Bilayer Permeability Research Articles

Light‐Activated Membrane Transport in Polymeric Cell‐Mimics

AbstractGiant polymersomes are versatile and stable biomimetic compartments that are ideal for building cell‐like systems. However, the transport of hydrophilic molecules across the membrane, which controls the function of cell‐like systems, is limited by the low permeability of polymeric bilayers. Therefore, mechanisms to control the permeability of polymersomes are necessary to create functional cell‐like systems. Here, we describe the design of giant polymersomes equipped with spiropyran‐based permeability modulators. Photo‐isomerization of the modulators leads to perturbation of the polymer membrane, resulting in increased permeability. The photoactivated polymersomes were used to construct two cell‐like systems controlled by light‐activated transport of hydrophilic molecules. First, we designed an enzymatic micro‐reactor activated by light irradiation. Second, we constructed a hybrid coacervate‐in‐polymersome system that mimics the adaptive formation of biological condensates in cells.

Shear stress induced lipid order and permeability changes of giant unilamellar vesicles

BackgroundThe permeability of a lipid bilayer is a function of its phase state and depends non-linearly on thermodynamic variables such as temperature, pressure or pH. We investigated how shear forces influence the phase state of giant unilamellar vesicles and their membrane permeability. MethodsWe determined the permeability of giant unilamellar vesicles composed of different phospholipid species under shear flow in a tube at various temperatures around and far off the melting point by analyzing the release of fluorescently labelled dextran. Furthermore, we quantified phase state changes of these vesicles under shear forces using spectral decomposition of the membrane embedded fluorescent dye Laurdan. ResultsWe observed that the membrane permeability follows a step function with increasing permeability at the transition from the gel to the fluid phase and vice versa. Second, there was an all-or-nothing permeabilization near the main phase transition temperature and a gradual dye release far off the melting transition. Third, the Laurdan phase state analysis suggests that shear forces induce a reversible melting temperature shift in giant unilamellar vesicle membranes. Major conclusionsThe observed effects can be explained best in a scenario in which shear forces directly induce membrane pores that possess relatively long pore lifetimes in proximity to the phase transition. General significanceOur study elucidates the release mechanism of thermo-responsive drug carriers as we found that liposome permeabilization is not continuous but quantized. Furthermore, the shear force induced melting temperature shift must be taken into consideration when thermo-responsive liposomes are designed.

Effect of cholesterol on nano-structural alteration of light-activatable liposomes via laser irradiation: Small angle neutron scattering study

Although the light-activated liposomes have been extensively studied for drug delivery applications, the fundamental mechanism of the drug release based on lipid compositions has not been fully understood. Especially, despite the extensive use of cholesterol in the lipid composition, the role of cholesterol in the light-activated drug release has not been studied. In this study, the influence of cholesterol on drug release from light-responsive drug-encapsulated liposomes after activated by near infrared (NIR) laser was investigated. We prepared methotrexate (MTX)-encapsulated DSPC liposomes consisting of 0 mol% (Chol) or 35 mol% cholesterol (+Chol), with (+Au) or without gold nanorods (Au) on the lipid bilayer to compare drug release, morphological changes, and nanostructures after laser irradiations. Transmission electron microscopy (TEM) and small angel neutron scattering (SANS) data revealed that only +Chol +Au liposomes showed partial aggregation of the liposomes after laser irradiation. Similar trends on the drug release and structural change were observed when the liposomes were heated to above chain-transition temperature. Overall, we have found that (1) inclusion of 35 mol% cholesterol enhanced the permeability of lipid bilayers above Tc; (2) the mechanism of laser-activated liposomal drug delivery is disrupting lipid bilayer membranes by the photothermal effect in the presence of plasmonic materials. By understanding the fundamentals of the technology, precise controlled drug release at a targeted site with great stability and repeatability is anticipated.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Spatial Confinement-Derived Double-Accelerated DNA Cascade Reaction for Ultrafast and Highly Sensitive In Situ Monitoring of Exosomal miRNA and Exosome Tracing.

Exosomal microRNAs (miRNAs) are reliable biomarkers of disease progression, allowing for non-invasive detection. However, detection of exosomal miRNAs in situ remains a challenge due to low abundance, poor permeability of the lipid bilayers, and slow kinetics of previous methods. Herein, an accelerated DNA nanoprobe was implemented for fast, in situ monitoring of miRNA in exosomes by employing a spatial confinement strategy. This nanoprobe not only detects miRNA in exosomes but also distinguishes tumor exosomes from those derived from normal cells with high accuracy, paving the way toward exosomal miRNA bioimaging and disease diagnosis. Furthermore, the fast response allows for this nanoprobe to be successfully utilized to monitor the process of exosomes endocytosis, making it also a tool to explore exosome biological functions.

