Permeability Of Lipid Bilayers Research Articles

Antibiotic Loaded Phytosomes as a Way to Develop Innovative Lipid Formulations of Polyene Macrolides.

The threat of antibiotic resistance of fungal pathogens and the high toxicity of the most effective drugs, polyene macrolides, force us to look for new ways to develop innovative antifungal formulations. The aim of this study was to determine how the sterol, phospholipid, and flavonoid composition of liposomal forms of polyene antibiotics, and in particular, amphotericin B (AmB), affects their ability to increase the permeability of lipid bilayers that mimic the membranes of mammalian and fungal cells. To monitor the membrane permeability induced by various polyene-based lipid formulations, a calcein leakage assay and the electrophysiological technique based on planar lipid bilayers were used. The replacement of cholesterol with its biosynthetic precursor, 7-dehydrocholesterol, led to a decrease in the ability of AmB-loaded liposomes to permeabilize lipid bilayers mimicking mammalian cell membranes. The inclusion of plant flavonoid phloretin in AmB-loaded liposomes increased the ability of the formulation to disengage a fluorescent marker from lipid vesicles mimicking the membranes of target fungi. I-V characteristics of the fungal-like lipid bilayers treated with the AmB phytosomes were symmetric, demonstrating the functioning of double-length AmB pores and assuming a decrease in the antibiotic threshold concentration. The therapeutic window of polyene lipid formulations might be expanded by varying their sterol composition. Polyene-loaded phytosomes might be considered as the prototypes for innovative lipid antibiotic formulations.

Distinguishing Molecular Mechanical Action from Photothermal and Photodynamic Behavior.

Molecular motors (MM) are molecular machines, or nanomachines, that rotate unidirectionally upon photostimulation and perform mechanical work on their environment. In the last several years, it has been shown that the photomechanical action of MM can be used to permeabilize lipid bilayers, thereby killing cancer cells and pathogenic microorganisms and controlling cell signaling. The work contributes to a growing acknowledgement that the molecular actuation characteristic of these systems is useful for various applications in biology. However, the mechanical effects of molecular motion on biological materials are difficult to disentangle from photodynamic and photothermal action, which are also present when a light-absorbing fluorophore is irradiated with light. Here, an overview of the key methods used by various research groups to distinguish the effects of photomechanical, photodynamic, and photothermal action is provided. It is anticipated that this discussion will be helpful to the community seeking to use MM to develop new and distinctive medical technologies that result from mechanical disruption of biological materials.

Martini 3 Coarse-Grained Force Field for Cholesterol.

Cholesterol plays a crucial role in biomembranes by regulating various properties, such as fluidity, rigidity, permeability, and organization of lipid bilayers. The latest version of the Martini model, Martini 3, offers significant improvements in interaction balance, molecular packing, and inclusion of new bead types and sizes. However, the release of the new model resulted in the need to reparameterize many core molecules, including cholesterol. Here, we describe the development and validation of a Martini 3 cholesterol model, addressing issues related to its bonded setup, shape, volume, and hydrophobicity. The proposed model mitigates some limitations of its Martini 2 predecessor while maintaining or improving the overall behavior.

Imaging Amyloid-β Membrane Interactions: Ion-Channel Pores and Lipid-Bilayer Permeability in Alzheimer's Disease.

The accumulation of the amyloid-β peptides (Aβ) is central to the development of Alzheimer's disease. The mechanism by which Aβ triggers a cascade of events that leads to dementia is a topic of intense investigation. Aβ self-associates into a series of complex assemblies with different structural and biophysical properties. It is the interaction of these oligomeric, protofibril and fibrillar assemblies with lipid membranes, or with membrane receptors, that results in membrane permeability and loss of cellular homeostasis, a key event in Alzheimer's disease pathology. Aβ can have an array of impacts on lipid membranes, reports have included: a carpeting effect; a detergent effect; and Aβ ion-channel pore formation. Recent advances imaging these interactions are providing a clearer picture of Aβ induced membrane disruption. Understanding the relationship between different Aβ structures and membrane permeability will inform therapeutics targeting Aβ cytotoxicity.

Imaging Amyloid-β Membrane Interactions: Ion-Channel Pores and Lipid-Bilayer Permeability in Alzheimer's Disease.

