Water as a Link between Membrane and Colloidal Theories for Cells.

This review is an attempt to incorporate water as a structural and thermodynamic component of biomembranes. With this purpose, the consideration of the membrane interphase as a bidimensional hydrated polar head group solution, coupled to the hydrocarbon region allows for the reconciliation of two theories on cells in dispute today: one considering the membrane as an essential part in terms of compartmentalization, and another in which lipid membranes are not necessary and cells can be treated as a colloidal system. The criterium followed is to describe the membrane state as an open, non-autonomous and responsive system using the approach of Thermodynamic of Irreversible Processes. The concept of an open/non-autonomous membrane system allows for the visualization of the interrelationship between metabolic events and membrane polymorphic changes. Therefore, the Association Induction Hypothesis (AIH) and lipid properties interplay should consider hydration in terms of free energy modulated by water activity and surface (lateral) pressure. Water in restricted regions at the lipid interphase has thermodynamic properties that explain the role of H-bonding networks in the propagation of events between membrane and cytoplasm that appears to be relevant in the context of crowded systems.

Effects of Phenylalanine on the Liquid-Expanded and Liquid-Condensed States of Phosphatidylcholine Monolayers.

BACKGROUND: Phenylalanine (Phe) is involved in physiological and pathological processes in cell membranes in which expanded and condensed states coexist. In this direction, it was reported that surface hydration is important for the binding affinity of the amino acid which significantly perturbs 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer structure and morphology. A deeper insight showed that Phe inserts in DPPC monolayer defects as a monomer at pH 5 and forms aggregates that adsorb to the membrane surface generating a reconfiguration of the lipid arrangement in areas of higher packing. This new arrangement in the monolayer causes the reorientation of dipoles of lipid and water molecules which is congruent with the dehydration and surface tension changes reported above. With this background, this article studies the affinity of Phe in liquid-expanded 1,2-dimyristoyl-sn-glycero-3 phosphocholine (LE DMPC) and liquid-condensed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (LC DPPC) monolayers and their effects on membrane properties. RESULTS: The adsorption of Phe can be described by a cooperative process in non-independent sites suggesting that Phe/lipid systems reorganize to form new structures at a high degree of coverage. Compressibility modulus and Brewster angle microscopy (BAM) images allow to propose that Phe causes a new phase in 1,2-dimyristoyl-sn-glycero-3 phosphocholine (DMPC) and DPPC. CONCLUSIONS: Phe imposes new arrangements in the lipid phase to form new structures with different compressibility behavior than lipid binary mixtures of DMPC and DPPC. Phe interaction with the LC and LE phases gives place to a process in which a synergistic effect between non-independent sites can be produced. These features of Phe/lipid interaction would be of great importance to understand the multiple effects of Phe on cell membranes.

Interaction of S-layer proteins of Lactobacillus kefir with model membranes and cells.

In previous works, it was shown that S-layer proteins from Lactobacillus kefir were able to recrystallize and stabilize liposomes, this feature reveling a great potential for developing liposomal-based carriers. Despite previous studies on this subject are important milestones, a number of questions remain unanswered. In this context, the feasibility of S-layer proteins as a biomaterial for drug delivery was evaluated in this work. First, S-layer proteins were fully characterized by electron microscopy, 2D-electrophoresis, and anionic exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Afterward, interactions of S-layer proteins with model lipid membranes were evaluated, showing that proteins adsorb to the lipid surface following a non-fickean or anomalous diffusion, when positively charged lipid were employed, suggesting that electrostatic interaction is a key factor in the recrystallization process on these proteins. Finally, the interaction of S-layer coated liposomes with Caco-2 cell line was assessed: First, cytotoxicity of formulations was tested showing no cytotoxic effects in S-layer coated vesicles. Second, by flow cytometry, it was observed an increased ability to transfer cargo molecules into Caco-2 cells from S-layer coated liposomes in comparison to control ones. All data put together, supports the idea that a combination of adhesive properties of S-layer proteins concomitant with higher stability of S-layer coated liposomes represents an exciting starting point in the development of new drug carriers.

Membrane Hydration: A Hint to a New Model for Biomembranes.

