Large Unilamellar Vesicles Research Articles

Rotaxanes with a photoresponsive macrocycle modulate the lipid bilayers of large and giant unilamellar vesicles

Rotaxanes equipped with actuators hold great potential for developing highly functional molecular machines. Such systems could significantly enhance our ability to study and manipulate biological and artificial membranes. Here, we introduce a rotaxane with a ring featuring two azobenzene photoswitches, which retain their photoreversibility and can be stochastically shuttled along the axle in solution. Studies in model bilayers, supported by molecular dynamics simulations, show how azobenzene photoswitching alters the interaction of rotaxanes with surrounding lipids, leading to changes in lipid packing. Such changes in the lipid bilayer were leveraged to induce the light-triggered release of sulforhodamine B from large unilamellar vesicles. Additionally, light activation of the rotaxanes is shown to induce reversible contraction and expansion of giant unilamellar vesicles. The results provide novel insights into the interactions and operation of rotaxanes in lipid bilayers and their impact on membrane properties. This will aid in developing systems for precise membrane manipulation for applications in biomedicine and bioengineering.

Ameloblastin binding to biomimetic models of cell membranes – A continuum of intrinsic disorder

ObjectiveA 37-residue amino acid sequence corresponding to the segment encoded by exon-5 of murine ameloblastin (Ambn), AB2 (Y67-Q103), has been implicated with membrane association, ameloblastin self-assembly, and amelogenin-binding. Our aim was to characterize, at the residue level, the structural behavior of AB2 bound to chemical mimics of biological membranes using NMR spectroscopy. DesignTo better define the structure of AB2 using NMR-based methods, recombinant 13C- and 15N-labelled AB2 (*AB2) was prepared and data collected free in solution and with deuterated dodecylphosphocholine (dPC) micelles, deuterated bicelles, and both small and large unilamellar vesicles. ResultsAmide chemical shift and intensity perturbations observed in 1H-15N HSQC spectra of *AB2 in the presence of bicelles and dPC micelles suggest that a region of *AB2, S6-E36 (murine Ambn S68 – E98), associates with the membrane biomimetics. A CSI-3 analysis of the NMR chemical shift assignments for *AB2 free in solution and bound to dPC micelles indicated the peptide remains disordered except for the adoption of a short, 12-residue α-helix, F10-G21 (murine Ambn F72-G83). In dPC micelles, the NOE NMR data was void of patterns characteristic of long-lived helical structure indicating this helix was transient in nature. ConclusionsA continuum of intrinsic disorder in the membrane-bound state may be responsible for ameloblastin’s ability to dynamically interact with multiple partners at the same site during amelogenesis.

Cholesterol Accelerates Aggregation of α-Synuclein Simultaneously Increasing the Toxicity of Amyloid Fibrils.

A hallmark of Parkinson disease (PD) is a progressive degeneration of neurons in the substantia nigra pars compacta, hypothalamus, and thalamus. Although the exact etiology of irreversible neuronal degeneration is unclear, a growing body of experimental evidence indicates that PD could be triggered by the abrupt aggregation of α-synuclein (α-Syn), a small membrane protein that is responsible for cell vesicle trafficking. Phospholipids uniquely alter the rate of α-Syn aggregation and, consequently, change the cytotoxicity of α-Syn oligomers and fibrils. However, the role of cholesterol in the aggregation of α-Syn remains unclear. In this study, we used Caenorhabditis elegans that overexpressed α-Syn to investigate the effect of low (15%), normal (30%), and high (60%) concentrations of cholesterol on α-Syn aggregation. We found that an increase in the concentration of cholesterol in diets substantially shortened the lifespan of C. elegans. Using biophysical methods, we also investigated the extent to which large unilamellar vesicles (LUVs) with low, normal, and high concentrations of cholesterol altered the rate of α-Syn aggregation. We found that only lipid membranes with a 60% concentration of cholesterol substantially accelerated the rate of protein aggregation. Cell assays revealed that α-Syn fibrils formed in the presence of LUVs with different concentrations of cholesterol exerted very similar levels of cytotoxicity to rat dopaminergic neurons. These results suggest that changes in the concentration of cholesterol in the plasma membrane, which in turn could be caused by nutritional preferences, could accelerate the onset and progression of PD.

