechanisms remain unclear. Here, we report the binding of non-myristoylated Gag to lipid membranes of different lipid compositions to dissect the influence of each component. We tested the contribution of phosphatidylserine, PI(4,5)P2, and cholesterol to membrane charge density and Gag affinity to membranes. Taking into account the influence of the membrane surface potential, we quantitatively characterized the adsorption of the protein onto model lipid membranes. The obtained Gag binding constants appeared to be the same regardless of the membrane charge. Furthermore, Gag adsorbed on uncharged membranes, suggesting a contribution of hydrophobic forces to the protein-lipid interaction. Charge-charge interactions resulted in an increase in protein concentration near the membrane surface. Lipid-specific interactions were observed in the presence of cholesterol, resulting in a two-fold increase in binding constants. The combination of cholesterol with PI(4,5)P2 showed cooperative effects on protein adsorption. Thus, we suggest that the affinity of Gag to lipid membranes results from a combination of electrostatic attraction to acidic lipids, providing different protein concentrations near the membrane surface, and specific hydrophobic interactions.

Frizzleds (ten paralogs: FZD 1-10 ) belong to the class F of G protein-coupled receptors (GPCRs), which remains poorly understood despite its crucial role in multiple key biological functions including embryonic development, stem cell regulation, and homeostasis in the adult. FZD 7 , one of the most studied members of the family, is more specifically involved in the migration of mesendoderm cells during the development and renewal of intestinal stem cells in adults. Moreover, FZD 7 has been highlighted for its involvement in tumor development predominantly in the gastrointestinal tract. This study reports the structure of inactive FZD 7 , without any stabilizing mutations, determined by cryo-electron microscopy (cryo-EM) at 1.9 Å resolution. We characterize a fluctuating water pocket in the core of the receptor important for FZD 7 dynamics. Molecular dynamics simulations are used to investigate the temporal distribution of those water molecules and their importance for potential conformational changes in FZD 7 . Moreover, we identify lipids interacting with the receptor core and a conserved cholesterol-binding site, which displays a key role in FZD 7 association with a transducer protein, Disheveled (DVL), and initiation of downstream signaling and signalosome formation., (© 2024. The Author(s).)

Introduction: Lipid-based nanoparticles (LNPs) is increasingly recognized for their potential in drug delivery, offering protection to hydrophobic drugs from degradation. Industrial synthesis of LNPs, exemplified by Pfizer-BioNTech and Moderna mRNA vaccines, utilizes flow chemistry or microfluidics, showcasing its scalability. This study explores the utilization of a novel design reactor, the vortex tube reactor, within flow chemistry for LNPs synthesis, aiming to optimize its conditions and compare them with batch synthesis., Methods: LNPs were synthesized using the vortex tube reactor, incorporating bovine serum albumin (BSA) as a model drug in the aqueous phase, alongside 1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol in the organic phase. Design of experiments (DoE), specifically Box-Behnken design, was employed to optimize parameters, including X 1 : the flow rate ratio (10-100 mL/min), X 2 : the aqueous-to-organic volumetric ratio (1:1-10:1), and X 3 : the number of reactor units (1-5 units). Responses evaluated encompassed physical properties and productivity. Optimized conditions were determined by minimizing particle size (Y 1 ), polydispersity index (Y 2 ), and zeta potential (Y 3 ), while maximizing entrapment efficiency (Y 4 ), drug loading (Y 5 ), and productivity (Y 5 )., Results: Results indicated that optimal conditions were achieved at X 1 of 100 mL/min, X 2 of 5.278, and X 3 of 1 unit. LNPs synthesized under these conditions exhibited favorable physical properties and productivity, with uniformity maintained across batches. The vortex tube reactor demonstrated superiority over batch synthesis, yielding smaller particles (166.23 ± 0.98 nm), more uniform nanoparticles (PDI 0.17 ± 0.01), and higher entrapment (67.75 ± 1.55%) and loading capacities (36.39 ± 0.83%), indicative of enhanced productivity (313.4 ± 12.88 mg/min)., Conclusion: This study elucidates the potential of flow chemistry, particularly utilizing the vortex tube reactor, for large-scale LNPs formulation, offering insights into parameter relationships and advancing nanoparticle synthesis for drug delivery applications., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Suwanpitak et al.)

The SARS-CoV-2 E protein is an enigmatic viral structural protein with reported viroporin activity associated with the acute respiratory symptoms of COVID-19, as well as the ability to deform cell membranes for viral budding. Like many viroporins, the E protein is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the structure of the protein complex have yielded inconclusive results, suggesting several possible oligomers, ranging from dimers to pentamers. Here, we combined patch-clamp, confocal fluorescence microscopy on giant unilamellar vesicles, and atomic force microscopy to show that E protein can exhibit two modes of membrane activity depending on membrane lipid composition. In the absence or the presence of a low content of cholesterol, the protein forms short-living transient pores, which are seen as semi-transmembrane defects in a membrane by atomic force microscopy. Approximately 30 mol% cholesterol is a threshold for the transition to the second mode of conductance, which could be a stable pentameric channel penetrating the entire lipid bilayer. Therefore, the E-protein has at least two different types of activity on membrane permeabilization, which are regulated by the amount of cholesterol in the membrane lipid composition and could be associated with different types of protein oligomers.