The classical view of a biological membrane is based on the Singer-Nicholson mosaic fluid model in which the lipid bilayer is the structural backbone. Under this paradigm, many studies of biological processes such as, permeability, active transport, enzyme activity and adhesion and fusion processes have been rationalized considering the lipid membrane as a low dielectric slab of hydrocarbon chains with polar head groups exposed to water at each side in which oil/water partition prevails. In spite of several analyses and evidence available in relation to membrane hydration, water is not taken into account as a functional component. For this purpose, new insights in the water organization in restricted environments and the thermodynamical and mechanical properties emerging from them are specifically analysed and correlated.This chapter summarizes the progress of the studies of water in membranes along the book in order to give a more realistic structural and dynamical picture accounting for the membrane functional properties.

Hydration in Lipid Monolayers: Correlation of Water Activity and Surface Pressure.

In order to give a physical meaning to each region of the membrane we define the interphase as the region in a lipid membrane corresponding to the polar head groups imbibed in water with net different properties than the hydrocarbon region and the water phase. The interphase region is analyzed under the scope of thermodynamics of surface and solutions based on the definition of Defay-Prigogine of an interphase and the derivation that it has in the understanding of membrane processeses in the context of biological response. In the view of this approach, the complete monolayer is considered as the lipid layer one molecule thick plus the bidimensional solution of the polar head groups inherent to it (the interphase region). Surface water activity appears as a common factor for the interaction of several aqueous soluble and surface active proteins with lipid membranes of different composition. Protein perturbation can be measured by changes in the surface pressure of lipid monolayers at different initial water surface activities. As predicted by solution chemistry, the increase of surface pressure is independent of the particle nature that dissolves. Therefore, membranes give a similar response in terms of the determined surface states given by water activity independent of the protein or peptide.

Molecular dynamics study of the interaction of arginine with phosphatidylcholine and phosphatidylethanolamine bilayers.

In this work, the differential interaction of zwitterionic arginines with fully hydrated dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers was analyzed by molecular dynamics simulations. In both systems, arginine binds to lipids with the carboxylate moiety oriented toward the aqueous phase, in agreement with previous experimental determinations of ζ potential of DMPC and DMPE liposomes. The guanidinium groups are found at different depths within the bilayers indicating that some arginines are buried, especially in DMPE. We observe, in the DMPE system, that the strongest interaction occurs between the guanidinium group and the carbonyl oxygen of the lipid. In the case of DMPC membranes, the strongest interaction is found between the guanidinium groups of the arginines and the phosphate groups of the lipids. Unexpectedly, arginine zwitterions are stabilized through the creation of hydrogen bonds (HB), either with water or with polar groups of the lipids. The mechanisms of interaction seem to be different in both membranes. In DMPE bilayers, arginines insert by breaking the inner HB network of the polar head groups, consequently increasing the occupied area per lipid molecule. In the DMPC bilayers the arginines insert by replacing the already present water molecules within the membrane, without significant effects on the area per lipid.

Coordination forces between lipid bilayers produced by ferricyanide and Ca2+.

Attractive forces usually invoked to take place in membrane-membrane contact in aggregation are hydrogen bonding cross-linkings and hydrophobic interactions between opposing surfaces. However, little is known in relation to the presence of coordination forces in the membrane-membrane interaction. These are understood as those that may be favoured by the formation or the participation of coordination complexes between surface specific groups. In this work, we have analyzed the formation of this type of aggregates between phosphatidylcholine vesicles mediated by a coadsorption of ferricyanide and Ca(2+) ions to the interface. The results obtained by surface potential measures, optical and electronic microscopy, FTIR and (1)H NMR spectroscopies indicate that ferricyanide [Fe(CN)(6)](3-) but not of ferrocyanide [Fe(CN)(6)](4-) can form the complex when Ca(2+) has been adsorbed previously to the membrane surface. In this condition, the anion is likely to act as a bridge between two opposing membranes causing a tight aggregation in which geometry and the polarizability of the ligands to Fe(3+) play a role.

Role of guanidinium group in the insertion of l-arginine in DMPE and DMPC lipid interphases.

l-Arginine (Arg) is a positively charged amino acid constituent of peptides and proteins, participating in diverse mechanisms of protein-membrane interaction. The effect of Arg on phosphatidylcholine (PC) membranes has been previously related to water structure changes and to the presence of water defects in the hydrocarbon region. However, no information is available with regard to phosphatidylethanolamine (PE), another important component of lipid membranes. For this reason, the aim of this study is to determine the effect of Arg on DMPE membranes and partially methylated PEs in comparison to DMPC. The adsorption of the amino acid onto the lipid membranes was followed by determining the changes in the surface potential as a function of the bulk amino acid concentrations. The effects of Arg on the surface properties were also measured by changes in the surface pressure and the dipole potential. The onset of the transition temperature was measured with a fluorophore anchored at the membrane interphase. The results provide a new insight on amino acid-PE interactions, which can be ascribed to specific perturbations in the head group region induced by the guanidinium residue.

