Supported phospholipid membranes: Comparison among different deposition methods for a phospholipid monolayer

Supported lipid membranes consisting of self-assembled alkanethiol and lipid monolayers on gold substrates could be produced by three different deposition methods: the Langmuir-Blodgett (L-B) technique, the painted method, and the paint-freeze method, By using cyclic voltammetry, chronoamperometry/chronocoulometry and a.c. impedance measurements, we demonstrated that lipid membranes prepared by these three deposition methods had obvious differences in specific capacitance, resistance and thickness. The specific capacitance of lipid membranes prepared by depositing an L-B monolayer on the alkanethiol alkylated surfaces was 0.53 mu Fcm(-2), 0.44 mu Fcm(-2) by the painted method and 0.68 mu Fcm(-2) by the paint-freeze method. The specific conductivity of lipid membranes prepared by the L-B method was over three times lower than that of the painted lipid membranes, while that of the paint-freeze method was the lowest. The difference among the three types of lipid membranes was ascribed to the influence of the organic solvent in lipid films and the changes in density of the films. The lipid membranes prepared by the usual painted method contained a trace amount of the organic solvent. The organic solvent existing in the hydrocarbon core of the membrane reduced the density of the membrane and increased the thickness of the membrane. The membrane prepared by depositing an L-B monolayer containing no solvent had higher density and the lowest fluidity, and the thickness of the membrane was smaller. The lipid membrane prepared by the paint-freeze method changed its structure sharply at the lower temperature. The organic solvent was frozen out of the membrane while the density of the membrane increased greatly. All these caused the membrane to exist in a ''tilted'' state and the thickness of this membrane was the smallest. The lipid membrane produced by the paint-freeze method was a membrane not containing organic solvent. This method was easier in manipulation and had better reproducibility than that of the usual painting method and the method of forming free-standing lipid film. The solvent-free membrane had a long lifetime and a higher mechanical stability. This model membrane would be useful in many areas of scientific research.

Reaction of a phospholipid monolayer with gas-phase ozone at the air-water interface: measurement of surface excess and surface pressure in real time

The reaction between gas-phase ozone and monolayers of the unsaturated lipid 1-palmitoy1-2-oleoyl-sn-glycero-3-phosphocholine, POPC, on aqueous solutions has been studied in real time using neutron reflection and surface pressure measurements. The reaction between ozone and lung surfactant, which contains POPC, leads to decreased pulmonary function, but little is known shout the changes that occur to the interfacial material as a result of oxidation. The results reveal that the initial reaction of ozone with POPC leads to a rapid increase in surface pressure followed by a slow decrease to very low values. The neutron reflection measurements, performed on an isotopologue of POPC with a selectively deuterated palmitoyl strand, reveal that the reaction leads to loss of this strand from the air-water interface. suggesting either solubilization of the product lipid or degradation of the palmitoyl strand by a reactive species. Reactions of H-1-POPC on D2O reveal that the headgroup region of the lipids in aqueous solution is not dramatically perturbed by the reaction of POPC monolayers with ozone supporting degradation of the palmitoyl strand rather than solubilization. The results are consistent with the reaction of ozone with the oleoyl strand of POPC at the air water interface leading to the formation of OH radicals. the highly reactive OH radicals produced can then go on to react with the saturated palmitoyl strands leading to the formation or oxidized lipids with shorter alkyl tails.

The role of protein hydrophobicity in thionin–phospholipid interactions: a comparison of α1 and α2-purothionin adsorbed anionic phospholipid monolayers

The plant defence proteins α1- and α2-purothionin (Pth) are type 1 thionins from common wheat (Triticum aestivum). These highly homologous proteins possess characteristics common amongst antimicrobial peptides and proteins, that is, cationic charge, amphiphilicity and hydrophobicity. Both α1- and α2-Pth possess the same net charge, but differ in relative hydrophobicity as determined by C18 reversed phase HPLC. Brewster angle microscopy, X-ray and neutron reflectometry, external reflection FTIR and associated surface pressure measurements demonstrated that α1 and α2-Pth interact strongly with condensed phase 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) monolayers at the air/liquid interface. Both thionins disrupted the in-plane structure of the anionic phospholipid monolayer, removing lipid during this process and both penetrated the lipid monolayer in addition to adsorbing as a single protein layer to the lipid head-group. However, analysis of the interfacial structures revealed that the α2-Pth showed faster disruption of the lipid film and removed more phospholipid (12%) from the interface than α1-Pth. Correlating the protein properties and lipid binding activity suggests that hydrophobicity plays a key role in the membrane lipid removal activity of thionins.

