Lipid Bilayer Research Articles

Background: Critical limb ischemia is a severe stage of peripheral artery disease. It causes claudication, ischemic pain, and ulceration. It is a serious condition that increases risks of limb amputation and death. While extracellular vesicles (EVs) secreted naturally from endothelial cells (ECs) or mesenchymal stem cells (MSCs) have shown promise for the treatment of ischemic limb diseases in mice, the clinical translation of EV therapy into patients has been limited by low yields from cultured cells. In this study, we evaluated the potential of xeno-transplanted nanovesicles (NVs) manufactured from human induced-pluripotent stem cells (hiPSCs) for the treatment of ischemic limb diseases in mice without administration of immunosuppressive drugs. Methods and Results: A hypo-immunogenic hiPSC line with β2-microglobulin knockout ( B2MKO hiPSCs) was used to manufacture NVs ( B2MKO hiPSC-NVs). NV size and concentration were measured using an Nanosight, NV morphology was imaged using an transmission electron microscope, and NV zeta potential was measured using the ZETASIZER Nano series. In vitro , the cytoprotective and proliferative effects of B2MKO hiPSC-NVs on human umbilical vein endothelial cells (HUVECs) were determined. In vivo , the therapeutic potential of B2MKO hiPSC-NVs was tested in a mouse model of hind limb ischemia, without administration of immunosuppressive drugs. Over 9,500 NVs could be manufactured from one hiPS cell. The zeta potential of B2MKO hiPSC-NVs was -16.7 mV, with a mean diameter of 115.9 ± 43.5 nm and bilayer lipid membranes. In vitro , B2MKO hiPSC-NVs protected HUVECs from hypoxic injury and promoted their proliferation. In vivo , B2MKO hiPSC-NV administration significantly improved blood perfusion, which was accompanied by significantly increased mouse EC proliferation and stimulated neovascularization in ischemic limbs, compared to control mice 14 days after treatment. Conclusions: B2MKO hiPSC-NVs hold significant potential for the treatment of ischemic limb diseases.

Dietary polyphenols, particularly flavonoids, have been extensively recognized for their role as a source of bioactive molecules that contribute to the prevention of various diseases, including cancer. This review aims to provide a comprehensive overview of dietary polyphenols by examining their sources, classification, mechanisms of action, and biological effects, with a particular emphasis on their nutritional and immunological roles. It also highlights the need for ongoing research into preventive strategies and the development of improved therapeutic options. Despite their broad spectrum of antioxidant, anti-inflammatory, neuroprotective, antimicrobial, anti-diabetic, and anti-cancer activities, the therapeutic application of polyphenols is significantly hindered by their inherently poor bioavailability. This limitation poses a substantial challenge, as it prevents polyphenols from achieving the systemic concentration necessary to elicit a therapeutic effect. This review critically evaluates current strategies, including nano- and liposomal-based delivery systems. Liposomal systems play a crucial role in enhancing the bioavailability of polyphenols by encapsulating these compounds in lipid bilayers. This encapsulation improves the solubility and stability of polyphenols, protects them from environmental degradation and rapid metabolism, and facilitates their controlled release and absorption in the body. Liposomes enable polyphenols to better traverse biological membranes and protect them from unfavorable conditions in the gastrointestinal tract, resulting in greater systemic availability and improved therapeutic efficacy compared to non-encapsulated forms. The current review also explores the modulatory impact of polyphenols on the immune system, their influence on gut microbiota, and their implications across various life stages, from infancy to aging, as well as in athletic performance and dermatological health. Future directions are proposed to optimize their clinical utility, including standardized dosing, improved delivery technologies, and targeted nutritional interventions. Ultimately, integrating polyphenols into daily dietary practices may offer promising avenues for enhancing immune resilience and preventing chronic diseases.

