Phospholipid Bilayers Research Articles

In Vivo and Ex Vivo Evaluation of a Novel Method for Topical Delivery of Macromolecules Through the Stratum Corneum for Cosmetic Applications.

Effective topical delivery of large/charged molecules into skin has always been challenging. Chemical penetration enhancers, organic substances that increase permeability of skin, have been in use for decades with variable success. One application of enhancers involves multilamellar vesicles composed of submicron emulsion droplets and micelles surrounded by concentric phospholipid bilayers. This report introduces the next generation of multilamellar vesicles, termed Tiered-Release Vesicles (TRVs), as a new platform for topical delivery of macromolecules such as peptides and hyaluronic acid (HA). Fluorescently labeled peptides and HA, diffusion cells, and confocal microscopy were employed to assess the penetration efficiency of macromolecules in TRV formulations using an ex vivo human skin model. Two in vivo studies utilized punch biopsies followed by histochemical staining and analysis. Based on fluorescent intensity, TRV formulations delivered a large peptide more completely (2-5 fold) into ex vivo human skin than optimized liposomes. The penetration of 2 HA species in TRV formulations was 3- to 13-fold higher than with a simple gel vehicle. In the case studies, reduction of solar elastosis was observed from a topical TRV formulation. Topical delivery of large peptides and HA into human skin using TRV technology has been demonstrated.

Spontaneous nanosized liposome formation from crude dried lecithin upon addition of glycerol

Nanosized liposomal vesicles (NLV) were successfully prepared using natural sunflower lecithin without the use of high-pressure homogenization or filtration. Upon glycerol addition to dispersions of lecithin multilamellar vesicles (MLVs), these broke down spontaneously to liposomes with diameters in the range of 100–200 nm. Static light scattering demonstrated that glycerol addition above 30% (w/w) induced the complete transformation of MLVs into NLVs. Langmuir trough compression experiments showed a two-region compressional behavior. Upon 62% (w/w) glycerol addition, the compressional modulus of the liposomes decreased from 18.5 to 8.13 mN/m. Water activity and pulse NMR measurements also showed a divergence in behavior above 30% (w/w) glycerol. Liposomes were not birefringent in water but became strongly birefringent at and above 30% (w/w) glycerol, as determined by polarized light microscopy, and lost all birefringence above 80% (w/w). This was interpreted as the induction of stress-birefringence in the phospholipid bilayers above 30% (w/w) glycerol, and a relaxation of such stress above 80% (w/w) glycerol. We hypothesize that the mixture of phospholipids in the lecithin results in an effective non-zero intrinsic curvature for the molecular mixture, which lowers the bending energy of the bilayer, allowing for an easier break-up upon mixing. Secondly, glycerol addition decreases attractive van der Waals’ interaction between lamellae in an MLV, thus weakening the multilamellar liposome walls. Glycerol also affects bilayer stability by strengthening the hydrogen bond network of water, which will affect phospholipid headgroup hydration. All these factors result in the spontaneous breakdown of MLVs into NLVs.

Interaction of meloxicam derivatives with phosphatidylcholine bilayers: A calorimetric study.

The drug interactions with the lipid membranes are crucial in many biochemical processes. Phospholipid model membranes are often used to assess such interactions. Our team has been researching new compounds with anti-inflammatory and analgesic effects for many years. Such compounds are derivatives of the well-known non-steroidal anti-inflammatory drug (NSAID) - meloxicam (MLX). Their biological target is cyclooxygenase (COX) - a membrane protein. The NSAIDs are mainly taken orally; therefore, drug-membrane interaction is a preliminary stage in the body. The purpose of the present work was to investigate the ability of 2 new MLX derivatives (compound PR51 and PR52) to interact with model membranes, in comparison to known NSAIDs medicine - MLX. The differential scanning calorimetry (DSC) method was used to study those interactions. As a model membrane, bilayers obtained from a phospholipid (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)) were used. Calorimetric measurements were performed using a differential scanning calorimeter DSC 214 Polyma equipped with an intracooler IC70. All examined compounds decreased the main transition temperature of DPPC in a concentration-dependent manner. The addition of studied compounds to DPPC also resulted in broadening of the transition peaks. Moreover, all examined compounds decreased the enthalpy of the DPPC main phase transition. For all DPPC gel-liquid crystalline phase transition parameters, the most pronounced effects were found for PR51 compound. We have shown that the above interactions depend on the chemical structure of individual compound. All studied compounds alter biophysical properties of phospholipid bilayer.