A Closed-Form Expression for Analysis of Dark State Exchange Saturation Transfer (DEST) NMR Experiments.

Closed-form expressions for the analysis of Dark state Exchange Saturation Transfer (DEST) NMR experiments, a powerful experimental tool for characterizing exchange processes involving the interaction of NMR visible species with very high molecular weight partners, is presented. Essentially identical exchange and relaxation parameters are derived from the analytical and numerical best fits of the DEST profiles obtained for a protein construct derived from huntingtin exon-1, comprising the N-terminal amphiphilic sequence followed by a seven-residue glutamine repeat, httNTQ7, in the presence of small (SUV) and large (LUV) unilamellar lipid vesicles. The use of analytical expressions significantly speeds up the fitting of experimental DEST profiles to a two-state exchange model and simplifies the analysis of the DEST effects.

Development and in vitro characterization of new carnosine-loaded liposomal formulations.

Carnosine is an endogenous dipeptide characterized by a multimodal mechanism of action. However, its clinical potential is limited by serum and cytosolic carnosinases, which significantly reduce its bioavailability. Based on that, different research groups have worked on the development of new strategies able not only to prevent its rapid metabolization but also to improve its distribution and specific targeting. In the present study, the development and in vitro characterization of new liposomal formulations loaded with carnosine are described. Nanoliposomes, produced through Thin-Layer Hydration followed by Extrusion method, were first investigated for their physicochemical stability. Photon correlation spectroscopy and electrophoretic light scattering, assessing the stability of the formulations, showed a strong homogeneity-oriented tendency for up to two months. Particle size, polydispersity index, and zeta potential were determined through dynamic light scattering and electrophoretic light scattering, demonstrating an almost neutral charge of the formulation and an effective encapsulation of carnosine. The morphology assessment performed via scanning electron microscopy showed good conformity and polydispersity. Differential scanning calorimetry measurements suggest the ability of carnosine to stabilize the large unilamellar vesicles. Lastly, the newly developed carnosine-loaded liposomal formulations also showed a good safety profile in human microglia.

Fluorescence Turn-ON Displacement Assays with Cucurbit[7]uril-Thiophenylpyridinium Complexes as Host-Dye Reporter Pairs.

The N-methyl-4-thiophenylpyridinium cation (ThioPy) is a high affinity (Kd ca. 5 nM), fast-exchanging fluorescent probe for cucurbit[7]uril (CB7). The CB7/ThioPy complex shows a unique fluorescence turn-ON response upon displacement by an analyte in sensing application. This enabled the development of a real-time fluorescence assay with the MRFA peptide for the protease thermolysin, which is also suitable for the cancer biomarker cathepsin B. Moreover, liposome encapsulation of CB7/ThioPy in large unilamellar vesicles (LUVs) provided mechanistic insight into intravesicular dye displacement reactions.

Interactions between gamma-aminobutyric capped silver nanoparticles and large unilamellar vesicles (LUVs) and their antimicrobial activities

Multidrug-resistant bacteria (MDR) and bacterial virulence pose significant challenges to global health. Nanotechnology offers exciting possibilities for developing innovative strategies to combat these issues. This study investigates the synergistic effects of silver nanoparticles (AgNPs) and gamma-aminobutyric acid (GABA) to address antibiotic resistance and bacterial virulence. We report the synthesis and characterization of GABA-coated AgNPs (GABAAgNPs) using UV–VIS and infrared spectroscopies, zeta potential, X-ray diffraction, and transmission electron microscopy. The interaction between GABAAgNPs and model membranes (large unilamellar vesicles, LUVs) was assessed, revealing the formation of liposomal-bound GABAAgNPs (LPGABAAgNPs). Antimicrobial activity against E. coli ATCC 25922 demonstrated that LPGABAAgNPs exhibit enhanced antibacterial efficacy compared to free GABAAgNPs. These findings suggest that liposomal delivery of GABAAgNPs is a promising approach for optimizing their antibacterial properties. Moreover, incorporating GABA into the nanoparticle system offers the potential to modulate bacterial virulence, providing a multifaceted strategy to combat infection.

