A. Jordanova

Rhamnolipid biosurfactants produced by Renibacterium salmoninarum 27BN during growth on n-hexadecane.

A new strain Renibacterium salmoninarum 27BN was isolated for its capacity to utilize nhexadecane as sole substrate. Growth on n-hexadecane was accompanied with the production of glycolipid surface active substances detected by surface pressure lowering and emulsifying activity. Glycolipid detection by thin layer chromatography and infrared spectra analyses showed for the first time that Renibacterium salmoninarum 27BN secretes the two rhamnolipids RLL and RRLL typical for Pseudomonas aeruginosa. Growth of Renibacterium salmoninarum 27BN on n-hexadecane depended on the bioavailability of the substrate and the secreted rhamnolipids appeared to be efficient in increasing hexadecane availability for the cells.

Interactions of Meibomian gland secretion with polar lipids in Langmuir monolayers

The surface interactions of Meibomian gland secretion (MGS) with polar lipid (PL), Egg Sphingomyelin (SM) or Dipalmitoylphosphatidylcholine (DPPC), are studied in mixed pseudo-binary films formed at the air/water interface of Langmuir surface balance. The behavior of the mixed films during slow quasi-equilibrium compression and during fast dynamic compression-decompression is registered by measurements of surface pressure and surface potential, and by monitoring film morphology with Brewster Angle Microscopy (BAM). Quasi-equilibrium compression isotherms are used to calculate the excess Gibbs and Helmholtz energy of mixing between MGS and PLs and thus to evaluate the interactions between the lipid compounds at the interface. The effects of PLs on the mixed films elastic moduli of area compressibility, morphology and capability to attain high surface pressures are also examined. PLs interact with MGS with different strength and in different manner: MGS-SM interaction is weak and might lead to interfacial disaggregation of the thick meibium domains when SM is in excess, while MGS-DPPC interaction is strong and results in the formation of thick lipid aggregates. Both PLs increase the mixed films reciprocal compressibility and capability to achieve higher surface pressures. The results demonstrate that in vitro studies of the surface interactions between MGS and PLs might be beneficial in the selection of PLs for artificial tear formulations and for examination on molecular scale of the possible role of PLs at the ocular surface.

Surface chemistry study of the interactions of benzalkonium chloride with films of meibum, corneal cells lipids, and whole tears.

PURPOSEnTo perform a surface chemistry study of the interactions between benzalkonium chloride (BAC), a common preservative used in ophthalmic formulations, and tear film (TF) constituents.nnnMETHODSnThe interactions between BAC and human tears, meibum, and rabbit corneal cell lipid extracts at the air-water interface were examined in vitro during controlled compression-expansion of the film area by a Langmuir surface balance, surface potential measurements, and pendant drop-axisymmetric drop shape analysis (PD-ADSA). Surface pressure-area isotherms and isocycles were used to assess the samples lateral elasticity and capability of compressing and spreading during dynamic area changes. Lipid film morphology was monitored by Brewster angle microscopy. The viability of BAC-treated Statens Seruminstitut rabbit cornea (SIRC) cell cultures was also examined. The BAC concentration was kept within the clinical range of 0.001% to 0.02%.nnnRESULTSnIn the Langmuir balance and PD-ADSA experiments, the interactions between BAC and lipids or tears resulted in (1) impaired lipid spread and formation of discontinuous nonuniform surface layers, (2) increased surface pressure-area hysteresis during compression and expansion, and (3) displacement of the lipids by BAC from the surface. A decrease (>50%) in SIRC cell viability was observed. The effects occurred within seconds after BAC exposure, and their magnitude increased with BAC concentration.nnnCONCLUSIONSnThe surface chemistry approach used in this study provided molecular-scale insights into the detrimental effect of BAC on TF, which well explain the TF instability and corneal epithelial barrier dysfunction after exposure to BAC in the in vivo human eye.

Formation of monolayers and bilayer foam films from lamellar, inverted hexagonal and cubic lipid phases.

Abstract. This study revealed large distinctions between the lamellar and non-lamellar liquid crystalline lipid phases in their spreading at the air/water interface and propensity to form bilayer foam films. Comparative measurements were made for the lamellar Lα, the inverted hexagonal HII and the bicontinuous cubic Pn3m phases of the phospholipid dipalmitoleoylphosphatidylethanolamine (DPoPE). With regard to monolayer formation, followed as the decrease of surface tension with time, the best spreading (lowest surface tension) was observed for the Lα phase, and poorest spreading (highest surface tension) was recorded for the HII phase. The cubic Pn3m phase of DPoPE, induced by temperature cycling, retained an intermediate position between the Lα and HII phases. According to their ability to lower surface tension and disintegrate at the air/water interface, the three phases thus order as Lα>Pn3m>HII. Clearly expressed threshold (minimum) bulk lipid concentrations, Ct, required for formation of stable foam bilayers from these phases, were determined and their values were found to correlate well with the bulk lipid phase behaviour. The Ct values for Lα and HII substantially increase with the temperature. Their Arrhenius plots, lnCt versus 1/T, are linear and intersect at ~36–37xa0°C, coinciding with the onset of the bulk Lα→HII phase transition, as determined by differential scanning calorimetry. However, the Ct value for the Pn3m phase, equal to 30xa0µg/mL, was found to be constant over the whole range investigated between 20xa0°C and 50xa0°C. The horizontal Ct versus T plot for the Pn3m phase crosses the respective plot for the Lα phase at the temperature bounding from below the hysteretic loop of the Lα↔HII transition (~26xa0°C), thus providing a certain insight about the thermodynamic stability of the Pn3m phase relative to the Lα phase. The established strong effect of the particular lipid phase on the formation of monolayers and stable black foam films should be of importance in various in vitro and in vivo systems, where lipid structures are in contact with interfaces and disintegrate there to different extents.

