Direct submolecular scale imaging of mesoscale molecular order in supported dipalmitoylphosphatidylcholine bilayers

Supported dipalmitoylphosphatidylcholine (DPPC) bilayers are widely used membrane systems in biophysical and biochemical studies. Previously, short-range positional and orientational order of lipid headgroups of supported DPPC bilayers was observed at room temperature using low deflection noise frequency modulation atomic force microscopy (FM-AFM). While this ordering was supported by X-ray diffraction studies, it conflicted with diffusion coefficient measurements of gel-phase bilayers determined from fluorescence photobleaching experiments. In this work, we have directly imaged mica-supported DPPC bilayers with submolecular resolution over scan ranges up to 146 nm using low deflection noise FM-AFM. Both orientational and positional molecular ordering were observed in the mesoscale, indicative of crystalline order. We discuss these results in relation to previous biophysical studies and propose that the mica support induces mesoscopic crystalline order of the DPPC bilayer at room temperature. This study also demonstrates the recent advance in the scan range of submolecular scale AFM imaging.     

NSAIDs interactions with membranes: a biophysical approach

This work focuses on the interaction of four representative NSAIDs (nimesulide, indomethacin, meloxicam, and piroxicam) with different membrane models (liposomes, monolayers, and supported lipid bilayers), at different pH values, that mimic the pH conditions of normal (pH 7.4) and inflamed cells (pH 5.0). All models are composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) which is a representative phospholipid of most cellular membranes. Several biophysical techniques were employed: Fluorescence steady-state anisotropy to study the effects of NSAIDs in membrane microviscosity and thus to assess the main phase transition of DPPC, surface pressure-area isotherms to evaluate the adsorption and penetration of NSAIDs into the membrane, IRRAS to acquire structural information of DPPC monolayers upon interaction with the drugs, and AFM to study the changes in surface topography of the lipid bilayers caused by the interaction with NSAIDs. The NSAIDs show pronounced interactions with the lipid membranes at both physiological and inflammatory conditions. Liposomes, monolayers, and supported lipid bilayers experiments allow the conclusion that the pH of the medium is an essential parameter when evaluating drug-membrane interactions, because it conditions the structure of the membrane and the ionization state of NSAIDs, thereby influencing the interactions between these drugs and the lipid membranes. The applied models and techniques provided detailed information about different aspects of the drug-membrane interaction offering valuable information to understand the effect of these drugs on their target membrane-associated enzymes and their side effects at the gastrointestinal level.     

Freeze-fracture electron microscopic and calorimetric studies on microscopic states of surface-modified liposomes with poly(ethylene glycol) chains

The differential scanning calorimetry (DSC) and the freeze-fracture electron microscopy of dipalmitoyl phosphatidylcholine (DPPC) liposomes containing distearoyl-N-monomethoxy poly(ethylene glycol)-succinyl-phosphatidylethanolamines (PEG-DSPE) were carried out. The DSC peak of DPPC liposomes containing PEG-DSPE had a shoulder. The main phase transition temperature of DPPC bilayer membranes containing PEG-DSPE whose molecular weight of PEG is less than 3000 was slightly shifted to a higher temperature, while that containing PEG-DSPE whose molecular weight of PEG is more than 5000 was slightly shifted to a lower temperature. The electron micrographs of freeze-fracture replicas of DPPC liposomes containing PEG-DSPE quenched from 37±2°C exhibited banded and planar textures, suggesting the lateral phase separation in the bilayer membranes.     

