Charged Bilayers Research Articles

Turning a negative into a positive.

Negatively charged lipid bilayers enhance the interaction between a chemokine and an atypical chemokine receptor.

Insertion of anionic synthetic clay in lamellar surfactant phases.

We describe the different mixed colloidal solutions that can be obtained when mixing equivalent quantities of a synthetic anionic clay to surfactants forming lamellar phases in the absence of added salt. The important quantity driving toward insertion or depletion is the osmotic pressure, of the lamellar phase and of the clay alone. Competition for water is the main driving force toward dispersion, inclusion or exclusion (phase separation). In the case of a nonionic surfactant ( ) mixed with Laponite, undulations quenched by the surfactant-decorated clay lead to swelling; inclusion is not observed due to differences in rigidity. Long-range order is weakened leading eventually to the exclusion of surfactant in excess. In the case of a double anionic system (AOT-Laponite), electrostatic is dominant and the three regimes are encountered. In the catanionic case, admixing the double chain cationic lipid DDAB to the clay (in large charge excess), the platelets are coated by a positively charged bilayer. Long-range order is very efficiently dampened. From a low threshold (2% by weight), there is exclusion of a clay-poor collapsed lamellar phase, detected by the swelling of the main phase. The cationized clay does not interfere with the molecular force balance: the location of the critical point is unchanged. At high Laponite concentration, a very puzzling microstructure is observed. Some phase diagrams as well as representative SANS and SAXS data are extracted from the complete results concerning the lyotropic lamellar phase mixing problem available with all measures and evaluations of osmotic pressures in the PhD of the late Isabelle Grillo.

Bioequivalence Analysis of Terazosin Hydrochloride Tablets Based on Parallel Artificial Membrane Permeability Analysis.

Parallel artificial membrane permeability analysis (PAMPA) is used to determine the permeability of compounds through concentrated negatively charged phospholipid bilayer barriers. We employed MacroFlux (a scaled-up version of PAMPA) to test the permeation rate of terazosin hydrochloride (TH) tablets and predict in vivo bioequivalence. The dissolution profiles and permeability of one reference formulation, and seven generic TH tablets, were compared. The dissolution profiles of these generic tablets were equivalent to that of the reference drug in four different media. However, the flux and the total permeated amount of some generic TH tablets were below the lower limit of the confidence interval of the original acceptance range in MacroFlux, which implied risk in the bioequivalence test in vivo. We further evaluated potential factors responsible for this discrepancy by µFlux, including active pharmaceutical ingredient (API) permeability and excipient prescriptions. The analysis showed that different properties of API were a main factor leading to biological inequivalence in the MacroFlux assay, while excipient prescriptions did not have an impact on bioequivalence risk. These data indicated that the flux assay may be a helpful as an auxiliary method for predicting bioequivalence of generic drugs and analyze the factors responsible for bioequivalence risk.

Resolving the interactions between hydrophilic CdTe quantum dots and positively charged membranes at the nanoscale

The use of quantum dot nanoparticles (QDs) in bio-applications has gained quite some interest and requires a deep understanding of their interactions with model cell membranes. This involves assessing the extent of nanoparticle disruption of the membrane and how it depends on both nanoparticle and membrane physicochemical properties. Surface charge plays an important role in nanoparticle adsorption, which is primarily driven by electrostatic interactions; yet, once adsorbed, most reported works overlook the subsequent spatial nanoparticle insertion and location within the membrane. There is therefore a need for studies to assess the mutual role of membrane and nanoparticle charge into membrane structure and stability at the nanoscale, with a view to better design and control the functionality of these nanomaterials. In this work, we have resolved the extent of the interactions between hydrophilic, negatively charged CdTe QDs and positively charged lipid bilayers. A multiscale combination of surface-sensitive techniques enabled probing how surface charge mediates QD adsorption and membrane reorganization. Increasing membrane surface charge results into a larger adsorption of oppositely charged QDs, concomitantly inducing structural changes. Hydration of the membrane hydrophobic parts by QDs goes deeper into the inner leaflet with increasing membrane charge, resulting in supported lipid bilayers with decreased nanomechanical stability.

Hierarchical Bilayer Polyelectrolyte Ion Paper Conductor for Moisture-Induced Power Generation.

