The lung surfactant activity probed with molecular dynamics simulations.

The surface of pulmonary alveolar subphase is covered with a mixture of lipids and proteins. This lung surfactant plays a crucial role in lung functioning. It shows a complex phase behavior which can be altered by the interaction with third molecules such as drugs or pollutants. For studying multicomponent biological systems, it is of interest to couple experimental approach with computational modelling yielding atomic-scale information. Simple two, three, or four-component model systems showed to be useful for getting more insight in the interaction between lipids, lipids and proteins or lipids and proteins with drugs and impurities. These systems were studied theoretically using molecular dynamic simulations and experimentally by means of the Langmuir technique. A better understanding of the structure and behavior of lung surfactants obtained from this research is relevant for developing new synthetic surfactants for efficient therapies, and may contribute to public health protection.

The Molecular Bases of the Interaction between a Saponin from the Roots of Gypsophila paniculata L. and Model Lipid Membranes.

In view of the possible medical applications of saponins, the molecular structure of a GOTCAB saponin from the roots of Gypsophila paniculata L. was determined by NMR. The biological activity of saponins may depend on the interaction with cell membranes. To obtain more insight in the mechanism of membrane-related saponin function, an experimental and theoretical study was conducted. Ternary lipid systems composed of sphingomyelin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and cholesterol were used as models of mammalian cell membranes. The membrane-saponin interaction was studied experimentally by monitoring surface pressure in the monomolecular films formed at the air-aqueous subphase interface. The behavior of GOTCAB saponin in a water box and model monolayer systems was characterized by molecular dynamics simulations. The results obtained showed that, in the systems used, cholesterol had a decisive effect on the interaction between GOTCAB and phosphocholine or sphingomyelin as well as on its location within the lipid film.

Lung surfactant monolayer - A good natural barrier against dibenzo-p-dioxins.

The present study deals with interaction of two air pollutants: dibenzodioxin, DD, and its' monochlorinated derivative, 2-chlorodibenzodioxin, 2CLDD, with models of the lung surfactant (LS) system. A monolayer composed of DPPC and POPC in 1:1 molar ratio was used as a model of LS. One component monolayers of DPPC and POPC were also examined, to model the interiors of LC and LE domains in LS, respectively. Molecular dynamics simulations and measurements of surface pressure isotherms, as well as polarization modulation-infrared reflection-absorption spectra were employed to study the influence of dioxins on the monolayers. We demonstrate, that both dioxins adsorb and accumulate in the hydrophobic parts of all three monolayers. DD molecules prefer flat orientation on the surface at large areas. Upon compression, they lift and orient perpendicularly to the monolayer. Flat orientation of DD molecules leads to their large surface area. In consequence they preferentially locate in vicinity of unsaturated chains of POPC - they are small enough to fill void spaces created by kinks in unsaturated chains. 2CLDD orient along monolayer normal already at the largest areas and preference for POPC was not observed for them. In laterally relaxed states, a condensing effect, connected with reduction of surface area available to the lipids was observed for both dioxins. In the case of 2CLDD, additional locally ordering influence of dioxin molecules was detected. In compressed states, the presence of dioxin molecules hinders alignment and uniform ordering of lipid chains.

Nanoscale investigation of the interaction of colistin with model phospholipid membranes by Langmuir technique, and combined infrared and force spectroscopies.

Colistin (Polymyxin E), an antimicrobial peptide, is increasingly put forward as salvage for severe multidrug-resistant infections. Unfortunately, colistin is potentially toxic to mammalian cells. A better understanding of the interaction with specific components of the cell membranes may be helpful in controlling the factors that may enhance toxicity. Here, we report a physico-chemical study of model phospholipid (PL) mono- and bilayers exposed to colistin at different concentrations by Langmuir technique, atomic force microscopy (AFM) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The effect of colistin on chosen PL monolayers was examined. Insights into the topographical and elastic changes in the PL bilayers within time after peptide injection are presented via AFM imaging and force spectra. Finally, changes in the PL bilayers' ATR-FTIR spectra as a function of time within three bilayer compositions, and the influence of colistin on their spectral fingerprint are examined together with the time-evolution of the Amide II and νCO band integrated intensity ratios. Our study reveals a great importance in the role of the PL composition as well as the peptide concentration on the action of colistin on PL model membranes.

Two antibacterial nalidixate calixarene derivatives in cholesterol monolayers: Molecular dynamics and physicochemical effects.