The interaction of an antiparasitic peptide active against African Sleeping Sickness with cell membrane models

Zwitterionic peptides with trypanocidal activity are promising lead compounds for the treatment of African Sleeping Sickness, and have motivated research into the design of compounds capable of disrupting the protozoan membrane. In this study, we use the Langmuir monolayer technique to investigate the surface properties of an antiparasitic peptide, namely S-(2,4-dinitrophenyl)glutathione di-2-propyl ester, and its interaction with a model membrane comprising a phospholipid monolayer. The drug formed stable Langmuir monolayers. whose main feature was a phase transition accompanied by a negative surface elasticity. This was attributed to aggregation upon compression due to intermolecular bond associations of the molecules, inferred from surface pressure and surface potential isotherms. Brewster angle microscopy (BAM) images, infrared spectroscopy and dynamic elasticity measurements. When co-spread with dipalmitoyl phosphatidyl choline (DPPC). the drug affected both the surface pressure and the monolayer morphology, even at high surface pressures and with low amounts of the drug. The results were interpreted by assuming a repulsive, cooperative interaction between the drug and DPPC molecules. Such repulsive interaction and the large changes in fluidity arising from drug aggregation may be related to the disruption of the membrane, which is key for the parasite killing property. (C) 2009 Elsevier B.V. All rights reserved.

Regulated localization of rab18 to lipid droplets - Effects of lipolytic stimulation and inhibition of lipid droplet catabolism

Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3(DGV), a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3(DGV) labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.

Lanthanide ion induced formation of stripes domain structure in phospholipid Langmuir-Blodgett monolayer film observed by atomic force microscopy

Long-range ordered stripes domain structures were observed in Dipalmitoylphosphatidylcholine (DPPC) Langmuir-Blodgett monolayer film which was spread on the subphase of lanthanide ion (Eu3+) solution and transferred to a freshly cleaved mica substrate by vertical deposition. This novel phenomenon was discussed in terms of the competitive interaction of dipole-dipole and electrostatic interactions of the DPPC molecules combined with lanthanide ions with those DPPC molecules free of lanthanide ions.

Stabilization of the bio-membrane by small molecules: interaction of trehalose with the phospholipid bilayer

Anhydrobiotic organisms undergo periods of acute dehydration during their life cycle. It is of interest to understand how the biomembrane remains intact through such stress. A disaccharide, trehalose, which is metabolised during anhydrobiosis is found to prevent disruption of model membrane systems. Molecular modelling techniques are used to investigate the possible mode of interaction of trehalose with a model monolayer. The objective is to maximise hydrogen bonding between the two systems. A phospholipid matrix consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) is chosen to represent the monolayer. The crystal structure of DMPC reveals that there are two distinct conformers designated as A and B. An expansion of the monolayer, coplanar with its surface, results in the trehalose molecule being accommodated in a pocket formed by four B conformers. One glucose ring of the sugar rests on the hydrophobic patch provided by the choline methyls of an A conformer. Five hydrogen bonds are formed involving the phosphate oxygens of three of the surrounding B conformers. The model will be discussed with reference to relevant experimental data on the interaction.

Self-assembled monolayer growth of phospholipids on hydrophobic surface toward mimetic biomembranes: Scanning probe microscopy study

Atomic force microscopy (AFM) and lateral force microscopy (LFM) were used simultaneously to analyze a model membrane bilayer structure consisting of a phospholipid outer monolayer deposited onto organosilane-derivatized mica surfaces, which were constructed by using painting and self-assembly methods. The phospholipid used as outer monolayer was dimyristoylphosphatidylcholine (DMPC). The hydrocarbon-covered substrate that formed the inner half bilayer was composed of a self-assembly monolayer (SAM) of octadecyltrichloroorganosilane (OTS) on mica. SAMs of DMPC were formed by exposing hydrophobic mica to a solution of DMPC in decane/isobutanol and subsequently immersing into pure water. AFM images of samples immersed in solution for varying exposure times showed that before forming a complete monolayer the molecules aggregated into dense islands (2.2-2.6 nm high) on the surface. The islands had a compact and rounded morphology. LFM, coupled with topographic data obtained with the atomic force mode, had made possible the distinction between DMPC and OTS. The rate constant of DMPC growth was calculated. This is the first systematic study of the SAM formation of DMPC by AFM and LFM imaging. It reveals more direct information about the film morphology than previous studies with conventional surface analytical techniques such as infrared spectroscopy, X-ray, or fluorescence microscopy.

