Influence of the lipid composition of biomimetic monolayers on the structure and orientation of the gp41 tryptophan-rich peptide from HIV-1

The tryptophan-rich peptide of gp41 (so-called gp41W), one of the two envelope glycoproteins of HIV-1, is known to play a crucial role in the fusion between this virus and the host cell membranes. The influence of lipids on this role was investigated using different lipid monolayers at the air-water interface. Gp41W affinity for the lipid monolayer was measured by following the peptide-induced variation in the lateral surface pressure and we demonstrated that gp41W binds to monolayers containing the saturated zwitterionic dipalmitoylphosphatidylcholine (DPPC) as well as to the anionic dipalmitoylphosphatidylglycerol (DPPG) and to mixed monolayers containing DPPC and cholesterol (Chol). The secondary structure of gp41W in the presence of these lipid monolayers was determined by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The data showed that gp41W was an oriented α-helix in the presence of DPPG. However this spectroscopic method was unable to detect the gp41W structure in the presence of DPPC and DPPC/Chol monolayer. The peptide-induced modifications of the DPPC/Chol, DPPC and DPPG monolayer morphology were analyzed by Brewster angle microscopy (BAM). The peptide-induced changes in the DPPG monolayer morphology suggest that gp41W disturbed the lipid intermolecular interactions. Furthermore the peptide delayed the condensed state of DPPC and DPPC/Chol, indicating that, although gp41W was not detected by PM-IRRAS, it was present in these lipid monolayers.     

Fluorescently labeled pulmonary surfactant protein C in spread phospholipid monolayers.

Pulmonary surfactant, a lipid-protein complex, secreted into the fluid lining of lungs prevents alveolar collapse at low lung volumes. Pulmonary surfactant protein C (SP-C), an acylated, hydrophobic, alpha-helical peptide, enhances the surface activity of pulmonary surfactant lipids. Fluorescein-labeled SP-C (F-SP-C) (3, 6, 12 wt%) in dipalmitoylphosphatidylcholine (DPPC), and DPPC:dipalmitoylphosphatidylglycerol (DPPG) [DPPC:DPPG 7:3 mol/mol] in spread monolayers was studied by epifluorescence microscopy. Mass spectometry of F-SP-C indicated that the protein is partially deacylated and labeled with 1 mol fluorescein/1 mol protein. The protein partitioned into the fluid, or liquid expanded, phase. Increasing amounts of F-SP-C in DPPC or DPPC:DPPG monolayers decreased the size and total amounts of the condensed phase at all surface pressures. Calcium (1.6 mM) increased the amount of the condensed phase in monolayers of DPPC:DPPG but not of DPPC alone, and such monolayers were also perturbed by F-SP-C. The study indicates that SP-C perturbs the packing of neutral and anionic phospholipid monolayers even when the latter systems are condensed by calcium, indicating that interactions between SP-C and the lipids are predominantly hydrophobic in nature.     

Chitosan as a lipid binder: a langmuir monolayer study of chitosan-lipid interactions

Owing to its distinct chemico-biological properties, chitosan, a cationic biopolymer, offers a great potential in multifarious bioapplications. One such application is as a dietary antilipidemic supplement to be used to reduce obesity/overweight and to lower cholesterol. The lipid-binding efficiency of chitosan, however, remains debatable. Accordingly, in this study we investigated the interactions of chitosan with selected lipids, cholesterol and fatty acids, the latter including saturated (stearic acid) and unsaturated (oleic, linoleic, alpha-linolenic) acids. The experiments were performed with the Langmuir monolayer technique, in which surface pressure-area isotherms were recorded for the lipid monolayers spread on the acetate buffer pH 4.0 subphase in the absence and presence of chitosan. We found that the presence of chitosan in the subphase strongly influenced the shape and location of the isotherms, proving that there existed attractions between chitosan and lipid molecules. The attractions were revealed by changes of the molecular organization of the monolayers. The common feature of these changes was that all the monolayers studied underwent expansion, in each case reaching saturation with increasing chitosan concentration. In agreement with the lipid molecular structures, the highest expansions were observed for the most unsaturated fatty acids, linoleic and alpha-linolenic, the lowest for stearic acid, with oleic acid and cholesterol being the intermediate cases. By contrast, the main distinguishing feature of these changes was that, although none of the monolayers studied changed its state when completely saturated with chitosan, compared to the parent ones the compactness of the monolayers was modified. The solid monolayers of stearic acid and cholesterol were loosened, whereas those of all the unsaturated acids, liquid in nature, were tightened. On the basis of these results we tentatively propose a mechanism of the chitosan action that includes both electrostatic and hydrophobic lipid-chitosan interactions as well as hydrogen bonding between them.     