The interaction of two antibacterial calixarene derivatives with cholesterol, a eukaryotic cell membrane lipid, was investigated with the aim to get more insight in the potential advers effects on our cells. The derivatives used had one or two nalidixic acid arms grafted on the lower rim of the calixarene aromatic crown. Monomolecular films spread at the air-water interface were used as model lipid membranes. Pure cholesterol and pure calixarene derivatives, as well as binary cholesterol - calixarene derivative mixtures were studied using surface pressure measurements, polarization-modulation infrared reflection absorption spectroscopy and molecular dynamics simulations. The properties of the mixed monolayers were described quantitatively using thermodynamic models. The analysis of surface pressure-area isotherms of mixed monolayers shows that cholesterol may form homogenous but metastable domains with both nalidixate derivatives. This phenomenon is more clearly observed with mono-substituted calixarene. A detailed modeling analysis indicates that cholesterol favors dehydration of the calixarene polar headgroups and transfer of the derivatives from the aqueous to the gas phase. This effect, more pronounced in the case of the monosubstituted calixarene, can be linked to the hydrophobic interaction with cholesterol. This observation may be useful for developing new calixarene derivatives allowing us to control disease-causing bacteria without harming our own cells.

Structure - membrane activity relationship in a family of peptide-based gemini amphiphiles: An insight from experimental and theoretical model systems.

A study of the interaction between five gemini amphiphilic valine-based pseudopeptides (GAPs) differing by the length of the central aliphatic spacer linking two amino acid subunits, and a model bacterial membrane lipid, 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG), is here presented. Pure DMPG, pure GAPs and mixed GAPs/DMPG monolayers were formed at the air-water interface using Langmuir technique. The properties of the Langmuir films were investigated using surface pressure measurements, polarization modulation-infrared reflection-absorption spectroscopy, and Brewster angle microscopy. The atomic level information concerning the orientation of molecules in the monolayer and hydration of the polar headgroups was obtained from molecular dynamics simulations. It was demonstrated that the length of the central spacer in the GAPs structure is important for the properties of the mixed films; the disorganization of the membrane increases with the length of the spacer. The latter point is important for developing possible antimicrobial agents based on GAPs.

The selective interactions of cationic tetra-p-guanidinoethylcalix[4]arene with lipid membranes: theoretical and experimental model studies.

Behavior of cationic tetra-p-guanidinoethylcalix[4]arene (CX1) and its building block, p-guanidinoethylphenol (mCX1) in model monolayer lipid membranes was investigated using all atom molecular dynamics simulations and surface pressure measurements. Members of two classes of lipids were taken into account: zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and anionic 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine sodium salt (DMPS) as models of eukaryotic and bacterial cell membranes, respectively. It was demonstrated that CX1 and mCX1 accumulate near the negatively charged DMPS monolayers. The adsorption to neutral monolayers was negligible. In contrast to mCX1, CX1 penetrated into the hydrophobic part of the monolayer. The latter effect, which is possible due to a flip-flop inversion of the CX1 orientation in the lipid layer compared to the aqueous phase, may be responsible for its antibacterial activity.

Impact of two different saponins on the organization of model lipid membranes.

Saponins, naturally occurring plant compounds are known for their biological and pharmacological activity. This activity is strongly related to the amphiphilic character of saponins that allows them to aggregate in aqueous solution and interact with membrane components. In this work, Langmuir monolayer techniques combined with polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and Brewster angle microscopy were used to study the interaction of selected saponins with lipid model membranes. Two structurally different saponins were used: digitonin and a commercial Merck Saponin. Membranes of different composition, namely, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) were formed at the air/water and air/saponin solution interfaces. The saponin-lipid interaction was characterized by changes in surface pressure, surface potential, surface morphology and PM-IRRAS signal. Both saponins interact with model membranes and change the physical state of membranes by perturbing the lipid acyl chain orientation. The changes in membrane fluidity were more significant upon the interaction with Merck Saponin. A higher affinity of saponins for cholesterol than phosphatidylglycerols was observed. Moreover, our results indicate that digitonin interacts strongly with cholesterol and solubilize the cholesterol monolayer at higher surface pressures. It was shown, that digitonin easily penetrate to the cholesterol monolayer and forms a hydrogen bond with the hydroxyl groups. These findings might be useful in further understanding of the saponin action at the membrane interface and of the mechanism of membrane lysis.

A Study of the Interaction between a Family of Gemini Amphiphilic Pseudopeptides and Model Monomolecular Film Membranes Formed with a Cardiolipin.

