A Langmuir film approach to elucidating interactions in lipid membranes: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine/cholesterol/metal cation systems

The interactions between two membrane lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and cholesterol (CHOL), were studied in Langmuir films using surface pressure isotherms and Brewster angle microscopy. The DPPE/CHOL interactions were probed for chosen monolayer and subphase (Na(+), Ca(2+)) composition at 20, 25, and 30 degrees C. The results obtained show that DPPE and CHOL are miscible for the cholesterol mol fractions x(CHOL)=0.3-0.5. Cholesterol induces condensation of the DPPE monolayers. The most significant condensation of the DPPE/CHOL monolayers was observed in the presence of Ca(2+) ions in the subphase at x(CHOL)=0.4. The negative deviation of the molecular surface area (MMA) additivity from the ideal behavior together with negative values of excess free enthalpy of mixing in the monolayers were interpreted in terms of attractive interactions between lipid molecules.     

Comparison of the interaction of tomatine with mixed monolayers containing phospholipid, egg sphingomyelin, and sterols

The interaction of the glycoalkaloid tomatine with monolayers of a phospholipid (dimyristoylphosphatidylcholine, DMPC), and sphingolipid (egg sphingomyelin), and cholesterol is compared. Using measurements of the surface pressure response as a function of the subphase concentration of tomatine, interfacial binding constants are estimated for mixed monolayers of DMPC and cholesterol and for those of egg sphingomyelin and cholesterol of mole ratio 7:3. The binding constants obtained suggest a stronger interaction of tomatine with DMPC and cholesterol mixed monolayers, reflecting easier displacement of cholesterol from its interaction with DMPC than from its interaction with egg sphingomyelin. Mixtures of tomatine and cholesterol are found to spread directly at the water-air interface and form stable monolayers, suggesting that cholesterol holds tomatine at the interface despite the absence of observed monolayer behavior for tomatine alone. The interaction of tomatine with DMPC and cholesterol monolayers is found to exhibit a pH dependence in agreement with previously reported results for its interaction with liposomes; in particular, the interaction is much less at pH 5 than at pH 7 or pH 9. It is found that while tomatine interacts strongly with monolayers containing sitosterol, it does not interact with monolayers containing sitosterol glucoside. The response of monolayers of varying composition of DMPC and cholesterol to tomatine is also examined. Brewster angle microscopy (BAM) reveals further evidence for formation of suspected islands of tomatine + cholesterol complexes upon interaction with mixed monolayers of lipid and sterol.     

Model study of interactions of high-molecular dextran sulfate with lipid monolayers and foam films

The interaction of high-molecular dextran sulfate (DS-5000) with dimyristoylphosphatidylcholine (DMPC) monolayers and foam films (FF) at the air–water interface in the presence of Ca²⁺ and Na⁺ ions was studied. DS-5000 was added in monolayer films (MF) and in FF as monomer molecules and in liposomal form. When added in liposomal form in FF, DS-5000 decreased the stability of DMPC common black films (CBF), and no formation of Newton black films (NBF) was observed. However, when included as monomer molecules in FF, DS-5000 caused film thinning, and drastically decreased the expansion rate of the black spots and transition of thick films to NBF, thus avoiding formation of CBF. The above effects were observed in both gel and liquid-crystalline phase states of DMPC in the presence of Ca²⁺ ions only, and not in the presence of Na⁺ ions. We postulate that the interaction of DMPC with DS-5000 in the plane of FF is mediated by Ca²⁺ bridges and results in dehydration of the DMPC polar heads. The interaction between DMPC and DS-5000 in monolayers resulted in slower adsorption and spreading of DMPC molecules at the interface, lower monolayer surface pressure, and penetration of DS-5000 molecules to DMPC monolayers when surface lipid density was higher than 50 Ų per DMPC molecule. The applicability of the FF model for studying the interactions of phospholipids with polysaccharides at interfaces surrounded by bulk solution, and for modeling such interactions in biological systems, e.g. LDL adhesion to the arterial walls, aggregation and fusion of liposomes, etc., is discussed.     

