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.     

Polymeric amphiphiles with hydrophilic main chain spacers: Studies in monolayers and Langmuir-Blodgett multilayers

A methacrylic lipid was polymerized to form an amphiphilic homopolymer and random copolymers with various levels of acrylic acid comonomers. The behavior of these polymeric lipids was investigated in monolayers at the air-water interface and in Langmuir-Blodgett multilayers on polymer foil supports. The acrylic acid acts as a hydrophilic spacer along the polymer backbone, and improves the ability of the polymer to self-organize into highly ordered monolayers and ultimately LB multilayers. It is shown that the quality and stability of the multilayers increase substantially with the increase in comonomer content.     

Molecular organization of a water-insoluble iridium(III) complex in mixed monolayers

In this work, organized mixed monolayers containing a cationic water-insoluble iridium(III) complex, Ir-dye, [Ir(ppy)(2)(tmphen)]PF(6), (tmphen = 3,4,7,8-tetramethyl-1,10-phenanthroline, and ppy = 2-phenylpyridine), and an anionic lipid matrix, DMPA, dimyristoyl-phosphatidic acid, with different molar proportions, were formed by the co-spreading method at the air-water interface. The presence of the dye at the interface, as well as the molecular organization of the mixed films, is deduced from surface techniques such as pi-A isotherms, Brewster angle microscopy (BAM) and reflection spectroscopy. The results obtained remark the formation of an equimolar mixed film, Ir-dye/DMPA = 1:1. BAM images reveal a whole homogeneous monolayer, with gradually increasing reflectivity along the compression process up to reaching the collapse of this equimolecular monolayer at pi approximately equal to 37 mNm(-1). Increasing the molar ratio of DMPA in the mixture, the excess of lipid molecules organizes themselves forming dark flower-like domains of pure DMPA at high surface pressures, coexisting with the mixed Ir-dye/DMPA = 1:1 monolayer. On the other hand, unstable mixed monolayers are obtained by using an initial dye surface concentration higher than the equimolecular one. These mixed Langmuir monolayers have been successfully transferred onto solid substrates by the LB (Langmuir-Blodgett) technique.     

Self-assembly of amphiphilic hexapyridinium cations at the air/water interface and on HOPG surfaces.

Mono- and multilayers of a novel amphiphilic hexapyridinium cation with six eicosyl chains (3) are spread at the air/water interface as well as on highly ordered pyrolytic graphite (HOPG). On water, the monolayer of 3 is investigated by recording surface pressure/area and surface potential/area isotherms, and by Brewster angle microscopy (BAM). Self-organized tubular micelles with an internal edge-on orientation of molecules form at the air/water interface at low surface pressure whereas multilayers are present at high surface pressure, after a phase transition. Packing motifs suggesting a tubular arrangement of the constituting molecules were gleaned from atomic force microscopy (AFM) investigations of Langmuir-Blodgett (LB) monolayers being transferred on HOPG at different surface pressures. These LB film structures are compared to the self-assembled monolayer (SAM) of 3 formed via adsorption from a supersaturated solution, which is studied by scanning tunnelling microscopy (STM). On HOPG the SAM of 3 consists of nanorods with a highly ordered edge-on packing of the aromatic rings and an arrangement of alkyl chains which resembles the packing of molecules at the air/water interface at low surface pressure. Additional details of the molecular packing were gleaned from single-crystal X-ray structure analysis of the hexapyridinium model compound 2b, which possesses methyl instead of eicosyl residues.     

