A. Tsanova

Foam film study of albumin inhibited lung surfactant preparations: effect of added hydrophilic polymers

In Adult and Acute Respiratory Distress Syndrome (ARDS) the concentration of albumin in the alveolar fluid reaches 25–100 mg ml−1. Due to its high adsorption rate albumin adsorbs at the air/water interface making it inaccessible for the lung surfactant (LS). LS inactivation can be prevented by hydrophilic polymers due to depletion attraction osmotic pressure or other specific action. Two commercially available lung surfactant preparations, LSP (Curosurf and Survanta), and four hydrophilic polymers (PEG, dextran, PVP, hyaluronic acid-HA) were investigated in control experiments at one (monolayer) and two interacting air/solution interfaces (foam film) under albumin free and albumin inactivated conditions. The established procedure to measure the minimal surface tension in successive compression–expansion cycles of monolayers revealed that minimal surface tension ≤10 mN m−1 for Survanta is achieved with PEG, PVP, and HA, while for Curosurf with dextran, PVP, and HA. This correlates with the observations of Lu et al., 2005. Foam film experiments with the microinterferometric method of Scheludko and Exerowa revealed the relative ability of the polymers to decrease the effect of albumin inhibition in restoring the formation of stable and homogeneous black films (thickness <17 nm). Stable black foam films are formed by Survanta with PEG, PVP, and HA, while by Curosurf with dextran, PVP, and HA. Kinetic data of Curosurf foam film thinning were interpreted to obtain values of the disjoining pressure. An interesting change from repulsion to attraction was observed at a thickness of appr. 100 nm. It may be explained by depletion attraction overcoming the steric repulsion. The magnitude of the depletion attraction was estimated.

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.

Interaction of methionine-enkephalins with raft-forming lipids: monolayers and BAM experiments.

Enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are opioid peptides with proven antinociceptive action in organism. They interact with opioid receptors belonging to G-protein coupled receptor superfamily. It is known that these receptors are located preferably in membrane rafts composed mainly of sphingomyelin (Sm), cholesterol (Cho), and phosphatidylcholine. In the present work, using Langmuir’s monolayer technique in combination with Wilhelmy’s method for measuring the surface pressure, the interaction of synthetic methionine–enkephalin and its amidated derivative with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Sm, and Cho, as well as with their double and triple mixtures, was studied. From the pressure/area isotherms measured, the compressional moduli of the lipids and lipid–peptide monolayers were determined. Our results showed that the addition of the synthetic enkephalins to the monolayers studied led to change in the lipid monolayers characteristics, which was more evident in enkephalinamide case. In addition, using Brewster angle microscopy (BAM), the surface morphology of the lipid monolayers, before and after the injection of both enkephalins, was determined. The BAM images showed an increase in surface density of the mixed surface lipids/enkephalins films, especially with double and triple component lipid mixtures. This effect was more pronounced for the enkephalinamide as well. These observations showed that there was an interaction between the peptides and the raft-forming lipids, which was stronger for the amidated peptide, suggesting a difference in folding of both enkephalins. Our research demonstrates the potential of lipid monolayers for elegant and simple membrane models to study lipid–peptide interactions at the plane of biomembranes.

Effects of Leucin-Enkephalins on Surface Characteristics and Morphology of Model Membranes Composed of Raft-Forming Lipids

During the last decades opioid peptides, like enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are subject to extensive studies due to their antinociceptive action in organism. According to the membrane catalysis theory, in order to adopt a proper conformation for binding to their receptors, opioid peptides interact with the lipid phase of the membrane receptor surrounding. With this regard, the aim of the present work was to study the effects of synthetic leucine-enkephalin and leucine-enkephalinamide on surface characteristics and morphology of lipid monolayers, composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin, and cholesterol alone and with their mixtures. The lipids were chosen to represent a model of a membrane raft, since it is known that G-protein-coupled receptors, including opioid receptors, are located preferably in membrane rafts. By using Langmuir’s monolayer method, the change in surface pressure of the model membranes before and after the addition of the synthetic enkephalins was studied, and the compressional moduli of the lipids and lipid–peptides monolayers were determined. In addition, by Brewster angle microscopy, the surface morphology of the lipid monolayers alone and after the injection of both enkephalins was monitored. Our results showed that both leucine-enkephalins affected the lipid monolayers surface characteristics, and led to an increase in surface density of the mixed surface lipids/enkephalins films at loose lipid packing. This effect was more pronounced for the enkephalinamide, suggesting a different mechanism of interaction for the amidated enkephalin with the lipid phase, as compared to leucine-enkephalin.

Interaction of monogalactosyldiacylglycerol with cytochrome b6f complex in surface films

The interaction of monogalactosyldiacylglycerol (MGDG) with cytochrome b(6)f complex (cyt b(6)f), a major component of the photosynthetic apparatus, was studied in Langmuir monolayers during compression/expansion cycling and at constant surface pressure mode. The surface pressure/area isotherms of the mixed films were analyzed in terms of surface compressional modulus and two-dimensional virial equation of state. The morphology and the surface potential of the monolayers were monitored by Brewster angle microscopy and vibrating plate sensor respectively. Our results suggested that there is a specific interaction between MGDG and cyt b(6)f which resulted in depletion of lipid molecules from the interface. The current work sheds light on the still unclear question how b(6)f complex gets in touch with the major compound of the thylakoid membranes, the non-charged lipid MGDG. The interaction occured even at very low sub-nanomolar concentration of the complex. This effect most probably could be attributed to hydrogen bonding between the galactose headgroup of the lipid and the protein moiety of cyt b(6)f.