Investigations of antimicrobial peptides in planar film systems

Planar systems - monolayers and films - constitute a useful platform for studying membrane-active peptides. Here, we summarize varied approaches for studying peptide organization and peptide-lipid interactions at the air/water interface, and focus on three representative antimicrobial membrane-associated peptides—alamethicin, gramicidin, and valinomycin. Experimental data, specifically surface pressure/area isotherms and Brewster angle microscopy images, provided information on peptide association and the effects of the lipid monolayers on peptide surface organization. In general, film analysis emphasized the effects of lipid layers in promoting peptide association and aggregation at the air/water interface. Importantly, the data demonstrated that in many cases peptide domains are phase-separated within the phospholipid monolayers, suggesting that this behavior contributes to the biological actions of membrane-active antimicrobial peptides.     

Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.     

Influence of surface chemistry on the structural organization of monomolecular protein layers adsorbed to functionalized aqueous interfaces.

The molecular organization of streptavidin (SA) bound to aqueous surface monolayers of biotin-functionalized lipids and binary lipid mixtures has been investigated with neutron reflectivity and electron and fluorescence microscopy. The substitution of deuterons (2H) for protons (1H), both in subphase water molecules and in the alkyl chains of the lipid surface monolayer, was utilized to determine the interface structure on the molecular length scale. In all cases studied, the protein forms monomolecular layers underneath the interface with thickness values of approximately 40 A. A systematic dependence of the structural properties of such self-assembled SA monolayers on the surface chemistry was observed: the lateral protein density depends on the length of the spacer connecting the biotin moiety and its hydrophobic anchor. The hydration of the lipid head groups in the protein-bound state depends on the dipole moment density at the interface.     

Investigations of antimicrobial peptides in planar film systems

Planar systems--monolayers and films--constitute a useful platform for studying membrane-active peptides. Here, we summarize varied approaches for studying peptide organization and peptide-lipid interactions at the air/water interface, and focus on three representative antimicrobial membrane--associated peptides-alamethicin, gramicidin, and valinomycin. Experimental data, specifically surface pressure/area isotherms and Brewster angle microscopy images, provided information on peptide association and the effects of the lipid monolayers on peptide surface organization. In general, film analysis emphasized the effects of lipid layers in promoting peptide association and aggregation at the air/water interface. Importantly, the data demonstrated that in many cases peptide domains are phase-separated within the phospholipid monolayers, suggesting that this behavior contributes to the biological actions of membrane-active antimicrobial peptides.     

Anaesthetic — phospholipid interaction

Binding of the positively charged drug chlorpromazine to phospholipid monolayers was investigated. A preferential uptake was observed near the phase transtion of the corresponding lipid. Cholesterol considerably diminishes the chlorpromazine uptake, again particularly near a lipid phase transition. The binding properties depend on the chlorpromazine concentration in the subphase. A critical concentration is 5·10^-5M, where higher uptake occurs in the liquid condensed than in the liquid expanded state of the monolayer at pressures of about 10 mN/m. Dipalmitoylphosphatidylcholine monolayers spread on a subphase containing chlorpromazine are comparable to monolayers at higher temperature but in the absence of chlorpromazine. These data are in agreement with previous fluorescence and electron paramagnetic resonance experiments on lipid bilayer membranes (Luxnat and Galla 1986).Abbreviations CPZ chlorpromazine - DPPC dipalmitoylphosphatidylcholine - DMPC dimyristoylphosphatidylcholine - LE liquid expanded - LC liquid condensed     

A phospholipid bilayer supported under a polymerized Langmuir film

Phospholipid single bilayers supported on a hydrophilic solid substrate are extensively used in the study of the interaction between model membranes and proteins or polypeptides. In this article, the formation of a single dimyristoylphosphatidylcholine (DMPC) bilayer under an octadecyltrimethoxysilane (OTMS) polymerized Langmuir monolayer at the air-water interface is followed by Brewster angle microscopy (BAM) and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). The formation of the bilayer is initiated by injection of dimyristoylphosphatidylcholine small unilamellar vesicles into the aqueous subphase. Brewster angle microscopy allows visualization of the kinetics of formation and the homogeneity of the bilayer. Spectral simulations of the polarization-modulated infrared reflection absorption spectroscopy spectra reveal that the bilayer thickness is 39 +/- 5 A. This system constitutes the first example of a phospholipid bilayer on a "nanoscopic" support and opens the way to studies involving supported bilayers using powerful experimental techniques such as x-ray reflectivity, vibrational spectroscopies, or Brewster angle microscopy.     