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.

Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix

The three-dimensional (3D) cell culture approach offers a means to study cells under conditions that mimic an inxa0vivo environment, thus avoiding the limitations imposed by the conventional two-dimensional (2D) monolayer cell cultures. By using this approach we demonstrated significant differences in the plasma membrane phospholipid composition and susceptibility to oxidation in cells cultured in three-dimensional environment compared to conventional monolayer cultures. The plasma membrane sphingomyelin (SM), which is a functionally active membrane phospholipid, was markedly increased in plasma membranes of 3D cells. To analyze the mechanisms underlying SM accumulation, we determined the activities of sphingolipid-metabolizing enzymes like neutral sphingomyelinase and ceramidase, which are also related to cellular redox homeostasis and to oxidative stress. Fibroblasts cultured in three-dimensional environment showed different redox potential and lower lipid susceptibility to oxidative damage compared to monolayer cells. The relative content of unsaturated fatty acids, which serve as targets of oxidative attack, was observed to be higher in major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, in plasma membranes of 3D cells. The possibility that the higher level of SM, might be responsible for the lower degree of oxidation of 3D phospholipids was tested by selective reduction of SM through treatment with exogenous sphingomyelinase. The results showed that the decrease of plasma membrane SM was accompanied by an increase of the lipid peroxides in both 2D and 3D cells. We presume that culturing as a monolayer is stressful for the cells and leads to activation of certain stress-related enzymes, resulting in reduction of the SM level. Our results show that the lower content of plasma membrane SM in cells cultured as a monolayer renders the phospholipid molecules more susceptible to oxidative stress.

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.

Properties of alkyl-phosphatidylcholine monolayers in the presence of surface-active three-block copolymers

The interaction of three-block copolymers (poloxamers) with dimyristoyl-phosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) was investigated in monolayer experiments. The poloxamers selected were BASF PLURONICS F-68, F-88 and F-98. All of them are water-soluble and possess two hydrophilic polyoxyethylene 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/expansion behaviour. Two different experimental protocols were used to determine the surface pressures (pi) and areas (A) at squeeze-out (compression) and insertion (expansion) of the soluble component in the surface layer. The two protocols yielded differences in the squeeze-out and insertion parameters of the DMPC (DPPC)/Poloxamer systems. The differences disappear when comparing the product piA (2-D - mechanical work) for the various combinations of lipids and poloxamers. In all cases (piA)(out)>(piA)(in). This approach reveals a possibility for adequate comparison of monolayer compression/expansion isotherms made by different experimental protocols.

Influence of surfactant protein C on the interfacial behavior of phosphatidylethanolamine monolayers

In the current work we study with monolayer tensiometry and Brewster angle microscopy (BAM) the surface properties of Dipalmitoleoylphosphatidylethanolamine (DPoPE) films at the air/water interface in presence and absence of specific surfactant protein C (SP-C). DPoPE is used, as it readily forms both lamellar (Lα) and non-lamellar inverted hexagonal (HII) phases and appears as a suitable model phospholipid for probing the interfacial properties of distinct lipid phases. At pure air/water interface Lα shows faster adsorption and better surface disintegration than HII phase. The interaction of DPoPE molecules with SP-C (predeposited at the interface) results in equalizing of the interfacial disintegration of the both phases (reaching approximately the same equilibrium surface tension) although the adsorption kinetics of the lamellar phase remains much faster. Monolayer compression/decompression cycling revealed that the effect of SP-C on dynamic surface tensions (γmax and γmin) of mixed films is remarkably different for the two phases. If γmax for Lα decreased from the first to the third cycle, the opposite effect is registered for HII where γmax increases during cycling. Also the significant decrease of γmin for Lα in SP-C presence is not observed for HII phase. BAM studies reveal the formation of more uniform and homogeneously packed DPoPE monolayers in the presence of SP-C.

Effects of the interaction and surface morphology of mixed DPoPE/Poloxamer 188 monolayers and thin liquid films

The aim of the present study is to investigate the kinetics and the penetration degree of Poloxamer 188 (P188) of different concentrations to Langmuir lipid monolayers at the air–water interface formed from dipalmitoleoylphosphatidylethanolamine (DPoPE) in different liquid crystalline phase states, lamellar Lα and non-lamellar (inverse hexagonal HII and inverse bicontinuous cubic QII) phases, in static and dynamic conditions. It was found that the penetration of P188 to the DPoPE monolayers in all three lipid phases is very fast, strong and concentration dependent, and the strongest effect was detected for the non-lamellar DPoPE monolayers. Moreover, the dynamic characteristics of the mixed DPoPE + P188 monolayers revealed stronger interactions between P188 and DPoPE in QII phase, than in the other two, and more packed surface structures. In addition, by using Brewster angle microscopy (BAM), the surface morphology of mixed DPoPE + P188 monolayers was determined. The BAM images showed that the addition of P188 to DPoPE monolayers led to decrease in the lipid domain size and to formation of more homogeneous films. Furthermore, the effects of P188 on the hydrodynamic properties and the stability of lipid thin liquid films (LTLFs) were studied. A strong stabilization effect of P188 to the LTLF films was proved. This effect was probably influenced by the appearance of steric repulsion (in addition to the electrostatic repulsion and van der Waals attractive forces). The results suggested that the non-lamellar lipid phases have higher potential to act as a “reservoir” for penetrating agents, and as a pool for membrane repairing substances such as P188.