Supported cell mimetic monolayers and their interaction with blood

Surface modification using supported monolayers of phosphorylcholine containing phospholipids has been an accepted strategy for developing blood-contacting materials. We present a detailed study of the blood compatibility of the supported monolayers of phospholipid, glycolipid, and cholesterol (Chol) binary and ternary lipid combinations using in vitro techniques. The packing and orientation of these monolayers have been correlated with the blood compatibility. We have used phosphatidylcholine (PTC) for phospholipid, galactocerebroside (Gal) for glycolipid, and Chol based on the headgroup structure to represent the major lipid components of the endothelial luminal cell membrane. The interfacial behavior of various combinations of PTC, Gal, and Chol monolayers have been studied at the air/water interface and deposited on hydrophobic polycarbonate (PC) polymer substrates with the help of the Langmuir-Blodgett trough. The packing and orientation of the supported monolayers have been varied by means of changing the lipid composition rather than the deposition parameters. This approach seems to be more similar to the in vivo conditions. The different supported monolayer surfaces prepared accordingly are (1) a closely packed ordered hydrophobic surface, PC modified with the combination PTC/Chol/Gal (1:0.35:0.125), (2) a loosely packed ordered hydrophobic surface, PC modified with the combination PTC/Chol (1:0.35), and (3) a closely packed ordered hydrophilic surface, PC modified with the combination PTC/Chol (1:0.7). An optimized modified surface (PTC/Chol/Gal, 1:0.35:0.125) has been identified on the basis of the maximum transfer ratio from the air/water interface and characterized by using atomic force microscopy. The concentration of Chol has been found to be an important parameter, which influences the transfer ratio. The Gal improves the monolayer integrity under a reduced Chol concentration. The blood compatibility of these supported monolayers was studied by protein adsorption, blood cell adhesion, and calcification. The tightly packed ordered hydrophobic surface (PTC/Chol/Gal, 1:0.35:0.125), has been found to be more blood compatible because of reduced blood cell adhesion and calcification. This surface also promotes albumin adsorption and may be the reason for the reduced platelet activation, while in the case of the loosely packed ordered hydrophobic surface (PTC/Chol, 1:0.35) the protein adsorption also has been reduced along with the blood cell adhesion and calcification. When the ordered hydrophilic surface (PTC/Chol, 1: 0.7) of the monolayer has been exposed, the blood cell adhesions as well as the overall protein adsorption were significantly reduced. However, the packing of the phosphorylcholine moieties of the polar headgroup has been affecting the calcification on the surface. We have observed an increase in calcification to the surface modified with the loosely packed polar headgroup, from a relative study on chitosan and chitosan modified with the monolayer of PTC. These findings are helpful for the surface modifications for blood-contacting materials using this strategy.     

Lateral reorganization of myelin lipid domains by myelin basic protein studied at the air-water interface

It has been speculated that adsorption of myelin basic protein (MBP) to the myelin lipid membrane leads to lateral reorganization of the lipid molecules within the myelin membrane. This hypothesis was tested in this study by surface pressure measurement and fluorescent imaging of a monolayer composed of a myelin lipid mixture. The properties of the lipid monolayer before and after addition of MBP into the subphase were monitored. Upon addition of MBP to the monolayer subphase, the surface pressure rose and significant rearrangement of the lipid domains was observed. These results suggest that binding and partial insertion of MBP into the lipid monolayer led to dramatic rearrangement and morphological changes of the lipid domains. A model of adsorption of MBP to the lipid domains and subsequent domain fusion promoted by minimization of electrostatic repulsion between the domains was proposed to account for the experimental observations. The significance of these results in light of the role of MBP in maintaining the myelin structural integrity is discussed.     

髓鞘碱性蛋白对细胞膜脂质分子DPPC、DPPE单层膜影响机理研究

本文通过Langmuir单层膜的表面压力-平均分子面积(π-A)曲线的测定与分析,分别对髓鞘碱性蛋白(MBP)与细胞膜中不同头部基团脂质分子二棕榈酰基磷脂胆碱(DPPC)和二棕榈酰基磷脂酰乙醇胺(DPPE)在空气/液体界面上的相互作用过程进行了系统研究.实验结果表明:(1)当界面上脂质含量一定时,亚相中随着MBP浓度的增大,DPPC、DPPE单层膜的等温线向平均分子面积较大的方向移动;(2)在单层膜表面压力为10 mN/m时,一个MBP分子分别结合140±3个DPPC分子和100±3个DPPE分子,随着表面压力增大,当MBP分子分别与两种磷脂分子相互作用时,MBP插入到磷脂单层界面的个数逐渐减少;(3)随着蛋白质浓度的增加,脂分子形成的单层膜变得较为疏松,且MBP分子易于插入到分子头部较小的DPPE单层膜中;(4)蛋白质的存在使DPPC单层膜的表面压力逐渐减小,且蛋白质浓度越大表面压力降低越多,DPPC被MBP带入到亚相中越多;(5)对于DPPE单层膜,蛋白质通过与DPPE相互作用插入到界面膜中,引起表面压力增大,且蛋白质浓度越高,压力变化量越大.     