Engineering Phosphatidylserine Containing Asymmetric Giant Unilamellar Vesicles.

The plasma membrane lipid distribution is asymmetric, with several anionic lipid species located in its inner leaflet. Among these, phosphatidylserine (PS) plays a crucial role in various important physiological functions. Over the last decade several methods have been developed that allow for the fabrication of large or giant unilamellar vesicles (GUVs) with an asymmetric lipid composition. Investigating the physicochemical properties of PS in such asymmetric lipid bilayers and studying its interactions with proteins necessitates the reliable fabrication of asymmetric GUVs (aGUVs) with a high degree of asymmetry that exhibit PS in the outer leaflet so that the interaction with peptides and proteins can be studied. Despite progress, achieving aGUVs with well-defined PS asymmetry remains challenging. Recently, a Ca2+-initiated hemifusion method has been introduced, utilizing the fusion of symmetric GUVs (sGUVs) with a supported lipid bilayer (SLB) for the fabrication of aGUVs. We extend this approach to create aGUVs with PS in the outer bilayer leaflet. Comparing the degree of asymmetry between aGUVs obtained via Ca2+ or Mg2+ initiated hemifusion of a phosphatidylcholine (PC) sGUVwith a PC/PS-supported lipid bilayer, we observe for both bivalent cations a significant number of aGUVs with near-complete asymmetry. The degree of asymmetry distribution is narrower for physiological salt conditions than at lower ionic strengths. While Ca2+ clusters PS in the SLB, macroscopic domain formation is absent in the presence of Mg2+. However, the clustering of PS upon the addition of Ca2+ is apparently too slow to have a negative effect on the quality of the obtained aGUVs. We introduce a data filtering method to select aGUVs that are best suited for further investigation.

Micro-scale quantitative analysis of sterol content in liposomes

The high complexity of biological membranes has driven the development and application of a wide range of model membrane systems. Among these models, liposomes are extensively used because of their versatility in mimicking cellular membranes with a wide range of lipid compositions. However, the accurate quantification of lipid components, such as sterols, within these models remains a critical requirement for validation, data interpretation, and comparison. Here, we present a reliable and sensitive colorimetric assay using the Zak color reaction, which we have specifically adapted for the quantification of sterols at the micro-scale level. The assay was evaluated using cholesterol, ergosterol, and sitosterol standards, reflecting the diversity of sterol species across organisms. The reaction mechanism involves the dehydration of sterols to form carbonium ions, which are oxidized to form various enylic carbonium ions with specific absorption peaks. Due to the different chemical structures of cholesterol, ergosterol, and sitosterol, the resulting spectra show that the colored reaction products are formed in different proportions. The stability and interconversion of these species over time were analyzed. Cholesterol and sitosterol showed a clear peak at 555 nm, while ergosterol had prominent peaks at shorter wavelengths. Sterol assays on liposomal preparations showed accurate sterol incorporation with minimal loss during processing steps. These results demonstrate that this assay provides a robust and accurate measurement of sterol content in large unilamellar vesicles, making it a valuable tool for liposomal studies.

Ticagrelor increases its own potency at the P2Y12 receptor by directly changing the plasma membrane lipid order in platelets.