In Vitro Application of Langmuir Monolayer Model to Study In Vivo Biological Systems

ABSTRACT Langmuir monolayer model is applied to study molecular interactions between phospholipids and surface active compounds and to determine the lateral elasticity and compressibility of the surface films at air-liquid interface. The interaction of the synthetic Methionine-enkephalin (Met-enk) and its amidated derivative (Met-enk-NH2) with monolayers of the zwitterionic dimyristoylphosphatidylcholine and the negatively charged dimyristoylphosphatidylglycerol were evaluated by measurements of surface tension (γ, mN/m) - time dependencies after injection of enkephalins under the Langmuir monolayer. The decrease of γ values during time 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. The behavior of tear film lipid layer (TFLL) is studied in dynamic conditions by compression/expansion cycling of Langmuir films. It allows determining reciprocal compressibility, CS−1, and reversibility, Rv, of the tear films at air-liquid interface. Our results showed very high lateral elasticity (i.e. low reciprocal compressibility) and high structural reversibility of the meibomian surface films which was confirmed by Brewster Angle Microscopy images. It is shown that even at low surface pressures (between 0.5 and 10 mN/m) human meibum forms rough continuous multilayer films at the air/water interface that get more uniform when surface pressure increases. Our results demonstrate the potential of lipid monolayers formed in Langmuirs through to be successfully used as an elegant and simple model to study molecular interactions and properties of surface films at the air/water interface.

Interfacial Tensiometry of Tracheal Aspirate from Infants with Neonatal Respiratory Distress Syndrome

The interfacial properties of tracheal aspirate from infants with untreated neonatal respiratory distress syndrome (NRDS), and NRDS infants after therapy with the exogenous surfactant Curosurf® were assessed. The interfacial characteristics of the aspirate (equilibrium surface tension, maximal and minimal surface tension during lateral compression-decompression cycles) were determined with the pendant drop method. Our results show that the tracheal aspirate of infants with untreated NRDS had high equilibrium, maximal and minimal surface tension values. In contrast, the samples from infants, treated with Curosurf®, showed lower surface tension values, suggesting that the application of Curosurf® improves the composition and the properties of the pulmonary surfactant in the infant lung.

Tuning of Surface Properties of Thin Lipid-Protein Films by Hydrophilic Non-Surface Active Polymers

ABSTRACT We study the capability of hydrophilic polymers (HPs) to reverse the inhibitory effect of albumin and to restore the surface activity of lipid-protein lung surfactants preparations (LSPs): Curosurf and Survanta. HPs implied are dextran and Polyethylene glycol 10000 (PEG10000). LSPs surface activity is evaluated in Thin Films (monolayer and foam) at the air/water interface. Albumin displaces LSP from the interface and inhibits their ability to sustain low surface tension in monolayers and to form stable thin foam films (FFs). When HPs are added to monolayers they restore the surface activity of LSPs and recover their capability to maintain low dynamic surface tensions. Similarly the inclusion of HPs in foam films results in neutralization of the effect of albumin and in formation of stable, thin (with thickness <17 nm) films with homogeneous surfaces, as in the case of pure LSPs. Our findings permit to search for the answer of the intriguing problem why PEG recovers better the surface activity of Survanta, while Dextran—of Curosurf. Such results might be of major importance for the treatment of Adult Respiratory Distress Syndrome.

Comparative Study of the Interaction Between Synthetic Methionine-Enkephalin and Monolayers of Zwitterionic and Negatively Charged Phospholipids

ABSTRACT Using Langmuirs monolayer technique, surface behaviour and the interaction between a synthetic analogue of endogenous neuropeptide Methionine-enkephalin (Met-enk) and zwitterionic dimyristoylphosphatidylcholine (DMPC), and the negatively charged dimyristoylphosphatidylglycerol (DMPG) at air/water interface are studied. By using the Wilhelmy method, surface tension (γ, mN/m) changes (after injection of the peptide under the interface) of DMPG and DMPC monolayers as a function of time at given molecular area are detected. The decrease in γ values shows that there is a strong penetration effect of Met-enk molecules into the monolayers. The comparison of the interaction between the hydrophobic pentapeptide and both phospholipid monolayers shows that the enkephalin interacts similarly with DMPG and DMPC molecules, and that the ability of Met-enk to penetrate correlates with the given surface area per lipid molecule. Based on the results obtained we suggest that in case of liquid-expanded monolayers the electrostatic attraction facilitates the interaction between the neuropeptide and the negatively charged phospholipids (DMPG), while at more compact ordering of lipid molecules the penetration of the enkephalin is preliminary due to hydrophobic interactions.

Investigation of the Interaction between Three-Block Copolymers with Phospholipid Monolayers and foam Films

ABSTRACT The interaction of three-block copolymers (poloxamers) with dimiristoyl-phosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) is investigated experimentally at one (monolayer) and two interacting air/water interfaces (foam film). The poloxamers selected are F-88 and F-98. Both of them possess two hydrophilic polyoxyethylene (POE) and one hydrophobic polyoxypropylene moiety but differ in molecular weight. These substances and their interfacial behaviour are interesting for the development of targeted drug delivery systems. Monolayer tensiometry revealed the degree of poloxamer penetration in PC monolayers and the changes in monolayer compression/decompression behaviour. Foam film experiments showed the effect of steric disjoining pressure increase in presence of F-88 and F-98. It resulted in changes of film structure and equivalent water thickness. It was found that the interaction between poloxamers and PC molecules in monolayers and foam films depends both on copolymer size and on the acyl chain length of phosphatidylcholines.