PEG-covered lipid surfaces: bilayers and monolayers

In this review paper we survey the ways in which various micropipet techniques have been used to study the mechanochemical and interactive features of lipid bilayer vesicles and monolayer-coated gas bubbles. Special emphasis will be made on characterizing the barrier properties of grafted PEG layers and how a hierarchical approach that uses a short barrier and extended ligand allows us to start to mimic nature's own solution to the problem of ubiquitous repulsion and specific attraction. The information gained from such studies not only characterizes the membrane and other lipid surfaces and their intersurface interactions from a fundamental materials science perspective, but also provides essential materials property data that are required for the successful design and deployment of lipid-based carriers and other capsules in applications involving this so-called ‘stealthy’ surface.     

Interaction studies of diacyl glycerol arginine-based surfactants with DPPC and DMPC monolayers, relation with antimicrobial activity

Antimicrobial agents have a major practical importance in food, pharmaceutical and cosmetic applications for preventing contamination. Our group has developed a novel class of cationic diacyl glycerol arginine-based surfactants denoted 1414RAc and 1212RAc. To assess the antimicrobial properties of these new surfactants we have studied how they interact with 1,2-dipalmitoyl-sn-glycero-3-phosphocoline (DPPC) and 1,2-dimiristoyl-sn-glycero-3-phosphocoline (DMPC) as model membranes, as well with living organisms endowed or not with external barriers, such as gram negative bacteria, the human protozoa Leishmania and mammalian cell line.

The structure and phase characteristics of mixed monolayers have been assessed through the analysis of static elasticity. The extent of component miscibility of surfactant and phospholipid in mixed monolayers has been studied using the additivity rule and the excess free energy of a mixture as a function of the phospholipid molar fraction for different surface pressure values. In all the mixtures studied, the mixed monolayer is thermodynamically favoured except for the 1212RAc/DMPC case that exhibits positive values of excess free energy.

The interaction with living cells has cytotoxic effects only in eukaryotic cells, by unspecific membrane permeabilization.     

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.     

Pressure-area relations for Langmuir monolayers

Pressure-area relations are derived for quasi-static compression of a Langmuir monolayer in the regimes of expanded and partially compressed chains. A monolayer is treated as an ensemble of flexible chains anchored at a flat water-air interface. Formation of surface pressure is attributed to excluded-volume interactions between segments of hydrophobic tail-groups. Close-form expressions are derived for the energy of interactions. Based on this relations, strength of excluded-volume interactions is evaluated by fitting pressure-area isotherms on stearic, behenic and pentacosadiynoic acids. It is found that the dimensionless strength of excluded-volume interactions is weakly affected by chain length and its value is close to .     

Properties of monolayers of crown ethers bearing an azo and azoxy group in the macrocycle

Two methods are described for preparing monomolecular layers of crown ethers with an azo or azoxy group in the macrocycle. When the molecules used to build the monolayer are soluble in aqueous solutions, adsorptive preconcentration on mercury electrodes was used to prepare the monolayer coating. The monolayer was electroactive due to the presence of the azo or azoxy unit in the molecules. Monolayers of crown ethers bearing an azo group in the macrocycle were shown to recognize alkali metal cations present in the solution. Changes of the parameters of the voltammetric reduction peaks - peak potential and peak width, served as an indication of specific interactions of the monolayer of 13-membered and 16-membered azocrown ethers with Na⁺ and K⁺ cations, respectively.The monolayers capable of recognizing cations have also been prepared on the aqueous solution-air interface, using the Langmuir technique. In this approach, amphiphilic derivatives of the azocrowns were synthesized and the monolayer has been assembled on the subphase containing metal cations. Binding of the cation by the macrocycle has a stabilizing effect on the monolayer and higher collapse pressures are achieved than on the pure water subphase. The monolayer was transferred from the air-water interface on the solid substrate using the Langmuir-Blodgett technique. Thin mercury film electrodes on the Ag substrate, or An films evaporated on glass slides were employed as the electrode substrates. The former gave monolayer modified electrodes of higher stability.Presented at the Sixth International Seminar on Inclusion Compounds, Istanbul, Turkey, 27–31 August, 1995.     