Configuration of carbonyl groups at the lipid interphases of different topological arrangements of lipid dispersions.

The purpose of this work is to analyze the conformation of the carbonyl groups of acyl phospholipids at the hydrocarbon-water interphase in different topological ensembles and phase states, such as micelles and bilayers. The separation of the band components in lipids dispersed in D(2)O is compared with that of PCs in a low hydrated state. When hydrated, the differences in the frequencies of the band components corresponding to the carbonyl groups identified as low hydrated and hydrated populations increase when dimyristoylphosphatidylcholine (DMPC) bilayers go from the lamellar gel to the ripple corrugated phase at the pretransition temperature. Below the pretransition, at which the membrane in the gel state is planar, the two components overlap making the deconvolution unreliable. A further analysis shows that the frequency of the highly hydrated population increases more noticeable than that corresponding to the low hydrated one following the sequence: micelles, fluid phase, ripple gel phase, and lamellar gel phase. This is confirmed by the increase in the separation of the band components when the liposomes are subjected to an osmotic dehydration suggesting that the hydrated population loses water and the dehydrated one partially hydrates. It is concluded that this behavior is a feature conferred by hydration of the different topological arrangements. The relevance of these results on the interphase properties of lipid membranes is discussed.

Critical water activity for the preservation of Lactobacillus bulgaricus by vacuum drying.

Lactobacillus delbrueckii subsp. bulgaricus was dried under vacuum at different temperatures and its preservation evaluated analyzing the evolution of three parameters throughout the process: lag time, percentage of membrane damage and zeta potential. Microorganisms were dehydrated at 30, 45 and 70 degrees C in a vacuum centrifuge for different times. The aw achieved for each time of drying was correlated with the cell recovery at all the temperatures assayed. The recovery of microorganisms was evaluated by means of: a) kinetics of growth in milk after drying, as a measure of the global damage; b) quantification of the membrane damage using the fluorescent dyes SYTO 9 and PI; c) determination of changes in the superficial charges (zeta potential) as measured of the increase in the hydrophobic residues exposed in the bacterial surface after dehydration. These changes correlate well with the bacterial damage occurred during the dehydration process. The Page's equation allowed fitting of aw and time of drying, thus making possible the determination of the appropriate dehydration conditions (time-temperature ratios) for which no cell damage occurs. The evaluation of three parameters (lag time, percentage of membrane damage and zeta potential) allowed us to conclude that at the lowest temperature of dehydration, the first target of damage is the cell membrane. However, this damage is not decisive for the bacterial recovery after rehydration, as are the increase in the lag time and the changes in the zeta potential, as was observed for L. bulgaricus dehydrated at 45 and 70 degrees C for larger times.

Effect of trehalose on the contributions to the dipole potential of lipid monolayers.

The dipole potential and the area changes induced by trehalose on dimyristoyl phosphatidylcholine (DMPC), 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (dietherPC), dimyristoyl phosphatidylethanolamine (DMPE), 1,2-di-O-tetradecyl-sn-glycero-3-phosphoethanolamine (dietherPE) monolayers have been studied at different temperatures. The insertion of trehalose into DMPC monolayers in the fluid and gel states requires of the presence of carbonyl groups. The area increase observed at 0.15M trehalose is congruent with the decrease in the dipole potential. However, in dietherPC, in which trehalose does not affect the area, a decrease in the dipole potential is also observed. This is interpreted as a result of the displacement of water from the phosphate groups exposed to the aqueous phase. In DMPE, trehalose also decreases the dipole potential without affecting the area of saturated monolayers and in dietherPE no effect on dipole potential and area was observed. It is concluded that the spacer effect of trehalose depends on the specific interaction with CO, which is modulated by the strength of the interaction of the PO groups with lateral NH groups. However, it is not the only contribution to the dipole potential decrease.

Effect of arbutin on the dipole potential and area per lipid of ester and ether phosphatidylcholine and phosphatidyl ethanolamine monolayers.

