Mixed Lipid Monolayers Research Articles

Waveguide evanescent field fluorescence microscopy: high contrast imaging of a domain forming mixed lipid Langmuir-Blodgett monolayer mimicking lung surfactant

Waveguide evanescent field fluorescence (WEFF) microscopy is a new development that allows the imaging of contact regions between biological cells and their substratum, as well as imaging of ultrathin films such as monomolecular Langmuir-Blodgett (LB) films. Mixed-lipid monolayer systems mimicking lung surfactant were fabricated on waveguides using the LB technique and imaged by both WEFF and standard wide field epi-fluorescence microscopy. These two technologies were compared with respect to contrast, photobleaching, and sensitivity. It was found that WEFF microscopy produced images with a much higher contrast, lower photobleaching, and higher sensitivity. In addition, fine structures in the lipidic domains were observed for the first time.

Folic acid conjugated nanoparticles of mixed lipid monolayer shell and biodegradable polymer core for targeted delivery of Docetaxel

A system of nanoparticles of mixed lipid monolayer shell and biodegradable polymer core was developed for targeted delivery of anticancer drugs with Docetaxel as a model drug, which provide targeting versatility with a quantitative control of the targeting effect by adjusting the lipid component ratio of the mixed lipid monolayer, and combine the advantages and avoid disadvantages of polymeric nanoparticles and liposomes in drug delivery. X-ray photoelectron spectroscopy (XPS) confirmed the coating of the mixed lipid monolayer on the polymeric core. Fluorescent microscopy proved the targeting efficacy of the folic acid conjugated on the mixed lipid monolayer for the cancer cells of over expression of folate receptors. The folic acid conjugated nanoparticles of mixed lipid monolayer shell and biodegradable polymer core were proved to possess sustainable, controlled and targeted delivery of anticancer drugs with Docetaxel as a model drug, which may provide a drug delivery system of precise control of the targeting effect.

Preparation and Characterization of Glycoacrylate-Based Polymer-Tethered Lipid Bilayers on Benzophenone-Modified Substrates

Polymer-tethered lipid bilayers are promising models for biological membranes as they may provide a soft, lubricating environment with sufficient spacing between the substrate and bilayer for incorporating transmembrane proteins. We present such a system that uses a glycoacrylate-based telechelic lipopolymer in combination with a lipid analogue. Characterization of the mixed monolayers of lipopolymers and free lipids at the air-water interface is used to examine the molecular organization that dictates the final assembly properties. Isotherms indicate that the source of the dominating interactions, whether polymer interactions in the subphase or alkyl chain interactions, depends on both the tethering density and area per molecule. Moreover, a critical composition exists at which the alkyl chain interactions dominate the monolayer behavior regardless of the area per molecule. Isobaric creep and hysteresis experiments suggest that permanent states due to irreversible polymer-polymer interactions are not created as the monolayer is compressed. These data, combined with theoretical polymer predictions, are used to understand the organization of the monolayers at the air-water interface and, hence, the separation distance between the bottom of the bilayer and substrate in the water-swollen state of the final bilayer assembly. Atomic force microscopy is used to confirm that the measured separation distance of 11.2 nm is on the order of what would be predicted using a theoretical analysis for a representative 5 mol % lipopolymer-tethered bilayer. Next, the homogeneity of the final bilayer is probed at multiple scales. Fluorescence microscopy is used to demonstrate that homogeneous and continuous bilayers can be formed (within the optical resolution limit of 500 nm) with all polymer tethering densities used in this study. Atomic force microscopy studies demonstrate that homogeneity comparable to that of a solid-supported lipid bilayer can be achieved for a representative 5 mol % lipopolymer-tethered bilayer. Langmuir-Blodgett transfer conditions for depositing monolayers that can be used to create homogeneous, fluid bilayers are also discussed. Finally, the distal leaflet lateral mobility is measured using fluorescence recovery after photobleaching experiments and shown to be a function of the tethering density. A possible model for the mobility data is developed in which the tethered lipids in the proximal leaflet act as immobile lipid obstacles that couple to distal leaflet lipids.