Interactions of Triton X-100 with sphingomyelin and phosphatidylcholine monolayers: influence of the cholesterol content

The presence of microdomains, called lipid rafts, in biological membranes is usually explained by lateral segregation between specific lipids and proteins. These rafts present similarities with the membrane domains isolated by their non-ionic detergent-resistance at 4 degrees C. They are enriched in sphingomyelin and cholesterol as compared with the outer leaflet of eukaryotic cell membranes. To understand the role played by the lipids enriched in rafts in their resistance to solubilization by detergents, the interactions between these lipids and the non-ionic detergent Triton X-100 were studied by using different lipid monolayers at the air-water interface. The influence of Triton X-100 on the Langmuir isotherms (i.e. surface pressure/area isotherms) of monolayers containing sphingomyelin and cholesterol at different mole ratios was analyzed and the results were compared with the influence of Triton X-100 on monolayers containing a phosphatidylcholine bearing a saturated and an unsaturated fatty acid (i.e. palmitoyloleylphosphatidylcholine) and cholesterol. This phosphatidylcholine was chosen since the phosphatidylcholines present in rafts isolated from bovine kidney could contain about 50% of saturated fatty acids. Triton X-100 induces an increase in the condensing effect observed as compared with ideal mixture of phospholipid/cholesterol. Triton X-100-induced changes in the morphology of the monolayers were visualized by Brewster angle microscopy, which confirmed the differences of behavior observed by analyzing the isotherms.     

Molecular interactions between lipid bilayers and a water-soluble polymer

Molecular interactions between lipid bilayers (liposomes) and chondroitin sulfate C (CS), a water soluble polymer, have been investigated in terms of zeta-potential, particle size, microscopic-viscosity, microscopic-polarity of liposomes and permeability of calcein. Microscopic morphology is dramatically changed by the addition of CS to the positively charged liposomes (Pos.L), while it is not changed by the addition to uncharged liposomes (Unc.L) or negatively charged liposomes (Neg.L). The absolute value of the particle size of Pos.L increases with the addition of CS, while the zeta- potential of Pos.L decreases. Permeability of Pos.L decreases with an increase in the concentration of CS. Phase transition temperature of Pos.L is changed after the addition of CS. These values, however, are not changed for the other liposomes by the addition of CS. The results of gel filtration chromatography show that CS is absorbed on the Pos.L surface. Microscopic viscosity is also increased by the addition of CS to Pos.L due to the adsorption of CS.     

Langmur monolayers of cerebroside with different head groups originated from sea cucumber: Binary systems with dipalmitoylphosphatidylcholine (DPPC)

Surface properties (Langmuir monolayer) of two different cerebrosides which are extracted from the sea cucumber (Bohadschia argus) were investigated. A main difference in chemical structure of cerebroside between BAC-2a and BAC-4 is their head groups (glucose and galactose, respectively). Furthermore, miscibility and interaction between dipalmitoylphosphatidylcholine (DPPC) and cerebrosides (BAC-2a and BAC-4) in the monolayer have been systematically examined. The surface pressure (π)−area (A), the surface potential (ΔV)−A, and the dipole moment (μ)−A isotherms for monolayers of DPPC, cerebrosides, and their binary combinations have been measured using the Wilhelmy method and the ionizing electrode method. BAC-4 forms a stable liquid-expanded (LE) monolayer, whereas BAC-2a has a first-order phase transition from the LE phase to the liquid-condensed (LC) state on 0.15 M NaCl at 298.2 K. The fundamental properties for each cerebroside monolayer were elucidated in terms of the surface dipole moment based on the three-layer model [R.J. Demchak, T. Fort Jr., J. Colloid Interface Sci. 46 (1974) 191–202] for both cerebrosides and the apparent molar quantity change (Δs^γ, Δh^γ, and Δu^γ) for BAC-2a. In addition, their miscibility with DPPC was examined by the variation of the molecular areas and the surface potentials as a function of cerebroside mole fractions, the additivity rule. The miscibility was also confirmed by constructing the two-dimensional phase diagrams. The phase diagrams for the both binary systems were of negative azeotropic type. That is, the two-component DPPC/BAC-2a and DPPC/BAC-4 monolayers are miscible. Furthermore, the Joos equation for the analysis of the collapse pressure of binary monolayers allowed calculation of the interaction parameter and the interaction energy between the DPPC and cerebroside monolayers. The miscibility in the monolayer state was also confirmed by the morphological observation with Brewster angle microscopy (BAM), fluorescence microscopy (FM), and atomic force microscopy (AFM).     

