On the low surface tension of lung surfactant

Natural lung surfactant contains less than 40% disaturated phospholipids, mainly dipalmitoylphosphatidylcholine (DPPC). The mechanism by which lung surfactant achieves very low near-zero surface tensions, well below its equilibrium value, is not fully understood. To date, the low surface tension of lung surfactant is usually explained by a squeeze-out model which predicts that upon film compression non-DPPC components are gradually excluded from the air-water interface into a surface-associated surfactant reservoir. However, detailed experimental evidence of the squeeze-out within the physiologically relevant high surface pressure range is still lacking. In the present work, we studied four animal-derived clinical surfactant preparations, including Survanta, Curosurf, Infasurf, and BLES. By comparing compression isotherms and lateral structures of these surfactant films obtained by atomic force microscopy within the physiologically relevant high surface pressure range, we have derived an updated squeeze-out model. Our model suggests that the squeeze-out originates from fluid phases of a phase-separated monolayer. The squeeze-out process follows a nucleation-growth model and only occurs within a narrow surface pressure range around the equilibrium spreading pressure of lung surfactant. After the squeeze-out, three-dimensional nuclei stop growing, thereby resulting in a DPPC-enriched interfacial monolayer to reduce the air-water surface tension to very low values.     

Thermodynamic and structural characterization of a mixed perfluorocarbon-phospholipid ternary monolayer surfactant system

Pulmonary lung surfactant is a mixture of surfactants that reduces surface tension during respiration. Perfluorinated surfactants have potential applications for artificial lung surfactant formulations, but the interactions that exist between these compounds and phospholipids in surfactant monolayer mixtures are poorly understood. We report here, for the first time, a detailed thermodynamic and structural characterization of a minimal pulmonary lung surfactant model system that is based on a ternary phospholipid-perfluorocarbon mixture. Langmuir and Langmuir-Blodgett monolayers of binary and ternary mixtures of the surfactants 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and perfluorooctadecanoic acid (C18F) have been studied in terms of miscibility, elasticity and film structure. The extent of surfactant miscibility and elasticity has been evaluated via Gibbs excess free energies of mixing and isothermal compressibilities. Film structure has been studied by a combination of atomic force microscopy and fluorescence microscopy. Combined thermodynamic and microscopy data indicate that the ternary monolayer films were fully miscible, with the mixed films being more stable than their pure individual components alone, and that film compressibility is minimally improved by the addition of perfluorocarbons to the phospholipids. The importance of these results is discussed in context of these mixtures' potential applications in pulmonary lung surfactant formulations.     

Effect of mycolic acid on surface activity of binary surfactant lipid monolayers

In pulmonary tuberculosis, Mycobacterium tuberculosis lies in close physical proximity to alveolar surfactant. Cell walls of the mycobacteria contain loosely bound, detachable surface-active lipids. In this study, the effect of mycolic acid (MA), the most abundant mycobacterial cell wall lipid, on the surface activity of phospholipid mixtures from lung surfactant was investigated using Langmuir monolayers and atomic force microscopy (AFM). In the presence of mycolic acid, all the surfactant lipid mixtures attained high minimum surface tensions (between 20 and 40 mN/m) and decreased surface compressibility moduli <50 mN/m. AFM images showed that the smooth surface topography of surfactant lipid monolayers was altered with addition of MA. Aggregates with diverse heights of at least two layer thicknesses were found in the presence of mycolic acid. Mycolic acids could aggregate within surfactant lipid monolayers and result in disturbed monolayer surface activity. The extent of the effect of mycolic acid depended on the initial state of the monolayer, with fluid films of DPPC-POPC and DPPC-CHOL being least affected. The results imply inhibitory effects of mycolic acid toward lung surfactant lipids and could be a mechanism of lung surfactant dysfunction in pulmonary tuberculosis.     