Harvesting energy from air water (atmospheric moisture) promises a sustainable self-powered system without any restrictions from specific environmental requirements (e.g., solar cells, hydroelectric, or thermoelectric devices). However, the present moisture-induced power devices traditionally generate intermittent or bursts of energy, especially for much lower current outputs (generally keeping at nA or μA levels) from the ambient environment, typically suffering from inferior ionic conductivity and poor hierarchical structure design for manipulating sustained air water and ion-charge transport. Here, we demonstrate a universal strategy to design a high-performance bilayer polyelectrolyte ion paper conductor for generating continuous electric power from ambient humidity. The generator can produce a continuous voltage of up to 0.74 V and also an exceptional current of 5.63 mA across a single 1.0 mm-thick ion paper conductor. We discover that the sandwiched LiCl-nanocellulose-engineered paper promises an ion-transport junction between the negatively and positively charged bilayer polyelectrolytes for application in MEGs with both high voltage and high current outputs. Moreover, we demonstrated the universality of this bilayer sandwich nanocellulose-salt engineering strategy with other anions and cations, exhibiting similar power generation ability, indicating that it could be the next generation of sustainable MEGs with low cost, easier operation, and high performance.

Impedance spectra of soft ionics

Impedance spectroscopy is widely adopted for probing the charge and charge mobility of soft ion-conducting media, such as synthetic membranes and biological tissue. The spectra exhibit a variety of distinctive signatures, but the physical basis of these is not well understood, e.g. models have not previously accounted for viscoelasticity, hydrodynamics or microstructural heterogeneity. This study explores a physically grounded continuum model that captures how these factors shape conductivity spectra. Nonlinear thermodynamics and linearised dynamics of a viscous electrolyte and compressible, elastic polymer network are coupled under the forcing of an oscillatory electric field. The model is solved in a one-dimensional spatially periodic unit cell, reporting conductivity and dielectric permittivity spectra, including Nyquist representations. Whereas rigid microstructures exhibit ion-diffusion-controlled relaxation, which manifests as a low-frequency dielectric ‘constant’, hydrodynamic and elastic forces contribute to a strongly diverging dielectric permittivity at low frequencies for heterogeneous anionic microstructures. The model also captures distinctive characteristics of experimentally reported impedance spectra for films bearing alternating layers of cationic and anionic charge, again highlighting the role of coupled hydrodynamic, elastic and electrical forces. Sufficiently thin and highly charged bilayers exhibit a notably low high-frequency conductivity. This is explained by strong low-frequency electrostatic polarisation and counter-ion release. The one-dimensional solutions computed herein provide a foundation for much more challenging computations in two and three dimensions.

Metal ion cofactors modulate integral enzyme activity by varying differential membrane curvature stress.

Metal ions are well-known cofactors of protein function and stability. In the case of the integral membrane enzyme OmpLA (outer membrane phospholipase A) the active dimer is stabilized by calcium ions. We studied the lipid hydrolysis kinetics of OmpLA in charge-neutral and charged membranes with symmetric or asymmetric transbilayer lipid distributions. In charge-neutral membranes, OmpLA was more active in symmetric bilayers due to the lower differential curvature stress between membrane leaflets. Strikingly, this behavior was completely reversed in charged bilayers. Measurements revealed intrinsic molecular shape changes in the charged lipids upon addition of calcium. This effectively reduces the differential curvature stress in charged asymmetric membranes leading to increased protein activity. This conclusion is further supported by similar effects observed upon the addition of sodium ions, which also alter the shape of the lipids, but do not specifically interact with the protein. Additional lipid-protein interactions likely contribute to this phenomenon. Our findings demonstrate that ion cofactors not only interact directly with membrane proteins but also modulate protein activity indirectly by altering the effective molecular shape of charged lipid species.

Guanidinium-Functionalized Polymer Dielectrics for Triboelectric Bacterial Detection.

Development of rapid detection strategies that target potentially pathogenic bacteria has gained increasing attention due to the increasing awareness for better health and safety. In this study, we evaluate an intrinsically antimicrobial polymer, 2Gdm, which is a poly(norbornene)-based functional polymer featuring guanidinium groups as side chains, for bacterial detection by the means of triboelectric nanogenerators (TENGs) and triboelectric nanosensors (TENSs). Attachment of bacteria to the sensing layer is anticipated to alter the overall triboelectric properties of the underlying polymer layer. The positively charged guanidinium functional groups can interact with the negatively charged phospholipid bilayer of bacteria and lead to bacterial death, which can then be detected by optical microscopy, X-ray photoelectron microscopy, and more advanced self-powered sensing techniques such as TENGs and TENSs. The double bonds present along the poly(norbornene) backbone allow for thermally induced cross-linking to obtain X-2Gdm and thus rendering materials remain stable in water. By monitoring the change in voltage output after immersion in various concentrations of Gram-negative Escherichia coli (E. coli) and Gram-positive Streptococcus pneumoniae (S. pneumoniae), we have demonstrated the utility of X-2Gdm as a new polymer dielectric for autonomous bacterial detection. As the bacterial concentration increases, the amount of adsorbed bacteria also increases, resulting in a decrease in the surface potential of the X-2Gdm thin film; this reduction in surface potential can cause a decrease in the triboelectric output for both TENGs and TENSs, which serves as a key working mechanism for facile bacterial detection. TENG and TENS systems are capable of detecting E. coli and S. pneumoniae within a range of 4 × 105 to 4 × 108 CFU/mL with a limit of detection of 106 CFU/mL. This report highlights the promising prospects of employing TENGs and TENSs as innovative sensing technologies for rapid bacterial detection by leveraging the electrostatic interactions between bacterial cell membranes and cationic groups present on polymer surfaces.