The interaction between five gemini amphiphilic pseudopeptides (GAPs) differing by the length of the central spacer and a model membrane lipid, 1,3-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (cardiolipin) were studied with the aim to evaluate their possible antimicrobial properties. To this end, monomolecular films were formed at the air/water interface with pure cardiolipin or cardiolipin/GAPs mixtures; film properties were determined using surface pressure and surface potential measurements, as well as polarization-modulation infrared reflection-absorption spectroscopy. Moreover, to better understand the GAPs-phospholipid interaction at the molecular level, molecular dynamics simulations were performed. The results obtained indicate that the length of the central spacer has an effect on the interaction of GAPs with cardiolipin and on the properties of the lipid film. The GAPs with the longer linkers can be expected to be useful for biological membrane modification and for possible antimicrobial applications.

Effect of products of PLA2 catalyzed hydrolysis of DLPC on motion of rising bubbles.

Local velocities of rising bubbles decrease with the increasing concentration in solution of surface-active, water-soluble species. Therefore, it is possible to use this phenomenon to monitor products of enzymatic reactions, which meet such criteria. In this study, hydrolysis of 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC) catalyzed by calcium-dependent phospholipase A2 (PLA2) (EC3.1.1.4) from porcine pancreas was used as model reaction. The products of this reaction are lauric acid (LA) and 1-lauroyl-2-hydroxy-sn-glycero-3-phosphatidylcholine (Lyso-PC). DLPC was dispersed in a chloroform/methanol mixture that was spread on a free PLA2 solution surface. Air bubbles were then formed at a capillary orifice and the local velocity of rising bubbles as a function of the distance from the capillary tip was monitored. Local velocity profiles were compared with profiles recorded for solutions of pure enzymatic reaction products and their mixtures. Our experiments showed that the product, which had a dominating effect on bubble motion retardation, was lyso-phosphatidylcholine. This can be explained by differences in the kinetics of lauric acid and lyso-phosphatidylcholine transfer from the spread layer to the solution.

Molecular organization of nalidixate conjugated calixarenes in bacterial model membranes probed by molecular dynamics simulation and Langmuir monolayer studies.

Two p-tert-butylcalix[4]arene derivatives bearing one or two nalidixic acid groups connected to the lower rim of p-tert-butylcalix[4]arene through the propylenic spacer were studied upon interaction with model bacterial membranes. Indeed, these derivatives were developed recently as new macrocyclic antibiotic carriers for antibacterial therapy. To obtain molecular level information about the interaction between the calixarene conjugates and a membrane lipid, atomistic molecular dynamics simulation, as well as surface pressure, surface potential, polarization modulation infrared reflection-absorption spectroscopy, and Brewster angle microscopy studies of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)-calixarene derivative films were performed. The results obtained indicate that the interaction between the calixarene derivatives and DMPE occurs via the phosphate and carbonyl groups present in the lipid. Although both calixarene derivatives increase the chain tilt and conformational disordering of the DMPE molecules, these effects are more important in the case of the monosubstituted derivative. Importantly, the two derivatives have an opposite impact on hydration of the phosphoglyceride polar head.

A Langmuir monolayer study of the action of phospholipase A2 on model phospholipid and mixed phospholipid-GM1 ganglioside membranes.

Polarization-modulation infrared reflection-absorption spectroscopy, surface pressure measurements and thermodynamic analysis were used to study enzymatic hydrolysis of lipid monolayers at the air/water interface. The Ca(2+)-requiring pork pancreatic phospholipase A2 was used as a catalyst. The substrates were pure 1,2-dilauroyl-sn-glycero-3-phosphocholine or mixed 1,2-dilauroyl-sn-glycero-3-phosphocholine - monosialotetrahexosylganglioside Langmuir films. The physicochemical properties of the monolayers were established with the aim of a correlation with enzyme activity. The infrared spectra were acquired upon the advancement of the catalysis; the latter was studied at a controlled surface pressure and area of the film. Changes of the intensity and frequency of different infrared signals characteristic for the two lipids were correlated with modification of the properties of the monolayer due to hydrolysis. The amide I signal characteristic for peptides permitted detecting the enzyme adsorbed at the interface. The thermodynamic and infrared results indicate that monosialotetrahexosylganglioside increases H-bonding of the lipid polar heads in the films. This effect, which may be responsible for the low activity of phospholipase A2 in the mixed films, could be used for developing enzyme-resistant lipid systems.