Real-time investigation of lung surfactant respreading with surface vibrational spectroscopy

The respreading of a lung surfactant monolayer at the air-water interface is investigated with broad bandwidth sum frequency generation (BBSFG) spectroscopy. The lung surfactant mixture contains chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62), palmitoyloleoylphosphatidylglycerol (POPG), palmitic acid (PA), and KL4 (a 21-residue polypeptide analogue to the surfactant protein SP-B). DPPC-d62 serves as a probe molecule for the spectroscopic investigation. The BBSFG spectra of DPPC-d62 in the lung surfactant mixture are obtained in the C-D stretching region in real-time during film compression and expansion in a Langmuir trough. The BBSFG intensity of the CD3 stretch peak from DPPC-d62 terminal methyl groups is used as a measure of the interfacial density of DPPC-d62 after careful consideration of orientation effects. For the first time, the interfacial loss of DPPC in a complex lung surfactant mixture is quantified. Spectroscopic results reveal that there is an 18% DPPC-d62 interfacial loss during film respreading. However, the surface pressure-area isotherm measurements demonstrate that there is a rather large trough area reduction (37%) during film expansion. The relatively small interfacial loss of DPPC-d62 and the rather large trough area reduction indicate that the respreading of DPPC and non-DPPC components in the lung surfactant is not uniform and a surface refinement process exists during film compression and expansion. This refinement process results in a DPPC-enriched monolayer with a significant depletion of non-DPPC components after film respreading. Implication for replacement surfactant design from this work is discussed.     

Electrical surface potential of pulmonary surfactant

Pulmonary surfactant is a mixed lipid protein substance of defined composition that self-assembles at the air-lung interface into a molecular film and thus reduces the interfacial tension to close to zero. A very low surface tension is required for maintaining the alveolar structure. The pulmonary surfactant film is also the first barrier for airborne particles entering the lung upon breathing. We explored by frequency modulation Kelvin probe force microscopy (FM-KPFM) the structure and local electrical surface potential of bovine lipid extract surfactant (BLES) films. BLES is a clinically used surfactant replacement and here served as a realistic model surfactant system. The films were distinguished by a pattern of molecular monolayer areas, separated by patches of lipid bilayer stacks. The stacks were at positive electrical potential with respect to the surrounding monolayer areas. We propose a particular molecular arrangement of the lipids and proteins in the film to explain the topographic and surface potential maps. We also discuss how this locally variable surface potential may influence the retention of charged or polar airborne particles in the lung.     

The model of the action mechanism of SP-C in the lung surfactant monolayers

The action mechanism of surfactant protein C (SP-C) in the lung surfactant monolayers is studied. On the basis of the SP-C molecular structure, a detailed interaction model is developed to describe the interaction of phospholipids/SP-C in the lung surfactant monolayers. It is supposed that: (1) in an alveolus monolayer, SP-C molecules are surrounded by phosphatidylglycerol (PG). When the monolayer is compressed, SP-C molecules can promote PG molecules to be squeezed out; (2) during compressing of the monolayer, unsaturated-PG molecules form a collapse pit firstly when liquid-expanded state (LE) components achieve the collapse pressure. Then, SP-C's alpha-helix is attracted by the collapse pit and both alpha-helix and PG molecules are squeezed out speedily. Finally, the squeezed-out matters can form a lipid-protein aggregation in the subphase. The lipid-protein aggregation, in the centre of which, there is the hydrophobic alpha-helix section surrounded by PG molecules; (3) during the monolayer expanding, because of the increasing of the monolayer's surface tension, the structure of the lipid-protein aggregation is disturbed and reinserts into the surface of the monolayer rapidly. On the basis of analyzing the energies change of the squeeze-out process, a mathematical model is obtained to calculate the squeezed-out number of DPPG molecules when a SP-C molecule squeezes out in a monolayer. According to the model, it is concluded that SP-C has the capability to promote the squeeze-out and the reinsertion of most of PG component in an alveolus monolayer, the prediction data agree well with the experimental data.     

Dynamic surface tension measurements on surface active materials

Surface tension measurements by the Wilhelmy plate method are being done at our laboratory using an automatic balance. Surface-active material (surfactant) is spread as a monolayer on an air-water interface and a Pt plate, suspended from a micro-balance, is brought vertically into contact with the interface. The water is contained in a trough, two opposite sides of which can move independently, one producing a large variation in surface area and the other generating a longitudinal wave in the monolayer. This set-up is a new, asymmetric variant of the Benjamins-De Feyter method. Results of measurements on visco-elastic interfacial properties of model compounds of lung surfactant are reported.     