The accumulation of the amyloid-β peptides (Aβ) is central to the development of Alzheimer's disease. The mechanism by which Aβ triggers a cascade of events that leads to dementia is a topic of intense investigation. Aβ self-associates into a series of complex assemblies with different structural and biophysical properties. It is the interaction of these oligomeric, protofibril and fibrillar assemblies with lipid membranes, or with membrane receptors, that results in membrane permeability and loss of cellular homeostasis, a key event in Alzheimer's disease pathology. Aβ can have an array of impacts on lipid membranes, reports have included: a carpeting effect; a detergent effect; and Aβ ion-channel pore formation. Recent advances imaging these interactions are providing a clearer picture of Aβ induced membrane disruption. Understanding the relationship between different Aβ structures and membrane permeability will inform therapeutics targeting Aβ cytotoxicity.

Computational Prodrug Design Methodology for Liposome Formulability Enhancement of Small-Molecule APIs.

Encapsulation into liposomes is a formulation strategy that can improve efficacy and reduce side effects of active pharmaceutical ingredients (APIs) that exhibit poor biodistribution or pharmacokinetics when administered alone. However, many APIs are unsuitable for liposomal formulations intended for parenteral administration due to their inherent physicochemical properties─lipid bilayer permeability and water-lipid equilibrium partitioning coefficient. Too high permeability results in premature leakage from liposomes, while too low permeability means the API is not able to pass across biological barriers. There are several options for solving this issue: (i) change of the lipid bilayer composition, (ii) addition of a permeability enhancer, or (iii) modification of the chemical structure of the API to design a prodrug. The latter approach was taken in the present work, and the effect of small changes in the molecular structure of the API on its permeation rate across a lipidic bilayer was systematically explored utilizing computer simulations. An in silico methodology for prodrug design based on the COSMOperm approach has been proposed and applied to four APIs (abiraterone, cytarabine, 5-fluorouracil, and paliperidone). It is shown that the addition of aliphatic hydrocarbon chains via ester or amide bonds can render the molecule more lipophilic and increase its permeability by approximately 1 order of magnitude for each 2 carbon atoms added, while the formation of fructose adducts can provide a more hydrophilic character to the molecule and reduce its lipid partitioning. While partitioning was found to depend only on the size and type of the added group, permeability was found to depend also on the added group location. Overall, it has been shown that both permeability and lipid partitioning coefficient can be systematically shifted into the desired liposome formulability window by appropriate group contributions to the parental drug. This can significantly increase the portfolio of APIs for which liposome or lipid nanoparticle formulations become feasible.

Effect of Cisplatin and Its Cationic Analogues in the Phase Behavior and Permeability of Model Lipid Bilayers

Increasing evidence suggests a critical role of lipids in both the mechanisms of toxicity and resistance of cells to platinum(II) complexes. In particular, cisplatin and other analogues were reported to interact with lipids and transiently promote lipid phase changes both in the bulk membranes and in specific membrane domains. However, these processes are complex and not fully understood. In this work, cisplatin and its cationic species formed at pH 7.4 in low chloride concentrations were tested for their ability to induce phase changes in model membranes with different lipid compositions. Fluorescent probes that partition to different lipid phases were used to report on the fluidity of the membrane, and a leakage assay was performed to evaluate the effect of cisplatin in the permeability of these vesicles. The results showed that platinum(II) complex effects on membrane fluidity depend on membrane lipid composition and properties, promoting a stronger decrease in the fluidity of membranes containing gel phase. Moreover, at high concentration, these complexes were prone to alter the permeability of lipid membranes without inducing their collapse or aggregation.

Label-free quantification of passive membrane permeability of cyclic peptides across lipid bilayers: penetration speed of cyclosporin A across lipid bilayers.

Cyclic peptides that passively penetrate cell membranes are under active investigation in drug discovery research. PAMPA (Parallel Artificial Membrane Permeability Assay) and Caco-2 assay are mainly used for permeability measurements in these studies. However, permeability rates across the artificial membrane and the cell monolayer used for these assays are intrinsically different from the ones across pure lipid bilayers. There are also membrane permeability assays for peptides using reconstructed lipid bilayers, but they require labeling for detection, and the absolute membrane permeability of the natural peptides themselves could not be determined. Here, we constructed a lipid bilayer permeability assay and realized the first label-free measurements of the lipid bilayer permeability of cyclic peptides. Quantitative permeability values across lipid bilayers were determined for model cyclic hexapeptides and an important natural product, cyclosporin A (CsA). The obtained quantitative permeability values will provide new and advanced knowledge about the passive permeability of cyclic peptides.

Each side chain of cyclosporin A is not essential for high passive permeability across lipid bilayers.

We compared the passive permeability of cyclosporin A (CsA) derivatives with side chain deletions across lipid bilayers. CsA maintained passive permeability after losing any one of the side chains, which suggests that the propensity of the backbone of CsA is an important component for high passive permeability.

Antimicrobial Peptide Mechanisms Studied by Whole-Cell Deuterium NMR.