Damage of the Phospholipid Bilayer by Aβ42 at Physiologically Relevant Peptide Concentrations.

Amyloid β (Aβ) aggregates are implicated in the pathology of several neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, and Parkinson's disease, and damage to membranes is considered one of the pathology-related effects of Aβ. Experiments in vitro indicate that Aβ can damage these membranes; however, such experiments were performed at Aβ concentrations in the micromolar range, several orders above the physiologically relevant conditions. Our studies with Aβ42 in the low nanomolar concentrations did not reveal any damage to the supported lipid bilayer, questioning this membrane damage mechanism of Aβ. However, the phospholipid composition can be a factor contributing to the interaction of Aβ with the membrane. Therefore, in this study, we investigated the interaction of 50 nM Aβ42 with supported lipid bilayers composed of equimolar ratios of POPS and POPC at phospholipid concentrations of 0.1 and 0.25 mg/mL. Using atomic force microscopy (AFM), we observed that Aβ42 induced damage to bilayers at 0.1 mg/mL, characterized by forming defects that grew in size and number over time. The defects penetrate only the upper leaflet of the bilayer, but no such defects were observed at 0.25 mg/mL phospholipid concentrations. We additionally determined Young's modulus of these bilayers as a measure of stiffness, and these values were 6.9 ± 3.6 MPa and 16.6 ± 5.3 MPa for the 0.1 mg/mL and the 0.25 mg/mL bilayers, respectively. These findings suggest that Aβ42's ability to induce bilayer damage depends on membrane stiffness, with softer bilayers (0.1 mg/mL) being more susceptible to Aβ42-induced damage. The results are discussed and compared with models in which Aβ42 oligomers create localized membrane damage. The implication of the results to the mechanisms of the Aβ42 oligomer pathology is discussed.

Thienothiophene-Benzopyran Derivative and AQ4N-Assembled Liposomes for Near-Infrared II Fluorescence Imaging-Guided Phototherapy, Chemotherapy, and Immune Activation.

Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has attracted wide attention in tumor treatment. However, the hypoxic tumor microenvironment and the heat shock proteins produced by tumor cells significantly reduce their efficacy. Developing effective phototherapy agents that have high reactive oxygen species generation efficiency and photothermal conversion efficiency (PCE) while simultaneously utilizing the hypoxic tumor microenvironment is of great importance. Here, a thienothiophene-benzopyran derivative, BTPIC4F-C10 is designed and synthesized, with near-infrared (NIR) absorption and fluorescence. Then the lipid nanoparticles (LipBFCA NPs) which encapsulated BTPIC4F-C10 in a phospholipid bilayer together with hypoxia-activated prodrug banoxanthrone (AQ4N) are constructed for NIR-II fluorescence imaging-guided synergistic PDT/PTT/chemotherapy and immune activation. Under 808nm laser irradiation, LipBFCA NPs is a high singlet oxygen quantum yield of 20.2% and PCE of 78.8%. With ultra-high photon energy utilization efficiency of 99%, LipBFCA NPs is an excellent phototherapy effect. The hypoxic environment caused by phototherapy can further activate AQ4N to transform into chemically toxic AQ4 radicals to kill tumor cells. Moreover, phototherapy can induce immunogenic cell death, release tumor-associated antigens, and activate immune responses. This work provides a new way for the clinical application of fluorescence imaging in guiding tumor diagnosis and treatment.

Microfluidic Fabrication of Oleosin-Coated Liposomes as Anticancer Drug Carriers with Enhanced Sustained Drug Release.

Microfluid-derived liposomes (M-Lipo) exhibit great potential as drug and functional substance carriers in pharmaceutical and food science. However, the low liposome membrane stability, attributed to the liquid core, limits their application range. Oleosin, a natural surfactant protein, can improve the stability of the lipid nanoparticle membrane against various environmental stresses, such as heat, drying, and pH change; in addition, it can enable sustained drug release. Here, we proposed the fabrication of oleosin-coated M-Lipo (OM-Lipo) through self-assembly on a microfluidic chip and the evaluation of its anticancer drug (carmustine) delivery efficiency. Nanoparticle characterization revealed that the oleosin coating slightly lowered the membrane potential of M-Lipo and greatly improved their dispersibility. Additionally, the in vitro drug release profile showed that the oleosin coating improved the sustained release of the hydrophobic drug from the phospholipid bilayer in body temperature. Our results suggest that OM-Lipo has sufficient potential in various fields, based on its easy production, excellent stability, and biocompatibility.