Environmental nanoplastics (ENPs) are generated from natural weathering of larger plastic waste. These nanoplastics are capable of disrupting normal cellular functions. Despite the growing threats of plastic pollution, >90% of current research on interactions between nanomaterials and biological systems employs pristine nanoparticles of uniform shape and size, most often polystyrene (PS) nanospheres. Pristine nanoparticles are incomplete models because true ENP waste is morphologically diverse. In this work, we describe how lipid composition and ENP shape affect particle-membrane interactions, using simulated environmental ENPs (sENPs) and giant unilammelar vesicles (GUVs) as models of plasma membranes. Critically, we provide the first systematic quantitative analysis of how ENP shape controls their capacity to permeabilize membranes. Compared to pristine spherical nanoparticles, sENPs damaged membranes with a wider variety of lipid types and charge states. The capacity to damage membranes is determined in large part by ENP shape: angular, more sharply cornered particles tend to increase membrane disruption, demonstrating how the true biophysical effects of ENP pollution cannot be fully captured using pristine materials alone.

Mapping Berberine distribution in liposomes: the role of drug-phospholipid interactions in localization and release.

Exosomes in Alzheimer's disease: From pathogenesis to therapeutics-A comprehensive review of diagnostic and drug delivery applications.

Headgroup-driven binding selectivity of alkylphospholipids to anionic lipid bilayers.

Structure and interfacial properties of phospholipid-containing sponge nanoparticles and their interaction with myoglobin.

The need for new strategies to reduce the susceptibility of polymeric materials to bacterial colonization is growing, especially with the emergence of drug-resistant bacterial strains. Antimicrobial agents used to modify polymers should not only be effective against microorganisms in both planktonic and biofilm states but also be safe and environmentally friendly. Phytochemicals, which are components of essential oils, may be a suitable choice to help combat microbial resistance to antibiotics. Furthermore, they meet the requirements of green chemistry. Essential oils synthesized by plants as secondary metabolites are capable of combating both Gram-positive and Gram-negative bacteria by disrupting lipid bilayers, affecting efflux pumps, compromising the integrity of bacterial cell membranes, and inhibiting the quorum-sensing system. They are also effective as adjuvants in antibiotic therapies. In this review, we outline the mechanism of action of various essential oil components that resulted in enhanced eradication of planktonic bacteria and biofilms. We summarize the use of these antimicrobial agents in macromolecular systems (nanovessels, fibers, nanocomposites, and blends) and provide an overview of the relationship between the chemical structure of phytochemicals and their antimicrobial activity, as well as their influence on the properties of polymeric systems, with a special focus on green active packaging materials.

Membrane mimicking protein nanodiscs are nanoscale structures composed of a lipid bilayer and a scaffold protein that forms a circular disc-like structure. These NDs are designed to mimic the natural cell membrane and are used as a platform to study membrane-associated proteins, such as those involved in signal transduction or drug transport. In cancer therapeutics, NDs have been developed as a promising nano-formulation for delivering macromolecules, such as drugs or nucleic acids, to cancer cells. The NDs can be functionalized with targeting ligands, such as antibodies or peptides, to specifically bind to cancer cells and deliver therapeutic payloads. One advantage of ND-based formulations is their ability to protect macromolecules from degradation and enhance their pharmacokinetics and bioavailability. Additionally, the use of NDs as a delivery vehicle allows for the precise control of drug release, which can improve efficacy while reducing toxic side effects. Overall, membrane mimicking protein NDs show great potential as a versatile platform for macromolecular delivery in cancer therapeutics, with the ability to precisely target cancer cells and enhance the therapeutic effect of drugs or nucleic acids. In this review, we discuss the structural components, stability issues, synthetic strategies, limitations, therapeutic advancements, and future challenges associated with the clinical implication of ND's anti-cancer therapies.