Insertion of Alzheimer's A beta 40 peptide into lipid monolayers

The amyloid beta (A beta) peptide is the major component found in the amyloid deposits in the brains of Alzheimer's disease patients. In vitro studies have demonstrated that the aggregation of A beta can take place at three orders of magnitude lower concentrations in the presence of phospholipid molecules compared to bulk peptide studies, suggesting that membrane lipids may mediate A beta toxicity. To understand the interaction of A beta with lipid membranes, we have examined A beta 40 with anionic dipalmitoylphosphatidylglycerol (DPPG), zwitterionic dipalmitoylphosphatidylcholine (DPPC), and cationic dipalmitoyltrimethylammonium propane (DPTAP) monolayers under different subphase conditions. We have used a constant surface pressure insertion assay to assess the degree of peptide insertion into the lipids. Simultaneously, we monitored the surface morphology of the monolayers with fluorescence microscopy. We have also performed dual-probe fluorescence measurements where both the peptide and lipid are tagged with chromophores. Isotherm measurements show that A beta inserts into both DPTAP and DPPG monolayers under physiologically relevant conditions. Insertion into DPPC occurs at lipid densities below that found in a bilayer. The level of insertion is inversely proportional to the lipid packing density. Our results indicate that lipids need not be anionic to interact with A beta. Electrostatic effects involved in A beta 40-lipid interaction are discussed.     

Studies on the antibacterial activities and mechanisms of chitosan obtained from cuticles of housefly larvae

Chitosan was obtained from cuticles of the housefly (Musca domestica) larvae. Antibacterial activities of different Mw chitosans were examined against six bacteria. Antibacterial mechanisms of chitosan were investigated by measuring permeability of bacterial cell membranes and observing integrity of bacterial cells. Results show that the antibacterial activity of chitosan decreased with increase in Mw. Chitosan showed higher antibacterial activity at low pH. Ca2+ and Mg2+ could markedly reduce the antibacterial activity of chitosan. The minimum inhibitory concentrations of chitosans ranged from 0.03% - 0.25% and varied with the type of bacteria and Mw of chitosan. Chitosan could cause leakage of cell contents of the bacteria and disrupt the cell wall.     

Interaction of the cholesterol reducing agent simvastatin with zwitterionic DPPC and charged DPPG phospholipid membranes

Simvastatin is a lipid-lowering drug in the pharmaceutical group statins. Interaction of a drug with lipids may define its role in the system and be critical for its pharmacological activity. We examined the interactions of simvastatin with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) and anionic dipalmitoyl phosphatidylglycerol (DPPG) multilamellar vesicles (MLVs) as a function of temperature at different simvastatin concentrations using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The FTIR results indicate that the effect of simvastatin on membrane structure and dynamics depends on the type of membrane lipids. In anionic DPPG MLVs, high simvastatin concentrations (12, 18, 24 mol%) change the position of the CH₂ antisymmetric stretching mode to lower wavenumber values, implying an ordering effect. However, in zwitterionic DPPC MLVs, high concentrations of simvastatin disorder systems both in the gel and liquid crystalline phases. Moreover, in DPPG and DPPC MLVs, simvastatin has opposite dual effects on membrane dynamics. The bandwidth of the CH₂ antisymmetric stretching modes increases in DPPG MLVs, implying an increase in the dynamics, whereas it decreases in DPPC MLVs. Simvastatin caused broadening of the phase transition peaks and formation of shoulders on the phase transition peaks in DSC curves, indicating multi-domain formations in the phospholipid membranes. Because physical features of membranes such as lipid order and fluidity may be changed with the bioactivity of drugs, opposing effects of simvastatin on the order and dynamics of neutral and charged phospholipids may be critical to deduce the action mechanism of the drug and estimate drug-membrane interactions.     