Human HSP70-escort protein 1 (hHep1) interacts with negatively charged lipid bilayers and cell membranes.

Human Hsp70-escort protein 1 (hHep1) is a cochaperone that assists in the function and stability of mitochondrial HSPA9. Similar to HSPA9, hHep1 is located outside the mitochondria and can interact with liposomes. In this study, we further investigated the structural and thermodynamic behavior of interactions between hHep1 and negatively charged liposomes, as well as interactions with cellular membranes. Our results showed that hHep1 interacts peripherally with liposomes formed by phosphatidylserine and cardiolipin and remains partially structured, exhibiting similar affinities for both. In addition, after being added to the cell membrane, recombinant hHep1 was incorporated by cells in a dose-dependent manner. Interestingly, the association of HSPA9 with hHep1 improved the incorporation of these proteins into the lipid bilayer. These results demonstrated that hHep1 can interact with lipids also present in the plasma membrane, indicating roles for this cochaperone outside of mitochondria.

Anomalous Diffusion of Polyelectrolyte Segments on Supported Charged Lipid Bilayers.

This work provides mesoscale models for the anomalous diffusion of a polymer chain on a heterogeneous surface with rearranging randomly distributed adsorption sites. Both the "bead-spring" model and oxDNA model were simulated on supported lipid bilayer membranes with various molar fractions of charged lipids, using Brownian dynamics method. Our simulation results demonstrate that "bead-spring" chains exhibit sub-diffusion on charged lipid bilayers which agrees with previous experimental observations for short-time dynamics of DNA segments on membranes. In addition, the non-Gaussian diffusive behaviors of DNA segments have not been observed in our simulations. However, a simulated 17 base pairs double stranded DNA, using oxDNA model, performs normal diffusion on supported cationic lipid bilayers. Due to the number of positively charged lipids attracted by short DNA is small, the energy landscape that the short DNA experiences during diffusion is not as heterogeneous as that experienced by long DNA chains, which results in normal diffusion rather than sub-diffusion for short DNA.

Coarse-Grained Molecular Dynamics of pH-Sensitive Lipids.

pH-sensitive lipids represent a class of lipids that can be protonated and destabilized in acidic environments, as they become positively charged in response to low-pH conditions. They can be incorporated into lipidic nanoparticles such as liposomes, which are able to change their properties and allow specific drug delivery at the acidic conditions encountered in some pathological microenvironments. In this work, we used coarse-grained molecular-dynamic simulations to study the stability of neutral and charged lipid bilayers containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and various kinds of ISUCA ((F)2-(imidazol-1-yl)succinic acid)-derived lipids, which can act as pH-sensitive molecules. In order to explore such systems, we used a MARTINI-derived forcefield, previously parameterized using all-atom simulation results. We calculated the average area per lipid, the second-rank order parameter and the lipid diffusion coefficient of both lipid bilayers made of pure components and mixtures of lipids in different proportions, under neutral or acidic conditions. The results show that the use of ISUCA-derived lipids disturbs the lipid bilayer structure, with the effect being particularly marked under acidic conditions. Although more-in depth studies on these systems must be carried out, these initial results are encouraging and the lipids designed in this research could be a good basis for developing new pH-sensitive liposomes.

Interactions of β-barrel assembly machinery protein D (BamD) with phospholipid bilayers.