Collective and single-molecule interactions of alpha5beta1 integrins

A novel biomimetic system was used to study collective and single-molecule interactions of the alpha5beta1 receptor-GRGDSP ligand system with an atomic force microscope (AFM). Bioartificial membranes, which display peptides that mimic the cell adhesion domain of the extracellular matrix protein fibronectin, are constructed from peptide-amphiphiles. The interaction measured with the immobilized alpha5beta1 integrins and GRGDSP peptide-amphiphiles is specifically related to the integrin-peptide binding. It is affected by divalent cations in a way that accurately mimics the adhesion function of the alpha5beta1 receptor. The recognition of the immobilized receptor was significantly increased for a surface that presented both the primary recognition site (GRGDSP) and the synergy site (PHSRN) compared to the adhesion measured with surfaces that displayed only the GRGDSP peptide. At the collective level, the separation process of the receptor-ligand pairs is a combination of multiple unbinding and stretching events that can accurately be described by the wormlike chain (WLC) model of polymer elasticity. In contrast, stretching was not observed at the single-molecule level. The dissociation of single alpha5beta1-GRGDSP pairs under loading rates of 1-305 nN/s revealed the presence of two activation energy barriers in the unbinding process. The high-strength regime above 59 nN/s maps the inner barrier at a distance of 0.09 nm along the direction of the force. Below 59 nN/s a low-strength regime appears with an outer barrier at 2.77 nm and a much slower transition rate that defines the dissociation rate (off-rate) in the absence of force (k(off) degrees = 0.015 s(-1)).     

Ethanol-induced reorganization of the liquid-ordered phase: enhancement of cholesterol-phospholipid association

This paper records what is believed to be the first evidence for the reorganization of the liquid-ordered phase by ethanol. Specifically, ethanol has been found to significantly enhance sterol-phospholipid association in liquid-ordered bilayers derived from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) plus cholesterol and also 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) plus cholesterol. The evidence for such reorganization comes from a series of nearest-neighbor recognition (NNR) experiments that have been carried out, where low concentrations of equilibrating lipid dimers (i.e., "reporter molecules") have been used to detect changes in the phase composition of host membranes made from varying mixtures of DPPC/cholesterol, and also DSPC/cholesterol, in the presence and in the absence of ethanol. These findings have important biological implications, which are briefly discussed.     

The biologically important surfactin lipopeptide induces nanoripples in supported lipid bilayers

Under specific conditions, lipid membranes form ripple phases with intriguing nanoscale undulations. Here, we show using in situ atomic force microscopy (AFM) that the biologically important surfactin lipopeptide induces nanoripples of 30 nm periodicity in dipalmitoyl phosphatidylcholine (DPPC) bilayers at 25 degrees (i.e. well below the pretransition temperature of DPPC). Whereas most undulations formed the classical straight orientation with characteristic angle changes of 120 degrees , some of them also displayed unusual circular orientations. Strikingly, ripple structures were formed at 15% surfactin but were rarely or never observed at 5 and 30% surfactin, emphasizing the important role played by the surfactin concentration. Theoretical simulations corroborated the AFM data by revealing the formation of stable surfactin/lipid assemblies with positive curvature.     

Nanoscale patterning in mixed fluorocarbon-hydrocarbon phospholipid bilayers

A growing body of literature suggests that fluorocarbons can direct self-assembly within hydrocarbon environments. We report here the fabrication and characterization of supported lipid bilayers (SLBs) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a synthetic, fluorocarbon-functionalized analogue, 1. AFM investigation of these model membranes reveals an intricate, composition-dependent domain structure consisting of approximately 50 nm stripes interspersed between approximately 1 microm sized domains. Although DSC of 1 showed a phase transition near room temperature, DSC of DPPC:1 mixtures exhibited complex phase behavior suggesting domain segregation. Finally, temperature-dependent AFM of DPPC:1 bilayers shows that, while the stripe structures can be melted above the Tm of 1, the stripes and domains result from immiscibility of the hydrocarbon and fluorocarbon lipid gel phases. Fluorination appears to be a promising strategy for chemical self-assembly in two dimensions. In particular, because no modification is made to the lipid headgroups, it may be useful for nanopatterning biologically relevant ligands on bilayers in vitro or in living cells.     