Although the amphiphilic nature of the widely used antithrombotic drug Ticagrelor is well known, it was never considered as a membranotropic agent capable of interacting with the lipid bilayer in a receptor-independent way. In this study, we investigated the influence of Ticagrelor on plasma membrane lipid order in platelets and if this modulates the potency of Ticagrelor at the P2Y12 receptor. We combined fluorescent in situ, in vitro and in silico approaches to probe the interactions between the plasma membrane of platelets and Ticagrelor. The influence of Ticagrelor on the lipid order of the platelet plasma membrane and large unilamellar vesicles was studied using the advanced fluorescent probe NR12S. Furthermore, the properties of model lipid bilayers in the presence of Ticagrelor were characterized by molecular dynamics simulations. Finally, the influence of an increased lipid order on the dose-response of platelets to Ticagrelor was studied. Ticagrelor incorporates spontaneously into lipid bilayers and affects the lipid order of the membranes of model vesicles and isolated platelets, in a nontrivial composition and concentration-dependent manner. We showed that higher plasma membrane lipid order in platelets leads to a lower IC50 value for Ticagrelor. It is shown that membrane incorporation of Ticagrelor increases its potency at the P2Y12 receptor, by increasing the order of the platelet plasma membrane. A novel dual mechanism of Ticagrelor action is suggested that combines direct binding to P2Y12 receptor with simultaneous modulation of receptor-lipid microenvironment.

Leveraging Machine Learning-Guided Molecular Simulations Coupled with Experimental Data to Decipher Membrane Binding Mechanisms of Aminosterols.

Understanding the molecular mechanisms of the interactions between specific compounds and cellular membranes is essential for numerous biotechnological applications, including targeted drug delivery, elucidation of the drug mechanism of action, pathogen identification, and novel antibiotic development. However, estimation of the free energy landscape associated with solute binding to realistic biological systems is still a challenging task. In this work, we leverage the Time-lagged Independent Component Analysis (TICA) in combination with neural networks (NN) through the Deep-TICA approach for determining the free energy associated with the membrane insertion processes of two natural aminosterol compounds, trodusquemine (TRO), and squalamine (SQ). These compounds are particularly noteworthy because they interact with the outer layer of neuron membranes, protecting them from the toxic action of misfolded proteins involved in neurodegenerative disorders, in both their monomeric and oligomeric forms. We demonstrate how this strategy could be used to generate an effective collective variable for describing solute absorption in the membrane and for estimating free energy landscape of translocation via on-the-fly probability enhanced sampling (OPES) method. In this context, the computational protocol allowed an exhaustive characterization of the aminosterol entry pathway into a neuron-like lipid bilayer. Furthermore, it provided accurate prediction of membrane binding affinities, in close agreement with the experimental binding data obtained by using fluorescently labeled aminosterols and large unilamellar vesicles (LUVs). The findings contribute significantly to our understanding of aminosterol entry pathways and aminosterol-lipid membrane interactions. Finally, the computational methods deployed in this study further demonstrate considerable potential for investigating membrane binding processes.

Modulating lipid bilayer permeability and structure: Impact of hydrophobic chain length, C-3 hydroxyl group, and double bond in sphingosine

Sphingosine, an amphiphilic molecule, plays a pivotal role as the core structure of sphingolipids, essential constituents of cell membranes. Its unique capability to enhance the permeability of lipid membranes profoundly influences crucial life processes. The molecular structure of sphingosine dictates its mode of entry into lipid bilayers and governs its interactions with lipids, thereby determining membrane permeability. However, the incomplete elucidation of the relationship between the molecular structure of sphingosine and the permeability of lipid membranes persists due to challenges associated with synthesizing sphingosine molecules. A series of sphingosine-derived molecules, featuring diverse hydrophobic chain lengths and distinct headgroup structure, were meticulously designed and successfully synthesized. These molecules were employed to investigate the permeability of large unilamellar vesicles, functioning as model lipid bilayers. With a decrease in the hydrophobic chain length of sphingosine from C15 to C11, the transient leakage ratio of vesicle contents escalated from ∼ 13 % to ∼ 28 %. Although the presence of double bond did not exert a pronounced influence on transient leakage, it significantly affected the continuous leakage ratio. Conversely, modifying the chirality of the C-3 hydroxyl group gives the opposite result. Notably, methylation at the C-3 hydroxyl significantly elevates transient leakage while suppressing the continuous leakage ratio. Additionally, sphingosines that significantly affect vesicle permeability tend to have a more pronounced impact on cell viability. Throughout this leakage process, the charge state of sphingosine-derived molecule aggregates in the solution emerged as a pivotal factor influencing vesicle permeability. Fluorescence lifetime experiments further revealed discernible variations in the effect of sphingosine molecular structure on the mobility of hydrophobic regions within lipid bilayers. These observed distinctions emphasize the impact of molecular structure on intermolecular interactions, extending to the microscopic architecture of membranes, and underscore the significance of subtle alterations in molecular structure and their associated aggregation behaviors in governing membrane permeability.