Regulation of the intermittent release of giant unilamellar vesicles under osmotic pressure

Osmotic pressure can break the fluid balance between intracellular and extracellular solutions. In hypo-osmotic solution, water molecules, which transfer into the cell and burst, are driven by the concentration difference of solute across the semi-permeable membrane. The complicated dynamic processes of intermittent bursts have been previously observed. However, the underlying physical mechanism has yet to be thoroughly explored and analyzed. Here, the intermittent release of inclusion in giant unilamellar vesicles was investigated quantitatively, applying the combination of experimental and theoretical methods in the hypo-osmotic medium. Experimentally, we adopted a highly sensitive electron multiplying charge-coupled device to acquire intermittent dynamic images. Notably, the component of the vesicle phospholipids affected the stretch velocity, and the prepared solution of vesicles adjusted the release time. Theoretically, we chose equations and numerical simulations to quantify the dynamic process in phases and explored the influences of physical parameters such as bilayer permeability and solution viscosity on the process. It was concluded that the time taken to achieve the balance of giant unilamellar vesicles was highly dependent on the molecular structure of the lipid. The pore lifetime was strongly related to the internal solution environment of giant unilamellar vesicles. The vesicles prepared in viscous solution were able to visualize long-lived pores. Furthermore, the line tension was measured quantitatively by the release velocity of inclusion, which was of the same order of magnitude as the theoretical simulation. In all, the experimental values well matched the theoretical values. Our investigation clarified the physical regulatory mechanism of intermittent pore formation and inclusion release, which provides an important reference for the development of novel technologies such as gene therapy based on transmembrane transport as well as controlled drug delivery based on liposomes.

Evaluating the cross-membrane dynamics of a charged molecule on lipid films with different surface curvature

Does the curvature of a phospholipid membrane influence the permeability of the lipid bilayers? This is a question of great importance yet hard to answer. In this work the permeability of a positively charged rod like probing molecule (D289 dye) on the bilayers of DOPG lipid vesicles was investigated using angle resolved second harmonic generation method. It was revealed that the permeability of D289 on the surface of small vesicles with ∼ 100 nm diameter was notably lower than that on giant vesicles with ∼ 1000 nm diameter. With the increasing of temperature or the introducing of dimethyl sulfoxide (DMSO) in the solutions, the D289 permeability of the lipid bilayers was notably enhanced as expected, on both the small and the giant vesicles. Still, the D289 permeability of the lipid film with more curvature is lower than the relatively flat film in all these cases. This work demonstrated a general protocol for the investigating of surface permeability of lipid films with various curvature.

Calcium Ion Binding at the Lipid-Water Interface Alters the Ion Permeability of Phospholipid Bilayers.

Calcium ions (Ca2+) play a fundamental role in membrane-associated physiological processes. Ca2+ can also significantly modulate the physicochemical properties of phospholipid bilayers, but whether this occurs at physiologically relevant concentrations is difficult to determine because of the uncertainty in the reported affinity of Ca2+ for phospholipid bilayers. In this article, we determine the apparent affinity of Ca2+ for zwitterionic phospholipid bilayers using tethered bilayer lipid membranes (tBLMs) used in conjunction with swept-frequency electrical impedance spectroscopy (EIS). We report that Ca2+ binds to phospholipid bilayers at physiologically relevant concentrations and modulates membrane permeability. We present direct experimental evidence that this effect is governed by specific interactions with select lipid headgroup moieties, which is supported by data from molecular dynamics (MD) simulations. This is the first reported use of tBLM/EIS to estimate cation-membrane affinity. Combined with MD simulations, this technique provides a novel methodology to elucidate the molecular details of cation-membrane interactions at the water-phospholipid interface.

O2 permeability of lipid bilayers is low, but increases with membrane cholesterol

Oxygen on its transport route from lung to tissue mitochondria has to cross several cell membranes. The permeability value of membranes for O2 (PO2), although of fundamental importance, is controversial. Previous studies by mostly indirect methods diverge between 0.6 and 125 cm/s. Here, we use a most direct approach by observing transmembrane O2 fluxes out of 100 nm liposomes at defined transmembrane O2 gradients in a stopped-flow system. Due to the small size of the liposomes intra- as well as extraliposomal diffusion processes do not affect the overall kinetics of the O2 release process. We find, for cholesterol-free liposomes, the unexpectedly low PO2 value of 0.03 cm/s at 35 °C. This PO2 would present a serious obstacle to O2 entering or leaving the erythrocyte. Cholesterol turns out to be a novel major modifier of PO2, able to increase PO2 by an order of magnitude. With a membrane cholesterol of 45 mol% as it occurs in erythrocytes, PO2 rises to 0.2 cm/s at 35 °C. This PO2 is just sufficient to ensure complete O2 loading during passage of erythrocytes through the lung’s capillary bed under the conditions of rest as well as maximal exercise.