Effects of gemini amphiphilic pseudopeptides on model lipid membranes: a Langmuir monolayer study.

Monolayers formed with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] at the air/water interface were used as model membranes for studying a potential biological activity of four newly synthesized gemini amphiphilic pseudopeptides (GAPs); some of the GAPs studied showed interesting self-assembly properties. The capacity of GAPs to self-assemble in different environments let us think that these molecules may find biomedical applications in, e.g., drug delivery or transfection. The surface pressure-area and surface potential-area compression isotherms, as well as Brewster angle microscopy and polarization-modulation infrared reflection-absorption spectroscopy were used to study monolayers formed with pure GAPs, pure lipids and lipid/GAPs mixtures. The results obtained show that all four GAPs studied can be incorporated in lipid monolayers. The monolayers containing GAPs are expanded and more liquid-like compared to pure lipids. The overall results indicate that the important changes of the properties induced in the model membranes by GAPs are related to their intrinsic conformational flexibility. This feature of GAPs can be easily adjusted by engineering the structure of the spacer present in the polar head, with the aim to modify lipid membranes in a controlled way.

Interaction of a ß-lactam calixarene derivative with a model eukaryotic membrane affects the activity of PLA2.

In this research, the interaction between a membrane phospholipid, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and a p-tert-butylcalix[4]arene derivative bearing 6-aminopenicillanic acid (Calix), conceived as a possible drug carrier, was studied. The Langmuir film balance technique was used to measure surface pressure and electrical surface potential of pure and mixed Calix/DLPC monolayers spread on water at different temperatures. Phospholipase A2 (PLA2) activity was used as well to detect the impact of the calixarene derivative on the monolayer properties. Interaction between the molecules in mixed monolayers has been described quantitatively using thermodynamic functions. Interestingly, low amounts of Calix introduce ordering in the lipid film. This effect may be analogous to that of cholesterol interacting with phospholipids. A lower activity of PLA2 observed with the Calix/DLPC films compared to pure DLPC may be related to structural modifications of the mixed systems.

Penetration of milk-derived antimicrobial peptides into phospholipid monolayers as model biomembranes.

Three antimicrobial peptides derived from bovine milk proteins were examined with regard to penetration into insoluble monolayers formed with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG). Effects on surface pressure (Π) and electric surface potential (ΔV) were measured, Π with a platinum Wilhelmy plate and ΔV with a vibrating plate. The penetration measurements were performed under stationary diffusion conditions and upon the compression of the monolayers. The two type measurements showed greatly different effects of the peptide-lipid interactions. Results of the stationary penetration show that the peptide interactions with DPPC monolayer are weak, repulsive, and nonspecific while the interactions with DPPG monolayer are significant, attractive, and specific. These results are in accord with the fact that antimicrobial peptides disrupt bacteria membranes (negative) while no significant effect on the host membranes (neutral) is observed. No such discrimination was revealed from the compression isotherms. The latter indicate that squeezing the penetrant out of the monolayer upon compression does not allow for establishing the penetration equilibrium, so the monolayer remains supersaturated with the penetrant and shows an under-equilibrium orientation within the entire compression range, practically.

Preparation of meloxicam-ß-cyclodextrin-polyethylene glycol 6000 ternary system: characterization, in vitro and in vivo bioavailability.

Ternary complexes of meloxicam (ML), a poorly water-soluble anti-inflammatory drug, with ß-cyclodextrin (ßCD) and polyethylene glycol (PEG) 6000 were prepared from an equimolar (ML-ßCD) and 10% of PEG. Characterization of the ternary complex was carried out by differential scanning calorimetry and X-ray diffractometry. The solubility of ML increased as a function of increasing the concentration of ßCD and PEG 6000. Ternary system increased significantly ML solubility in water. Ternary complexes improved drug release compared with ML and ML-ßCD. The oral bioavailability of ML-ßCD-PEG was investigated by administration to rat and compared with ML and ML-ßCD. The results confirmed that the oral bioavailability of ML was significantly improved by complexation with ßCD in the presence of PEG.

Membrane activity of tetra-p-guanidinoethylcalix[4]arene as a possible reason for its antibacterial properties.