Exploring the effects of silver, silica-coated silver, and gold nanoparticles on lipid vesicles: Insights from LUVs and GUVs

Understanding the interaction between nanoparticles (NP) and lipid membranes is crucial for various biomedical applications. In this study, we investigated the effects of silver nanoparticles (AgNP), silica-coated silver nanoparticles (Ag@SiO2), and gold nanoparticles (AuNP) on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles. The dynamic light scattering (DLS) and zeta potential measurements were employed to evaluate DOPC large unilamellar vesicles (LUVs) exposed to AgNP, AgNP@SiO2, or AuNP for 180 min. The DOPC giant unilamellar vesicles (GUVs) exposed to the same NP were evaluated by phase-contrast microscopy. The AgNP showed higher reactivity along the exposition time, leading the vesicles to rupture (LUVs and GUVs). The SiO2 coating from Ag@SiO2 decreases the rupture effects compared to uncovered AgNP, showing surface adsorption and, consequently, vesicle morphological changes. The AuNP showed a partial wrapping on the GUVs bilayer and permeation on the LUVs, however the integrity of the bilayer structure is maintained in both GUVs and LUVs. These findings highlight the varying effects of AgNP, Ag@SiO2, and AuNP on lipid vesicles, underscoring the importance of nanoparticle material and coating in their interactions with biological membranes.

Flow-based bioconjugation of coumarin phosphatidylethanolamine probes: Optimised synthesis and membrane molecular dynamics studies

A series of phosphatidylethanolamine fluorescent probes head-labelled with 3-carboxycoumarin was prepared by an improved bioconjugation approach through continuous flow synthesis. The established procedure, supported by a design of experiment (DoE) set-up, resulted in a significant reduction in the reaction time compared to the conventional batch method, in addition to a minor yield increase. The characterization of these probes was enhanced by an in-depth molecular dynamics (MD) study of the behaviour of a representative probe of this family, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine labelled with 3-carboxycoumarin (POPE-COUM), in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) 2:1, mimicking the composition of the egg yolk lecithin membranes recently used experimentally by our group to study POPE-COUM as a biomarker of the oxidation state and integrity of large unilamellar vesicles (LUVs). The MD simulations revealed that the coumarin group is oriented towards the bilayer interior, leading to a relatively internal location, in agreement with what is observed in the nitrobenzoxadiazole fluorophore of commercial head-labelled NBD-PE probes. This behaviour is consistent with the previously stated hypothesis that POPE-COUM is entirely located within the LUVs structure. Hence, the delay on the oxidation of the probe in the oxygen radical absorbance capacity (ORAC) assays performed is related with the inaccessibility of the probe until alteration of the LUV structure occurs. Furthermore, our simulations show that POPE-COUM exerts very little global and local perturbation on the host bilayer, as evaluated by key properties of the unlabelled lipids. Together, our findings establish PE-COUM as suitable fluorescent lipid analogue probes.