Interfacial Approach to Polyaromatic Hydrocarbon Toxicity: Phosphoglyceride and Cholesterol Monolayer Response to Phenantrene, Anthracene, Pyrene, Chrysene, and Benzo[a]pyrene

Interactions of phenantrene, anthracene, pyrene, chrysene, and benzo[a]pyrene (polyaromatic hydrocarbons) with model phospholipid membranes were probed using the Langmuir technique. The lipid monolayers were prepared using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoserine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, and cholesterol. Surface pressure and electrical surface potential were measured on mixed phospholipid/PAH monolayers spread on a pure water subphase. The morphology of the mixed monolayers was followed with Brewster angle microscopy. Polarization-modulation infrared reflection-absorption spectroscopy spectra obtained on DPPE/benzo[a]pyrene showed that the latter interacts with the carbonyl groups of the phospholipid. On the other hand, the activity of phospholipase A2 toward DLPC used as a probe to locate benzo[a]pyrene in the monolayers indicates that the polyaromatic hydrocarbons are not accessible to the enzyme. The results obtained show that all PAHs studied affect the properties of the pure lipid, albeit in different ways. The most notable effects, namely, film fluidization and morphology changes, were observed with benzo[a]pyrene. In contrast, the complexity of mixed lipid monolayers makes the effect of PAHs difficult to detect. It can be assumed that the differences observed between PAHs in monolayers correlate with their toxicity.

Hydrogen Bonding and Binding of Polybasic Residues with Negatively Charged Mixed Lipid Monolayers

Phosphoinositides, phosphorylated products of phosphatidylinositol, are a family of phospholipids present in tiny amounts (1% or less) in the cytosolic surface of cell membranes, yet they play an astonishingly rich regulatory role, particularly in signaling processes. In this letter, we use molecular dynamics simulations on a model system of mixed lipid monolayers to investigate the interaction of phosphatidylinositol 4,5-bisphosphate (PIP2), the most common of the phosphoinositides, with a polybasic peptide consisting of 13 lysines. Our results show that the polybasic peptide sequesters three PIP2 molecules, forming a complex stabilized by the formation of multiple hydrogen bonds between PIP2 and the Lys residues. We also show that the polybasic peptide does not sequester other charged phospholipids such as phosphatidylserine because of the inability to form long-lived stable hydrogen bonds.

Sum-Frequency Spectroscopy Analysis of Two-Component Langmuir Monolayers and the Associated Interfacial Water Structure

Sum-frequency spectroscopy (SFS) in the CH and OH stretching regions was employed to obtain structural information about Langmuir monolayers on the H(2)O subphase of the model lipid dioctadecyldimethylammonium bromide (DOMA) and of the neutral surfactant methyl stearate (SME) and their mixtures and about the interfacial water structure underneath the films. These results were compared with the sum-frequency spectra of the interface between Langmuir monolayers of stearic acid and stearic acid-DOMA monolayers and water to prove that the uncompensated headgroup charge of DOMA at the interface is the reason for structuring of interfacial water close to the studied monomolecular films. Sum-frequency spectra on D(2)O subphase were also studied to account for the interference between the CH and OH spectral signatures because of the coherent nature of the SFS signals. Interfacial water structure proved to be a determining factor in the behavior of the mixed lipid monolayers. A mixing induced amplification in the surface potential DeltaV observed in our previous work was explained with total increase of the dipole moment for the mixed films, bigger than the arithmetic average for DOMA and SME monolayers alone. The increase is due to the better packing of the molecules in the mixed films and to the decrease in the interfacial water dipole moment arising from a more disordered water structure underneath the mixed monolayers.