The influence of an antitumor lipid – erucylphosphocholine – on artificial lipid raft system modeled as Langmuir monolayer

Abstract

Outer layer of cellular membrane contains ordered domains enriched in cholesterol and sphingolipids, called ‘lipid rafts’, which play various biological roles, i.e., are involved in the induction of cell death by apoptosis. Recent studies have shown that these domains may constitute binding sites for selected drugs. For example alkylphosphocholines (APCs), which are new-generation antitumor agents characterized by high selectivity and broad spectrum of activity, are known to have their molecular targets located at cellular membrane and their selective accumulation in tumor cells has been hypothesized to be linked with the alternation of biophysical properties of lipid rafts. To get a deeper insight into this issue, interactions between representative APC: erucylphosphocholine, and artificial lipid raft system, modeled as Langmuir monolayer (composed of cholesterol and sphingomyelin mixed in 1:2 proportion) were investigated. The Langmuir monolayer experiments, based on recording surface pressure-area isotherms, were complemented with Brewster angle microscopy results, which enabled direct visualization of the monolayers structure. In addition, the investigated monolayers were transferred onto solid supports and studied with AFM. The interactions between model raft system and erucylphosphocholine were analyzed qualitatively (with mean molecular area values) as well as quantitatively (with ΔG^exc function). The obtained results indicate that erucylphosphocholine introduced to raft-mimicking model membrane causes fluidizing effect and weakens the interactions between cholesterol and sphingomyelin, which results in phase separation at high surface pressures. This leads to the redistribution of cholesterol molecules in model raft, which confirms the results observed in biological studies.     

Equivalent Aqueous Phase Modulation of Domain Segregation in Myelin Monolayers and Bilayer Vesicles

Purified myelin can be spread as monomolecular films at the air/aqueous interface. These films were visualized by fluorescence and Brewster angle microscopy, showing phase coexistence at low and medium surface pressures (<20-30 mN/m). Beyond this threshold, the film becomes homogeneous or not, depending on the aqueous subphase composition. Pure water as well as sucrose, glycerol, dimethylsulfoxide, and dimethylformamide solutions (20% in water) produced monolayers that become homogeneous at high surface pressures; on the other hand, the presence of salts (NaCl, CaCl₂) in Ringer's and physiological solution leads to phase domain microheterogeneity over the whole compression isotherm. These results show that surface heterogeneity is favored by the ionic milieu. The modulation of the phase-mixing behavior in monolayers is paralleled by the behavior of multilamellar vesicles as determined by small-angle and wide-angle x-ray scattering. The correspondence of the behavior of monolayers and multilayers is achieved only at high surface pressures near the equilibrium adsorption surface pressure; at lower surface pressures, the correspondence breaks down. The equilibrium surface tension on all subphases corresponds to that of the air/alkane interface (27 mN/m), independently on the surface tension of the clean subphase.     

Comparative study of the interaction of synthetic methionine-enkephalin and its amidated derivate with monolayers of zwitterionic and negatively charged phospholipids

Using Langmuir’s monolayer technique, the surface behavior and the interaction of the synthetic neuropeptide methionine-enkephalin (Met-enk) and its amidated derivate (Met-enk-NH₂) with monolayers of the zwitterionic dimyristoylphosphatidylcholine (DMPC) and the negatively charged dimyristoylphosphatidylglycerol (DMPG) were studied. The surface tension (γ, mN/m) of DMPG and DMPC monolayers as a function of time (after injection of the peptide under the interface) was detected. The decrease in γ values showed that there was a strong penetration effect of both types of Met-enk molecules into the monolayers, being significantly stronger for the amidated derivate, Met-enk-NH₂. We suggest that the interaction between the neuropeptides and DMPC was predominantly determined by peptides amphiphilicity, while the electrostatic forces play significant role for the insertion of the cationic Met-enk-NH₂ in DMPG monolayers, especially at high packing densities. Our results demonstrate the potential of lipid monolayers formed in Langmuir’s trough to be successfully used as an elegant and simple membrane models to study lipid–peptide interactions at the air/water interface.     