Adsorption-penetration studies of glucose oxidase into phospholipid monolayers at the 1,2-dichloroethane/water interface.

The interaction between glucose oxidase (GOx) and phospholipid monolayers is studied at the 1,2-dichloroethane/water interface by electrochemical impedance spectroscopy. Electrochemical experiments show that the presence of GOx induces changes in the capacitance curves at both negative and positive potentials, which are successfully explained by a theoretical model based on the solution of the Poisson-Boltzmann equation. These changes are ascribed to a reduced partition coefficient of GOx and an increase of the permittivity of the lipid hydrocarbon domain. Our results show that the presence of lipid molecules enhances the adsorption of GOx molecules at the liquid/liquid interface. At low lipid concentrations, the adsorption of GOx is probably the first step preceding its penetration into the lipid monolayer. The experimental results indicate that GOx penetrates better and forms more stable monolayers for lipids with longer hydrophobic tails. At high GOx concentrations, the formation of multilayers is observed. The phenomenon described here is strongly dependent on 1) the GOx and lipid concentrations, 2) the nature of the lipid, and 3) the potential drop across the interface.     

Adsorption of detergent-solubilized and phospholipase C-solubilized alkaline phosphatase at air/liquid interfaces

To investigate the influence of a hydrophobic anchor on protein adsorption, equilibrium and dynamic aspects of the adsorption of two different solubilized forms of rat osseous plate alkaline phosphatase on Langmuir monolayers of dimyristoylphosphatidic acid (DMPA) were studied. Surface pressure and surface potential measurements at air/liquid interfaces were carried out using the detergent-solubilized form (DSAP) of alkaline phosphatase, which holds a glycosylphosphatidylinositol (GPI) hydrophobic anchor, and the glycosylphosphatidylinositol-specific phospholipase C-solubilized form (PLSAP), lacking the GPI anchor. Similar surface transitions observed for both DMPA and DMPA/PLSAP mixed monolayers indicate that the presence of PLSAP does not promote significant changes in surface packing of the DMPA monolayer. However, PLSAP interacts with the polar portion of the phospholipid even at high lateral compression. The presence of the GPI anchor increases the adsorption of DSAP at a plain air/liquid interface and also enables the penetration of the protein into the DMPA monolayers. The penetration is dependent on both time and surface pressure. Up to 20 mN/m, the surface pressure increases smoothly indicating a diffusion followed by an adsorption process. Above 20 mN/m, after a fast increase, the surface pressure slowly decays to equilibrium values quite close to the initial surface pressures. The results indicate that the molecular packing of the lipid layer drives the enzyme adsorption to the interface either through the GPI anchor or by the polypeptide moiety.     

Interaction of bovine myelin basic protein with triphosphoinositide

Despite the essential role played by myelin basic protein (MBP) in stabilizing the multilamellar structure of the myelin membrane, attempts at deciphering the structure of MPB have so far failed. Given that MBP is known to specifically interact with the membrane's lipid components, this study was designed to explore the effects of these lipids on the conformation of the protein by examining its interaction with the lipid triphosphoinositide (TPI). MBP was identified by high-performance liquid chromatography (HPLC) in myelin extracted from cow's brain and its molecular weight determined. In aqueous solution, MBP showed a random coil structure confirmed by its circular dichroism (CD) spectra. Possible structural changes to the protein induced by different proportions of TPI were also explored. The CD spectra of these mixtures indicated that this lipid fails to induce the adoption of a secondary structure by MBP. We then performed monolayer experiments to evaluate the type of interaction that occurs between MBP and TPI. First, the molecular weight of the protein sample was established to determine the state of the MBP within the monolayer by applying the equation for gases to the so-called gaseous zone of the monolayer for the conditions under which the expression holds. The similar molecular weights yielded by HPLC performed on dissolved samples and by the monolayers suggests that, as in solution, in the membrane the protein exists as a monomer. Monolayer data suggest forces of attraction between the two components and, through thermodynamic calculations, it was established that this interaction is spontaneous and the interaction is of the electrostatic type.     