Interactions of Alkylphosphocholines with Model Membranes—The Langmuir Monolayer Study

Alkylphosphocholines (APCs) belong to a class of synthetic antitumor lipids, which are new-generation anticancer agents. In contrast to traditional antitumor drugs, they do not attack the cell nucleus but, rather, the cellular membrane; however, their mechanism of action is not fully understood. This work compared the interactions of selected APCs [namely, hexadecylphosphocholine (miltefosine), octadecylphosphocholine and erucylphosphocholine] with the most important membrane lipids [cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] and examined their influence on a model membrane of tumor and normal cells. As a simple model of membranes, Langmuir monolayers prepared by mixing cholesterol either with a saturated phosphatidylcholine (DPPC), for a normal cell membrane, or with an unsaturated one (POPC), for a tumor cell membrane, have been applied. The APC–lipid interactions, based on experimental surface pressure (π) versus mean molecular area (A) isotherms, were analyzed qualitatively (with mean molecular area values) as well as quantitatively (with the ΔG ^exc function). Strong attractive interactions were observed for mixtures of APCs with cholesterol, contrary to the investigated phosphatidylcholines, for which the interactions were found to be weak with a tendency to separation of film components. In ternary monolayers it has been found that the investigated model systems (cholesterol/DPPC/APC vs cholesterol/POPC/APC) differ significantly as regards the interactions between film-forming molecules. The results demonstrate stronger interactions between the components of cholesterol/POPC/APC monolayers compared to cholesterol/POPC film, mimicking tumor cell membranes. In contrast, the interactions in cholesterol/DPPC/APC films were found to be weaker than those in the cholesterol/DPPC system, serving as a model of healthy cell membranes, thus proving that the incorporation of APCs is, from a thermodynamic point of view, unfavorable for binary cholesterol/DPPC monolayers. It can be concluded that the composition of healthy cell membranes is a natural barrier preventing the incorporation of APCs into normal cells.     

Interaction of chitosan with cell membrane models at the air-water interface

In this paper we employed phospholipid Langmuir monolayers as membrane models to probe interactions with chitosan. Using a combination of surface pressure--area and surface potential--area isotherms and rheological measurements with the pendent drop technique, we observed that chitosan interacts with phospholipid molecules at the air-water interface. We propose a model in which chitosan interacts with the phospholipids mainly through electrostatic interactions, but also including H-bonding and hydrophobic forces, depending on the phospholipid packing density. At large areas per molecule, chitosan in the subphase adsorbs onto the monolayer, expanding it. At small areas per molecule, chitosan is located in the subsurface. Indeed, a mixed chitosan-phospholipid monolayer can be transferred onto solid supports, even at high surface pressures. The effects of chitosan on the viscoelastic properties of phospholipid monolayers may be taken as evidence for the ability of chitosan to disrupt cell membranes.     