Phospholipid dynamics in Aspergillus species: relations between biological membrane composition and cellular morphology.

Biological membranes, primarily composed of phospholipid bilayers, are essential structures that compartmentalize the cell from the extracellular environment. The biosynthesis and regulation of membrane lipids have been extensively studied in model organisms such as Saccharomyces cerevisiae and mammalian cells. However, our understanding of biological membrane regulation in filamentous fungi, some of which are significant in medicine, pharmacy, and agriculture, remains limited. This minireview provides a comprehensive overview of the latest knowledge, focusing on filamentous fungi of Aspergillus species. Recent progress in understanding dynamic changes in membrane lipid profiles, driven by improvements in analytical techniques for lipidomics, is also presented. Furthermore, known that the cell morphology of filamentous fungi is closely linked to its harmful and beneficial characteristics, the influence of membrane composition on cell morphology is discussed. The integration of these findings will further enhance our understanding of the biological functions of membranes in filamentous fungi.

Suppressing Effect of Flavonoid Compounds on Lipids Photooxidation of Sheep Red Blood Cells and Oleic Acid Photooxidation

ABSTRACTPhotosensitizers and pigments in raw meat such as porphyrins, riboflavin, and myoglobin after incorporation with light beam prompt the generation of singlet oxygen (1O2) from triplet oxygen (3O2) and cause oxidative rancidity of meat products. In this study, the results of photooxidation reactions of sheep erythrocyte (red blood cell) model as a model rich in hemoglobin and phospholipids bilayer, and oleic acid model were obtained by 1H NMR spectroscopy, TBARS assay, and iodometric titration. In both models, the rate of lipid photooxidation in the presence of hydroalcoholic extracts of Turmeric (Curcuma longa L.) and Cumin (Cuminum cyminum L.) as natural antioxidants, Butyl hydroxytoluene (BHT) as a synthetic antioxidant, and sodium azide (NaN3) as a well‐known 1O2 scavenger were decreased in the order of NaN3 > Turmeric > Cumin > BHT. It was proven that during the photooxidation process, there is a direct association between the amount of flavonoid compounds and 1O2 scavenging.

Leveraging machine learning to streamline the development of liposomal drug delivery systems

Drug delivery systems efficiently and safely administer therapeutic agents to specific body sites. Liposomes, spherical vesicles made of phospholipid bilayers, have become a powerful tool in this field, especially with the rise of microfluidic manufacturing during the COVID-19 pandemic. Despite its efficiency, microfluidic liposomal production poses challenges, often requiring laborious, optimization on a case-by-case basis. This is due to a lack of comprehensive understanding and robust methodologies, compounded by limited data on microfluidic production with varying lipids. Artificial intelligence offers promise in predicting lipid behaviour during microfluidic production, with the still unexploited potential of streamlining development. Herein we employ machine learning to predict critical quality attributes and process parameters for microfluidic-based liposome production. Validated models predict liposome formation, size, and production parameters, significantly advancing our understanding of lipid behaviour. Extensive model analysis enhanced interpretability and investigated underlying mechanisms, supporting the transition to microfluidic production. Unlocking the potential of machine learning in drug development can accelerate pharmaceutical innovation, making drug delivery systems more adaptable and accessible.

Towards Understanding the Role of the Glycosylation of Proteins Present in Extracellular Vesicles in Urinary Tract Diseases: Contributions to Cancer and Beyond.

Extracellular vesicles (EVs) are a population of nanoscale particles surrounded by a phospholipid bilayer, enabling intercellular transfer of bioactive molecules. Once released from the parental cell, EVs can be found in most biological fluids in the human body and can be isolated from them. For this reason, EVs have significant diagnostic potential and can serve as an excellent source of circulating disease biomarkers. Protein glycosylation plays a key role in many biological processes, and aberrant glycosylation is a hallmark of various diseases. EVs have been shown to carry multiple glycoproteins, but little is known about the specific biological roles of these glycoproteins in the context of EVs. Moreover, specific changes in EV glycosylation have been described for several diseases, including cancers and metabolic, cardiovascular, neurological or kidney diseases. Urine is the richest source of EVs, providing almost unlimited (in terms of volume) opportunities for non-invasive EV isolation. Recent studies have also revealed a pathological link between urinary EV glycosylation and urological cancers, as well as other pathologies of the urinary tract. In this review, we discuss recent research advances in this field and the diagnostic/prognostic potential of urinary EV glycosylation. In addition, we summarize common methods for isolating EVs from urine and techniques used to study their glycosylation.