Properties of Mixed Lipid Monolayers Assembled on Hydrophobic Surfaces through Vesicle Adsorption

Supported lipid films are becoming increasingly important tools for the study of membrane protein function because of the availability of high-sensitivity surface analytical and patterning techniques. In this study, we have characterized the physical chemical properties of lipid films assembled on hydrophobic surfaces through the spontaneous adsorption of large unilamellar lipid vesicles composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). The density of the lipid films was measured with surface plasmon resonance spectroscopy as the lipid composition of the vesicles and ionic concentration were varied. As expected, monolayer films were formed, but the density of the monolayers was found to be weakly dependent on the lipid composition of the vesicles and strongly dependent on the ionic concentration of the solution in contact with the monolayer. Atomic force microscopy (AFM) images of the lipid films indicate that they are composed of a homogeneous monolayer. Surface force measurements were used to determine the surface charge and DOPG density of the monolayers. The DOPG content of the films was found to be weakly dependent on the DOPG composition of the vesicles and strongly dependent on the salt concentration of the environment. A model has been developed to describe the behavior of the lipid composition of the films in terms of the hydrophobic, electrostatic, and steric forces acting on the lipid monolayer on the hydrophobic surface.

Area Expansion and Adsorption Isotherm in Protein Binding to Mixed Lipid Membranes

We present a thermodynamic analysis of protein adsorption to a mixed lipid monolayer containing a binding component and a nonbinding component at constant surface pressure. We derive the general equations for the adsorption isotherm and for the area expansion of the monolayer. In the limits of either low or high surface coverage, simplified forms of these equations are obtained. We construct simple thermodynamic models to examine the crossover from low to high surface coverage.

Functional microdomains of glycolipids with partially fluorinated membrane anchors: impact on cell adhesion.

Functional microdomains of glycolipids were designed by mixing neoglycolipids with partially fluorinated alkyl (F-alkyl) chains and matrix lipids with alkyl chains. Fluorescence images of the mixed lipid monolayers at the air-water interface demonstrated that it is possible to control both size and distribution of the microdomains by means of the strong demixing of alkyl and F-alkyl membrane anchors, while the carbohydrate head groups seemed to play a rather minor role. These microdomains in monolayers could be transferred onto hydrophobized substrates and subjected to experiments in a dynamic flow chamber. The results obtained here clearly indicated that the dynamic adhesion of Chinese hamster ovarial cells expressing E-selectin (CHO-E cells) on a lipid monolayer containing microdomains of sialyl Lewisx (sLex) can be both enhanced and reduced by controlled demixing of ligands and matrices. Moreover, the same clusters of sLex could also be formed in giant lipid vesicles, which can be used as a model cell that locally expresses biospecific functions.

Monolayer techniques for studying lipase kinetics.

Publisher Summary This chapter discusses the why lipid monolayers is used as lipase substrates for studying lipase kinetics. The monolayer is a suitable model system to study the enzymatic reactions carried out at the interfaces in a heterogeneous medium. There are at least five major reasons for using lipid monolayers as substrates for lipolytic enzymes. To use the monolayer technique for lipase kinetics, one necessary condition is that the substrate must be insoluble in the aqueous subphase and must form stable monomolecular films. The monolayer technique is ideally suited for studying the mode of action of lipolytic enzymes at interfaces using controlled mixtures of lipids. Two methods exist for forming mixed lipid monolayers at the air/water interface: either by spreading a mixture of water-insoluble lipids from a volatile organic solvent, or by injecting a micellar detergent solution into the aqueous subphase covered with preformed insoluble lipid monolayers.