Lipid headgroup discrimination by antimicrobial peptide LL-37: insight into mechanism of action

Interaction of the human antimicrobial peptide LL-37 with lipid monolayers has been investigated by a range of complementary techniques including pressure-area isotherms, insertion assay, epifluorescence microscopy, and synchrotron x-ray scattering, to analyze its mechanism of action. Lipid monolayers were formed at the air-liquid interface to mimic the surface of the bacterial cell wall and the outer leaflet of erythrocyte cell membrane by using phosphatidylglycerol (DPPG), phosphatidylcholine (DPPC), and phosphatidylethanolamine (DPPE) lipids. LL-37 is found to readily insert into DPPG monolayers, disrupting their structure and thus indicating bactericidal action. In contrast, DPPC and DPPE monolayers remained virtually unaffected by LL-37, demonstrating its nonhemolytic activity and lipid discrimination. Specular x-ray reflectivity data yielded considerable differences in layer thickness and electron-density profile after addition of the peptide to DPPG monolayers, but little change was seen after peptide injection when probing monolayers composed of DPPC and DPPE. Grazing incidence x-ray diffraction demonstrated significant peptide insertion and lateral packing order disruption of the DPPG monolayer by LL-37 insertion. Epifluorescence microscopy data support these findings.     

Interaction of two phenothiazine derivatives with phospholipid monolayers

This paper addresses the cooperative interaction of two phenothiazine drugs, viz. trifluoperazine (TFP) and chlorpromazine (CPZ), with phospholipid monolayers as the model membrane system. Surface pressure and surface potential isotherms were obtained for mixed Langmuir monolayers of either dipalmitoyl-phosphatidyl-choline (DPPC) or dipalmitoyl-phosphatidyl-glycerol (DPPG) co-spread with TFP or CPZ. The changes in monolayer behavior caused by incorporation of a few molar ratio of drug molecules were practically within the experimental dispersion for the zwitterionic DPPC, and therefore a more refined analysis will be required to probe the interactions in an unequivocal way. For the charged DPPG, on the other hand, the surface pressure and the dipole moment were significantly affected even for TFP or CPZ concentrations as low as 0.002 molar ratio. Overall, the effects from CPZ and TFP are similar, but small differences exist which are probably due to the different protonation properties of the two drugs. For both drugs, changes are more prominent at the liftoff of the surface pressure, i.e. at the gas-condensed phase transition, with the surface pressure and surface potential isotherms becoming more expanded with the drug incorporation. With DPPG/CPZ monolayers, in particular, an additional phase transition appears at higher CPZ concentrations, which resembles the effects from increasing the subphase temperature for a pure DPPG monolayer. The dipole moment for DPPG/CPZ and DPPG/TFP monolayers decreases with the drug concentration, which means that the effects from the charged drugs are not associated with changes in the double-layer potential. Otherwise, the effective dipole moment should increase with the drug concentration. The changes caused in surface pressure and dipole moment by small concentrations of TFP or CPZ can only be explained by some cooperative effect through which the contribution from DPPG molecules changes considerably, i.e. even DPPG molecules that are not neighbor to a CPZ or TFP molecule are also affected. Such changes may occur either through a significant reorientation of the DPPG molecules or to a change in their hydration state. We discuss the cooperativity semi-quantitatively by estimating the number of lipid molecules affected by the drug interaction. CPZ and TFP also affect the morphology of DPPG monolayers, which was confirmed with Brewster angle microscopy. The biological implications from the cooperative, non-specific interaction of CPZ and TFP with membranes are also commented upon.     

Aggregation of puroindoline in phospholipid monolayers spread at the air-liquid interface

Puroindolines, cationic and cystine-rich low molecular weight lipid binding proteins from wheat seeds, display unique foaming properties and antimicrobial activity. To unravel the mechanism involved in these properties, the interaction of puroindoline-a (PIN-a) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers was studied by coupling Langmuir-Blodgett and imaging techniques. Compression isotherms of PIN-a/phospholipid monolayers and adsorption of PIN-a to lipid monolayers showed that the protein interacted strongly with phospholipids, especially with the anionic DPPG. The electrostatic contribution led to the formation of a highly stable lipoprotein monolayer. Confocal laser scanning microscopy and atomic force microscopy showed that PIN-a was mainly inserted in the liquid-expanded phase of the DPPC, where it formed an aggregated protein network and induced the fusion of liquid-condensed domains. For DPPG, the protein partitioned in both the liquid-expanded and liquid-condensed phases, where it was aggregated. The extent of protein aggregation was related both to the physical state of phospholipids, i.e., condensed or expanded, and to the electrostatic interactions between lipids and PIN-a. Aggregation of PIN-a at air-liquid and lipid interfaces could account for the biological and technological properties of this wheat lipid binding protein.     