Understanding how membrane lipids interact with small-molecule drugs is crucial for applications in anesthetics, antibacterial agents, surfactants, and biopesticides. Key questions include identifying the factors that modulate drug affinity for target membranes and elucidating the mechanisms underlying their interactions and translocations across lipid bilayers. Molecular dynamics simulations are a widely used tool to characterize these interactions at a high resolution. Given the finely tuned lipid composition of different cell types and subcellular compartments, membrane models must account for this diversity to accurately represent the chemical, structural, and dynamic environments of the membrane of interest. Nonetheless, most simulation studies model drug-bilayer interactions using small patches of pure or binary lipid mixtures, with about 20-120 lipids per leaflet. In this study, we systematically investigate the effect of the model membrane patch size on the biophysical properties of symmetric bilayers composed of complex lipid mixtures. We used a mixture of POPC, SOPS, SOPE, PSM, SAPI, and cholesterol to model eukaryotic cell membranes and determine the minimum bilayer patch size required to accurately reproduce key biophysical properties. Systems were constructed in six sizes, ranging from 50 to 300 lipids per leaflet, at two cholesterol concentrations, resulting in 36 trajectories totaling 11.6 μs of simulation trajectories. Each system was simulated for at least four times longer than the time required to reach thermal equilibrium, ensuring sufficient sampling for analysis. Using metrics such as area per lipid, bilayer thickness, lipid headgroup tilt, 2D radial distribution functions, lipid tail splay angle, lipid packing defects, area compressibility, and lateral pressure profiles, we determined that a bilayer patch of approximately 8 × 8 nm, containing at least 150 lipids per leaflet, provides robust and efficient sampling of membrane structure and dynamics as well as sufficient surface area to capture interactions between membrane lipids and small molecules.

Eukaryogenesis remains one of biology's most intriguing transitions, yet the events driving the emergence of the eukaryotic cell membrane have not been sufficiently explored. Canonical membrane fusion models are not appropriate to explain the transition from an archaeal cell membrane to a bacterial one via heterochiral intermediates. Here, we show that a noncanonical, lipid-mediated mechanism spontaneously generates closed bilayers of mixed bacterial and archaeal lipids. Using as a proxy a combination of enhanced-sampling and unbiased molecular dynamics simulations, we demonstrate that transient edge-mediated archaeal intermediates merge with bacterial vesicles without the formation or expansion of a fusion pore. Key indicators include reduced energy barriers and significant membrane stability postfusion. The edge-induced route bridges the inconsistencies found in protocell models and protein-dependent pathways, proposing an unconventional explanation that describes how early eukaryotic systems achieved membrane continuity. These findings provide a plausible biophysical basis for the origin of the eukaryotic plasma membrane.

During the biogenesis of most eukaryotic integral membrane proteins (IMPs), transmembrane domains are inserted into the endoplasmic reticulum membrane by a dedicated insertase or the SEC61 translocon. The SRP-independent (SND) pathway is the least understood route into the membrane, despite catering for a broad range of IMP types. Here, we show that Chaetomium thermophilum SND3 is a membrane insertase with an atypical fold. We further present a cryo-electron microscopy structure of a ribosome-associated SND3 translocon complex involved in co-translational IMP insertion. The structure reveals that the SND3 translocon additionally comprises the complete SEC61 translocon, CCDC47 and TRAPɑ. Here, the SEC61β N-terminus works together with CCDC47 to prevent substrate access to the translocon. Instead, molecular dynamics simulations show that SND3 disrupts the lipid bilayer to promote IMP insertion via its membrane-embedded hydrophilic groove. Structural and sequence comparisons indicate that the SND3 translocon is a distinct multipass translocon in fungi, euglenozoan parasites and other eukaryotic taxa.

The term extracellular vesicles (EVs) includes a variety of anucleated, non-self-replicative particles released by cells, whose cargo content is compartmentalized by a lipidic bilayer membrane and includes proteins, DNA, and RNA (both coding and non-coding) molecules. MicroRNAs (miRs) are small non-coding RNA involved in gene expression regulation that functionally participate in inter-cellular communication as EV cargo. Natural Killer (NK) cells are innate immunity lymphocytes specialized in the killing of cancer cells and virally infected cells. Increasing evidence shows that NK cell-derived EVs contribute to the anti-tumoral activity of NK cells and that such effects are, at least in part, mediated by the miR cargo of these EVs. Conversely, cancer cells release EVs whose cargo includes proteins and miRs that impair NK cell function. These interactions highlight a central role for EV miRs both in the NK-mediated cytotoxicity and as a major immune-escape mechanism for cancer cells, ultimately contributing to the overall success or failure of NK cells in eliciting their anti-tumoral activity.