Polymer nanomicelles have the advantages of small particle size, improved drug solubility, retention effect and enhanced permeability, so they can be used in the treatment of tumour diseases. The aim of this study was to prepare and optimise a nanomicelle which can improve the solubility of insoluble drugs. Firstly, the carboxyl group of cholesterol succinic acid monoester was grafted with the side chain amino group of O-carboxymethyl chitosan-g-cholesterol succinic acid monoester (CCMC), and its structure was characterized by fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance ( 1 H-NMR). Particle size has an important impact on tissue distribution, cell uptake, permeability and inhibition of tumour tissue. In this study, particle size and polydispersity index (PDI) were selected as indexes to optimise the preparation process of CCMC nanomicelles through single factor experiment, Plackett-Burman experiment, the steepest climbing experiment and response surface design experiment. The optimised CCMC nanomicelles showed an average particle size of 173.9 ± 2.3 nm and a PDI of 0.170 ± 0.053. The Cell Counting Kit-8 assay showed no significant effect on cell viability in the range of 0-1000 μg ml -1 concentration. Coumarin-6 (C6) was used as a fluorescent probe to investigate the drug-carrying ability of CCMC nanomicelles. C6-CCMC showed 86.35 ± 0.56% encapsulation efficiency with a drug loading of 9.18 ± 0.32%. Both CCMC and C6-CCMC demonstrated excellent stability in different media. Moreover, under the same conditions, the absorption effect of C6 in C6-CCMC nanomicelles was significantly higher than that of free C6 while also exhibiting good sustained-release properties. Therefore, this study demonstrates CCMC nanomicelles as a promising new drug carrier that can significantly improve insoluble drug absorption., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

The ATP-binding cassette transporter P-glycoprotein (P-gp) is a multidrug efflux pump that is overexpressed in a variety of cancers and associated with the drug-resistance phenomenon. P-gp structures were previously determined in detergent and in nanodiscs, in which different transmembrane helix conformations were found, "straight" and "kinked," respectively, indicating a possible role of the lipid environment on the P-gp structural ensemble. Here, we investigate the dynamic conformational ensembles and protein-lipid interactions of two human P-gp inward-open conformers, straight and kinked, employing all-atom molecular dynamics (MD) simulations in asymmetric multicomponent lipid bilayers that mimic the highly specialized hepatocyte membrane in which P-gp is expressed. The two conformers are found to differ in terms of the accessibility of the substrate cavity. The MD simulations show how cholesterol and different lipid species wedge, snorkel, and partially enter into the cavity of the straight P-gp conformer solved in detergent. However, access to the cavity of the kinked P-gp conformer solved in nanodiscs is restricted. Furthermore, the volume and dynamic fluctuations of the substrate cavity largely differ between the two P-gp conformers and are modulated by the presence (or absence) of cholesterol in the membrane and/or of ATP. From the mechanistic perspective, the findings indicate that the straight conformer likely precedes the kinked conformer in the functional working cycle of P-gp, with the latter conformation representing a post substrate-bound state. The inaccessibility of the main transmembrane cavity in the kinked conformer might be crucial in preventing substrate disengagement and transport withdrawal. Remarkably, in our unbiased MD simulations, one transmembrane helix (TM10) of the straight conformer underwent a spontaneous transition to a kinked conformation, underlining the relevance of both conformations in a native phospholipid environment and revealing structural descriptors defining the transition between the two P-gp conformers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Liposomes are one of the most important drug delivery vectors, nowadays used in clinics. In general, polyethylene glycol (PEG) is used to ensure the stealth properties of the liposomes. Here, we have employed hydrophilic, biocompatible and highly non-fouling N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers containing hydrophobic cholesterol anchors for the surface modification of liposomes, which were prepared by the method of lipid film hydration and extrusion through 100 nm polycarbonate filters. Efficient surface modification of liposomes was confirmed by transmission electron microscopy, atomic force microscopy, and gradient ultracentrifugation. The ability of long-term circulation in the vascular bed was demonstrated in rabbits after i.v. application of fluorescently labelled liposomes. Compared to PEGylated liposomes, HPMA-based copolymer-modified liposomes did not induce specific antibody formation and did not activate murine and human complement. Compared with PEGylated liposomes, HPMA-based copolymer-modified liposomes showed a better long-circulating effect after repeated administration. HPMA-based copolymer-modified liposomes thus represent suitable new candidates for a generation of safer and improved liposomal drug delivery platforms., 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.)

Felodipine has proven to be effective as an atherosclerosis therapy because it increases blood flow to the vessel wall. However, the poor solubility, low bioavailability, and hepatic first-pass metabolism of oral felodipine compromise its therapeutic effectiveness. The study's goal is to create a nasal pH-sensitive hydrogel of felodipine-loaded invasomes (IPHFI) that will improve felodipine's release, permeation, bioavailability, and efficacy as a potential diabetes-associated atherosclerosis therapy. According to the pre-formulation study, the felodipine-loaded invasomes formulation composed of phospholipid (3%w/v), cholesterol (0.16%w/v), ethanol (3%v/v) and cineole (1%v/v) was chosen as the optimum formulation. The optimum formulation was characterized in vitro and then mixed with a mixture of chitosan and glyceryl monooleate to make the IPHFI formulation. The IPHFI formulation enhanced the release and permeation of felodipine by 2.99 and 3-fold, respectively. To assess the efficacy and bioavailability of the IPHFI formulation, it was studied in vivo using an experimental atherosclerosis rat model. Compared to oral free felodipine, the nasal administration of the IPHFI formulation increased the bioavailability by 3.37-fold and decreased the serum cholesterol, triglycerides, LDL, and calcification score by 1.56, 1.53, 1.80, and 1.18 ratios, respectively. Thus, nasal IPHFI formulation may represent a promising diabetes-associated atherosclerosis therapy., 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.)