Naphthylated LEGO-lipophosphonoxin antibiotics used as a fluorescent tool for the observation of target membrane perturbations preceding its disruption

Linker-Evolved-Group-Optimized-Lipophosphonoxins (LEGO-LPPO) are small synthetic modular peptidomimetics with promising antimicrobial activity. The LEGO-LPPO mechanism of antibacterial action has been determined to be the depolarization and disruption of bacterial membranes. Their modular nature is advantageous for fine tuning their biological properties. In order to optimize the structure of LEGO-LPPO even further, it is important to understand the interaction of LEGO-LPPO with bacterial membranes at the molecular level. In this work, we present the synthesis of five LEGO-LPPO (designated as1_naph2-4-G to5_naph2-4-G) molecules bearing fluorescent naphtylethyl moieties and their usage in the study of LEGO-LPPO behaviour in the membrane. Our goal was to characterize fluorescently labelled LEGO-LPPO under conditions that do not completely disrupt the membrane, mostly in the form of membrane-bound monomers. We observed the intramolecular interactions of hydrophobic modules of1_naph2-4-G in the buffer by detecting dynamic naphthyl excimers and their disappearance after1_naph2-4-G bind into the membranes. In the membrane, the molecule1_naph2-4-G slightly affects the membrane fluidity of DOPG membranes above the phase transition. The naphthyl fluorophore itself has fast and almost unrestricted rotation around ethylene linking groups (rinf= 0.010), which indicates a considerable chaotropic effect of the hydrophobic modules of1_naph2-4-G at the given depth of the membrane.1_naph2-4-G proved to be a useful model for observing the interaction of LEGO-LPPO antibiotics with the phospholipid bilayer enabling us to decipher its effects on membrane state and dynamics; its binding and penetration into the membrane, its structure and the particular depth that it occupies.

Highly Resistive Biomembranes Coupled to Organic Transistors enable Ion‐Channel Mediated Neuromorphic Synapses

AbstractOrganic electrochemical transistors (OECTs) functionalized with lipid membranes could enable new hybrid synapses for sensing and neuromorphic computing in biological media. However, prior attempts to pair these components resulted in low quality membranes formed on the OECT surface. We present a new method for forming a highly‐resistive phospholipid bilayer coupled to a poly(3,4‐ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) OECT that avoids the disruptive effects of the rough polymer surface. Transistor coupled droplet bilayers (TCDBs) formed from diphytanoyl phosphatidylcholine lipids exhibit an average specific resistance that is 1000X higher than values reported for solid‐supported lipid membranes assembled on PEDOT:PSS. High membrane resistance and the addition of voltage‐activated ionophores enable us to demonstrate that selective ion transport and spontaneous membrane resealing in response to dynamic gate voltage imparts selective programming and memory‐storage capability to an OECT without chemical modification of the PEDOT:PSS. These capabilities enable paired‐pulse facilitation and depression with writing speeds and memory retentions that are both >10X higher than previously reported with membrane‐coated OECTs. The high resistance of the TCDB establishes a basis for hybrid biomolecular synapses that can integrate stimuli‐responsive membranes and organic electronics for future applications in sensing, signal processing, and neuromorphic computing at the edge of biology.

Ligand-Based Radical Reactivity of Metal Thiosemicarbazones Prompts the Identification of Platinum(II)-Based Cytoprotectants.

CuATSM, a copper(II) complex of a bis(thiosemicarbazone) of diacetyl, prevents oxidative cell death and acts as a neuroprotectant in vivo, prompting its evaluation to treat amyotrophic lateral sclerosis and other neurodegenerative conditions in the clinic. We recently demonstrated that CuATSM functions as a potent radical-trapping antioxidant (RTA), inhibiting lipid peroxidation and associated ferroptotic cell death by a noncanonical mechanism based on radical addition to the ligand backbone. Herein we report our investigations of the generality of this reactivity, which include studies of corresponding complexes of various other metals, including Co, Ru, Ni, Pd, Pt, and Au. Inhibited autoxidations of styrene and dioxane reveal that most of these complexes exhibit RTA activity, consistent with ligand-based reactivity, but the identity of the metal atom nevertheless plays a role. In particular, analyses of the electronic structures of the complexes of metals within the same group (i.e., the group 10 metals Ni, Pd and Pt) highlight how the metal atom can modulate the ligand-based reactivity by enabling spin delocalization to the other thiosemicarbazone moiety. The RTA activity determined in organic solution largely translates to phospholipid bilayers and mammalian cells, where most complexes inhibited lipid peroxidation and associated ferroptotic cell death. A preliminary structure-activity study revealed Pt complexes with potencies eclipsing those of archetype ferroptosis inhibitors ferrostatin-1 and liproxstatin-1, suggesting that Pt (and to a lesser extent Ni) bis(thiosemicarbazone)s may be better suited to optimization for therapeutic development than those based on Cu.