Comparative study of the interaction of fullerenol nanoparticles with eukaryotic and bacterial model membranes using solid-state NMR and FTIR spectroscopy

Native fullerene is notoriously insoluble in water and forms aggregates toxic to cell membranes, thus limiting its use in nanomedicine. In contrast, water-soluble fullerenol is compatible with biological systems and shows low in vivo toxicity on human cell lines. The interaction mechanism between these hydrophilic nanoparticles and biological membranes is however not well understood. Therefore, in this work, the effect of fullerenol on model eukaryotic and bacterial membranes was investigated using (31)P- and (2)H solid-state NMR as well as FTIR spectroscopy. DPPC/cholesterol and DPPC/DPPG bilayers were used to mimic eukaryotic and bacterial cell membranes, respectively. Our results show low affinity of fullerenol for DPPC/cholesterol bilayers but a clear interaction with model bacterial membranes. A preferential affinity of fullerenol for the anionic phospholipids DPPG in DPPC/DPPG membranes is also observed. Our data suggest that fullerenol remains at the water/bilayer interface of eukaryote-like membranes. They also indicate that the presence of a polar group such as DPPG's hydroxyl moiety at the bilayer surface plays a key role in the interaction of fullerenol with membranes. Hydrogen bonding of fullerenol nanoparticles with DPPGs' OH groups is most likely responsible for inducing lipid segregation in the lipid bilayer. Moreover, the location of the nanoparticles in the polar region of DPPG-rich regions appears to disturb the acyl chain packing and increase the membrane fluidity. The preferential interaction of fullerenol with lipids mostly found in bacterial membranes is of great interest for the design of new antibiotics.     

Preparation and antibacterial activity of chitosan nanoparticles

Chitosan nanoparticles, such as those prepared in this study, may exhibit potential antibacterial activity as their unique character. The purpose of this study was to evaluate the in vitro antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against various microorganisms. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. Copper ions were adsorbed onto the chitosan nanoparticles mainly by ion-exchange resins and surface chelation to form copper-loaded nanoparticles. The physicochemical properties of the nanoparticles were determined by size and zeta potential analysis, atomic force microscopy (AFM), FTIR analysis, and XRD pattern. The antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against E. coli, S. choleraesuis, S. typhimurium, and S. aureus was evaluated by calculation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results show that chitosan nanoparticles and copper-loaded nanoparticles could inhibit the growth of various bacteria tested. Their MIC values were less than 0.25 microg/mL, and the MBC values of nanoparticles reached 1 microg/mL. AFM revealed that the exposure of S. choleraesuis to the chitosan nanoparticles led to the disruption of cell membranes and the leakage of cytoplasm.     

Blending chitosan with polycaprolactone: effects on physicochemical and antibacterial properties

Chitosan is a well sought-after polysaccharide in biomedical applications and has been blended with various macromolecules to mitigate undesirable properties. However, the effects of blending on the unique antibacterial activity of chitosan as well as changes in fatigue and degradation properties are not well understood. The aim of this work was to evaluate the anti-bacterial properties and changes in physicochemical properties of chitosan upon blending with synthetic polyester poly(epsilon-caprolactone) (PCL). Chitosan and PCL were homogeneously dissolved in varying mass ratios in a unique 77% acetic acid in water mixture and processed into uniform membranes. When subjected to uniaxial cyclical loading in wet conditions, these membranes sustained 10 cycles of predetermined loads up to 1 MPa without break. Chitosan was anti-adhesive to Gram-positive Streptococcus mutans and Gram-negative Actinobacillus actinomycetemcomitans bacteria. Presence of PCL compromised the antibacterial property of chitosan. Four-week degradation studies in PBS/lysozyme at 37 degrees C showed initial weight loss due to chitosan after which no significant changes were observed. Molecular interactions between chitosan and PCL were investigated using Fourier transform infrared spectroscopy (FTIR) which showed no chemical bond formations in the prepared blends. Investigation by wide-angle X-ray diffraction (WAXD) indicated that the crystal structure of individual polymers was unchanged in the blends. Dynamic mechanical and thermal analysis (DMTA) indicated that the crystallinity of PCL was suppressed and its storage modulus increased with the addition of chitosan. Analysis of surface topography by atomic force microscopy (AFM) showed a significant increase in roughness of all blends relative to chitosan. Observed differences in biological and anti-bacterial properties of blends could be primarily attributed to surface topographical changes.     