Exosomes have garnered significant interest in biomedical research due to their potential therapeutic applications. They are extracellular vesicles secreted by cells, distinguished by a lipid bilayer membrane that encases various biological substances, including nucleic acids and proteins, within their lumen or lipid bilayer. They offer several advantages, like superior compatibility and targeted delivery capability, enabling innovative therapeutic development and efficient drug transport across cellular barriers, including the BBB. They also provide long circulation times, low toxicity, and protection from degradation. They play critical roles in cell communication, tissue repair, facilitating immune response, modulating inflammation, homeostasis, transferring molecules between cells, and specifically homing to tumor sites. Techniques such as microfluidic-based isolation and surface modification are advancing the production of clinical-grade exosomes. Exosomes have shown potential in delivering drugs for a wide range of diseases, including cancer, neurological disorders, infectious diseases, and cardiovascular issues. Challenges such as low yield during isolation, difficulty in large-scale production, heterogeneity of exosome populations, and maintaining stability during storage hinder their practical use. Multidisciplinary research is needed to overcome these limitations and unlock their potential in early disease detection and therapy, with future applications expected in advanced drug delivery, diagnostic biomarkers, and disease prognosis. Regulatory considerations, including rigorous preclinical and clinical trials, are crucial for translating these innovations into approved therapies. This review highlights the emerging development of exosomes in therapeutic drug delivery systems. The loading technique is discussed in a detailed manner through which the drugs are attached to exosomes and delivered to the target site.

A ubiquitous problem in protein analytics and medical biotechnology is assessing the interaction of a membrane protein receptor with its cognate protein ligand. This task generally requires transferring the receptor from native membranes or other expression host systems into supported lipid bilayers, liposomes, or nanodiscs. Such a reintegration process necessitates multiple steps for protein solubilization, renaturing, and functional reconstitution. Here, we opportunistically show that biolayer interferometry (BLI) can be directly utilized to evaluate the pre-equilibrium binding kinetics of a membrane protein receptor with its protein ligand in a label-free and membraneless setting. We present real-time measurements probing the association and dissociation phases of these transient complexes, conducted at a high signal-to-noise ratio using free proteomicelles in solution. As a proof-of-concept, we employ a subset of synthetic membrane proteins equipped with a programmable antibody mimetic binder that targets a specific protein ligand. Proteomicelles containing these binder-equipped membrane proteins exhibit high-affinity interactions with ligands attached to the sensor surface. These determinations are further validated by closely related surface plasmon resonance (SPR) measurements of the binder-ligand and proteomicelle-ligand interactions. Finally, this approach is amenable to high-throughput data collection, and its conceptual formulation is potentially extendable to other membrane proteins.

Terephthyl-linked acyl hydrazone-based K+/Cl- ion pair symporter: synthesis, mechanistic insights, and in vitro cytotoxicity evaluation.

Molecular dynamics simulations play an important role in investigating biological systems. However, simulating large-scale systems can be computationally expensive, which can be improved by the employment of a coarse-graining force field. This study focuses on the automated parametrization of small molecules within the CGCompiler framework. This optimization approach utilizes a mixed-variable particle swarm algorithm to avoid the manual tweaking of parameters. Particularly, the optimization focuses on matching experimentally known log P values of partitioning in water-octanol phases, reproducing atomistic density profiles in lipid bilayers, and optimizing overall shape and volume aspects of the modeled atomistic molecules. After the atomistic to coarse-grained mapping, the model’s accuracy is evaluated through a fitness function, which combines structural and dynamic targets, to accurately capture the shape and behavior of the small molecule in question. Through the investigation of the interactions between small molecules and cellular membranes, this optimization process supports the development of accurate coarse-grained models for small molecules relevant to drug discovery. Our work demonstrates promising results in automating the high-fidelity parametrization of small molecules using the Martini 3 force-field guided by experimental log P values.