K+ sensors based on supported alkanethiol/phospholipid bilayers

A novel kind of K+ sensor with valinomycin-incorporated bilayers supported on a gold electrode consisting of self-assembled alkanethiol monolayers (SAMs) and a lipid monolayer has been fabricated successfully. The lipid monolayer is deposited on the alkylated surface of the first alkanethiol monolayer through three different methods, such as the Langmuir-Blodgett (LB) technique, painted method and painted-frozen method. The response of K + sensors produced by a painted or painted-frozen lipid monolayer on an alkanethiol alkylated gold electrode is larger than that by the LB method, which is due to the difference in fluidity of the three kinds of bilayers. Selectivity coefficients KK+, Na+, KK+, Li+, KK+, Ca2+ and KK+, Mg2+ are 10(-4), 10(-4), 2 x 10(-5) and 3 x 10(-5) respectively, and there is no obvious difference among different fabricating methods. A linear response toward the potassium ion was found in the range from 10(-1) M to 10(-5) M with the detection limit of 10(-6) M. The sensor has a slope of 60 mV per decade. Meanwhile, the longevity of the sensor was improved obviously for at least two months at about -10 degrees C. The higher stability shows the possibility to fabricate a practical biosensor.

The ion selectivity of monensin incorporated phospholipid/alkanethiol bilayers

Monensin was incorporated into phospholipid/alkanethiol bilayers on the gold electrode surface by a new, paint-freeze method to deposit a lipid monolayer on the self-assembled monolayers (SAMs) of alkanethiol. The advantages of this assembly system with a suitable function for investigating the ion selective transfer across the mimetic biomembrane are based on the characteristics of SAMs of alkanethiols and monensin. On the one hand, the SAMs of alkanethiols bring out their efficiency of packing and coverage of the metal substrate and relatively long-term stability; on the other hand, monensin improves the ion selectivity noticeably. The selectivity coefficients K-Na+,K-K+, K-Na+,K-Rb+ and K-Na+,K-Ag+ are 6 x 10(-2), 7.2 x 10(-3) and 30 respectively. However, the selectivity coefficient K-Na+,K-Li+ could not be obtained by a potentiometric method due to the specific interaction between Li+ and phospholipid and the lower degree of complexion between Li+ and monensin. The potential response of this bilayer system to monovalent ions is fairly good. For example, the slope of the response to Na+ is close to 60 mV per decade and its linearity range is from 10(-1) to 10(-5) M with a detection limit of 2 x 10(-6) M, The bilayer is stable for at least two months without changing its properties. This monensin incorporated lipid/alkanethiol bilayer is a good mimetic biomembrane system, which provides great promise for investigating the ion transfer mechanism across the biomembrane and developing a practical biosensor.

Nanopatterning and functionalization of phospholipid-based Langmuir-Blodgett and Langmuir-Schaefer films