Study of the cholesterol-GM3 ganglioside interaction by surface pressure measurements and fluorescence microscopy

The nature of the cholesterol/glycolipid interaction in rafts being poorly understood, the interaction of cholesterol with the GM₃ ganglioside has been studied by surface pressure measurements and fluorescence microscopy. Results have been compared to those obtained with sphingomyelin (SM)-cholesterol and palmitoyl-oleoyl-phosphatidylcholine (POPC)–cholesterol monolayers. The analysis of (π–A) isotherms of mixed monolayers show a condensing effect of cholesterol on GM₃ molecules, in the same range than the effect observed with POPC and higher than the effect on SM. This is likely due to the similar state of GM₃ and POPC, since both molecules are in liquid expanded phases in our experimental conditions. The study of the cholesterol desorption induced by β-cyclodextrin suggests also that the GM₃–cholesterol interaction is rather weak as in the case of POPC–cholesterol interaction, and clearly lower than SM–cholesterol one. This lack of interaction is discussed in terms of nature of lipid chains and molecular shape, and suggests that no hydrogen bond is formed between GM₃ and cholesterol polar heads. Fluorescence microscopy performed on mixed GM₃–cholesterol monolayers shows the presence, at surface pressure higher than 10 mN/m, of particular blurring patterns without defined boundary, which could be due to a partial solubilization in one phase of different phases observed at lower surface pressure, whereas SM–cholesterol and POPC–cholesterol monolayers are homogeneous at the lateral resolution of our microscopy set-up.     

Compressibility study of quaternary phospholipid blend monolayers

The mechanical properties of liposome membranes are strongly dependent on type and ratio of lipid compounds, which can have important role in drug targeting and release processes when liposome is used as drug carrier. In this work we have used Brewster's angle microscopy to monitor the lateral compression process of lipid monolayers containing as helper lipids either distearoyl phosphatidylethanolamine (DSPE) or dioleoyl phophatidylethanolamine (DOPE) molecules on the Langmuir trough. The compressibility coefficient was determined for lipid blend monolayers containing the helper lipids above, cholesterol, distearoyl phosphatidylcholine (DSPC) and pegylated-DSPE at room temperature. Two variables, the cholesterol fraction and the ratio ρ between the helper lipid (either DSPE or DOPE) and the reference lipid DSPC, were studied by multivariate analysis to evaluate their impact on the compressibility coefficient of the monolayers. The cholesterol level was found to be the most significant variable for DSPE blends while the ratio ρ was the most significant one for DOPE blend monolayers. It was also found that these two variables can exhibit positive interaction and the same compressibility value can be obtained with different blend compositions.     

Studies on curcumin and curcuminoids. XXXIV. Photophysical properties of a symmetrical, non-substituted curcumin analogue

Curcumin is the main constituent of curry. In its ground state it shows chemo-preventive, chemo-therapeutic, anti-inflammatory and immune stimulating effects, and it is considered as a drug or drug model in the treatment of AIDS and cystic fibrosis. Further biological activity is induced in curcumin by light exposure: cytotoxicity is enhanced and photosensitized antibacterial effects are achieved. For the curcumin cis enol conformer, the fastest deactivation mechanism of the first excited singlet state is an excited-state intra-molecular proton transfer, which brings curcumin back to the ground state. This mechanism, as well as reketonization, interaction with the solvent and photodegradation, compete with the phototherapeutic action. The native compound curcumin carries phenolic hydroxyl and methoxy groups that influence the molecular charge distribution and hence the excited-state intra-molecular proton transfer rate in an unpredictable way. In this work we study static and time-resolved spectroscopic properties of a non-substituted curcuminoid that lacks both the phenolic hydroxyl and the phenolic methoxy groups. The photophysical properties of this compound are compared to those of native curcumin, in order to provide a rationale to the design of curcuminoids with molecular structures optimized for a photosensitizer.     

Probing the interplay between amyloidogenic proteins and membranes using lipid monolayers and bilayers

Many degenerative diseases such as Alzheimer's and Parkinson's involve proteins that have a tendency to misfold and aggregate eventually forming amyloid fibers. This review describes the use of monolayers, bilayers, supported membranes, and vesicles as model systems that have helped elucidate the mechanisms and consequences of the interactions between amyloidogenic proteins and membranes. These are twofold: membranes favor the formation of amyloid structures and these induce damage in those membranes. We describe studies that show how interfaces, especially charged ones, favor amyloidogenic protein aggregation by several means. First, surfaces increase the effective protein concentration reducing a three-dimensional system to a two-dimensional one. Second, charged surfaces allow electrostatic interactions with the protein. Anionic lipids as well as rafts, rich in cholesterol and gangliosides, prove to play an especially important role. Finally, these amphipathic systems also offer a hydrophobic environment favoring conformational changes, oligomerization, and eventual formation of mature fibers. In addition, we examine several models for membrane permeabilization: protein pores, leakage induced by extraction of lipids, chaotic pores, and membrane tension, presenting illustrative examples of experimental evidence in support of these models. The picture that emerges from recent work is one where more than one mechanism is in play. Which mechanism prevails depends on the protein, its aggregation state, and the lipid environment in which the interactions occur.     

