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The study and characterization of the biophysical properties of membranes and drug-membrane interactions represent a critical step in drug development, as biological membranes act as a barrier that the drug must overcome to reach its active site. Liposomes are widely used in drug delivery to circumvent the poor aqueous solubility of most drugs, improving systemic bioavailability and pharmacokinetics. Further, they can be targeted to deliver to specific disease sites, thus decreasing drug load, and reducing side effects and poor adherence to treatment. To improve drug solubility during liposome preparation, DMSO is the most widely used solvent. This raises concern about the potential effect of DMSO on membranes and leads us to investigate, using DSC and EPR, the influence of DMSO on the behavior of lipid model membranes of DMPC and DPPC. In addition, we tested the influence of DMSO on drug-membrane interaction, using compounds with different hydrophobicity and varying DMSO content, using the same experimental techniques. Overall, it was found that with up to 10% DMSO, changes in the bilayer fluidity or the thermotropic properties of the studied liposomes were not significant, within the experimental uncertainty. For higher concentrations of DMSO, there is a stabilization of both the gel and the rippled gel phases, and increased bilayer fluidity of DMPC and DPPC liposomes leading to an increase in membrane permeability.

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Classification of the category of diabetes is extremely important for clinicians to diagnose and select the correct treatment plan. Glycosylation, oxidation and other post-translational modifications of membrane and transmembrane proteins, as well as impairment in cholesterol homeostasis, can alter lipid density, packing, and interactions of Red blood cells (RBC) plasma membranes in type 1 and type 2 diabetes, thus varying their membrane micropolarity. This can be estimated, at a submicrometric scale, by determining the membrane relative permittivity, which is the factor by which the electric field between the charges is decreased relative to vacuum. Here, we employed a membrane micropolarity sensitive probe to monitor variations in red blood cells of healthy subjects (n=16) and patients affected by type 1 (T1DM, n=10) and type 2 diabetes mellitus (T2DM, n=24) to provide a cost-effective and supplementary indicator for diabetes classification. We find a less polar membrane microenvironment in T2DM patients, and a more polar membrane microenvironment in T1DM patients compared to control healthy patients. The differences in micropolarity are statistically significant among the three groups (p<0.01). The role of serum cholesterol pool in determining these differences was investigated, and other factors potentially altering the response of the probe were considered in view of developing a clinical assay based on RBC membrane micropolarity. These preliminary data pave the way for the development of an innovative assay which could become a tool for diagnosis and progression monitoring of type 1 and type 2 diabetes.

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Ibuprofen is a non-steroidal anti-inflammatory drug widely used to treat inflammatory diseases, and for its analgesic and antipyretic activity. Although operating as a protein inhibitor, it is also known to interact with lipid membranes. We combined calorimetry, electron spin resonance, attenuated total reflectance-Fourier transform infrared and molecular docking to characterize the interaction of ibuprofen with dimyristyolphosphatidylcholine (DMPC) bilayers, as a function of temperature and drug concentration. At increasing concentration, ibuprofen first perturbs and then suppresses the DMPC pre-transition, stabilizes the fluid state, and favours gel-fluid phase coexistence. The drug decreases the molecular packing of the polar heads and of the first methylene segments of lipid membranes in the gel phase, whereas it leaves unperturbed the chain flexibility in the liquid-crystalline phase. The action of ibuprofen also leads to a higher degree of hydration of the bilayer polar heads and favours hydrogen bond formation with solvent molecules. The overall results reveal that ibuprofen affects a number of key molecular properties of DMPC bilayers by binding through non-specific interactions at the polar/apolar interface.

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BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is a major lipid second messenger in insulin-mediated signalling towards the metabolic actions of this hormone in muscle and fat. PURPOSE: Assessing the intracellular transport of exogenous PIP3 attached to a polymeric carrier in an attempt to overcome cellular insulin resistance. METHODS: Artificial chromatic bio-mimetic membrane vesicles composed of dimyristoylphosphatidylcholine and polydiacetylene were applied to screen the polymeric carriers. PIP3 cellular localization and bio-activity was assessed by fluorescent and live-cell microscopy in L6 muscle cells and in 3T3-L1 adipocytes. RESULTS AND DISCUSSION: We demonstrate that a specific-branched polyethylenimine (PEI-25, 25 kDa) carrier forms complexes with PIP3 that interact with the bio-mimetic membrane vesicles in a manner predictive of their interaction with cells: In L6 muscle cells, PEI-25/fluorescent-PIP3 complexes are retarded at the cell perimeter. PEI-25/PIP3 complexes retain their bio-activity, engaging signalling steps downstream of PIP3, even in muscle cells rendered insulin resistant by exposure to high glucose/high insulin. CONCLUSIONS: Inducing insulin actions by intracellular PIP3 delivery (PEI-25/PIP3 complexes) in some forms of insulin-resistant cells provides the first proof-of-principle for the potential therapeutic use of PIP3 in a "second-messenger agonist" approach. In addition, utilization of an artificial bio-mimetic membrane platform to screen for highly efficient PIP3 delivery predicts biological function in cells.