Durant les dernières décennies, la technique Langmuir-Blodgett (LB) s’est beaucoup développée dans l’approche « bottom-up » pour la création de couches ultra minces nanostructurées. Des patrons constitués de stries parallèles d’environ 100 à 200 nm de largeur ont été générés avec la technique de déposition LB de monocouches mixtes de 1,2-dilauroyl-sn-glycéro-3-phosphatidylcholine (DLPC) et de 1,2-dipalmitoyl-sn-glycéro-3-phosphatidylcholine (DPPC) sur des substrats de silicium et de mica. Afin d’amplifier la fonctionnalité de ces patrons, la 1-palmitoyl-2-(16-(S-methyldithio)hexadécanoyl)-sn-glycéro-3-phosphatidylcholine (DSDPPC) et la 1-lauroyl-2-(12-(S-methyldithio)dodédecanoyl)-sn-glycéro-3-phosphatidylcholine (DSDLPC) ont été employées pour la préparation de monocouches chimiquement hétérogènes. Ces analogues de phospholipide possèdent un groupement fonctionnel méthyldisulfide qui est attaché à la fin de l’une des chaînes alkyles. Une étude exhaustive sur la structure de la phase des monocouches Langmuir, Langmuir-Schaefer (LS) et LB de la DSDPPC et de la DSDLPC et leurs différents mélanges avec la DPPC ou la DLPC est présentée dans cette thèse. Tout d’abord, un contrôle limité de la périodicité et de la taille des motifs des stries parallèles de DPPC/DLPC a été obtenu en variant la composition lipidique, la pression de surface et la vitesse de déposition. Dans un mélange binaire de fraction molaire plus grande de lipide condensé que de lipide étendu, une vitesse de déposition plus lente et une plus basse pression de surface ont généré des stries plus continues et larges. L’addition d’un tensioactif, le cholestérol, au mélange binaire équimolaire de la DPPC/DLPC a permis la formation de stries parallèles à de plus hautes pressions de surface. La caractérisation des propriétés physiques des analogues de phospholipides a été nécessaire. La température de transition de phase de la DSDPPC de 44.5 ± 1.5 °C comparativement à 41.5 ± 0.3 °C pour la DPPC. L’isotherme de la DSDPPC est semblable à celui de la DPPC. La monocouche subit une transition de phase liquide-étendue-à-condensée (LE-C) à une pression de surface légèrement supérieure à celle de la DPPC (6 mN m-1 vs. 4 mN m-1) Tout comme la DLPC, la DSDLPC demeure dans la phase LE jusqu’à la rupture de la monocouche. Ces analogues de phospholipide existent dans un état plus étendu tout au long de la compression de la monocouche et montrent des pressions de surface de rupture plus basses que les phospholipides non-modifiés. La morphologie des domaines de monocouches Langmuir de la DPPC et de la DSDPPC à l’interface eau/air a été comparée par la microscopie à angle de Brewster (BAM). La DPPC forme une monocouche homogène à une pression de surface (π) > 10 mN/m, alors que des domaines en forme de fleurs sont formés dans la monocouche de DSDPPC jusqu’à une π ~ 30 mN m-1. La caractérisation de monocouches sur substrat solide a permis de démontrer que le patron de stries parallèles préalablement obtenu avec la DPPC/DLPC était reproduit en utilisant des mélanges de la DSDPPC/DLPC ou de la DPPC/DSDLPC donnant ainsi lieu à des patrons chimiquement hétérogènes. En général, pour obtenir le même état de phase que la DPPC, la monocouche de DSDPPC doit être comprimée à de plus hautes pressions de surface. Le groupement disulfide de ces analogues de phospholipide a été exploité, afin de (i) former des monocouches auto-assemblées sur l’or et de (ii) démontrer la métallisation sélective des terminaisons fonctionnalisées des stries. La spectrométrie de photoélectrons induits par rayons X (XPS) a confirmé que la monocouche modifiée réagit avec la vapeur d’or pour former des thiolates d’or. L’adsorption de l’Au, de l’Ag et du Cu thermiquement évaporé démontre une adsorption préférentielle de la vapeur de métal sur la phase fonctionnalisée de disulfide seulement à des recouvrements sub-monocouche.

Cholesterol Mediates Chitosan Activity on Phospholipid Monolayers and Langmuir-Blodgett Films

The polysaccharide chitosan has been largely used in many biological applications as a fat and cholesterol reducer, bactericide agent, and wound healing material. While the efficacy for some of such uses is proven, little is known about the molecular-level interactions involved in these applications. In this study, we employ mixed Langmuir and Langmuir-Blodgett (LB) films of negatively charged dimyristoyl phosphatidic acid (DMPA) anti cholesterol as cell membrane models to investigate the role of cholesterol in the molecular-level action of chitosan. Chitosan does not remove cholesterol froth the monolayer. The interaction with chitosan tends to expand the DMPA monolayer due to its interpenetration within the film. On the other hand, cholesterol induces condensation of the DMPA monolayer. The competing effects cause the surface pressure isotherms of mixed DMPA-cholesterol films on a chitosan subphase to be unaffected by the cholesterol mole fraction, due to distinct degrees of chitosan penetration into the film in the presence of cholesterol. By combining polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and sum-frequency generation spectroscopy (SFG), we showed that chitosan induces order into negatively charged phospholipid layers, whereas the opposite occurs for cholesterol. In conclusion, chitosan has its penetration in the film modulated by cholesterol, and electrostatic interactions with negatively charged phospholipids, such as DMPA, are crucial for the action of chitosan.