The Influence of Lipid Dipole Moment and Interfacial Water Structure on Protein Adsorption to Mixed Lipid Monolayers

AbstractProtein interactions with a heterogeneous surface, such as a two‐component lipid film, often deviate from simple additivity of the adsorption to the respective single component, “homogenous” films. This behavior was highlighted in previous research where adsorption of the negatively charged protein ferritin to mixed cationic / non‐ionic lipid films was greater than adsorption to either of the single component lipid films [Britt et al., Phys. Chem. Chem. Phys., 2, 4594‐4599, 2000]. Here we investigate mixing induced changes in lipid dipole moments and local water structure as factors leading to amplification of monolayer charge and subsequent enhanced protein adsorption to the mixed lipid films. Surface potential and Fourier transform infrared spectroscopy (FTIR) revealed a reduction in both lipid tilt angle and gauche defects in the mixtures that would lead to a greater contribution of lipid alkyl‐tail dipoles to the measured (normal) surface potential; however, as the orientation of the alkyl‐tail dipole is from water (negative) to air (positive) the greater dipolar contribution for reduced tilt angles actually opposes adsorption of a negatively charged protein from the subphase. FTIR also revealed a mixing induced disordering of water solvating the cationic lipid head group, suggesting water dipoles were less effective in screening charge, which in turn would enhance the electrostatic interactions between anionic protein and the mixed cationic / nonionic lipid film.

Langmuir monolayers to study interactions at model membrane surfaces

Langmuir monolayers at the liquid–air interface are well-defined interfacial systems and, therefore, excellent model systems to learn about interactions at interfaces beyond the classical DLVO description. Many parameters can be independently varied over a broad range and the structure can be analyzed with Å precision. In the first part of the paper, the rich polymorphism in monolayers composed of amphiphilic molecules is demonstrated. Using homologues series generic phase diagrams can be derived. The delicate interplay of interactions causes a richness of phases which in turn can be used to measure fine variations in these interactions. Based on the understanding of the polymorphism in pure or mixed lipid monolayers, one can study the interaction of molecules dissolved in the subphase with monolayers. Samples presented are chemical reactions catalyzed by enzymes and coupling of polyelectrolytes to oppositely charged monolayers. To relate structure and reactivity, the activity of enzymes at the interface can be studied, predominantly combining X-ray diffraction and FTIR-spectroscopy. It is shown that the activity depends on monolayer structure. In one case, the reaction product leads to structural changes in the monolayer and stops the reaction, hence, indicating a subtle case of product inhibition via the membrane. On the other hand it has become possible to manipulate the organization of polyelectrolytes at interfaces via lipid charge density and ionic strength. In the most important case of DNA interacting with a membrane surface we show that DNA arranges at the interface in a lamellar manner, and the intermolecular distances, measured by Synchrotron X-ray diffraction can be varied by the lipid density.

Shape Instabilities in Charged Lipid Domains

A theoretical model describing the formation and shapes of domains in amphiphilic monolayers driven by screened electrostatic interactions is presented. The model is related to previous studies of pattern formation in magnetic systems in Hele-Shaw cells. Using quantitative estimates of line tension, charge density, and ionic strength appropriate for biological systems, this model accounts for the appearance of circular and irregular domains in mixed lipid monolayer systems containing the anionic lipid phosphatidylinositol monophosphate. The results may have implications for pattern formation in both synthetic materials and biological membranes.

Imaging mixed lipid monolayers by dynamic atomic force microscopy

Phase imaging with tapping mode atomic force microscopy (AFM) and force modulation microscopy were used to probe the mechanical properties of phase-separated lipid monolayers made of a mixture (0.25:0.75) of the surface-active lipopeptide surfactin and of dipalmitoylphosphatidylcholine (DPPC). The π– A isotherms and the result of a molecular modeling study revealed a loose, 2-D liquid-like organization for the surfactin molecules and a closely packed, 2-D solid-like organization for DPPC molecules. This difference in molecular organization was responsible for a significant contrast in height, tapping mode phase and force modulation amplitude images. Phase imaging at light tapping, i.e., with a ratio of the set-point tapping amplitude with respect to the free amplitude A sp/ A 0≈0.9, showed larger phase shifts on the solid-like DPPC domains attributed to larger Young’s modulus. However, contrast inversion was observed for A sp/ A 0<0.7, suggesting that at moderate and hard tapping the image contrast was dominated by the probe–sample contact area. Surprisingly, force modulation amplitude images showed larger stiffness for the liquid-like surfactin domains, suggesting that the contrast was dominated by contact area effects rather than by Young’s modulus. These data emphasize the complex nature of the contrast mechanisms of dynamic AFM images recorded on mixed lipid monolayers.