Influence of levofloxacin and clarithromycin on the structure of DPPC monolayers

Research on lipid/drug interactions at the nanoscale underpins the emergence of synergistic mechanisms for topical drug administration. The structural understanding of bio-mimetic systems employing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as a lung surfactant model mixed with antibiotics, as well as their biophysical properties, is of critical importance to modulate the effectiveness of therapeutic agents released directly to the airways. In this paper, we investigate the structural details of the interaction between Levofloxacin, ‘a respiratory quinolone’, and the macrolide Clarithromycin, with DPPC monolayers at the air-water interface, using a combination of Brewster angle microscopy, polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS), surface pressure isotherms and neutron reflectometry (NR) to describe the structural details of this interaction. The results allowed association of changes in the π-A isotherm profile with changes in the molecular organization and the co-localization of the antibiotics within the lipid monolayer by NR measurements. Overall, both antibiotics are able to increase the thickness of the acyl tails in DPPC monolayers with a corresponding reduction in tail tilt as well as to interact with the phospholipid headgroups as shown by PM-IRRAS experiments. The effects on the DPPC monolayers are correlated with the physical-chemical properties of each antibiotic and dependent on its concentration.     

Surface interactions determined by stereostructure on the example of 7-hydroxycholesterol epimers – The Langmuir monolayer study

Stereoselective interactions are pivotal for molecular recognition between biomolecules and lipid surfaces. The aim of this study was to determine factors differencing molecular interactions between 7-hydroxycholesterol epimers (oxysterols, which excessively appear in pathological processes in human body) and natural membrane phospholipids. Two-component systems of different mutual proportions of 7-hydroxycholesterol (7α-hydroxycholesterol or 7-β-hydroxycholesterol, in short 7α-OH or 7β-OH) and membrane lipids (POPC, DPPC, DPPE, DPPS, SM) were systematically analyzed in artificial membranes modeled as Langmuir monolayers. Classical surface pressure measurements were complemented with direct visualization of films texture both in situ (with Brewster angle microscopy, BAM) and after their transfer onto solid supports (with Atomic Force Microscopy, AFM). Our results clearly show striking differences in surface properties of the studied binary mixtures, emphasizing distinct effects of both 7-hydroxycholesterol epimers on the organization of lipid layers. Systematic study allowed to conclude that the structure of polar head group and interfacial region of the molecule play important role in oxysterol-phospholipid interactions, while the hydrophobic region is significantly less important in this respect.     

Mitochondrial creatine kinase interaction with heterogeneous monolayers: Effect on lipid lateral organization

Our study highlights the tight relationship between protein binding to monolayers and the phase-state of the phospholipids. Interaction of mitochondrial creatine kinase with phospholipidic membranes was analysed using a two-phase monolayer system containing anionic phospholipids under chain mismatch conditions. Monolayers were made up of mixtures of DMPC/DPPG or DPPC/DMPG containing 40% negatively charged phospholipids which is approximately the negative charge content of the mitochondrial inner membrane. Langmuir isotherms of these monolayers showed that they underwent a phase transition from a liquid expanded state to a liquid-condensed phase at about 2 mN/m and 5 mN/m respectively. Interface morphology modifications caused by injection of mtCK under these monolayers at low or high surface pressure were monitored by Brewster angle microscopy. This work provides evidence that the presence at the air/water interface of discrete domains with increased charge density, may lead to difference in partition of soluble proteins such as mtCK, interacting with the lipid monolayer. Conversely these proteins may help to organize charged phospholipid domains in a membrane.     

Mitochondrial Creatine Kinase Binding to Phospholipid Monolayers Induces Cardiolipin Segregation

It is well established that the octameric mitochondrial form of creatine kinase (mtCK) binds to the outer face of the inner mitochondrial membrane mainly via electrostatic interactions with cardiolipin (CL). However, little is known about the consequences of these interactions on membrane and protein levels. Brewster angle microscopy investigations provide, for the first time to our knowledge, images indicating that mtCK binding induced cluster formation on CL monolayers. The thickness of the clusters (10-12 nm) corresponds to the theoretical height of the mtCK-CL complex. Protein insertion into a condensed CL film, together with monolayer stabilization after protein addition, was observed by means of differential capacity measurements. Polarization modulation infrared reflection-absorption spectroscopy showed that the mean orientation of α-helices within the protein shifted upon CL binding from 30° to 45° with respect to the interface plane, demonstrating protein domain movements. A comparison of data obtained with CL and phosphatidylcholine/phosphatidylethanolamine/CL (2:1:1) monolayers indicates that mtCK is able to selectively recruit CL molecules within the mixed monolayer, consolidating and changing the morphology of the interfacial film. Therefore, CL-rich domains induced by mtCK binding could modulate mitochondrial inner membrane morphology into a raft-like organization and influence essential steps of mitochondria-mediated apoptosis.     