Immobilization of Alcohol Dehydrogenase in Phospholipid Langmuir-Blodgett Films To Detect Ethanol

Enzyme immobilization in nanostructured films may be useful for a number of biomimetic systems, particularly if suitable matrixes are identified. Here we show that alcohol dehydrogenase (ADH) has high affinity toward a negatively charged phospholipid, dimyristoylphosphatidic acid (DMPA), which forms a Langmuir monolayer at an air-water interface. Incorporation of ADH into the DMPA monolayer was monitored with Surface pressure measurements; and polarization-modulation infrared reflection absorption spectroscopy, with the alpha-helices from ADH being mainly oriented parallel to the water surface. ADH remained at the interface even at high surface pressures, thus allowing deposition of Langmuir-Blodgett (LB) films from the DMPA-ADH film. Indeed, interaction with DMPA enhances the transfer of ADH, where the mass transferred onto a solid support increased from 134 ng for ADH on a Gibbs monolayer to 178 ng for an LB film with DMPA. With fluorescence spectroscopy it was possible to confirm that the ADH structure was preserved even after one month of the LB deposition. ADH-containing films deposited onto gold-interdigitated electrodes were employed in a sensor array capable of detecting ethanol at concentrations down to 10 ppb (in volume), using impedance spectroscopy as the method of detection.

Interaction of small amounts of bovine serum albumin with phospholipid monolayers investigated by surface pressure and atomic force microscopy

The influence of small amounts of bovine serum albumin (BSA) (nM concentration) on the lateral organization of phospholipid monolayers at the air-water interface and transferred onto solid substrates as one-layer Langmuir-Blodgett (LB) films was investigated. The kinetics of adsorption of BSA onto the phospholipid monolayers was monitored with surface pressure isotherms in a Langmuir trough, for the zwitterionic dipalmitoylphosphatidyl ethanolamine (N,N-dimethyl-PE) and the anionic dimyristoylphosphatidic acid (DMPA). A monolayer of N,N-dimethyl-PE or DMPA incorporating BSA was transferred onto a solid substrate using the Langmuir-Blodgett technique. Atomic force microscopy (AFM) images of one-layer LB films displayed protein-phospholipid domains, whose morphology was characterized using dynamic scaling theories to calculate roughness exponents. For DMPA-BSA films the surface is characteristic of self-affine fractals, which may be described with the Kardar-Parisi-Zhang (KPZ) equation. on the other hand, for N,N-dimethyl-PE-BSA films, the results indicate a relatively flat surface within the globule. The height profile and the number and size of globules varied with the type of phospholipid. The overall results, from kinetics of adsorption on Langmuir monolayers and surface morphology in LB films, could be interpreted in terms of the higher affinity of BSA to the anionic DMPA than to the zwitterionic N,N-dimethyl-PE. Furthermore, the effects from such small amounts of BSA in the monolayer point to a cooperative response of DMPA and N,N-dimethyl-PE monolayers to the protein. (c) 2005 Elsevier B.V. All rights reserved.

pH-dependent distribution of chlorin e6 derivatives across phospholipid bilayers probed by NMR spectroscopy

The pH-dependent membrane adsorption and distribution of three chlorin derivatives, chlorin e6 (CE), rhodin G7 (RG), and monoaspartyl-chlorin e6 (MACE), in the physiological pH range (pH 6-8) were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. The chlorin derivatives were characterized with respect to their aggregation behavior, the pK(a) values of individual carboxylate groups, the extent of membrane adsorption, and their flip-flop rates across the bilayer membrane for pH 6-8. External membrane adsorption was found to be lower for RG than for CE and MACE. Both electrostatic interactions and the extent of aggregation seemed to be the main determinants of membrane adsorption. Rate constants for chlorin transfer across the membrane were found to correlate strongly with the pH of the surrounding medium, in particular, for CE and RG. In acidic solution, CE and RG transfer across the membrane was strongly accelerated, and in basic solution, all compounds were retained, mostly in the outer monolayer. In contrast, MACE flip-flop across the membrane remained very low even at pH 6. The protonation of ionizable groups is suggested to be a major determinant of chlorin transfer rates across the bilayer. pK(a) values of CE and RG were found to be between 6 and 8, and two of the carboxylate groups in MACE had pK(a) values below 6. For CE and RG, the kinetic profiles at acidic pH indicated that the initial fast membrane distribution was followed by secondary steps that are discussed in this article.