Incorporation of dehydrodieugenol,a neolignan isolated from Nectandra leucantha (Lauraceae), in lipid Langmuir monolayers as biomembrane models

Dehydrodieugenol, a neolignan isolated from the Brazilian plant Nectandra leucantha (Lauraceae) with reported antiprotozoal and anticancer activity, was incorporated in Langmuir monolayers of selected lipids as cell membrane models, aiming to comprehend its action mechanism at the molecular level. The interaction of this compound with the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), and dipalmitoylphosphatidylglycerol (DPPG) was inferred through tensiometry, infrared spectroscopy, and Brewster angle microscopy. The interactions had different effects depending on the chemical nature of the lipid polar head, with expansion for DPPC monolayers, condensation for DPPE, and expansion (at low surface pressures) followed by the overlap of the isotherms (at high surface pressure values) for DPPS and DPPG. Effects caused by dehydrodieugenol in the negatively charged lipids were distinctive, which was also reflected in the hysteresis assays, surface potential-area isotherms, and rheological measurements. Infrared spectroscopy indicated that the drug interaction with the monolayer affects not only the polar groups, but also the acyl lipid chains for all lipids. These results pointed to the fact that the interaction of the drug with lipid monolayers at the air-water interface is modulated by the lipid composition, mainly considering the polar head of the lipids, as well as the hydrophobicity of the lipids and the drug. As negatively charged lipids pointed to distinctive interaction, we believe this can be related to the antiprotozoal and anticancer properties of the compound.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: I. Monolayers of pulmonary surfactant protein SP-B and phospholipids.

The effects of pulmonary surfactant protein SP-B on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), and a mixture of DPPC:DPPG (7:3, mol:mol) were studied using spread films at the air-water interface. The addition of SP-B to the phospholipid monolayers gave positive deviations from additivity of the mean areas in the films. At low protein concentrations (less than 45% amino acid residues which corresponds to 0.5 mol% or 10 weight% SP-B) monolayers of SP-B/DPPC, SP-B/DPPG and SP-B/(DPPC:DPPG) collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At higher concentrations of SP-B in the protein-lipid monolayers, kink points appeared in the isotherms at about 40-45 mN.m-1, implying possible exclusion of material from the films, hence, changes in the original monolayer compositions. Calculated analyses of the monolayer compositions as a function of surface pressure indicated that nearly pure SP-B, associated with small amounts of phospholipid (2-3 lipid molecules per SP-B dimer), was lost from SP-B/DPPC, SP-B/DPPG, and SP-B/(DPPC:DPPG) films at surface pressures higher than 40-45 mN.m-1. The results are consistent with a low effectiveness of SP-B in removing saturated phospholipids, DPPC or DPPG, from the spread SP-B/phospholipid films.     