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Of group 12 metals, zinc is an essential element to maintain our life, but other metals such as cadmium and mercury are toxic in cellular activities. Interactions of these metals with biomembranes are important to understand their effects on our living cells. Here, we describe the membrane perturbations induced by these metals in human erythrocytes. Of these metals, Zn2+ ions only induced the erythrocyte agglutination. Histidine residues in extracellular domains of band 3 participated in Zn2+-induced agglutination. Interestingly, it was found that band 3-cytoskeleton interactions play an important role in Zn2+-induced agglutination. In contrast with Hg2+ and Cd2+ ions, Zn2+ ions greatly suppressed pressure-induced hemolysis by cell agglutination. Such a suppression was removed upon dissociation of agglutinated erythrocytes by washing, indicating the reversible interactions of Zn2+ ions with erythrocyte membranes. Excimer fluorescence of pyrene indicated that spectrin is denatured by a pressure of 200 MPa irrespective of hemolysis suppression. Taken together, these results suggest that the agglutination of erythrocytes due to the interactions of Zn2+ ions with band 3 is stable under pressure, but spectrin, cytoskeletal protein, is denatured by pressure.

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Interactions between paclitaxel and its squalenoyl prodrug with dimyristoylphosphatidylcholine (DMPC) monolayer at the air/water interface were studied. Paclitaxel is an antineoplastic drug, largely used as anti-cancer agents. Because its low aqueous solubility, Cremophor EL is used as excipient for its formulation. However, it has been shown that Cremophor causes serious side effects. Several attempts have been made to develop a safer formulation such as the synthesis of lipophilic prodrug. In particular we have synthesized a paclitaxel prodrug obtained by conjugation with 1,1,2-trisnorsqualenoic acid to improve the physico-chemical properties of this antineoplastic drug. The miscibility of these compounds with DMPC monolayer were studied analyzing thermodynamic properties as well as excess Gibbs free energies, compressibility modulus and mixed monolayer isotherms. The results allowed to evaluate the spatial organization of the compounds and suggested that the prodrug can efficiently be incorporated in the DMPC monolayer.

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We have used systematic structure-based coarse graining to derive effective site-site potentials for a 10-site coarse-grained dimyristoylphosphatidylcholine (DMPC) lipid model and investigated their state point dependence. The potentials provide for the coarse-grained model the same site-site radial distribution functions, bond and angle distributions as those computed in atomistic simulations carried out at four different lipid-water molar ratios. It was shown that there is a non-negligible dependence of the effective potentials on the concentration at which they were generated, which is also manifested in the properties of the lipid bilayers simulated using these potentials. Thus, effective potentials computed at low lipid concentration favor to more condensed and ordered structure of the bilayer with lower average area per lipid, while potentials obtained at higher lipid concentrations provide more fluid-like structure. The best agreement with the reference data and experiment was achieved using the set of potentials derived from atomistic simulations at 1:30 lipid:water molar ratio providing fully saturated hydration of DMPC lipids. Despite theoretical limitations of pairwise coarse-grained potentials expressed in their state point dependence, all the resulting potentials provide a stable bilayer structure with correct partitioning of different lipid groups across the bilayer as well as acceptable values of the average lipid area, compressibility and orientational ordering. In addition to bilayer simulations, the model has proven its robustness in modeling of self-aggregation of lipids from randomly dispersed solution to ordered bilayer structures, bicelles, and vesicles.

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The fusion peptide of Ebola virus comprises a highly hydrophobic sequence located downstream from the N-terminus of the glycoprotein GP2 responsible for virus-host membrane fusion. The internal fusion peptide of GP2 inserts into membranes of infected cell to mediate the viral and the host cell membrane fusion. Since the sequence length of Ebola fusion peptide is still not clear, we study in the present work the behavior of two fusion peptides of different lengths which were named EBO17 and EBO24 referring to their amino acid length. The secondary structure and orientation of both peptides in lipid model systems made of DMPC:DMPG:cholesterol:DMPE (6:2:5:3) were investigated using PMIRRAS and polarized ATR spectroscopy coupled with Brewster angle microscopy. The infrared results showed a structural flexibility of both fusion peptides which are able to transit reversibly from an α-helix to antiparallel ß-sheets. Ellipsometry results corroborate together with isotherm measurements that EBO peptides interacting with lipid monolayer highly affected the lipid organization. When interacting with a single lipid bilayer, at low peptide content, EBO peptides insert as mostly α-helices mainly perpendicular into the lipid membrane thus tend to organize the lipid acyl chains. Inserted in multilamellar vesicles at higher peptide content, EBO peptides are mostly in ß-sheet structures and induce a disorganization of the lipid chain order. In this paper, we show that the secondary structure of the Ebola fusion peptide is reversibly flexible between α-helical and ß-sheet conformations, this feature being dependent on its concentration in lipids, eventually inducing membrane fusion.