Interfacial interaction between dextran sulfate and lipid monolayers: an electrochemical study

The interaction between dextran sulfate (DS) with zwitterionic dipalmitoylphosphatidylcholine (DPPC) and negatively charged dipalmitoylphosphatidic acid monolayers at different surface pressures at air-liquid and liquid-liquid interfaces was studied using Langmuir-Blodgett (LB) and electrochemical techniques. The negatively charged DS can bind to phospholipids via calcium ions. To investigate the mechanism of the adsorption of DS on lipid monolayers, compression isotherms (pi-A) and capacitance-potential curves were measured, and a theoretical model was developed to interpret the capacitance data. The compression of lipid monolayers in the presence of DS led to a more condensed hybrid layer, removing the LE-LC phase transition of DPPC. Lower surface pressures improved the binding of DS on the lipid monolayers via calcium bridges due to the electrostatic attraction. Alternating current voltammetry and cyclic voltammetry were used to monitor the transfer of a cationic beta-blocker (metoprolol) across lipid monolayers in the absence and presence of the polyelectrolyte and to compare with the transfer of the standard probe, tetraethylammonium cation. Results showed a strong dependence on (i) the surface pressure, (ii) the applied potential, and, (iii) in the case of the hybrid layer, the charge of the phospholipid headgroup. Finally, results were also confirmed by attenuated total reflection Fourier transform infrared spectroscopy, performed after transferring lipid multilayers onto a solid substrate by the LB method.     

Chitosan as a removing agent of beta-lactoglobulin from membrane models

Many chitosan biological activities depend on the interaction with biomembranes, but so far it has not been possible to obtain molecular-level evidence of chitosan action. In this article, we employ Langmuir phospholipid monolayers as cell membrane models and show that chitosan is able to remove beta-lactoglobulin (BLG) from negatively charged dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG). This was shown with surface pressure isotherms and elasticity and PM-IRRAS measurements in the Langmuir monolayers, in addition to quartz crystal microbalance and fluorescence spectroscopy measurements for Langmuir-Blodgett (LB) films transferred onto solid substrates. Some specificity was noted in the removal action because chitosan was unable to remove BLG incorporated into neutral dipalmitoyl phosphatidyl choline (DPPC) and cholesterol monolayers and had no effect on horseradish peroxidase and urease interacting with DMPA. An obvious biological implication of these findings is to offer reasons that chitosan can remove BLG from lipophilic environments, as reported in the recent literature.     

The Langmur-blodgett monolayer dipole potential: a smeared dipole model for a lipid array,and pulsing of the potential by direct subphase infusion of immunochemical and lecti/polysaccharide complexes

An important contribution to the surface potential of lipid bilayers and monolayers comes from the intrinsic dipole moment of the lipid molecules. A theoretical model of the monolayer which involves a smeared dipole sheet approximation is introduced. This model is used to explore the nature and origins of the surface potential. In addition, the potential associated with phosphatidyl choline/cholesterol monolayers compressed on a Langmuir-Blodgett trough was measured with a non-contacting electrostatic voltmeter. A trough infusion configuration was fabricated to perform dynamic subphase experiments with compressed films in place. The potential/time response of monolayers to selective bimolecular systems such as antibody-antigen and concanavalin A-saccharide pairs was examined. These reactions induce spontaneous transients in dipole potential of magnitude 20–80 mV and duration of less than 1 s. The potential transients are attributed to local perturbation of lipid orientation and introduction of protein dipole fields caused by the formation of aggregates at the monolayer/water interface.     