Synthesis and luminescent properties of a silylated-terpyridine derivative and its metalated complexes in solutions and self-assembled monolayers

A silylated-terpyridine(Si TPy) derivative was newly synthesized and reacted with various transition metal ions in the solutions and self-assembled monolayers(SAMs).Composition and morphology of the SAMs were characterized by using absorption spectra,X-ray photoelectron spectra and atomic force microscope.The silylated-TPy compound gave off a luminescent emission at about 456 nm,which slightly shifted to 452 nm in the Zn2+-Si TPy and Fe2+-Si TPy metalated complexes.The absorbed energy can be further transferred to lanthanide ions(Tb3+and Eu3+) to give off the typical emissions of the lanthanide complexes together with an emission of the silylated-TPy at about 363 nm.     

Interaction of the antimalarial agents halofantrine and lumefantrine with lipid bilayers

The effects of antimalarial drugs halofantrine and lumefantrine on the fluoresence anisotropy of diphenylhexatriene (DPH)-containing phospholipid vesicles have been examined. Lumefantrine increases DPH anisotropy, indicating a condensing effect on bilayers of dipalmitoylphosphatidyl choline (DPPC), dioleoylphosphatidylcholine (DOPC), egg lecithin and mouse erythrocyte membranes (including membranes isolated from plasmodial-infected mice). Its condensing effect is more pronounced in bilayers of lower microviscosity. In contrast, increases or decreases in DPH anisotropy are observed with halofantrine, depending on the nature of the lipid. Decreases in anisotropy, which reflect a perturbing effect, are observed in bilayers of high microviscosity (for example, gel state of DPPC bilayers). Increases in anisotropy are observed in bilayers of low microviscosity (such as DOPC and egg lecithin bilayers). The perturbing effect of halofantrine is further confirmed by the increases in permeability of calcein-containing DPPC vesicles in the presence of the drug. However the perturbative effects of halofantrine are observed to the same magnitude in uninfected and plasmodial-infected erythrocyte membranes, and may not be relevant to the antimalarial action of the drug. In contrast, the condensing effect of lumefantrine is significantly greater in infected erythrocyte membranes and may contribute to its antimalarial action.     

Patterned supported lipid bilayers and monolayers on poly(dimethylsiloxane)

A simple and practical method for patterning supported lipid bilayers on poly(dimethylsiloxane) is presented. By using electron microscopy grids to laterally control the extent of plasma oxidation, the substrate is partitioned into regions of different hydrophilicities. Addition of vesicles then results in the spontaneous formation of lipid bilayers and monolayers side-by-side on the surface, separated by regions that contain no lipid and/or a region with adhering vesicles. By using millimeter-sized plastic masks we are able to control the formation of these lipid structures on macroscopic patches by simply varying the plasma-cleaning time. For the first time, we are able to influence, in a controlled fashion, the chemical composition of a substrate in such a way that it supports fluid lipid monolayers, rejects lipid adhesion, adsorbs intact lipid vesicles, or supports fluid bilayers.     

Interaction of antimicrobial arginine-based cationic surfactants with liposomes and lipid monolayers

The present work examines the relationship between the antimicrobial activity of novel arginine-based cationic surfactants and the physicochemical process involved in the perturbation of the cell membrane. To this end, the interaction of these surfactants with two biomembrane models, namely, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar lipid vesicles (MLVs) and monolayers of DPPC, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DPPG), and Escherichia coli total lipid extract, was investigated. For the sake of comparison, this study included two commercial antimicrobial agents, hexadecyltrimethylammonium bromide and chlorhexidine dihydrochloride. Changes in the thermotropic phase transition parameters of DPPC MLVs in the presence of the compounds were studied by differential scanning calorimetry analysis. The results show that variations in both the transition temperature (Tm) and the transition width at half-height of the heat absorption peak (deltaT1/2) were consistent with the antimicrobial activity of the compounds. Penetration kinetics and compression isotherm studies performed with DPPC, DPPG, and E. coli total lipid extract monolayers indicated that both steric hindrance effects and electrostatic forces explained the antimicrobial agent-lipid interaction. Overall, in DPPC monolayers single-chain surfactants had the highest penetration capacity, whereas gemini surfactants were the most active in DPPG systems. The compression isotherms showed an expansion of the monolayers compared with that of pure lipids, indicating an insertion of the compounds into the lipid molecules. Owing to their cationic character, they are incorporated better into the negatively charged DPPG than into zwitterionic DPPC lipid monolayers.     