Effect of adsorption of bovine serum albumin on liposomal membrane characteristics

The effect of adsorption of bovine serum albumin (BSA) on the membrane characteristics of liposomes at pH 7.4 was examined in terms of zeta potential, micropolarity, microfluidity and permeability of liposomal bilayer membranes, where negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG)/L-alpha-dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP)/DPPC and positively charged stearylamine (SA)/DPPC mixed liposomes were used. BSA with negative charges adsorbed on negatively charged DPPG/DPPC mixed liposomes but did not adsorb on negatively charged DCP/DPPC and positively charged SA/DPPC mixed liposomes. Furthermore, the adsorption amount of BSA on the mixed DPPG/DPPC liposomes increased with increasing the mole fraction of DPPG in spite of a possible electrostatic repulsion between BSA and DPPG. Thus, the adsorption of BSA on liposomes was likely to be related to the hydrophobic interaction between BSA and liposomes. The microfluidity of liposomal bilayer membranes near the bilayer center decreased by the adsorption of BSA, while the permeability of liposomal bilayer membranes increased by the adsorption of BSA on liposomes. These results are considered to be due to that the adsorption of BSA brings about a phase separation in liposomes and that a temporary gap is consequently formed in the liposomal bilayer membranes, thereby the permeability of liposomal bilayer membranes increases by the adsorption of BSA.     

Chemical details on nucleolipid supramolecular architecture: molecular modeling and physicochemical studies

Nucleolipids are currently under investigation as vectors for oligonucleotides (ON) delivery thanks to their supramolecular organization properties and their ability to develop specific interactions (i.e., stacking and potential Watson and Crick hydrogen bonds) for lipoplexes formation. To investigate the factors that govern the interaction events at a molecular level and optimize nucleolipid chemical structures, physicochemical experiments (tensiometry, AFM, BAM, and ellipsometry) combined with molecular dynamics simulation were performed on a series of zwitterionic nucleolipids (PUPC, DPUPC, PAPC) featuring a phosphocholine chain (PC). After construction and initial equilibration, simulations of pure nucleolipid bilayers were run for 100 ns at constant temperature and pressure, and their properties were compared to experimental data and to natural dipalmitoylphosphatidylcholine (DPPC) bilayers. Nucleolipid-based membranes are significantly more ordered and compact than DPPC bilayers mainly due to the presence of many intermolecular interactions between nucleoside polar heads. The hydrophilic phosphocholine moieties connected to the 5' hydroxyls are located above the bilayers, penalizing nucleic bases accessibility for further interactions with ON. Hence, a neutral nucleolipid (PUOH) without hydrophilic phosphocholine was inserted in the membranes. Simulations and experimental analysis of nucleolipid membranes in interaction with a single strand RNA structure indicate that PUOH interacts with ON in the subphase. This study demonstrates that molecular modeling can be used to determine the interactions between oligonucleotide and nucleolipids.     

Vesicle—vesicle interaction and forces between bilayers in phospholipid systems incorporating phosphatidylinositol

Sonicated vesicles have been prepared from mixtures of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylinositol (PI) covering a range of composition. The effect of temperature on the rates of aggregation of the vesicles on addition of calcium and magnesium ions has been investigated. Apart from pure PI vesicles the rates of aggregation decrease dramatically as the temperature approaches the gel-to-liquid crystalline phase transition temperature of DPPC. Low angle X-ray analysis of lamellar phases of DPPC-PI (75:25 wt%) in the presence of Ca²⁺ ions shows that between 35 and 45°C the repeat distance goes from 136 to 68 Å. It is suggested that below the chain-melting temperature of DPPC Ca²⁺ ions induce lateral phase separation of PI giving a lamellar repeat distance corresponding to the thickness of two bilayers.The net repulsive pressure between DPPC-PI bilayers has been measured by a vapour pressure technique as a function of temperature. At close apposition (<15 Å) the pressure is characteristic of hydration repulsion and increases with temperature. The repulsive force between the bilayers, lateral pressure and compressibility of the bilayers have also been determined. On progressively removing water from between the bilayers 5–14% of the work done goes into bilayer deformation, the remaining 86–95% being required to bring the bilayers together.     