New insights into lung surfactant monolayers using vibrational sum frequency generation spectroscopy

At the air-water interface, interfacial molecular structure, intermolecular interactions, film relaxation and film respreading of model lung surfactant monolayers were studied using vibrational sum frequency generation (VSFG) spectroscopy combined with a Langmuir film balance. Chain-perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62), palmitoyloleoyl-phosphatidylglycerol (POPG), palmitic acid (PA) and tripalmitin were investigated. In the DPPC-d62-PA binary monolayer, PA showed a condensing effect on the DPPC chains. On the contrary, in the DPPC-d62-POPG binary monolayer, POPG showed a fluidizing effect on the DPPC chains. In the ternary monolayer system of DPPC-d62-POPG-PA, the balance between the fluidizing and the condensing effect was also observed. In addition, the film relaxation behavior of DPPC-d62 and the enhanced film stability of DPPC-d62 caused by the addition of tripalmitin were observed. Real-time VSFG was also employed to study the respreading properties of a complex lung surfactant mixture containing DPPC-d62, POPG, PA and KL4 (a mimic of SP-B) peptide, which revealed DPPC enrichment after film compression.     

Properties of oxidized phospholipid monolayers: An atomistic molecular dynamics study

Non-saturated lipids in the lung surfactant are prone to oxidation by oxidative species present in air. In this Letter, oxidized monolayers of dioleoylphosphatidylcholine (DOPC) were studied by means of both molecular dynamics and potential of mean force calculations. Structural changes of the monolayer following oxidation were revealed, including orientational reversal of oxidized chains. Accumulation of oxidized lipids in the monolayer is observed for moderate oxidation ratios, whereas removal of the short-chain oxidation product is predicted at long timescales. Massive oxidation leads to the loss of the well-ordered monolayer structure and partial solubilization of the oxidized lipids in the aqueous subphase.     

Molecular interactions of cord factor with dipalmitoylphosphatidylcholine monolayers: implications for lung surfactant dysfunction in pulmonary tuberculosis

Molecular interactions between mycobacterial cell wall lipid, cord factor (CF) and the abundant surfactant lipid, dipalmitoylphosphatidylcholine (DPPC) were investigated using Langmuir monolayers at physiological temperatures (37 degrees C). Surface topography of the films was visualized by atomic force microscopy (AFM). Thermodynamic behavior of the mixed monolayers was evaluated by investigating the molecular area excess, excess Gibbs free energy of mixing and maximum compressibility modulus (SCM(max)). Cord factor formed immiscible and thermodynamically unstable monolayers with DPPC. Monolayer presence of cord factor altered the physical state of DPPC monolayers from liquid condensed to liquid expanded with the lowering of SCM(max) from 160 to 40 mN/m, respectively. AFM imaging exhibited smooth homogenous surface topography of DPPC films which in the presence of cord factor was markedly altered with the appearance of aggregates and increased surface roughness. The results highlight the capacity of cord factor to disturb DPPC monolayer organization and structure. Interfacial presence of cord factor results in DPPC monolayer fluidization. Lung surfactant function is attributed to its ability to form well packed low compressibility films. Such molecular interactions suggest a dysfunction of lung surfactant in pulmonary tuberculosis due to surfactant monolayer fluidization.     

Reactive Uptake of N(2)O(5) on Aqueous H(2)SO(4) Solutions Coated with 1-Component and 2-Component Monolayers

Reactive uptake of N(2)O(5) on aqueous sulfuric acid solutions was studied in the presence of 1-component (octadecanol) and 2-component (octadecanol + phytanic acid) monolayers. In the 1-component monolayer experiments, the reactive uptake coefficient depended strongly on the molecular surface area of the surfactant. Also, the 1-component monolayer showed significant resistance to mass transfer even when the fractional surface coverage of the surfactant was less than 1. For example, a monolayer of 1-octadecanol with a fractional surface coverage of 0.75 decreased the reactive uptake coefficient by a factor of 10. This is consistent with previous studies. In the 2-component monolayer experiments, the reactive uptake coefficient depended strongly on the composition of the monolayer. When the monolayer contained only straight-chain molecules (1-octadecanol), the reactive uptake coefficient decreased by a factor of 42 due to the presence of the monolayer. However, when the monolayer contained 0.20 mole fraction of a branched surfactant (phytanic acid) the reactive uptake coefficient only decreased by a factor of 2. Hence, a small amount of branched surfactant drastically changes the overall resistance to reactive uptake. Also, our results show that the overall resistance to reactive uptake of 2-component monolayers can be predicted reasonably accurately by a model that assumes the resistances to mass transfer can be combined in parallel.     