Interaction of rabbit C-reactive protein with phospholipid monolayers at air/water interface

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. The structure of CRP has been previously established by crystallography, and the significance of its interaction with lipid membranes is accepted in the literature. However, the nature of the interaction between CRP and phospholipids is not yet well understood. In this paper we use monolayer technique to study the characteristics of the interaction of rabbit C-reactive protein (rCRP) with the phospholipid membranes. The results show that rCRP is surface active and can spontaneously insert into the lipid monolayers. The critical pressure for rCRP inserting into the phospholipid monolayers is about 34.5 mN/m, which is not sensitive to the types of the lipid headgroups and the presence of calcium ions in the subphase. The findings of this paper may provide a clue to the further understanding of the mechanism of the interactions between rCRP and the biological membranes.     

Effect of dipolar moments in domain sizes of lipid bilayers and monolayers

Lipid domains are found in systems such as multicomponent bilayer membranes and single component monolayers at the air-water interface. It was shown by Keller et al. [J. Phys. Chem. 91, 6417 (1987)] that in monolayers, the size of the domains results from balancing the line tension, which favors the formation of a large single circular domain, against the electrostatic cost of assembling the dipolar moments of the lipids. In this paper, we present an exact analytical expression for the electric potential, ion distribution, and electrostatic free energy for different problems consisting of three different slabs with different dielectric constants and Debye lengths, with a circular homogeneous dipolar density in the middle slab. From these solutions, we extend the calculation of domain sizes for monolayers to include the effects of finite ionic strength, dielectric discontinuities (or image charges), and the polarizability of the dipoles and further generalize the calculations to account for domains in lipid bilayers. In monolayers, the size of the domains is dependent on the different dielectric constants but independent of ionic strength. In asymmetric bilayers, where the inner and outer leaflets have different dipolar densities, domains show a strong size dependence with ionic strength, with molecular-sized domains that grow to macroscopic phase separation with increasing ionic strength. We discuss the implications of the results for experiments and briefly consider their relation to other two dimensional systems such as Wigner crystals or heteroepitaxial growth.     

The study of the interaction of a model alpha-helical peptide with lipid bilayers and monolayers

We studied the interaction of the alpha-helical peptide acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)) with tethered bilayer lipid membranes (tBLM) and lipid monolayers formed at an air-water interface. The interaction of L(24) with tBLM resulted in adsorption of the peptide to the surface of the bilayer, characterized by a binding constant K(c)=2.4+/-0.6 microM(-1). The peptide L(24) an induced decrease of the elasticity modulus of the tBLM in a direction perpendicular to the membrane surface, E(radial). The decrease of E(radial) with increasing peptide concentration can be connected with a disordering effect of the peptide to the tBLM structure. The pure peptide formed a stable monolayer at the air/water interface. The pressure-area isotherms were characterized by a transition of the peptide monolayer, which probably corresponds of the partial intercalation of the alpha-helixes at higher surface pressure. Interaction of the peptide molecules with lipid monolayers resulted in an increase of the mean molecular area of phospholipids both in the gel and liquid crystalline states. With increasing peptide concentration, the temperature of the phase transition of the monolayer shifted toward lower temperatures. The analysis showed that the peptide-lipid monolayer is not an ideally miscible system and that the peptide molecules form aggregates in the monolayer.     

Preparation and structure evolution of bowknot-like calcium carbonate particles in the presence of poly(sodium 4-styrene sulfate)

We determined how glucose or insulin interacts with a phospholipid monolayer at the air/water interface and explained these mechanisms from a physico-chemical point of view. The 1,2-dipalmitoyl-2-sn-glycero-3-phosphatidylcholine (DPPC) monolayer at an air/water interface acted as a model membrane, which allowed the effect of the molecular packing density in the monolayer on the interactions to be determined. The interaction of glucose, insulin, and a mixture of glucose and insulin to the DPPC monolayer were investigated via surface pressure-area per molecule Langmuir isotherms and fluorescence microscopy. Glucose adsorbed to the underside of the DPPC monolayer, while insulin was able to penetrate through the monolayer when the phospholipid molecules were not densely packed. The presence of a mixture of insulin and glucose affected the molecular packing in the DPPC monolayer differently than the pure insulin or glucose solutions, and the glucose-insulin mixture was seen to be able to penetrate through the monolayer. These results indicated that glucose and insulin interact with one another, giving a material that may then transported through a pore in the monolayer or through the spaces between the molecules of the monolayer.