The β-barrel assembly machinery (BAM) complex from Escherichia coli consists of the transmembrane protein BamA and four peripheral proteins, BamB, BamC, BamD and BamE. We investigated the lipid interactions of BamD (also called YfiO), which is essential for E. coli using site-directed fluorescence spectroscopy. To prepare a range of single-cysteine mutants of BamD for fluorescence labelling at the cysteine, we selected nine amino acid residues that were distributed over the entire surface of BamD. Expression plasmids were prepared and the mutants were then isolated from transformed E. coli strains to analyze interactions of BamD with bilayers composed of DLPC or of DLPG or of mixtures of DLPG and DLPE at a ratio of 4:1. BamD mutants were labeled with the fluorophore IANBD to examine BamD-lipid interactions by fluorescence spectroscopy. Higher fluorescence intensities and blue shifts of the emission spectra indicated that the entire surface of BamD interacted with the lipid bilayer. The fluorescence maxima of IANBD-labeled mutants were shifted towards shorter wavelength by ∼ 6 to 18 nm. Intensities of IANBD fluorescence increased most strongly for negatively charged DLPG bilayers, suggesting stronger binding due to electrostatic interactions. The weakest interactions were observed for lipid bilayers composed of DLPE and DLPG at a ratio of 4:1. For these bilayers, the IANBD-fluorescence spectra were most strongly changed for the IANBD labeled mutants A37C-BamD, G153C-BamD, and V181C-BamD. Fluorescence of all labeled mutants, except G200C-BamD, was quenched when 14-Doxyl-PC was used as a quencher, indicating penetration of BamD into the lipid bilayer. Fluorescence spectra of L75C-BamD and of V181C-BamD also indicated interactions with BamA in the absence of lipid bilayers.

Lactaderhin's C1 domain restricts protein's ensemble as it binds to charged membranes.

Lactadherin's (Lact) binding to phosphotidylserine (PS) enriched cell surfaces is critical for controlling our blood coagulation. Although structural and mutagenesis studies indicate important binding regions of this protein, they are not sufficient to construct a detailed, membrane-bound model especially since they mainly consider only one of its two globular domains, namely C2. We thus use molecular dynamics (MD) simulations and the AlphaFold program to generate an ensemble of Lact encounter complexes with a negatively charged bilayer consistent with biochemical studies. This was accomplished by first using the highly mobile membrane mimetic (HMMM) to efficiently simulate its C2 domain binding to PS lipids. We then converted the HMMM model to standard full-tail lipid membranes to further sample and obtain converged bound structures that we call the “vertical” and “side-lying” ensembles. From these bilayer-bound poses, we infer which one allows for attachment of its other C1 domain and use AlphaFold to reconstruct it and further simulate to capture equilibrium complexes. Modeling revealed that C1 domain restricts the dynamics structural ensemble of Lact bound to membranes. We also report Lact's full dynamic structure in the presence of negatively charged lipids and compare them with other homologous blood coagulation proteins to provide a rationale for its competitive binding. These results shed light on the structural mechanism of blood coagulation by considering its binding in a membrane environment which can serve as a general framework for studying these critical protein-membrane interactions. They also provide a structural basis for further understanding the significance that PS lipids play in regulating blood coagulation processes.

Coarse-grained molecular dynamics simulation of cation distribution profiles on negatively charged lipid membranes during phase separation.

Revealing the ion distributions on a charged lipid membrane in aqueous solution under the influence of long-range interactions is essential for understanding the origin of the stability of the bilayer structure and the interaction between biomembranes and various electrolytes. However, the ion distributions and their dynamics associated with the phase separation process of the lipid bilayer membrane are still unclear. We perform coarse-grained molecular dynamics simulations to reveal the Na+ and Cl- distributions on charged phospholipid bilayer membranes during phase separation. During the phase separation, cations closely follow the position of negatively charged lipids on a microsecond timescale and are rapidly redistributed parallel to the lipid bilayer. In the homogenous mixture of zwitterionic and negatively charged lipids, cations weakly follow negatively charged lipids, indicating the strong interaction between cations and negatively charged lipids. We also compare cation concentrations as a function of surface charge density obtained by our simulation with those obtained by a modified Poisson-Boltzmann theory. Including the ion finite size makes the statistical results consistent, suggesting the importance of the ion-ion interactions in aqueous solution. Our simulation results advance our understanding of ion distribution during phase separation.

Pro-inflammatory protein S100A9 alters membrane organization by dispersing ordered domains

Pro-inflammatory, calcium-binding protein S100A9 is localized in the cytoplasm of many cells and regulates several intracellular and extracellular processes. S100A9 is involved in neuroinflammation associated with the pathogenesis of Alzheimer's disease (AD). The number of studies on the impact of S100A9 in co-aggregation processes with amyloid-like proteins is increasing. However, there is still a lack of data on how this protein interacts with lipid membranes. We employed atomic force microscopy (AFM), dynamic light scattering (DLS), and fluorescence measurements (Laurdan and Thioflavin-T) to study the interaction between protein and the membrane surface. We used lipid vesicles in bulk and planar tethered lipid bilayers as biomimetic membrane models. We demonstrated that the protein accumulates on negatively charged lipid bilayers but with no further loss of the bilayer's integrity. The most important result is that the initial adsorption and accumulation of apo-form of S100A9 on the lipid membrane surface is lipid phase-sensitive. The breaking down of raft-like and disappearance of gel-like domains indicate that protein incorporates into the hydrophobic part of the lipid bilayer. We observed the most noticeable loss of integrity in lipid bilayers constructed from a lipid mixture (brain total lipid extract). Understanding the function and interactions of these proteins in cellular environments might expand the development of new diagnostic and therapeutic approaches for AD or other related diseases.