Atomistic simulations modify interpretation of spin-label oximetry data. Part 1: intensified water-lipid interfacial resistances.

The role of membrane cholesterol in cellular function and dysfunction has been the subject of much inquiry. A few studies have suggested that cholesterol may slow oxygen diffusive transport, altering membrane physical properties and reducing oxygen permeability. The primary experimental technique used in recent years to study membrane oxygen transport is saturation-recovery electron paramagnetic resonance (EPR) oximetry, using spin-label probes targeted to specific regions of a lipid bilayer. The technique has been used, in particular, to assess the influence of cholesterol on oxygen transport and membrane permeability. The reliability of such EPR recordings at the water-lipid interface near the phospholipid headgroups has been challenged by all-atom molecular dynamics (MD) simulation data that show substantive agreement with spin-label probe measurements throughout much of the bilayer. This work uses further MD simulations, with an updated oxygen model, to determine the location of the maximum resistance to permeation and the rate-limiting barrier to oxygen permeation in 1-palmitoyl,2-oleoylphosphatidylcholine (POPC) and POPC/cholesterol bilayers at 25 and 35°C. The current simulations show a spike of resistance to permeation in the headgroup region that was not detected by EPR but was predicted in early theoretical work by Diamond and Katz. Published experimental nuclear magnetic resonance (NMR) oxygen measurements provide key validation of the MD models and indicate that the positions and relative magnitudes of the phosphatidylcholine resistance peaks are accurate. Consideration of the headgroup-region resistances predicts bilayer permeability coefficients lower than estimated in EPR studies, giving permeabilities lower than the permeability of unstirred water layers of the same thickness. Here, the permeability of POPC at 35°C is estimated to be 13 cm/s, compared with 10 cm/s for POPC/cholesterol and 118 cm/s for simulation water layers of similar thickness. The value for POPC is 12 times lower than estimated from EPR measurements, while the value for POPC/cholesterol is 5 times lower. These findings underscore the value of atomic resolution models for guiding the interpretation of experimental probe-based measurements.

Взаимодействие катионных антисептиков с кардиолипинсодержащей модельной бактериальной мембраной

Plasma membrane is one of the major targets for cationic antiseptics (CA). The study was aimed to assess molecular effects of CAs of different chemical classes on cardiolipin-containing regions of bacterial plasma membranes. The study was carried out using coarse-grained molecular modeling. Interaction of CAs, such as miramistin, chlorhexidine, picloxidine, and octenidine, with cardiolipin-containing bilayer was assessed based on the CA coarse-grained models. CAs reduced lipid lateral diffusion coefficients and increased the membrane area per lipid. All CAs, except miramistin, reduced the lipid fatty acid chain order parameters. Adding octenidine at a CA : lipid ratio of 1 : 4 resulted in cardiolipin clustering with subsequent pulling the neutral phosphatidylethanolamine molecules out of the model bilayer. It was found that CАs have the potential for sorption to lipid bilayer, causing clustering of negatively charged lipids. Antiseptic octenidine causes formation of cardiolipin microdomains. Abnormal lateral lipid distribution together with pulling out phosphatidylethanolamine molecules can result in increased lipid bilayer permeability. The most significant reduction of cardiolipin lateral diffusion coefficient by 2.8 ± 0.4 times was observed in the presence of CA chlorhexidine at an antiseptic : lipid ratio of 1 : 4.

The role of the envelope protein in the stability of a coronavirus model membrane against an ethanolic disinfectant.

Ethanol is highly effective against various enveloped viruses and can disable the virus by disintegrating the protective envelope surrounding it. The interactions between the coronavirus envelope (E) protein and its membrane environment play key roles in the stability and function of the viral envelope. By using molecular dynamics simulation, we explore the underlying mechanism of ethanol-induced disruption of a model coronavirus membrane and, in detail, interactions of the E-protein and lipids. We model the membrane bilayer as N-palmitoyl-sphingomyelin and 1-palmitoyl-2-oleoylphosphatidylcholine lipids and the coronavirus E-protein. The study reveals that ethanol causes an increase in the lateral area of the bilayer along with thinning of the bilayer membrane and orientational disordering of lipid tails. Ethanol resides at the head-tail region of the membrane and enhances bilayer permeability. We found an envelope-protein-mediated increase in the ordering of lipid tails. Our simulations also provide important insights into the orientation of the envelope protein in a model membrane environment. At ∼25 mol. % of ethanol in the surrounding ethanol-water phase, we observe disintegration of the lipid bilayer and dislocation of the E-protein from the membrane environment.

Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics.

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.

Visualizing the osmotic water permeability of a lipid bilayer under measured bilayer tension using a moving membrane method

The water flux across the cell membrane is a fundamental physiological process ubiquitous to all cell types. For evaluating water permeability (Pf), volume changes of cells and vesicles have been employed to measure water flux. However, upon volume expansion, the membrane stretches, and Pf could vary with membrane tension changes. Moreover, local concentration polarization of solutes occurs at the membrane interface due to bulk water flux across the membrane (unstirred layer; UL), which hampers evaluation of Pf values. Here, we establish an accurate method for evaluating the osmotic Pf of lipid bilayers under constant bilayer tension and known unstirred layer. A lipid bilayer is formed across a glass capillary by docking monolayers at oil-water interfaces, and osmotically driven bulk water flux across the membrane yields translational membrane movements along the capillary (moving membrane method). Integrated electrical and image analyses allowed a quantitative evaluation of membrane tension, which remained constant during measurements. In parallel, the unstirred layer was quantified at the nearest vicinity of the membrane using the gramicidin channel as a sensor. Accordingly, Pf values without and with cholesterol were evaluated under known bilayer tension and unstirred layers. Pf of the gramicidin A channel was also measured.

On-chip label-free impedance-based detection of antibiotic permeation.

Biosensors are analytical tools used for the analysis of biomaterial samples and provide an understanding about the biocomposition, structure, and function of biomolecules and/or biomechanisms by converting the biological response into an electrical and/or optical signal. In particular, with the rise in antibiotic resistance amongst pathogenic bacteria, the study of antibiotic activity and transport across cell membranes in the field of biosensors has been gaining widespread importance. Herein, for the rapid and label‐free detection of antibiotic permeation across a membrane, a microelectrode integrated microfluidic device is presented. The integrated chip consists of polydimethylsiloxane based microfluidic channels bonded onto microelectrodes on‐glass and enables us to recognize the antibiotic permeation across a membrane into the model membranes based on electrical impedance measurement, while also allowing optical monitoring. Impedance testing is label free and therefore allows the detection of both fluorescent and non‐fluorescent antibiotics. As a model membrane, Giant Unilamellar Vesicles (GUVs) are used and impedance measurements were performed by a precision inductance, capacitance, and resistance metre. The measured signal recorded from the device was used to determine the change in concentration inside and outside of the GUVs. We have found that permeation of antibiotic molecules can be easily monitored over time using the proposed integrated device. The results also show a clear difference between bilayer permeation that occurs through the lipidic bilayer and porin‐mediated permeation through the porin channels inserted in the lipid bilayer.

Liposome Fusion Mediated by Hydrophobic Magnetic Nanoparticles Stabilized with Oleic Acid and Modulated by an External Magnetic Field.

Membrane fusion is considered relevant in countless scientific areas and biotechnological processes, ranging from vital life events to biomedicine, pharmaceuticals, and materials engineering, among others. In this study, we employed hydrophobic oleic acid (OA)-coated magnetite (Fe3O4) nanoparticles (MNP-OA) as a platform to induce the fusion of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes [large unilamellar vesicles (LUVs)] in a colloidal dispersion. This fusion was monitored through dynamic light scattering, turbidimetry, and fluorescence assay using the well-known Tb/dipicolinic acid (DPA) complex formation assay. MNP-OA have shown to be able to induce fusion with the mixing of liposomal inner content with direct dependence on the nanoparticle concentration added to the LUVs. Moreover, changes in the permeability of the liposome bilayer, upon the addition of MNP-OA to liposomes, were evaluated by studying the leakage of carboxyfluorescein and of the co-encapsulated Tb/DPA complex. These assays allowed us to determine that MNP-OA did not significantly modify liposome permeability during the fusion process. Transmission electron microscopy and confocal microscopy revealed that MNP-OA remained embedded in the lipid bilayer without producing membrane rupture, liposome deformation, or destruction. In addition, we evaluated the effect of applying a low-intensity magnetic field to the LUVs/MNP-OA system and observed that the nanoparticles considerably increased their fusogenic activity under this external stimulus, as well as they are capable of responding to low magnetic fields of around 0.45 mT. These results revealed the potential of hydrophobic magnetic nanoparticles, stabilized with OA, to act as a fusogen, thus representing a valuable tool for biotechnological applications.