Spontaneous formation of asymmetric lipid bilayers by adsorption of vesicles

We have investigated the adsorption of phospholipid mixtures using neutron reflection. Small sonicated unilamellar vesicles (SUV) composed of DOPC and d(62)-DPPC were incubated at 50 degrees C in contact with a silica surface using a method commonly employed to form supported model membranes. The composition of the mixed supported bilayer was found to be substantially different from that of the bulk vesicles in a direction indicating a higher affinity of DPPC for the silica surface. Formation of an asymmetric bilayer arrangement was also discovered in all the cases studied. DPPC tended to dominate the composition of the leaflet next to silica, while the outer leaflet was generally closer to the bulk composition. The supported bilayers also exhibited increasing interfacial roughness in the outer membrane leaflet in the region of the DOPC-DPPC gel-liquid immiscibility region. To our knowledge, this is the first time that both the structure and the absolute composition of a mixed-lipid supported bilayer have been resolved, and the results raise a number of questions regarding the adsorption of vesicles and the properties of supported bilayers, which are discussed in terms of the bulk phase diagram of DOPC and DPPC.     

Remodeling of ordered membrane domains by GPI-anchored intestinal alkaline phosphatase

Glycosylphosphatidyl-inositol (GPI)-anchored proteins preferentially localize in the most ordered regions of the cell plasma membrane. Acyl and alkyl chain composition of GPI anchors influence the association with the ordered domains. This suggests that, conversely, changes in the fluid and in the ordered domains lipid composition affect the interaction of GPI-anchored proteins with membrane microdomains. Validity of this hypothesis was examined by investigating the spontaneous insertion of the GPI-anchored intestinal alkaline phophatase (BIAP) into the solid (gel) phase domains of preformed supported membranes made of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC), DOPC/sphingomyelin (DOPC/SM), and palmitoyloleoylphosphatidylcholine/SM (POPC/SM). Atomic force microscopy (AFM) showed that BIAP inserted in the gel phases of the three mixtures. However, changes in the lipid composition of membranes had a marked effect on the protein containing bilayer topography. Moreover, BIAP insertion was associated with a net transfer of phospholipids from the fluid to the gel (DOPC/DPPC) or from the gel to the fluid (POPC/SM) phases. For DOPC/SM bilayers, transfer of lipids was dependent on the homogeneity of the gel SM phase. The data strongly suggest that BIAP interacts with the most ordered lipid species present in the gel phases of phase-separated membranes. They also suggest that GPI-anchored proteins might contribute to the selection of their own microdomain environment.     

Dissipation-enhanced quartz crystal microbalance studies on the experimental parameters controlling the formation of supported lipid bilayers

We report on the investigations of the transformation of spherically closed lipid bilayers to supported lipid bilayers in aqueous media in contact with SiO(2) surfaces. The adsorption kinetics of small unilamellar vesicles composed of dimyristoyl- (DMPC) and dipalmitoylphosphatidylcholine (DPPC) mixtures on SiO(2) surfaces were investigated using a dissipation-enhanced quartz crystal microbalance (QCM-D) as a function of buffer (composition and pH), lipid concentration (0.01-1.0 mg/mL), temperature (15-37 degrees C), and lipid composition (DMPC and DMPC/DPPC mixtures). The lipid mixtures used here possess a phase transition temperature (T(m)) of 24-33 degrees C, which is close to the ambient temperature or above and thus considerably higher than most other systems studied by QCM-D. With HEPES or Tris.HCl containing sodium chloride (150 mM) and/or calcium chloride (2 mM), intact vesicles adsorb on the surface until a critical density ((c)) is reached. At close vesicle contact the transformation from vesicles to supported phospholipid bilayers (SPBs) occurs. In absence of CaCl(2), the kinetics of the SPB formation process are slowed, but the passage through (c) is still observed. The latter disappears when buffers with low ionic strength were used. SPB formation was studied in a pH range of 3-10, yet the passage through (c) is obtained only for pH values above to the physiological pH (7.4-10). With an increasing vesicle concentration, (c) is reached after shorter exposure times. At a vesicle concentration of 0.01-1 mg/mL, vesicle fusion on SiO(2) proceeds with the same pathway and accelerates roughly proportionally. In contrast, the pathway of vesicle fusion is strongly influenced by the temperature in the vicinity of T(m). Above and around the T(m), transformation of vesicles to SPB proceeds smoothly, while below, a large number of nonruptured vesicles coexist with SPB. As expected, the physical state of the membrane controls the interaction with both surface and neighboring vesicles.     