Enhanced in vivo Stability and Antitumor Efficacy of PEGylated Liposomes of Paclitaxel Palmitate Prodrug.

The clinical use of paclitaxel (PTX) in cancer treatment is limited by its poor water solubility, significant toxicity, and adverse effects. This study aimed to propose a straightforward and efficient approach to enhance PTX loading and stability, thereby offering insights for targeted therapy against tumors. We synthesized a paclitaxel palmitate (PTX-PA) prodrug by conjugating palmitic acid (PA) to PTX and encapsulating it into liposomal vehicles using a nano delivery system. Subsequently, we investigated the in vitro and in vivo performance as well as the underlying mechanisms of PTX-PA liposomes (PTX-PA-L). PTX had a remarkable antitumor effect in vivo and significantly decreased the myelosuppressive toxicity of PTX. Moreover, the introduction of PA increased the lipid solubility of PTX, forming a phospholipid bilayer as a membrane stabilizer, prolonging the circulation time of the drug and indirectly increasing the accumulation of liposomes at the tumor site. Our in vivo imaging experiments demonstrated that PTX-PA-L labeled with DiR has greater stability in vivo than blank liposomes and that PTX-PA-L can target drugs to the tumor site and efficiently release PTX to exert antitumor effects. In a mouse model, the concentration of PTX at the tumor site in the PTX-PA-L group was approximately twofold greater than that of Taxol. However, in a nude mouse model, the concentration of PTX at the tumor site in the PTX-PA-L group was only approximately 0.8-fold greater than that of Taxol. Furthermore, the originally observed favorable pharmacodynamics in normal mice were reversed following immunosuppression. This may be caused by differences in esterase distribution and immunity. This prodrug technology combined with liposomes is a simple and effective therapeutic strategy with promising developmental prospects in tumor-targeted therapy owing to its ability to convert PTX into a long-circulating nano drug with low toxicity, high pharmacodynamics, and good stability in vivo.

Construction and Evaluation of Hepatic Targeted Drug Delivery System with Hydroxycamptothecin in Stem Cell-Derived Exosomes.

Hydroxycamptothecin (HCPT) is commonly used in the treatment of liver cancer; however, its low water solubility and poor stability significantly limit its clinical application. In recent years, research on exosomes has deepened considerably. Exosomes possess a unique phospholipid bilayer structure, enabling them to traverse tissue barriers, which provides natural advantages as drug carriers. Nevertheless, delivering exosomes safely and efficiently to target cells remains a major challenge. In this study, we utilized the affinity of the SP94 peptide for human liver cancer cell receptors. HCPT was coated with exosomes in our experimental design, and the exosome membrane was modified with SP94 peptide to facilitate drug delivery to liver cancer cells. Exosomes were purified from bone marrow mesenchymal stem cells, and targeted peptides were attached to their surfaces via post-insertion techniques. Subsequently, HCPT was incorporated into the exosomes through electroporation. Using the HepG2 hepatoma cell line, we evaluated a series of in vitro pharmacodynamics and studied pharmacokinetics and tissue distribution in animal models. The results indicated that ligand-targeted, modified drug-carrying exosomes significantly enhance drug bioavailability, prolong retention time in vivo, and facilitate liver targeting. Moreover, this approach reduces drug nephrotoxicity, enhances anti-tumor efficacy, and lays the groundwork for the development of novel liver cancer-targeting agents.