Tetra-p-guanidinoethylcalix[4]arene trifluoroacetate salt (CX1) was synthesized recently as an antibacterial agent. It showed to be active in vitro against various Gram-positive and Gram-negative bacteria. To get more insight in the mechanism of the biological activity of this derivative, it was studied upon interactions with model lipid membranes. Langmuir monolayers were formed with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, and with anionic 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) and 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine. The two classes of lipids were used, respectively, as model lipids of the eukaryotic and bacterial cell membranes. The monolayers were exposed to CX1 at different concentrations around the minimum inhibitory concentration found for E. coli . The surface pressure-area and surface potential-area compression isotherms, as well as Brewster angle microscopy and polarization-modulation infrared reflection-absorption spectroscopy, were employed to study the monolayers. The results obtained show a higher affinity of CX1 for the anionic lipids, indicating importance of charge-charge interactions. On the basis of a comparative study of the behavior of CX1 and that of p-guanidinoethylphenol trifluoroacetate salt, we propose that interplay of charge-charge and apolar interactions between CX1 and lipids is responsible for the important reorganization of model membranes. This proposal may be helpful in developing new antibacterial calixarene derivatives.

Glycolipid-cholesterol monolayers: towards a better understanding of the interaction between the membrane components.

In this work, the interaction between a synthetic analog of archaeal lipids and cholesterol was studied using Langmuir technique. The lipid, ß-Mal(3)O(C(16+4))(2), contained phytanyl chains attached via two ether bonds to the sn-2 carbon of the glycerol backbone. The preliminary studies showed that monolayers formed with the pure lipid have a liquid-like character; here, a hypothesis that admixing cholesterol to ß-Mal(3)O(C(16+4))(2) could confer a higher rigidity on the films was tested. To check this proposal, two-dimensional miscibility of cholesterol and ß-Mal(3)O(C(16+4))(2) in monomolecular films was studied using surface pressure and surface potential measurements, as well as Brewster angle microscopy and polarization-modulation infrared reflection absorption spectroscopy. The stability of the monomolecular films was evaluated based on thermodynamics of mixing of cholesterol and ß-Mal(3)O(C(16+4))(2). Atomic level information concerning the orientation of molecules and the degree of hydration of polar headgroups was obtained from molecular dynamics simulations.

Enzymatic probing of model lipid membranes: phospholipase A2 activity toward monolayers modified by oxicam NSAIDs.

Three nonsteroidal anti-inflammatory oxicam drugs, namely meloxicam, piroxicam, and tenoxicam, were used to modify the properties of monomolecular films formed with 1,2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, or 1,2-dilauroyl-sn-glycero-3-phospho-(1-rac-glycerol). These systems were examined via surface pressure and surface electrical potential measurements, polarization modulation infrared reflection absorption spectroscopy, and Brewster angle microscopy. Moreover, phospholipase A2 activity was used to differentiate between the three drugs. Our results reveal that the oxicams studied modify membrane properties, namely hydration of the lipid polar heads, orientation of the molecules, and morphology of the domains. Phospholipase A2 was shown to be sensitive to the presence of the drugs in the systems studied; the activity of the enzyme correlates with the effect of meloxicam, piroxicam, and tenoxicam on the monolayer properties. The latter indicates that the anti-inflammatory action of oxicams may be related to interference with phospholipase activity in addition to cyclooxygenase inhibition.

Structuring of supported hybrid phospholipid bilayers on electrodes with phospholipase A2.

The effect of a lipolytic enzyme, pork pancreatic phospholipase A(2), on hybrid bilayer membranes was monitored using voltammetry, impedance spectroscopy and surface plasmon resonance. The hybrid bilayers were prepared by Langmuir-Schaefer transfer of lipid monolayers onto gold electrodes modified with self-assembled alkanethiol monolayers, or by liposome spreading. The electrodes were immersed in the phospholipase aqueous solution to allow adsorption of the enzyme and cleavage of the ester bond in the sn-2 position of phospholipids in the outer leaflet of the hybrid layers. The action of phospholipase A(2) led to perforation of the lipid films. Impedance spectroscopy and surface plasmon resonance were used for monitoring enzyme adsorption, phospholipid hydrolysis and product desorption. The results obtained show that transport efficiency of an electroactive probe, ferrocyanate, and of an electroactive drug, doxorubicin, through the bilayer depends on the action of the enzyme; the state of the lipid layer covering the electrode surface depends on the latter as well. Cyclic voltammetry and electrochemical impedance spectroscopy were used to study this effect. The doxorubicin reduction/oxidation signals appearing at potentials close to those observed using a bare gold electrode indicated facilitated penetration of the drug into the layer. The results obtained were interpreted in terms of pore formation in the lipid matrix; phospholipase A(2) can be considered as a nano-device for high precision perforation of the lipid layer.