Importance of annexin V N-terminus for 2D crystal formation and quick purification protocol of recombinant annexin V.

Annexin V forms trimeric structures which further assemble into two-dimensional crystal (2D crystal) lattices on negatively charged phospholipid bilayer in a Ca2+-dependent manner. It is also known that annexin V 2D crystals show two types of symmetric patterns with six-fold symmetry (p6) and three-fold symmetry (p3). The p6 lattice also contains additional trimers in the gaps between the p6 axes, which are also referred to as non-p6 trimers because they do not participate in the formation of the p6 lattice. We here show that the annexin V N-terminal has significant influence on 2D crystal formation using high-speed atomic force microscopy (HS-AFM) observations. We also present a quick purification method to purify recombinant annexin V without any residual affinity tag after protein purification in ~3h.

Single-Molecule Trapping and Measurement in a Nanostructured Lipid Bilayer System.

The repulsive electrostatic force between a biomolecule and a like-charged surface can be geometrically tailored to create spatial traps for charged molecules in solution. Using a parallel-plate system composed of silicon dioxide surfaces, we recently demonstrated single-molecule trapping and high precision molecular charge measurements in a nanostructured free energy landscape. Here we show that surfaces coated with charged lipid bilayers provide a system with tunable surface properties for molecular electrometry experiments. Working with molecular species whose effective charge and geometry are well-defined, we demonstrate the ability to quantitatively probe the electrical charge density of a supported lipid bilayer. Our findings indicate that the fraction of charged lipids in nanoslit lipid bilayers can be significantly different from that in the precursor lipid mixtures used to generate them. We also explore the temporal stability of bilayer properties in nanofluidic systems. Beyond their relevance in molecular measurement, such experimental systems offer the opportunity to examine lipid bilayer formation and wetting dynamics on nanostructured surfaces.

A THEORETICAL MODEL FOR DESCRIPTION OF WORK FUNCTION LOWERING FOR SEMICONDUCTOR / INSULATOR UNDER THE INFLUENCE OF THE CHARGED BILAYER IN THE SURFACE REGION

We develop a simple theoretical model, connecting a lowering of the electron affinity of a semiconductor (or insulator) with such parameters as the density of surface charge, localized on surface states or adsorbed atoms, and the volume density of charge within the space charge region, both of which form a charged bilayer on the surface. Our model allows to estimate the perspectiveness of various materials as films for creating modern photocathodes or effective field emission cathodes with substantially lowered or zero work function.

Investigations into the membrane activity of arenicin antimicrobial peptide AA139

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3.

Surface Characteristics of Antibacterial Polystyrene Nanoparticles Synthesized Using Cationic Initiator and Comonomers.

Polymer nanoparticles have attracted attention as antibacterial materials, but the function of the polymer itself has not yet been clarified sufficiently. To estimate the essential surface properties of antibacterial polymer nanoparticles, herein, we synthesized cationic polystyrene (PSt) nanoparticles via soap-free emulsion polymerization using 2,2'-azobis-[2-(1,3-dimethyl-4,5-dihydro-1H-imidazol-3-ium-2-yl)]propane triflate (ADIP) as initiator. The conversion of total monomers was drastically increased through the addition of the commoner (vinylbenzyl)trimethylammonium chloride (VBTMAC), where unimodal size distributions (Cv ≤ 10%) were obtained at comonomer molar ratios between 0.0083 and 0.0323. The adsorption behavior of a solvatochromic anionic fluorescent dye revealed the surface charge density (σ) and affinity with anionic molecules (K) of PSt nanoparticles. The PSt nanoparticles with increased K values exhibited antibacterial activity against Staphylococcus epidermidis, with a minimum inhibitory concentration of at least 0.69 mg/mL. To determine a plausible mechanism for the antibacterial activity, the membrane damage induced by PSt nanoparticles was evaluated using an assay utilizing polydiacetylene vesicles as the model for negatively charged bilayer membranes. The PSt nanoparticles exhibiting large K values disturbed the bilayer structure of the model membrane system, suggesting that the synthesized PSt nanoparticles could be utilized as a contact-killing antibacterial agent.