Much of the work probing antimicrobial peptide (AMP) mechanisms has focussed on how these molecules permeabilize lipid bilayers. However, AMPs must also traverse a variety of non-lipid cell envelope components before they reach the lipid bilayer. Additionally, there is a growing list of AMPs with non-lipid targets inside the cell. It is thus useful to extend the biophysical methods that have been traditionally applied to study AMP mechanisms in liposomes to the full bacteria, where the lipids are present along with the full complexity of the rest of the bacterium. This review focusses on what can be learned about AMP mechanisms from solid-state NMR of AMP-treated intact bacteria. It also touches on flow cytometry as a complementary method for measuring permeabilization of bacterial lipid membranes in whole bacteria.

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.

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.

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 Disordering Effect of Plant Metabolites on Model Lipid Membranes of Various Thickness

This study was focused on the effect of plant metabolites (phloretin, capsaicin, digitonin, diosgenin and betulin) on the model lipid membranes. The methods of assessing the permeability of lipid bilayers based on measuring the leakage of a fluorescent marker (calcein) from liposomes and differential scanning microcalorimetry of vesicle suspension were used. It was found that the release rate of calcein from 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) liposomes with test compounds added to the suspension at a ratio with lipid 1 : 50 decreased in the order capsaicin > phloretin >> betulin ≈ diosgenin ≈ digitonin. In the case of cholesterol- and ergosterol-containing POPC liposomes, the activity decreased as diosgenin ≈ digitonin > betulin > capsaicin > phloretin. It was demonstrated that phloretin and capsaicin significantly reduce the melting temperature (Tm) and to increase the half-width of the main peak on the endotherm (T1/2) of dipalmitoylphosphocholine (DPPC), distearoylphosphocholine (DSPC) and diarachidoylphosphocholine (DAPC). These results show that the incorporation of these small molecules between the polar “heads” of phosphocholine. It was found that the increase in the length of saturated chains of membrane-forming lipids (from 16 to 20 hydrocarbon units), the absolute values of ΔTm and ΔT1/2 decreased in the presence of phloretin and increased with capsaicin. This may be the result of differences in the localization of phloretin and capsaicin in the membrane. Steroid saponins exhibited a weak effect on the thermotropic behavior of phosphocholines. The absolute values of ΔTm and ΔT1/2 decreased in the order DPPC, DSPC, and DAPC and increased in the order betulin, diosgenin, and digitonin. Steroid saponins are characterized by a more pronounced effect on the thermotropic behavior of the sterol–phospholipid mixture. The findings are consistent with the assumption of a high affinity of the tested saponins for sterol-containing membranes.

Interaction of the Anti-Proliferative GPER Inverse Agonist ERα17p with the Breast Cancer Cell Plasma Membrane: From Biophysics to Biology.

The peptide ERα17p, which corresponds to the 295-311 fragment of the hinge/AF2 domains of the human estrogen receptor α (ERα), exerts apoptosis in breast cancer cells through a mechanism involving the G protein-coupled estrogen-dependent receptor GPER. Besides this receptor-mediated mechanism, we have detected a direct interaction (Kd value in the micromolar range) of this peptide with lipid vesicles mimicking the plasma membrane of eukaryotes. The reversible and not reversible pools of interacting peptide may correspond to soluble and aggregated membrane-interacting peptide populations, respectively. By using circular dichroism (CD) spectroscopy, we have shown that the interaction of the peptide with this membrane model was associated with its folding into β sheet. A slight leakage of the 5(6)-fluorescein was also observed, indicating lipid bilayer permeability. When the peptide was incubated with living breast cancer cells at the active concentration of 10 μM, aggregates were detected at the plasma membrane under the form of spheres. This insoluble pool of peptide, which seems to result from a fibrillation process, is internalized in micrometric vacuoles under the form of fibrils, without evidence of cytotoxicity, at least at the microscopic level. This study provides new information on the interaction of ERα17p with breast cancer cell membranes as well as on its mechanism of action, with respect to direct membrane effects.

In silico modeling of izoniazid-steroid conjugates interactions with cytochromes P450 of mycobacteria and their bioconversion in vitro by the cells

Molecular docking of four hydrazones of isoniazid with steroids (dehydroepiandrosterone, pregnenolone, 16α,17α-epoxypregnenolone, cholestenone) - IDHEA, IPRE, IEP5, ICHN, to mycobacterial cytochromes P450 was performed. The in silico study has shown than these hydrazones can be effectively bound to CYP121, CYP124, CYP125, CYP126A1, CYP130, and CYP51 with binding energy ranged from -9 kcal/mol to -12 kcal/mol. Calculations also demonstrated enhancement of passive lipid bilayer permeability with respect to isoniazid. In vitro IDHEA, IPRE, IEPR were found to undergo bioconversion into their 3-keto-4-en derivatives. This suggests their ability to penetrate into M. tuberculosis H37Rv cells. The results of this study are important in the context of understanding of specificity of binding of synthetic steroid derivatives to mycobacterial CYPs and indicate the possibility of using the steroid compounds studied by us as new ligands for these enzymes.