Formation of 2D colloidal crystals by the Langmuir-Blodgett technique monitored in situ by Brewster angle microscopy

We report a method that combines Brewster angle microscopy and Langmuir-Blodgett films technique to obtain highly ordered 2D colloidal crystals of nanospheres. The deposition of Langmuir-Blodgett films of silica spheres monitored by Brewster angle microscopy allows to determine with accuracy the best physical conditions to transfer highly ordered monolayers of nanoparticles.     

Incorporation conditions guiding the aggregation of a glycosylphosphatidyl inositol (GPI)-anchored protein in Langmuir monolayers

This work investigates the process of incorporation of a glycosylphosphatidyl inositol (GPI)-anchored alkaline phosphatase into Langmuir monolayers of dimyristoyl phosphatidic acid (DMPA). Three different methods of protein incorporation were assayed. When the protein solution was injected below the air–water interface after formation of the lipid monolayer a micro-heterogeneous distribution of alkaline phosphatase throughout the interface was observed. Adsorption kinetics studied by fluorescence microscopy, associated with surface pressure measurements, led to the proposition of a model in which the protein penetration is modulated by the surface packing of the monolayer and intermolecular interactions occurring between the phospholipid and the protein. At initial surface pressures higher than 20 mN m⁻¹, the protein is quickly adsorbed on the interface and the lateral diffusion drives the alkyl chains to turn towards the air phase while the polypeptide moiety faces the aqueous subphase.     

Probing chitosan and phospholipid interactions using Langmuir and Langmuir-Blodgett films as cell membrane models

The interaction between chitosan and Langmuir and Langmuir-Blodgett (LB) films of dimyristoyl phosphatidic acid (DMPA) is investigated, with the films serving as simplified cell membrane models. At the air-water interface, chitosan modulates the structural properties of DMPA monolayers, causing expansion and decreasing the monolayer elasticity. As the surface pressure increased, some chitosan molecules remained at the interface, but others were expelled. Chitosan could be transferred onto solid supports alongside DMPA using the LB technique, as confirmed by infrared spectroscopy and quartz crystal microbalance measurements. The analysis of sum-frequency vibration spectroscopy data for the LB films combined with surface potential measurements for the monolayers pointed to chitosan inducing the ordering of the DMPA alkyl chains. Furthermore, the morphology of DMPA LB films, studied with atomic force microscopy, was affected significantly by the incorporation of chitosan, with the mixed chitosan-DMPA films displaying considerably higher thickness and roughness, in addition to chitosan aggregates. Because chitosan affected DMPA films even at pressures characteristic of cell membranes, we believe this study may help elucidate the role of chitosan in biological systems.     

Influence of Poly(ethylenimine) on the Monolayer of Oleic Acid at the Air/Water Interface

The effect of poly(ethylenimine) (PEI) dissolved in water on the surface pressure-area isotherm of oleic acid (OA) at the air/water interface was investigated. On a concentrated PEI solution, the isotherm of the OA monolayers exhibited a noticeable difference as a function of subphase pH. PEI caused the collapse pressure of the OA monolayer to increase up to 45 mN/m, due to a stronger acid-base-type interaction occurring between the amine group of the PEI and the carboxyl group of OA; on a pure water subphase, the collapse pressure was;28 mN/m. On the other hand, owing to a stronger OA-PEI interaction, the OA monolayers favored a liquid-expanded state more on the PEI-containing water subphase than on the pure water. From the QCM measurement, each OA molecule appeared to interact, on average, with 4.3-5.8 ethylenimine repeating units at basic pHs. We also found that OA multilayers could be assembled on a hydrophilic substrate by a Z-type Langmuir-Blodgett (LB) deposition in a PEI-containing subphase at basic pHs. The ATR-IR spectral data revealed that, in a Z-type LB film, the headgroup of OA was mostly present as carboxylate, interacting in an ionic state with the protonated amine groups of PEI. In acidic conditions, neither a Y-type nor a Z-type deposition was really accomplished. Nonetheless, the ATR-IR spectral data suggested that OA molecules should exist in a monomeric state in a LB film assembled at acidic pHs without PEI while they would form intermolecular hydrogen bridges and/or dimers in the presence of PEI. Copyright 2000 Academic Press.     