Benefits of Combined Fluorescence Lifetime Imaging Microscopy and Fluorescence Correlation Spectroscopy for Biomedical Studies Demonstrated by Using a Liposome Model System

Drug delivery systems play a pivotal role in targeted pharmaceutical transport and controlled release at specific sites. Liposomes, commonly used as drug carriers, constitute a fundamental part of these systems. Moreover, the drug–liposome model serves as a robust platform for investigating interaction processes at both cellular and molecular levels. To advance our understanding of drug carrier uptake mechanisms, we employed fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), leveraging the unique benefits of two-photon (2P) excitation. Our approach utilized giant unilamellar vesicles (GUVs) as a simplified model system for cell membranes, labelled with the amphiphilic fluorescent dye 3,3′-dioctadecyloxa-carbocyanine (DiOC18(3)). Additionally, large unilamellar vesicles (LUVs) functioned as a drug carrier system, incorporating the spectrally distinct fluorescent sulforhodamine 101 (SRh101) as a surrogate drug. The investigation emphasized the diverse interactions between GUVs and LUVs based on the charged lipids employed. We examined the exchange kinetics and structural alterations of liposome carriers during the uptake process. Our study underscores the significance of employing 2P excitation in conjunction with FLIM and FCS. This powerful combination offers a valuable methodological approach for studying liposome interactions, positioning them as an exceptionally versatile model system with a distinct technical advantage.

High throughput microfluidics-based synthesis of PEGylated liposomes for precise size control and efficient drug encapsulation

The low scalability and reproducibility of existing synthesis methods have hindered the translation of liposome nanoparticles as carriers for targeted drug delivery from conventional laboratory techniques to mass production. To this end, in this study, we present a high-throughput microfluidics-based approach for the synthesis of PEGylated liposomes with a primary focus on achieving precise size control and efficient encapsulation of hydrophobic drug molecules. In this platform, liposomes were self-assembled through a controllable mixing of lipids (EYPC, cholesterol, and DSPE-PEG 2000) dissolved in ethanol and an aqueous solution. The key parameters, including the chip design, total flow rate, flow rate ratio, lipid concentrations, as well as variations in buffer (HEPES and NaCl) and solvent composition (commercial and reagent-grade ethanol) were explored in detail. Through comprehensive parametric studies, we gained valuable insights into the influence of these variables on the size distribution of liposomes and succeeded in producing highly reproducible liposomes ranging from approximately 60 nm (corresponding to small unilamellar vesicles) to 150 nm (representing large unilamellar vesicles), all while maintaining a polydispersity index (PDI) of less than 0.2. To assess the encapsulation efficiency of hydrophobic drug molecules, Nile red (NR) was employed as a surrogate. We meticulously examined the impact of NR concentration on the drug encapsulation process, resulting in up to 74% drug encapsulation efficiency within the PEGylated liposomes. This research offers crucial advances in liposome synthesis and drug delivery, providing a high-throughput, controllable method for PEGylated liposomes with potential in pharmaceutical and biomedical fields.

A novel approach to pH-Responsive targeted cancer Therapy: Inhibition of FaDu cancer cell proliferation with a pH low insertion Peptide-Conjugated DGAT1 inhibitor

Targeting enzymes involved in lipid metabolism is increasingly recognized as a promising anticancer strategy. Efficient inhibition of diacylglycerol O-transferase 1 (DGAT1) can block fatty acid (FA) storage. This, in turn, triggers an increase in free polyunsaturated FA concentration, leading to peroxidation and ferroptosis. In this study, we report the development of a pH-sensitive peptide (pHLIP)-drug conjugate designed to selectively deliver DGAT1 inhibitors to cancer cells nested within the acidic microenvironment of tumors. We utilized two previously established pHLIP sequences for coupling with drugs. The study of DGAT1 conjugates in large unilamellar vesicles (LUVs) of different compositions did not reveal enhanced pH-dependent insertion compared to POPC LUVs. However, using in vitro 3D tumor spheroids, significant antiproliferative effects were observed upon exposure to pHLIP-T863 (DGAT1 inhibitor) conjugates, surpassing the inhibitory activity of T863 alone. In conclusion, our study provides the first evidence that pHLIP-based conjugates with DGAT1 inhibitors have the potential to specifically target the acidic compartment of tumors. Moreover, it sheds light on the limitations of LUV models in capturing the pH-dependency of such conjugates.