Line tension of alkane lenses on aqueous surfactant solutions at phase transitions of coexisting interfaces

Alkane droplets on aqueous solutions of surfactants exhibit a first-order wetting transition as the concentration of surfactant is increased. The low-concentration or “partial wetting” state corresponds to an oil lens in equilibrium with a two-dimensional dilute gas of oil and surfactant molecules. The high-concentration or “pseudo-partial wetting” state consists of an oil lens in equilibrium with a mixed monolayer of surfactant and oil. Depending on the combination of surfactant and oil, these mixed monolayers undergo a thermal phase transition upon cooling, either to a frozen mixed monolayer or to an unusual bilayer structure in which the upper leaflet is a solid layer of pure alkane with hexagonal packing and upright chains while the lower leaflet remains a disordered liquid-like mixed monolayer. Additionally, certain long-chain alkanes exhibit a surface freezing transition at the air–oil interface where the top monolayer of oil freezes above its melting point. In this review, we summarize our previous studies and discuss how these wetting and surface freezing transitions influence the line tension of oil lenses from both an experimental and theoretical perspective.     

基于表面活性剂单分子层修饰碳糊电极的一氧化氮电化学传感器及其应用

报道了一种表面活性剂单分子层修饰碳糊电极,并用于NO的高灵敏电化学检测。研究表明,表面活性剂通过烷基链在电极表面形成的疏水性单分子层微环境对NO的电化学响应具有较好的促进作用。其中,阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)对NO的电化学氧化表现出最强的催化活性和增敏作用。在Nafion膜覆盖的CTAB修饰碳糊电极上,NO的安培响应与其浓度在3.6×10-8～1.8×10-5mol/L范围内呈良好的线性关系,检出限为1.8×10-8mol/L。该电极作为低成本、高灵敏的NO电化学传感器,被成功应用于大鼠肺组织细胞中NO释放的实时监测。     

High-resolution studies of lung surfactant collapse

Survanta is a replacement lung surfactant (LS) used in the treatment of respiratory distress syndrome (RDS), the fourth leading cause of infant mortality in the United States. It consists of purified LS from bovine sources and retains the surfactant proteins (SP) SP-B and SP-C, both thought to be important in proper respiratory function. As such, it provides a useful and biologically relevant model system to probe the structure and function of natural LS. Here, we report results from high-resolution studies on model monolayers formed from Survanta to probe the mechanism of collapse at high surface pressure. Our results show the formation of two different collapse structures. At 62 mN/m, slightly below the collapse pressure, monolayer collapse occurs through buckling. Confocal fluorescence measurements on supported films reveal regions of overlapping phase structure in the films that mark the transition from monolayer to multilayer. Simultaneous near-field scanning optical microscopy fluorescence and force measurements show that the transition seen in the fluorescence measurements accompanies corresponding approximately 4-5 nm changes in membrane topography. This change in height is consistent with bilayer formation on monolayer collapse. Analysis of the phase structure near the transitions also suggests that the buckling occurs from a continuous film. However, when the film is compressed to its collapse pressure of 65 mN/m, buckling is no longer evident in the collapsed region. In addition, multilayers and lipid-protein aggregates that are up to 40 nm higher than the monolayer are observed in the collapsed film at this pressure.     

A double injection ADSA-CSD methodology for lung surfactant inhibition and reversal studies

This paper presents a continuation of the development of a drop shape method for film studies, ADSA-CSD (Axisymmetric Drop Shape Analysis-Constrained Sessile Drop). ADSA-CSD has certain advantages over conventional methods. The development presented here allows complete exchange of the subphase of a spread or adsorbed film. This feature allows certain studies relevant to lung surfactant research that cannot be readily performed by other means. The key feature of the design is a second capillary into the bulk of the drop to facilitate addition or removal of a secondary liquid. The development will be illustrated through studies concerning lung surfactant inhibition. After forming a sessile drop of a basic lung surfactant preparation, the bulk phase can be removed and exchanged for one containing different inhibitors. Such studies mimic the leakage of plasma and blood proteins into the alveolar spaces altering the surface activity of lung surfactant in a phenomenon called surfactant inhibition. The resistance of the lung surfactant to specific inhibitors can be readily evaluated using the method. The new method is also useful for surfactant reversal studies, i.e. the ability to restore the normal surface activity of an inhibited lung surfactant film by using special additives. Results show a distinctive difference between the inhibition when an inhibitor is mixed with and when it is injected under a preformed surfactant film. None of the inhibitors studied (serum, albumin, fibrinogen, and cholesterol) were able to penetrate a preexisting film formed by the basic preparation (BLES and protasan), while all of them can alter the surface activity of such preparation when mixed with the preparation. Preliminary results show that reversal of serum inhibition can be easily achieved and evaluated using the modified methodology.     