We report on development of a vibrational probe (N3–C12H25, az12) suitable for reportingon the rigidityand fluidity of the interfacial region in lipid bilayers. Such probecan help assess critical biological functions of cell membranes, includingmembrane permeability. We demonstrated a high sensitivity of the az12probe to the rigidity of the interfacial region, manifested in thesensitivity of the width of the N3 moiety asymmetric stretchingmode absorption peak. The structural and dynamic changes associatedwith the gel–liquid-crystal phase transition (Lβ-Lα)for three different lipids dipalmitoylphosphatidylcholine (DPPC),dipalmitoylphosphatidylglycerol (DPPG), and egg sphingomyelin (SM)were studied using FTIR and two-dimensional infrared (2DIR) spectroscopies.In addition to the new az12 probe targeting the interfacial region,the N3-(CH2)11–CN probe (az11CN),recently developed to examine the hydrophobic region of bilayers,was also used, showing characteristic phase transitions for each bilayerat their characteristic phase transition temperatures. An order parameter, SN3, corrected for the difference in the polaritiesof the interfacial and hydrophobic regions, was constructed. It showshow the overall rigidity of the bilayer is divided between the interfacialand hydrophobic regions, emphasizing their correlations. 2DIR spectraldiffusion data were acquired for az12 and az11CN probes in the bilayerat various temperatures, reporting on inhomogeneous and homogeneousline width contributions (rigidity) and correlation times (fluidity)for the interfacial and hydrophobic regions. The spectral diffusiondata for the interfacial region for all three bilayer types show largestatic inhomogeneous contributions, which are absent in the hydrophobicregion data. The spectral diffusion data revealed differences fordifferent bilayers, most apparent for the interfacial region. A greaterincrease in a homogeneous line width for SM with temperature, comparedto that for DPPC and DPPG, could be linked to an increase in waterpermeability to the interfacial region of SM at higher temperatures.We found that the az12 probe constitutes a powerful reporter to measurerigidity (exerted angular constraints) and fluidity (time responsesof the environment) in the interfacial region of a bilayer.

The SARS‐CoV‐2 virus—responsible for the COVID‐19 pandemic—requires a replication–transcription complex (RTC) for efficient RNA synthesis and viral propagation. One critical RTC component is nonstructural protein 4 (nsp4), a multipass transmembrane (TM) protein implicated in endoplasmic reticulum (ER) membrane rearrangements and double‐membrane vesicle (DMV) formation. The membrane topology and functional role of nsp4 in SARS‐CoV‐2 remain unclear. Here we determined that SARS‐CoV‐2 nsp4 contains a partially cleaved signal peptide, three TM segments, an extracellularly oriented N‐terminus (towards the ER lumen in human cells), and a cytoplasm‐facing C‐terminus. The non‐canonical glycosylation sequon (N131IC) in nsp4 is not glycosylated in mammalian cells. Molecular dynamics simulations based on these findings refined the structural predictions and folding of individual nsp4 molecules in lipid bilayers, elucidating its membrane disposition. This study advances our understanding of nsp4 membrane topology and its contributions to the formation of DMV and double‐membrane‐spanning pores, which are essential for viral RNA transport.

In this cryo-electron microscopy study, we provide mechanistic insights into how an archetypical β-barrel pore-forming toxin (β-PFT), Vibrio cholerae Cytolysin (VCC), ruptures the membrane lipid bilayer by inducing membrane curvature. We demonstrate how VCC oligomers cluster together and drastically increase local membrane curvature, thereby causing membrane blebbing. In addition, we also show how these PFTs, after rupturing the host membrane, tend to form symmetric supermolecular assemblies to stabilize their hydrophobic transmembrane rim domains. We further provide another example of membrane rupture with gamma hemolysin, a Staphylococcal bicomponent β-PFT. These insights will usher in new studies on membrane curvature due to protein crowding and broaden our mechanistic understanding of how this largest class of bacterial protein toxins induces host cellular death.