Streptavidin arrays as supramolecular architectures in surface-plasmon optical sensor formats

We describe a series of interfacial biomolecular architectures employed for studies aimed at elucidating the structural and dynamical factors governing biorecognition and binding reactions between surface-attached biotin-derivatives and analogues and streptavidin from solution. We compare binding matrices based on mixed lipid monolayers transferred to a solid support from the water/air-interface and surface coatings based on binary mixed self-assembled monolayers. The analytical techniques used to monitor the various layers and the binding events are based on surface-plasmon spectroscopy and microscopy, in some cases in combination with electrochemical methods controlling, in particular, the surface potential of electrode surfaces in contact with aqueous electrolyte solutions. Finally, an example is given for the sensitivity enhancement obtainable by combining the field-enhancement of surface-plasmon resonances with fluorescence detection schemes.

Modulation of phospholipase A2 by electrostatic fields and dipole potential of glycosphingolipids in monolayers

Phospholipase A2 activity against mixed monolayers of dilauroylphosphatidic acid or dilauroylphosphatidylcholine with glycosphingolipids can be reversibly modulated by external constant electrostatic fields. The changes of enzymatic activity are correlated to the depolarization or hyperpolarization of the film caused by specific dipolar properties of glycosphingolipids. Hyperpolarizing fields enhance the enzymatic activity against pure dilauroylphosphatidic acid while depolarizing fields induce a decrease of activity. Compared to the pure substrate, the interface of mixed films containing neutral glycosphingolipids or gangliosides is already partially depolarized and the magnitude of activation induced by an external hyperpolarizing field is decreased; conversely, depolarizing fields cause an increased inhibition of activity. Differing from gangliosides, sulfatides bring about a hyperpolarization of the mixed lipid monolayer and external hyperpolarizing or depolarizing fields cause enhanced activation and reduced inhibition, respectively. The effects of glycosphingolipids depend on their relative proportion in the monolayer. Results were similar with dilauroylphosphosphatidylcholine but the field effects were less than half of those found with dilauroylphosphatidic acid. Our work shows that the activity of phospholipase A2 in addition to responding reversibly to external electrostatic fields, is directly modulated by the polarity and magnitude of the lipid polar head group dipole moments.—Maggio, B. Modulation of phospholipase A2 by electrostatic fields and dipole potential of glycosphingolipids in monolayers. J. Lipid Res. 1999. 40: 930–939.

A Neutron Reflectivity Study of Polymer-Modified Phospholipid Monolayers at the Solid-Solution Interface: Polyethylene Glycol-Lipids on Silane-Modified Substrates

The structure of polymer-decorated phospholipid monolayers at the solid-solution interface was investigated using neutron reflectometry. The monolayers were composed of distearoylphosphatidylethanolamine (DSPE) matrixed with varying amounts of DSPE-PEG (DSPE with polyethylene glycol covalently grafted to its headgroup). Mixed lipid monolayers were Langmuir-Blodgett deposited onto hydrophobic quartz or silicon substrates, previously hydrophobized by chemically grafting a robust monolayer of octadecyltrichlorosilane (OTS). We show that this method results in homogeneous and continuous phospholipid monolayers on the silanated substrates and determine that the grafted PEG chains extend away from the monolayers into the solvent phase as a function of their density, as expected from scaling theories. In addition, ligands were coupled to the end of the PEG chains and selective binding was demonstrated using fluorescence microscopy. Our results demonstrate that these constructs are ideal for further characterization and studies with well-defined monomolecular films.