Increase in the current variance in bilayer lipid membranes near phase transition as a result of the occurrence of hydrophobic defects

Most researchers associate the increase in the permeability of lipid bilayers of artificial and biological membranes observed in various experiments with the formation of hypothetical hydrophobic and hydrophilic pores. Although the existence of hydrophobic defects, as the first stage of the formation of a hydrophilic pore, was hypothesized decades ago from electroporation experiments, the difficulty of describing this stage is determined by the lack of experimental data confirming the existence or at least associated with hydrophobic pores. We explored the increase in the current variance through the lipid membrane, observed when approaching the phase transition from the side of high temperatures, and have associated it with capacitive currents arising in response to the formation of hydrophobic pores. Assuming that the number of hydrophobic pores in a membrane follows a Poisson distribution, and thus, the mean number of hydrophobic pores is equal to the variance of that number, we used the measurements of the membrane current variance to evaluate the number of hydrophobic pores. Analysis of experimental data within this model allows us to estimate the number of hydrophobic pores at or above the phase transition and shows that the number of hydrophobic pores in a membrane close to the phase transition increased 20 times compared to the number of hydrophobic pores existing in the membrane far from the melting transition.

High Permeability of Polyunsaturated Lipid Bilayers As Applied to Attoliter Enzyme Reactors.

Phospholipid vesicles encapsulated with enzymes have potential applications for artificial organelles. A critical problem associated with the compartmentalized enzymes is their low reactivity because of the permeability resistance of lipid bilayers to substrates. In the present work, the polyunsaturated bilayers of 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3-PC) were elucidated to be highly permeable to 5(6)-carboxyfluorescein at high temperatures up to 60 °C and applied to fabricate vesicle-based reactive enzyme reactors. d-Amino acid oxidase (DAO) from porcine kidney was encapsulated in 18:3-PC vesicles with each aqueous volume of 3.4 × 10-21 m3 (=3.4 aL). The DAO-containing vesicles were highly reactive at 40 °C toward d-alanine being added to bulk solution at pH 9.0 and stably catalyzed following two types of reactions. One is the DAO-catalyzed continuous production of H2O2 in the vesicles for 30 min being detected by the free peroxidase-catalyzed oxidation of o-dianisidine in bulk solution. The other is the cascade reaction in the vesicles coencapsulating DAO and catalase being followed for 5 h on the basis of the concentration of unreacted d-alanine. In the latter reaction, the intermediate product H2O2 was decomposed by catalase producing oxygen allowing its cyclical use for the DAO-catalyzed oxidation. Furthermore, thanks to the highly temperature-dependent permeability of 18:3-PC bilayers, on/off-like switching in the activity could be induced with respect to the vesicle-confined enzyme by shifting the reaction temperature between 20 and 40 °C. The above reactive vesicles can offer the opportunity of the H2O2-based reliable detection of d-amino acids and the continuous optical resolution of racemic mixtures of amino acids.

Proton leakage across lipid bilayers: Oxygen atoms of phospholipid ester linkers align water molecules into transmembrane water wires

Up to half of the cellular energy gets lost owing to membrane proton leakage. The permeability of lipid bilayers to protons is by several orders of magnitude higher than to other cations, which implies efficient proton-specific passages. The nature of these passages remains obscure. By combining experimental measurements of proton flow across phosphatidylcholine vesicles, steered molecular dynamics (MD) simulations of phosphatidylcholine bilayers and kinetic modelling, we have analyzed whether protons could pass between opposite phospholipid molecules when they sporadically converge. The MD simulations showed that each time, when the phosphorus atoms of the two phosphatidylcholine molecules got closer than 1.6 nm, the eight oxygen atoms of their ester linkages could form a transmembrane ‘oxygen passage’ along which several water molecules aligned into a water wire. Proton permeability along such water wires would be limited by rearrangement of oxygen atoms, which could explain the experimentally shown independence of the proton permeability of pH, H2O/D2O substitution, and membrane dipole potential. We suggest that protons can cross lipid bilayers by moving along short, self-sustaining water wires supported by oxygen atoms of lipid ester linkages.