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This report presents evidence that the following Solanum steroids: solasodine, diosgenin and solanine interact with human erythrocytes and molecular models of their membranes as follows: a) X-ray diffraction studies showed that the compounds at low molar ratios (0.1-10.0mol%) induced increasing structural perturbation to dimyristoylphosphatidylcholine bilayers and to a considerable lower extent to those of dimyristoylphosphatidylethanolamine; b) differential scanning calorimetry data showed that the compounds were able to alter the cooperativity of dimyristoylphosphatidylcholine, dimyristoylphosphatidylethanolamine and dimyristoylphosphatidylserine phase transitions in a concentration-dependent manner; c) in the presence of steroids, the fluorescence of Merocyanine 540 incorporated to the membranes decreased suggesting a fluidization of the lipid system; d) scanning electron microscopy observations showed that all steroids altered the normal shape of human erythrocytes inducing mainly echinocytosis, characterized by the formation of blebs in their surfaces, an indication that their molecules are located into the outer monolayer of the erythrocyte membrane.

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In the present work we have analyzed the effect of StAsp-PSI (plant-specific insert of potato aspartic protease) on the structural and thermotropic properties of the major phospholipid types of bacterial and animal cells. Results obtained suggest that StAsp-PSI induces a destabilization of the membrane bilayers, depending on the time of interaction between the protein and the bilayers, rather than on its concentration. This temporal delay would be consistent with a lateral diffusion of StAsp-PSI monomers to assemble into aggregates to form pores. Like with the results previously reported for the StAsp-PSI circular dichroism, data obtained here from IR spectroscopy show that there are slight changes in the StAsp-PSI secondary structure in the presence of lipid membranes; suggesting that these changes could be related with the StAsp-PSI self-association. Results obtained from steady-state fluorescence anisotropy and differential scanning calorimetry assays suggest that StAsp-PSI interacts with both uncharged and negatively charged phospholipids, modulates the phase polymorphic behavior of model membranes and partitions and buries differentially in the membrane depending on the presence of negatively charged phospholipids.

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This work analyzes the surface properties of PE-containing membranes modified at the head group region by the addition of methyl and ethyl residues at or near the amine group. These residues alter the lipid-lipid and lipid-water interactions by changes in the hydrogen bonding capability and the charge density of the amine group thus affecting the electrostatic interaction. The results obtained by measuring the dipole potential, the zeta potential, the area per lipid and the compressibility properties allow to conclude that the H-bonding capability prevails in the lipid-lipid interaction. The non polar groups attached to the C2-carbon of the ethanolamine chain introduces a steric hindrance against compression and increases the dipole potential. The analysis of areas suggests that lipids with methylated head groups have a much larger compressibility at expense of the elimination of hydration water, which is congruent with the broader extent of the hysteresis loop.

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In this study, we report the effect of cholesterol content on the dynamic and structural properties of a dimyristoyl-phosphatidylcholine and distearoyl-phosphatidylcholine mixture in large unilamellar vesicles. The range of cholesterol concentrations studied varied around approximately 33.3mol%, where it has been postulated that an abrupt change in bilayer organization occurs. Steady-state fluorescence measurements demonstrated a typical behavior; at low temperatures in the main phase transition, the cholesterol concentration did not affect the gel phase, but at 37.5°C (phase coexistence) and in the liquid crystalline phase, the presence of cholesterol produced an increase in the fluorescence anisotropy of DPH and the generalized polarization of Laurdan. The greater effect was observed in the liquid crystalline phase, in which the bilayer became a mixture of fluid-like and liquid-ordered phases. The results obtained at approximately 33.3mol% of Cholesterol demonstrated that the Generalized Polarization of Laurdan, the DPH lifetime, the limiting anisotropy and the rotational correlation time, as well as the fluorescence quenching of DPH by TEMPO, are at maxima, while the fluorescence intensity of dehydroergosterol and the lipid solubility in TritonX-100 are at minima. These results correlate well with the hypothesis of domain segregation in the DMPC/DSPC/Cholesterol LUV system. In this context, we postulate that at 33.3mol% of Cho, the proportion of ordered domains reaches a maximum.

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Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.

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The quaternary structure of the homodimeric small multidrug resistance protein EmrE has been studied intensely over the past decade. Structural models derived from both two- and three-dimensional crystals show EmrE as an anti-parallel homodimer. However, the resolution of the structures is rather low and their relevance for the in vivo situation has been questioned. Here, we have challenged the available structural models by a comprehensive in vivo Trp scanning of all four transmembrane helices in EmrE. The results are in close agreement with the degree of lipid exposure of individual residues predicted from coarse-grained molecular dynamics simulations of the anti-parallel dimeric structure obtained by X-ray crystallography, strongly suggesting that the X-ray structure provides a good representation of the active in vivo form of EmrE.

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