Effects of albumin and erythrocyte membranes on spread monolayers of lung surfactant lipids

Dipalmitoyl phosphatidylcholine (DPPC), one of the main constituents of lung surfactant is mainly responsible for reduction of surface tension to near 0 mN/m during expiration, resisting alveolar collapse. Other unsaturated phospholipids like palmitoyloleoyl phosphatidylglycerol (PG), palmitoyloleoyl phosphatidylcholine (POPC) and neutral lipids help in adsorption of lung surfactant to the air-aqueous interface. Lung surfactant lipids may interact with plasma proteins and hematological agents flooding the alveoli in diseased states. In this study, we evaluated the effects of albumin and erythrocyte membranes on spread films of DPPC alone and mixtures of DPPC with each of PG, POPC, palmitoyloleoyl phosphatidylethanolamine (PE), cholesterol (CHOL) and palmitic acid (PA) in 9:1 molar ratios. Surface tension-area isotherms were recorded using a Langmuir-Blodgett (LB) trough at 37 degrees C with 0.9% saline as the sub-phase. In the presence of erythrocyte membranes, DPPC and DPPC+PA monolayers reached minimum surface tensions of 7.3+/-0.9 and 9.6+/-1.4 mN/m, respectively. Other lipid combinations reached significantly higher minimum surface tensions >18 mN/m in presence of membranes (Newman Keul's test, p<0.05). The relative susceptibility to membrane inhibition was [(DPPC+PG, 7:3)=(DPPC+PG, 9:1)=(DPPC+POPC)=(DPPC+PE)=(DPPC+CHOL)]>[(DPPC+PA)=(DPPC)]. The differential response was more pronounced in case of albumin with DPPC and DPPC+PA monolayers reaching minimum surface tensions less than 2.4 mN/m in presence of albumin, whereas DPPC+PG and DPPC+POPC reached minimum surface tensions of around 20 mN/m in presence of albumin. Descending order of susceptibility of the spread monolayers of lipid mixtures to albumin destabilization was as follows: [(DPPC+PG, 7:3)=(DPPC+PG, 9:1)=(DPPC+POPC)]>[(DPPC+PE)=(DPPC+CHOL)]>[(DPPC+PA)=(DPPC)] The increase in minimum surface tension in presence of albumin and erythrocyte membranes was accompanied by sudden increases in compressibility at surface tensions of 15-30 mN/m. This suggests a monolayer destabilization and could be indicative of phase transitions in the mixed lipid films due to the presence of the hydrophobic constituents of erythrocyte membranes.     

Chain-melting phase transition and short-range molecular interactions in phospholipid foam bilayers