Extracellular vesicles characterization in patients with hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is a genetic disorder, diagnosed according to the presence of phenotypical traits of the myocardium. A specific biomarker which can associate with the severity of HCM is lacking. Extracellular Vesicles (EVs), nanoparticles released by cells into biological fluids, hold promise as accessible diagnostic tools, as their abundance and molecular composition can reflect the severity of cardiac diseases (Rizzuto 2024). We aim at characterising plasma-derived EVs isolated from 28 HCM patients and 18 healthy volunteers (CTR). Methods The whole cohort underwent transthoracic echocardiography, whereas magnetic resonance and exome sequencing was performed on HCM patients. EVs were isolated via ultracentrifugation from plasma of both groups. Quantitative and qualitative assessments of EVs were performed by nanoparticle tracking analysis, transmission electron microscopy, Western blot, targeted (Olink) and untargeted (nLC-MS/MS) proteomics and FACS analysis. Plasma-derived EV subpopulations were characterised according to their cellular origin. Data are expressed as median and interquartile range (25th, 75th). Results Most patients were male (68% HCM and 66% CTR) with a median age of 58 (49-69) years (HCM) and 47 (44-52) (CTR). Among HCM patients, missense mutations in the MYH7 gene were the most identified. The median maximum wall thickness (WTMax) in HCM was 16 mm (15-19) vs 8 mm (7-9) in CTR. The isolated EVs expressed tetraspanins (CD9, CD63 and CD81), Alix and β-integrin and showed an intact phospholipid bilayer of 100 nm in size. EV concentration was reduced in HCM vs CTRL, respectively 2.8*109 EV/ml/cell count (2*109-4*109) and 4.6*10⁹ EV/ml/cell count (3*109-6*109). Among 15 plasma-derived EV subpopulations studied, those released from platelets (CD41a) and activated platelets (CD62P and CD40L) were increased in HCM patients vs CTR by 1.7 fold. Negative associations were found between E/e’, parameter of diastolic function, and cardiomyocyte-derived EVs (r= -0.2658), and between left ventricle ejection fraction and platelet-derived EVs (r=-0.2189); a positive correlation was found between WTMax and EVs derived from activated endothelial cells (r=0.2330). The proteomic analysis of EVs shows an enrichment in proteins related to platelets adhesion and inflammation in HCM patients. Conclusions HCM patients present a peculiar phenotypic pattern of EVs that may associate with diagnostic clinical features of HCM.

High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array.

Extracellular vesicles (EVs) are small particles with phospholipid bilayers that carry a diverse range of cargoes including nucleic acids, proteins and metabolites. EVs have important roles in various cellular processes and are increasingly recognized for their ubiquitous role in cell-cell communications and potential applications in therapeutics and diagnostics. Although many methods have been developed for the characterization and measurement of EVs, analyzing them from biofluids remains a challenge with regard to throughput and sensitivity. Recently, we introduced an approach to facilitate high-throughput analysis of EVs from trace amounts of sample. In this method, an amphiphile-dendrimer supramolecular probe (ADSP) is coated onto a nitrocellulose membrane for array-based capture and to enable an in situ immunoblotting assay. Here, we describe the protocol for our array-based method of EV profiling. We describe an enhanced version of the method that incorporates an automated printing workstation, ensuring high throughput and reproducibility. We further demonstrate the use of our array to profile specific glycosylations on the EV surface using click chemistry of an azide group introduced by metabolic labeling. In this protocol, the synthesis of ADSP and the fabrication of ADSP nitrocellulose membrane array can be completed on the same day. EVs are efficiently captured from biological or clinical samples through a 30-min incubation, followed by an immunoblotting assay within a 3-h window, thus providing a high-throughput platform for EV isolation and in situ targeted analysis of EV proteins and their modifications.

Multiplex antimicrobial activities of the self-assembled amphiphilic polypeptide β nanofiber KF-5 against vaginal pathogens