Interaction between chitosan and bovine lung extract surfactants

The interaction between a cationic polyelectrolyte, chitosan, and an exogenous bovine lung extract surfactant (BLES) was studied using dynamic compression/expansion cycles of dilute BLES preparations in a Constrained Sessile Drop (CSD) device equipped with an environmental chamber conditioned at 37 degrees C and 100% R.H. air. Under these conditions, dilute BLES preparations tend to produce variable and relatively high minimum surface tensions. Upon addition of "low" chitosan to BLES ratios, the minimum surface tension of BLES-chitosan preparations were consistently low (i.e. <5 mJ/m2), and the resulting surfactant monolayers (adsorbed at the air-water interface) were highly elastic and stable. However, the use of "high" chitosan to BLES ratios induced the collapse of the surfactant monolayer at high minimum surface tensions (i.e. >15 mJ/m2). The zeta potential of the lung surfactant aggregates in the subphase suggests that chitosan binds to the anionic lipids (phosphatidyl glycerols) in BLES, and that this binding is ultimately responsible for the changes in the surface activity (elasticity and stability) of these surfactant-polyelectrolyte mixtures. Furthermore the transition from "low" to "high" chitosan to BLES ratios correlates with the flocculation and de-flocculation of surfactant aggregates in the subphase. It is proposed that the aggregation/segregation of "patches" of anionic lipids in the surfactant monolayer produced at different chitosan to BLES ratios explains the enhancing/inhibitory effects of chitosan. These observations highlight the importance of electrostatic interactions in lung surfactant systems.     

Interactions of ciprofloxacin with DPPC and DPPG: fluorescence anisotropy, ATR-FTIR and 31P NMR spectroscopies and conformational analysis

The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T(m)) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and (31)P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K(app) were in the order of 10(5) M(-1) and the affinity appeared dependent on the negative charge of liposomes: DPPG>DOPC:DPPG (1:1; M:M)>DPPC>DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Deltasigma) values determined by (31)P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T(m) of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T(m) and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that ciprofloxacin interacts preferentially with anionic lipid compounds, like phosphatidylglycerol, present at a high content in these membranes.     

Influence of lipid saturation grade and headgroup charge: a refined lung surfactant adsorption model

Rapid adsorption of surfactant material to the air/liquid interface of the lung is essential for maintaining normal lung function. The detailed mechanism of this process, however, remains unclear. In this study, we elucidate the influence of lipid saturation grade and headgroup charge of surface layer lipids on surfactant protein (SP)-induced vesicle insertion into monolayers spread at the air/water interface of a film balance. We used dipalmitoylphosphatidlycholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) as monolayer lipids doped with either hydrophobic surfactant-specific protein SP-B or SP-C (0.2 and 0.4 mol %, respectively). Vesicles consisting of DPPC/DPPG (4:1, mol ratio) were injected into a stirred subphase to quantify adsorption kinetics. Based on kinetic film balance and fluorescence measurements, a refined model describing distinct steps of vesicle adsorption to surfactant monolayers is presented. First, in a protein-independent step, lipids from vesicles bridged to the interfacial film by Ca²⁺ ions are inserted into defects of a disordered monolayer at low surface pressures. Second, in a SP-facilitated step, active material insertion involving an SP-B- or SP-C-induced flip-flop of lipids occurs at higher surface pressures. Negatively charged lipids obviously influence the threshold pressures at which this second protein-mediated adsorption mechanism takes place.     

Study in mono and bilayers of the interaction of hepatitis G virus (GBV-C/HGV) synthetic antigen E2(99-118) with cell membrane phospholipids

The interaction of the hepatitis G synthetic peptide E2(99-118) with cell membrane phospholipids of different characteristics such as dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) was studied by Langmuir isotherms. Epifluorescence microscopy and Atomic force microscopy (AFM) was also used to study interactions with DPPC. Compression isotherms of DPPC/E2(99-118) and DPPG/E2(99-118) mixed monolayers showed negative deviation from ideallity consistent with the existence of attractive interactions. The incorporation of the peptide in DPPC monolayer was also confirmed in epifluorescence microscopy and AFM studies. The peptide retarded the formation of DPPC domains and did not let the phospholipid get organized. No important differences in the interactions with DPPC (neutral) or DPPG (anionic) were found, thus suggesting that electrostatics forces do not have a predominant influence in these interactions.