Occurrence of two-dimensional chain melting phase transition in foam bilayers was established for the first time. Microscopic horizontal foam bilayers [Newton black films (NBF)] were investigated by the microinterferometric method of Scheludko-Exerowa. The foam bilayers were formed from water-ethanol solutions of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine (Egg PC) and samples of amniotic fluid (AF) at different temperatures. The influence of temperature on the foam bilayer thickness h(w) and on the critical concentration Cc for formation of foam bilayer was studied. It was shown that in the range of the main phase transition the temperature dependence of h(w) and C(c) changed specifically in the case of DMPC and DPPC foam bilayers. The thickness of the foam bilayers increased with decreasing temperature in the range of the main phase transition due to the melting of hydrocarbon tails of phospholipid molecules. These changes took place at the temperatures of the bulk chain-melting phase transitions, as determined by differential scanning calorimetry (DSC) for both aqueous, and water/ethanol DMPC, DPPC, and DPPC dispersions. An effect of the 'disperse medium' on h(w) was found for foam bilayers from DPPC. The results that foam bilayers could have different thickness at different temperatures disproved the current concept that NBF acquired constant thickness at concentrations higher than C(el,cr). The data for Cc were analysed on the basis of the hole-nucleation theory of bilayer stability of Kashchiev and Exerowa. This theory considered the amphiphile bilayer as a two-dimensional ordered system with short-range molecular interactions between the first neighbour molecules (as in a crystal). The short-range molecular interactions were presented by the parameter binding energy Q of an amphiphile molecule in the bilayer. The binding energy Q of two neighbouring phospholipids was calculated for the gel (30-60 kT) and liquid crystalline state (16-18 kT) of the bilayers from DMPC, DPPC, Egg PC, AF. Concentration/temperature phase diagram of DPPC foam bilayers that defined regions of gaseous (ruptured), gel and liquid crystalline foam bilayers were drawn. The values of Q obtained for various samples were very close and vary from 5.3 x 10(-20) to 9.4 x 10(-20) (approx. 13-22 kT) which indicated that in all cases the foam bilayers were in liquid-crystalline state. This is an important result since the parameter studied-threshold concentration (threshold dilution) is crucial for a very successful assessment of the risk for respiratory distress syndrome (RDS) in newborns and could be employed in medicine for assessment of other respiratory disturbances. It is to be expected that foam bilayers from phospholipids could be used as a model for investigation of short-range forces in biological structures, of interaction between membranes, etc.     

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Tilted peptides are known to insert in lipid bilayers with an oblique orientation, thereby destabilizing membranes and facilitating membrane fusion processes. Here, we report the first direct visualization of the interaction of tilted peptides with lipid membranes using in situ atomic force microscopy (AFM) imaging. Phase-separated supported dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers were prepared by fusion of small unilamellar vesicles and imaged in buffer solution, in the absence and in the presence of the simian immunodeficiency virus (SIV) peptide. The SIV peptide was shown to induce the rapid appearance of nanometer scale bilayer holes within the DPPC gel domains, while keeping the domain shape unaltered. We attribute this behavior to a local weakening and destabilization of the DPPC domains due to the oblique insertion of the peptide molecules. These results were directly correlated with the fusogenic activity of the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By contrast, the nontilted ApoE peptide did not promote liposome fusion and did not induce bilayer holes but caused slight erosion of the DPPC domains. In conclusion, this work provides the first direct evidence for the production of stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a behavior that we have shown to be specifically related to the tilted character of the peptide. A molecular mechanism underlying spontaneous insertion of the SIV peptide within lipid bilayers and the subsequent removal of bilayer patches is proposed, and its relevance to membrane fusion processes is discussed.     

Membrane interactions of ternary phospholipid/cholesterol bilayers and encapsulation efficiencies of a RIP II protein

Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P(min), the bilayer lysis tensions gamma(l) were determined. The increase in P(min) and gamma(l) with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron microscopy (Cryo-TEM) images show that beside spherical, single-walled liposomes, there is a considerable fraction of discoidally deformed vesicles. Based on our results and those found in the literature, we speculate that the flattening of the vesicles is a consequence of lipid phase separation and the formation of condensed complexes and areas of different bending elasticities. This phenomenon eventually leads to agglomeration of deformed liposomal structures, becoming more pronounced with the increase in the relative amount of saturated fatty acids, presumably caused by hydrophobic interaction. For the same lipid mixture aggregation correlated linearly with the ML content. Finally, tested liposomal samples were kept at 4 degrees C to examine their stability. Only slight fluctuations in diameter and the increase in polydispersity after 3 weeks of storage occurred, with no statistically significant evidence of drug leakage during a time period of 12 days, illustrating physical stability of liposomes.     