The present results report for the first time a systematic study of the effect of arbutin on the dipole potential of lipid membranes. The dipole potential and the area per lipid were measured in monolayers of dimyristoylphosphatidylcholine (DMPC), 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (dietherPC), dimyristoylphosphatidylethanolamine (DMPE) and 1,2-di-O-tetradecyl-sn-glycero-3-phosphoethanolamine (dietherPE), spread on aqueous solutions of different concentrations of arbutin. The decrease of the dipole potential of DMPC, both in condensed and expanded monolayers, is parallel to an increase in the area per lipid. In contrast, for dietherPC, the area per lipid is not affected, in spite of the fact that arbutin is also able to decrease the dipole potential in a less drastic extent. In the case of DMPE, the response is similar to that observed with dietherPC: the dipole potential decreases, while the area per lipid remains unchanged. However, when the carbonyl groups are absent in phosphatidylethanolamine derivatives such as the dietherPE, the dipole potential is not affected by arbutin, with a small decrease in the area. The effect of arbutin on the dipole potential differs from that of sucrose, trehalose and phloretin and is congruent with previous results obtained by FTIR on its interaction with the CO groups. Arbutin binding is interpreted in terms of the exposure to water of the phosphate and carbonyl groups at the membrane interface of the different monolayers.

Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus.

Changes in water permeability and membrane packing were measured in cells of Lactobacillus bulgaricus and in vesicles prepared with lipids extracted from them. The osmotic response of whole cells and vesicles is compared with the one of bacteria grown in a high osmolal medium. Both bacteria and vesicles, behave as osmometers. This means that the volume decrease is promoted by the outflow of water, driven by the NaCl concentration difference, arguing that neither Na+ nor Cl- permeates the cell or the lipid membrane in these conditions. Therefore, the volume changes can be correlated with the rate of water permeation across the cell or the vesicle membranes. The permeation of water was analyzed as a function of the lipid species by measuring the volume changes and the saturation ratio of the lipids. To put into relevance the membrane processes, the permeation properties of lipid vesicles prepared with lipids extracted from bacteria grown in normal and high osmolality conditions were also analyzed. The permeation response was correlated with the physical properties of the membrane of whole cells and vesicles, by means of fluorescence anisotropy of diphenyl hexatriene (DPH). The modifications in membrane properties are related with the changes in the membrane composition triggered by the growth in a high osmolal medium. The changes appear related to an increase in the sugar content of the whole pool of lipids and in the saturated fatty acid residues.

Effect of phloretin on the dipole potential of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol monolayers.

The effect of phloretin on the potential of phosphatidylcholine (PC), phosphatidylethanolamine (PE,) and phosphatidylglycerol (PG) monolayers below and above the phase transition in mixtures of different PC/PE ratios with and without cholesterol of ester and ether phospholipids have been determined. The effectiveness of phloretin to decrease the dipole potential of monolayers in the fluid state is lessened by the moieties esterified to the phosphate group in the sequence choline > ethanolamine > glycerol. These effects on the dipole potential of monolayers are independent of the presence of carbonyls. In addition, in the gel state phloretin does not affect the dipole potential on dimyristoylphosphatidylethanolamine, although it is very pronounced in dimyristoylphosphatidylcholine. The changes of the dipole potential induced by phloretin were correlated with the packing of the lipids and with the formation of intermolecular hydrogen bonds between adjacent phospholipid molecules. These results may be indicative of the different distribution of polarized water around the phosphate groups imposed by the surrounding environment.

Molecular dynamics simulation study of the interaction of trehalose with lipid membranes.

The interactions of the cryoprotective agent trehalose with a lipid membrane made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine at 323 K were studied by means of molecular dynamics simulations. It was observed that trehalose binds to the phospholipid headgroups with its main axis parallel to the membrane normal. Trehalose establishes hydrogen bonds with the carbonyl and phosphate groups and replaces water molecules from the lipid headgroup. Notably, the number of hydrogen bonds (HBs) that the membrane made with its environment was conserved after trehalose binding. The HBs between lipid and trehalose have a longer lifetime than those established between lipid and water. The binding of the sugar does not produce changes either in the lipid area or in the lipid order parameter. The effect of trehalose on the dipole potential is in agreement with experimental results. The contribution of the different components to the membrane dipole potential was analyzed. It was observed that the binding of trehalose produces changes in the different components and the sugar itself contributes to the surface potential due to the polarization of its hydroxyl in the interface.