BackgroundVaginal infections caused by multidrug-resistant pathogens such as Candida albicans and Gardnerella spp. represent a significant health challenge. Current treatments often fail because of resistance and toxicity. This study aimed to synthesize and characterize a novel amphiphilic polypeptide, KF-5, and evaluate its antibacterial and antifungal activities, biocompatibility, and potential mechanisms of action.ResultsThe KF-5 peptide was synthesized via solid-phase peptide synthesis and self-assembled into nanostructures with filamentous and hydrogel-like configurations. Characterization by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) confirmed the unique nanostructural properties of KF-5. KF-5 (125, 250, or 500 µg/ml) demonstrated potent antibacterial and antifungal activities, with significant inhibitory effects on drug-resistant Candida albicans and Gardnerella spp. (P < 0.05). In vitro assays revealed that 500 µg/ml KF-5 disrupted microbial cell membranes, increased membrane permeability, and induced lipid oxidation, leading to cell death (P < 0.05). Cytotoxicity tests revealed minimal toxicity in human vaginal epithelial cells, keratinocytes, and macrophages, with over 95% viability at high concentrations. Molecular dynamics simulations indicated that KF-5 interacts with phospholipid bilayers through electrostatic interactions, causing membrane disruption. In vivo studies using a mouse model of vaginal infection revealed that 0.5, 1, and 2 mg/ml KF-5 significantly reduced fungal burden and inflammation, and histological analysis confirmed the restoration of vaginal mucosal integrity (P < 0.01). Compared with conventional antifungal treatments such as miconazole, KF-5 exhibited superior efficacy (P < 0.01).ConclusionsKF-5 demonstrates significant potential as a safe and effective antimicrobial agent for treating vaginal infections. Its ability to disrupt microbial membranes while maintaining biocompatibility with human cells highlights its potential for clinical application. These findings provide a foundation for further development of KF-5 as a therapeutic option for combating drug-resistant infections.

A green approach coupled with molecular dynamics simulations and toxicity assays to infer the mode of action for organophosphate esters

Organophosphate esters (OPEs) have attracted extensive attention due to their toxic effects on human health and biological systems as plasticizers and flame retardants. This study focused on exploring a green approach to get toxicity mechanisms that used less solvents or organisms. The toxicity values of selected organophosphate esters including tri (2-chloroethyl) phosphate (TCEP), tri (1, 3-dichloro-2-propyl) phosphate (TDCP), tripropyl phosphate (TPrP), tri-n-butyl phosphate (TnBP), and tritolyl Phosphate (TCrP) to Photobacterium phosphoreum were determined. Their EC50 range was between 7.27 × 10−8–5.12 × 10−6 mol/L. Based on molecular dynamics simulation the data of binding affinity between OPEs and n-octanol / phospholipid bilayer were calculated respectively. Coupling with binding affinity energies and toxicity values, the mode of action (MOA) of OPEs including types of reactive or anesthetic mechanism could be fast deduced. The proposed hypothesis of n-octanol (C8H18O) as a virtual biofilm to replace phospholipid bilayer was verified. The alternative green approach could simplify toxicity assay and realize fast predicting MOA for different chemicals and model organisms.

Molecular engineering of tethered bilayer lipid membranes for impedimetric detection of pore-forming toxins

Tethered bilayer lipid membranes (tBLMs) with two different molecular architectures were investigated in the context of their utility for biosensing of pore-forming toxins (PFTs). The different architectures were achieved by using two back-filler molecules of different chain length to act as surface diluents for the molecular anchors [20-tetradecyloxy-3,6,9,12,15,18,22-heptaoxahexatricontane-1-thiol (WC14)] that ensure immobilization of the phospholipid bilayers on a solid conductive (metal, such as gold) support. The back-filler molecules were β-mercaptoethanol (BME) and HS(CH2)3(CH2CH2O)5H thiol (C3EO5H). In the case of BME, the submembrane space separating solid surface from attached bilayer (typically 1–2 nm thick) is populated with the ice-like, presumably low entropy and low dielectric constant water, however, the water fraction in the submembrane is high. Much bulkier C3EO5H back-filler renders less space for water in the submembrane, however, it is populated with more mobile water molecules resulting in higher dielectric constant. These two opposing effects result in similar submembrane specific conductances, i.e., the specific electrochemical impedance spectral (EIS) characteristics of the tBLMs and their variation, upon exposure to and reorganization with, the pore-forming toxin (PFT), alpha-hemolysin, occur in the same frequency range for both back-fillers. Significantly, the sensitivity of the C3EO5H-containing tBLMs to PFT is approximately 7–8 times higher than that of the BME-containing surface constructs. At least two physical phenomena contribute to this difference. The bulkier C3EO5H back-fillers facilitate the formation of nanoclusters of the molecular anchors, WC14, with cluster sizes ranging from 20 to 120 nm. These highly anchor-clustered, “patchy” tBLMs, consequently exhibit other domains devoid, or nearly so, of anchors, resulting in lowering the tBLM global 2-D viscosity by a factor of 3. Thus, a major conclusion of this study is that lower tBLM 2D fluidity increases the sensitivity of tBLMs to PFTs.