Specific effect of polyunsaturated fatty acids on the cholesterol-poor membrane domain in a model membrane

To understand more fully the effect of polyunsaturated fatty acids (PUFAs) on lipid bilayers, we investigated the effects of treatment with fatty acids on the properties of a model membrane. Three kinds of liposomes comprising dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC), and cholesterol (Ch) were used as the model membrane, and the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and detergent insolubility were determined. Characterization of the liposomes clarified that DPPC, DPPC/Ch, and DPPC/DOPC/Ch existed as solid-ordered phase (L beta), liquid-ordered phase (l o), and a mixture of l o and liquid-disordered phase (L alpha) membranes at room temperature. Treatment with unsaturated fatty acids such as oleic acid (OA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) markedly decreased the fluorescence anisotropy value and detergent insolubility. PUFAs and OA had different effects on the model membranes. In DPPC liposomes, the most prominent change was induced by PUFAs, whereas, in DPPC/Ch and DPPC/DOPC/Ch liposomes, OA had a stronger effect than PUFAs. The effect of PUFAs was strongly affected by the amount of Ch in the membrane, which confirmed a specific effect of PUFAs on the Ch-poor membrane domain. We further explored the effect of fatty acids dispersed in a water-in-oil-in-water multiple emulsion and found that unsaturated fatty acids acted on the membranes even when incorporated in emulsion form. These findings suggest that treatment with PUFAs increases the segregation of ordered and disordered phase domains in membranes.     

Influence of perfluorinated compounds on the properties of model lipid membranes

The influence of selected perfluorinated compounds (PFCs), perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid (PFOS), on the structure and organization of lipid membranes was investigated using model membranes-lipid monolayers and bilayers. The simplest model--a lipid monolayer--was studied at the air-water interface using the Langmuir-Blodgett technique with surface pressure and surface potential measurements. Lipid bilayers were characterized by NMR techniques and molecular dynamics simulations. Two phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), characterized by different surface properties have been chosen as components of the model membranes. For a DPPC monolayer, a phase transition from the liquid-expanded state to the liquid-condensed state can be observed upon compression at room temperature, while a DMPC monolayer under the same conditions remains in the liquid-expanded state. For each of the two lipids, the presence of both PFOA and PFOS leads to the formation of a more fluidic layer at the air-water interface. Pulsed field gradient NMR measurements of the lateral diffusion coefficient (DL) of DMPC and PFOA in oriented bilayers reveal that, upon addition of PFOA to DMPC bilayers, DL of DMPC decreases for small amounts of PFOA, while larger additions produce an increased DL. The DL values of PFOA were found to be slightly larger than those for DMPC, probably as a consequence of the water solubility of PFOA. Furthermore, 31P and 2H NMR showed that the gel-liquid crystalline phase transition temperature decreased by the addition of PFOA for concentrations of 5 mol % and above, indicating a destabilizing effect of PFOA on the membranes. Deuterium order parameters of deuterated DMPC were found to increase slightly upon increasing the PFOA concentration. The monolayer experiments reveal that PFOS also penetrates slowly into already preformed lipid layers, leading to a change of their properties with time. These experimental observations are in qualitative agreement with the computational results obtained from the molecular dynamics simulations showing a slow migration of PFCs from the surrounding water phase into DPPC and DMPC bilayers.     

Layer-by-layer assembled membranes of protonated 18-azacrown-6 and polyvinylsulfate and their application for highly efficient anion separation

Ultrathin separation membranes were prepared upon alternating electrostatic adsorption of 1,4,7,10,13,16-hexaazacyclooctadecane (18-azacrown-6, hexacyclen, aza6) and polyvinylsulfate on porous polyacrylonitrile/polyethylene terephthalate (PAN/PET) substrates. The resulting composite membranes were highly permeable for electrolyte solutions, but the selectivity in ion transport was poor (alpha(Cl-/SO42-) = 2). However, after a treatment of the membrane with 0.1 M aqueous copper(II) acetate solution, the rejection of divalent anions was strongly enhanced. Separation factors alpha(Cl-/SO42-) = 110 and alpha(Cl-/SO32-) = 1420 were found. Spectroscopic studies indicate a highly specific complex formation of aza6 and copper acetate in the membrane. Treatment of the membranes with cobalt(II) and nickel(II) acetate also led to an enhancement of the separation factors, but the effect was much smaller. Detailed studies of the influence of the pH of the dipping solutions and number of deposited bilayers are also reported. A model is presented describing the origin of the high transport selectivity.