Preparation and Properties of Functional Mixed-Lipid Liposomes by γ-Ray Irradiation

The feature of mixed-lipid liposomes such as polymerization and polymerized liposomes stability were investigated to find means for producing red cells containing hemoglobin inside the liposomes. The surface pressure-area isotherm values of the mixed-lipid monolayer indicated 1-stearoyl-2- (2, 4-octadecadienoyl) -glycero-3-phosphocholine (SOPC) to be immiscible in cholesterol (Chol) and stearic acid (SA), and each component to contain separate domains in the bilayer membrane of liposomes. Radiation induced polymerization of mixed-SOPC liposomes was carried out using γ-rays from60 Co at 4°C to stabilize lipid bilayers. The polymer yield increased significantly by adding Chol and SA to SOPC. The rate of polymerization of SOPC Liposomes increased linearly with increasing of dose rate. The molecular weight of the polymer decreased with an increase in irradiation time. Irradiated SOPC/Chol/SA liposome vesicle size was affected by freeze-thawing. The vesicle size did not change when SOPC/Chol/SA was present in the system due to the addition of immiscible saturated 1, 2-dipalmi-toyl-glycero-3-phosphocholine (DPPC).

Mutual modulation of sphingomyelinase and phospholipase A2 activities against mixed lipid monolayers by their lipid intermediates and glycosphingolipids.

Sphingomyelinase activity against pure sphingomyelin monolayers is constant up to a surface pressure of 18 mN/m and falls above it. Sphingomyelinase- and phospholipase A2-mediated phosphohydrolytic pathways are mutually modulated by the presence of their respective substrates and products. At 15 mN/m non-substrate lipids such as ceramide at a mole fraction of 0.1 in mixed films with the pure substrate, inhibit the sphingomyelinase activity. Ganglioside GM1, another ceramide-containing complex sphingolipid, also inhibits sphingomyelinase activity, while a chemically related glycosphingolipid such as asialo-GM1 has no effect. The activity is unaltered by dipalmitoylphosphatidylcholine and by an equimolar mixture of its products of hydrolysis by phospholipase A2, fatty acid and lysoderivative, but it is inhibited by only one of them or by dilauroylphosphatidylcholine. Phospholipase A2 is inhibited by sphingomyelin, and activated by ceramide and by palmitic acid, one of the products of its own phosphohydrolytic reaction.

Stability of Chlorophyll a at the Gas−Water Interface in Pure and Mixed Monolayers. An Evaluation of Interfacial pH

The stability of chlorophyll a, Chl a, in neat and mixed lipid monolayers at the nitrogen−water interface has been measured using Langmuir trough and HPLC techniques. In neat monolayers, at subphase pH less than 8, Chl a degrades rapidly to produce pheophytin a, Phe a. Above this pH, Chl a appears to hydrolyze, giving a new product, Chl a hyd, spectroscopically similar to the parent compound. This latter process appears to involve alteration at the pentanone ring peripheral to the porphyrin structure. For mixed monolayer studies, four oleoyl-bearing lipids with headgroups differing in charge have been used to explore the influence of such headgroups on these two pH dependent processes. Consistent with a mechanism by which a negatively charged headgroup may enhance H3O+ concentration in the monolayer region, it has been shown that l-α-dioleoylphosphatidyl-dl-glycerol markedly increases pheophytinization while eliminating the formation of Chl a hyd. By contrast, positively charged dioleoyl-N-(3-trimethylammoniopropyl)carbamoylglycerol serves to generate formation of Chl a hyd with no production of Phe a. In neutral but polar 1,2-dioleoyl-sn-glycerol monolayers, product yields were observed that are consistent with the larger acidity in the region of Chl a. Finally, l-α-dioleoylphosphatidylcholine, which bears a zwitterionic headgroup, significantly enhanced apparent Chl a hydrolysis. These results are interpreted in terms of alterations by the host lipid in pH local to Chl a.