Effect of helper lipids on the interaction of DNA with cationic lipid monolayers studied by specular neutron reflection

The interaction of DNA with monolayers of the cationic lipid dimethyldioctadecylammonium bromide, with/without 50 mol % of a neutral "helper" lipid, either dioleoylphosphatidylethanolamine or cholesterol, has been studied using specular neutron reflection, surface pressure-area isotherms, and Brewster angle microscopy. The amount of DNA bound to the lipid head groups has been comprehensively quantified in the range of 8-39 vol% of DNA with respect to the monolayer composition (monolayers composed of dimethyldioctadecylammonium bromide binding the most DNA and monolayers containing dioleoylphosphatidylethanolamine binding the least) and surface pressure (DNA binding being greatest at highest surface pressures). Surprisingly, regardless of these variables, the thickness of the DNA-containing layer remained approximately constant between 18 and 25 ?. This systematic study is the first direct quantification of the binding of DNA with two different helper-lipid-containing multicomponent monolayers, an important step toward understanding interaction parameters in more realistic models of gene delivery systems.     

Incorporation of beta-lactoglobulin in a lipid/porphyrin monolayer at the air--water interface

A catanionic lipid/porphyrin monolayer was formed at the air-water interface by the tetra-anionic porphyrin, tetra-sodium-meso-tetra(4-sulfonatophenyl)porphine (TSPP), mixed with the cationic lipid dioctadecyldimethylammonium bromide (DODAB) in a 1:4 molar ratio. This binary mixture (TSPP/4DODAB) was used as the incorporation matrix of beta-lactoglobulin (betaLG). Binary and ternary systems (TSPP/4DODAB/zbetaLG, where z stands for the number of protein residues per TSPP) were characterized by surface pressure versus area (pi-A) measurements and by Brewster angle microscopy (BAM) observation at the air-water interface. Pi-A measurements and BAM images show that protein is incorporated in the expanded regime of the monolayer and is gradually expelled upon compression at high surface pressures. The successive compression-expansion cycles indicate that the protein under adsorbed to the floating film is reincorporated after the expansion of the monolayer. At low subphase pH, TSPP tends to aggregate decreasing the interaction with DODAB molecules. Electrostatic and hydrophobic interactions are responsible for the presence of betaLG at the interfacial film.     

Domain structure and molecular conformation in annexin V/1,2-dimyristoyl-sn-glycero-3-phosphate/Ca2+ aqueous monolayers: a Brewster angle microscopy/infrared reflection-absorption spectroscopy study.

Annexins comprise a family of proteins that exhibit a Ca2+-dependent binding to phospholipid membranes that is possibly relevant to their in vivo function. Although substantial structural information about the ternary (protein/lipid/Ca2+) interaction in bulk phases has been derived from a variety of techniques, little is known about the temporal and spatial organization of ternary monolayer films. The effect of Ca2+ on the interactions between annexin V (AxV) and anionic DMPA monolayers was therefore investigated using three complementary approaches: surface pressure measurements, infrared reflection-absorption spectroscopy (IRRAS), and Brewster angle microscopy (BAM). In the absence of Ca2+, the injection of AxV into an aqueous subphase beneath a DMPA monolayer initially in a liquid expanded phase produced BAM images revealing domains of protein presumably surrounded by liquid-expanded lipid. The protein-rich areas expanded with time, resulting in reduction of the area available to the DMPA and, eventually, in the formation of condensed lipid domains in spatial regions separate from the protein film. There was thus no evidence for a specific binary AxV/lipid interaction. In contrast, injection of AxV/Ca2+ at a total Ca2+ concentration of 10 microM beneath a DMPA monolayer revealed no pure protein domains, but rather the slow formation of pinhead structures. This was followed by slow (>2 h) rigidification of the whole film accompanied by an increase in surface pressure, and connection of solid domains to form a structure resembling strings of pearls. These changes were characteristic of this specific ternary interaction. Acyl chain conformational order of the DMPA, as measured by nu(sym)CH2 near 2850 cm(-1), was increased in both the AxV/DMPA and AxV/DMPA/Ca2+ monolayers compared to either DMPA monolayers alone or in the presence of Ca2+. The utility of the combined structural and temporal information derived from these three complementary techniques for the study of monolayers in situ at the air/water interface is evident from this work.