Self-organization of a wedge-shaped surfactant in monolayers and multilayers

The self-organization behavior of a wedge-shaped surfactant, disodium-3,4,5-tris(dodecyloxy)phenylmethylphosphonate, was studied in Langmuir monolayers (at the air-water interface), Langmuir-Blodgett (LB) monolayers and multilayers, and films adsorbed spontaneously from isooctane solution onto a mica substrate (self-assembled films). This compound forms an inverted hexagonal lyotropic liquid crystal phase in the bulk and in thick adsorbed films. Surface pressure isotherm and Brewster angle microscope (BAM) studies of Langmuir monolayers revealed three phases: gas (G), liquid expanded (LE), and liquid condensed (LC). The surface pressure-temperature phase diagram was determined in detail; a triple point was found at approximately 10 degrees C. Atomic force microscope (AFM) images of LB monolayers transferred from various regions of the phase diagram were consistent with the BAM images and indicated that the LE regions are approximately 0.5 nm thinner than the LC regions. AFM images were also obtained of self-assembled films after various adsorption times. For short adsorption times, when monolayer self-assembly was incomplete, the film topography indicated the coexistence of two distinct monolayer phases. The height difference between these two phases was again 0.5 nm, suggesting a correspondence with the LE/LC coexistence observed in the Langmuir monolayers. For longer immersion times, adsorbed multilayers assembled into highly organized periodic arrays of inverse cylindrical micelles. Similar periodic structures, with the same repeat distance of 4.5 nm, were also observed in three-layer LB films. However, the regions of organized periodic structure were much smaller and more poorly correlated in the LB multilayers than in the films adsorbed from solution. Collectively, these observations indicate a high degree of similarity between the molecular organization in Langmuir layers/LB films and adsorbed self-assembled films. In both cases, monolayers progress through an LE phase, into LE/LC coexistence, and finally into LC phase as surface density increases. Following the deposition of an additional bilayer, the film reorganizes to form an array of inverted cylindrical micelles.     

Towards a fluorescent chemoreceptive lipid membrane-based optode

Lipid membranes have been deposited on the surface of glass slides and quartz fibres for the development of optical chemical sensors. The fluorescence properties of probes embedded within ordered lipid matrices were sensitive to physical and electrostatic environmental alterations of the membrane caused by various non-selective processes. This work reports criteria for surface deposition of monolayers of phosphatidylcholine-steroid mixtures and stearic acid by Langmuir-Blodgett techniques. The fluorophores 1-anilinonaphthalene-8-sulphonate and 12-(9-anthroyloxy)stearic acid were incorporated into the monolayers for determination of the physical characteristics of the membranes. Interactions of the membranes with the perturbing species phloretin and valinomycin in aqueous solution, and with chloroform, n-hexane and N,N-dimethylaniline in the gas phase, are described. An evanescent-wave intrinsic fibre-optic sensor based on lipid membrane excitation is reported.     

Monolayers of reactive cellulose derivatives

Functional cellulose derivatives are very versatile materials for the creation of mono- and multilayer systems. Hydrophobic alkyl and trimethylsilyl celluloses form highly ordered Langmuir-Blodgett multilayers on hydrophobic substrates. Cellulose thiosulfates and methyl thio ethers were self-assembled on gold and silver surfaces to form hydrophilic monolayers. Cellulose layer systems are capable for chemical transformations under conservation of the structural order. They are suitable platforms for the investigation of molecular recognition at surfaces and the construction of sensor devices. Both biological ligands, e.g. biotin, and enzymes, e.g. horse radish peroxidase, could be attached to cellulose under conservation of their biological function.