Glycerophospholipid polyunsaturation modulates resveratrol action on biomimetic membranes

The phytoalexin resveratrol has received increasing attention for its potential to prevent oxidative damages in human organism. To shed further light on molecular mechanisms of its interaction with lipid membranes we study resveratrol influence on the organisation and mechanical properties of biomimetic lipid systems composed of synthetic phosphatidylcholines with mixed aliphatic chains and different degree of unsaturation at sn-2 position (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, PDPC). High-sensitivity isothermal titration calorimetric measurements reveal stronger spontaneous resveratrol association to polyunsaturated phosphatidylcholine bilayers compared to the monounsaturated ones resulting from hydrophobic interactions, conformational changes of the interacting species and desolvation of molecular surfaces. The latter is supported by the results from Laurdan spectroscopy of large unilamellar vesicles providing data on hydration at the glycerol backbones of glycerophospholipides. Higher degree of lipid order is reported for POPC membranes compared to PDPC. While resveratrol mostly enhances the hydration of PDPC membranes, increasing POPC dehydration is reported upon treatment with the polyphenol. Dehydration of the polyunsaturated lipid bilayers is measured only at the highest phytoalexin content studied (resveratrol/lipid 0.5 mol/mol) and is less pronounced than the effect reported for POPC membranes. The polyphenol effect on membrane mechanics is probed by thermal shape fluctuation analysis of quasispherical giant unilamellar vesicles. Markedly different trend of the bending elasticity with increasing resveratrol concentration is reported for the two types of phospholipid bilayers studied. POPC membranes become more rigid in the presence of resveratrol, whereas PDPC-containing bilayers exhibit softening at lower concentrations of the polyphenol followed by a slight growth without bilayer stiffening even at the highest resveratrol content explored. The new data on the structural organization and membrane properties of resveratrol-treated phosphatidylcholine membranes may underpin the development of future liposomal applications of the polyphenol in medicinal chemistry.

Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles.

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs aretrapped in microfluidic devices for analysis by optical microscopy.

Assessing the negative impact of chlorantraniliprole, isoxaflutole, and simazine pesticides on phospholipid membrane models and tilapia gill tissues

The indiscriminate and, very often, incorrect use of pesticides in Brazil, as well as in other countries, results in severe levels of environmental pollution and intoxication of human life. Herein, we studied plasma membrane models (monolayer and bilayer) of the phospholipid Dioleoyl-sn-glycerol-3-phosphocholine (DOPC) using Langmuir films, and large (LUVs) and giant (GUVs) unilamellar vesicles, to determine the effect of the pesticides chlorantraniliprole (CLTP), isoxaflutole (ISF), and simazine (SMZ), used in sugarcane. CLTP affects the lipid organization of the bioinspired models of DOPC π-A isotherms, while ISF and SMZ pesticides significantly affect the LUVs and GUVs. Furthermore, the in vivo study of the gill tissue in fish in the presence of pesticides (2.0 × 10−10 mol/L for CLTP, 8.3 × 10−9 mol/L for ISF, and SMZ at 9.9 × 10−9 mol/L) was performed using optical and fluorescence images. This investigation was motivated by the gill lipid membranes, which are vital for regulating transporter activity through transmembrane proteins, crucial for maintaining ionic balance in fish gills. In this way, the presence of phospholipids in gills offers a model for understanding their effects on fish health. Histological results show that exposure to CLTP, ISF, and SMZ may interfere with vital gill functions, leading to respiratory disorders and osmoregulation dysfunction. The results indicate that exposure to pesticides caused severe morphological alterations in fish, which could be correlated with their impact on the bioinspired membrane models. Moreover, the effect does not depend on the exposure period (24h and 96h), showing that animals exposed to pesticides for a short period suffer irreparable damage to gill tissue. In summary, we can conclude that the harm caused by pesticides, both in membrane models and in fish gills, occurs due to contamination of the aquatic system with pesticides. Therefore, water quality is vital for the preservation of ecosystems.