Updated Evaluation of Cholesterol's Influence on Membrane Oxygen Permeability.

There is a surprising gap in knowledge regarding the mechanism of oxygen (O2) diffusional delivery at the level of tissues and cells. Yet, the effectiveness of tumor radiotherapy, the success of tissue engineering, and healthy metabolism all require ample intracellular oxygen. Tissue-level diffusion takes place in a complex and crowded macromolecular environment. Cholesterol-rich cellular membranes have been thought to reduce oxygen flux. Here, we use atomistic molecular dynamics simulations to update prior estimates of bilayer permeability and related parameters for 1-palmitoyl,2-oleoylphosphatidylcholine (POPC) and POPC/cholesterol bilayers, using a modified O2 model with improved membrane-water partitioning behavior. This work estimates an oxygen permeability coefficient of 15±1cm/s for POPC and 11.5±0.4cm/s for POPC/cholesterol (1:1 molecular ratio) at 37°C. The permeability of POPC is found to be ~1/3 that of a water layer of similar thickness, and the permeability of POPC/cholesterol is estimated to be 20-30% below that of POPC. Void pathway visualization and free energy data support channeling of oxygen toward the center of cholesterol-incorporating membranes, while partition coefficient data suggest reduced membrane solubility of oxygen due to cholesterol. Further study is needed to understand whether diffusion pathway changes due to cholesterol and other molecular compositional factors influence oxygen availability within tissue.

Regulation of Lipid Bilayer Ion Permeability by Antibacterial Polymethyloxazoline-Polyethyleneimine Copolymers.

Amphiphilic antimicrobial polymers display activity against the outer bacterial cell membrane, triggering various physiological effects. We investigated the regulation of ion transport across the lipid bilayer to understand differences in biological activity for a series of amphiphilic polymethyloxazoline - polyethyleneimine copolymers. The results confirmed that the tested structures were able to increase the permeability of the lipid bilayer (LB) membrane or its rupture. Black lipid membrane (BLM) experiments show that the triggered conductance profile and its character is strongly correlated with the polymer structure and zeta potential. The polymer exhibiting the highest antimicrobial activity promotes ion transport by using a unique mechanism and step-like characteristics with well-defined discreet openings and closings. The molecule was incorporated into the membrane in a reproducible way, and the observed channel-like activity could be responsible for the antibacterial activity of this molecule.

Photolipid Bilayer Permeability is Controlled by Transient Pore Formation.

Controlling the release or uptake of (bio-) molecules and drugs from liposomes is critically important for a range of applications in bioengineering, synthetic biology, and drug delivery. In this paper, we report how the reversible photoswitching of synthetic lipid bilayer membranes made from azobenzene-containing phosphatidylcholine (azo-PC) molecules (photolipids) leads to increased membrane permeability. We show that cell-sized, giant unilamellar vesicles (GUVs) prepared from photolipids display leakage of fluorescent dyes after irradiation with UV-A and visible light. Langmuir-Blodgett and patch-clamp measurements show that the permeability is the result of transient pore formation. By comparing the trans-to-cis and cis-to-trans isomerization process, we find that this pore formation is the result of area fluctuations and a change of the area cross-section between both photolipid isomers.

Organic Cation Transporters in Human Physiology, Pharmacology, and Toxicology.

Individual cells and epithelia control the chemical exchange with the surrounding environment by the fine-tuned expression, localization, and function of an array of transmembrane proteins that dictate the selective permeability of the lipid bilayer to small molecules, as actual gatekeepers to the interface with the extracellular space. Among the variety of channels, transporters, and pumps that localize to cell membrane, organic cation transporters (OCTs) are considered to be extremely relevant in the transport across the plasma membrane of the majority of the endogenous substances and drugs that are positively charged near or at physiological pH. In humans, the following six organic cation transporters have been characterized in regards to their respective substrates, all belonging to the solute carrier 22 (SLC22) family: the organic cation transporters 1, 2, and 3 (OCT1–3); the organic cation/carnitine transporter novel 1 and 2 (OCTN1 and N2); and the organic cation transporter 6 (OCT6). OCTs are highly expressed on the plasma membrane of polarized epithelia, thus, playing a key role in intestinal absorption and renal reabsorption of nutrients (e.g., choline and carnitine), in the elimination of waste products (e.g., trimethylamine and trimethylamine N-oxide), and in the kinetic profile and therapeutic index of several drugs (e.g., metformin and platinum derivatives). As part of the Special Issue Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations, this article critically presents the physio-pathological, pharmacological, and toxicological roles of OCTs in the tissues in which they are primarily expressed.