Competitive adsorption of fibrinogen and dipalmitoylphosphatidylcholine at the air/aqueous interface

The competitive adsorption of fibrinogen (FB) and DPPC at the air/aqueous interface, in phosphate buffer saline at 25 degrees C, was studied with tensiometry, infrared reflection absorption spectroscopy (IRRAS), and ellipsometry. For FB/DPPC mixtures with 750 ppm (0.075 wt%) FB and 1000 ppm (0.10 wt%) DPPC, the tension behavior was found to be similar to that of FB when alone, even with DPPC and FB being at the interface. Thus, FB interferes with adsorption of DPPC and inhibits its surface tension lowering ability. When FB protein is introduced in the solution after a DPPC monolayer has formed, the adsorption of FB is inhibited by the DPPC monolayer. When a DPPC monolayer is spread onto a solution with a preadsorbed FB layer, the DPPC monolayer excludes FB from the surface and controls the tension behavior with little inhibition by FB. When a DPPC dispersion is introduced with the Trurnit method, or sprayed dropwise, onto an aqueous FB/DPPC surfaces, the DPPC layer formed on the surface prevents the adsorption of FB and dominates the surface tension behavior. These results have implications in controlling the inhibition of lung surfactant tension behavior by serum proteins, when they leak at the alveolar lining layer, and in developing surfactant replacement therapies for alveolar respiratory diseases.     

Complex layering observed in high internal phase emulsions at a silicon surface by neutron reflectometry

The neutron reflectivity profiles from the interface between silicon and aqueous phase-in-oil high internal phase emulsions of steadily increasing surfactant hydrophilicity, are reported for two isotopic contrasts for each surfactant. Layered models are required to fit the structured reflectivity profiles that demonstrate that the oxidised top layer of the silicon is always covered by a surfactant monolayer. Interposed between the surfactant monolayer and the bulk emulsion is a layer of oil--a geometric effect caused by reorganisation of the aqueous droplets. As the surfactant hydrophilicity increases, alternating aqueous and oil+surfactant layers are inserted between this topmost oil layer and the oxide attached surfactant monolayer. The resulting structures have compositions and layer spacings suggestive of sections from lamellar phases. This increase in layer ordering with increasing surfactant hydrophilicity is expected. The bulk emulsions are observed to exhibit lamellar or sponge phases increasingly as surfactant hydrophilicity increases.     

Anomalous behavior of amine oxide surfactants at the air/water interface

A commonly stated requirement for the preparation of stable Langmuir monolayers of amphiphilic molecules at an air/water interface is that the surfactant must be insoluble in the subphase solution; however, a few prior studies have reported that some soluble surfactants can, under certain conditions, be compressed. The anomalous compression of soluble amphiphiles is extremely interesting and important, as it presents the possibility of greatly increasing the number of candidate compounds suitable for Langmuir monolayer studies and Langmuir-Blodgett deposition. The aim of this work was to obtain a better understanding of the factors that determine whether monolayers of a given water-soluble surfactant can be compressed. A series of amine oxide surfactants, including a novel gemini surfactant, were studied to explore the relationship between molecular structure and behavior at the air/water interface. Amine oxides are an especially interesting class of surfactants because their self-assembly in solution and at interfaces is pH-sensitive. Surface pressure-area isotherms show that the solubility of a surfactant in the subphase solution is not, in and of itself, a useful parameter in predicting whether the monolayer is compressible. Molecular modeling calculations suggest that the tendency of molecules to self-assemble plays a much more important role than solubility in this regard. The effect of pH was also investigated. We present a hypothesis that formation of dimers or small clusters of molecules at the interface inhibits the dissolution of these species into the subphase, and as a consequence the monolayer can be compressed.     

The impact of cigarette/e‐cigarette vapour on simulated pulmonary surfactant monolayers under physiologically relevant conditions

Deviation in pulmonary surfactant structure–function activity can impair airway patency and lead to respiratory disorders. This novel study aims to evaluate the influence cigarette/e‐cigarette vapour has on model surfactant films located within a simulated pulmonary environment using a lung biosimulator. Chromatographic analysis confirmed that nicotine levels were consistent with the sampling regimen employed. On exposure to smoke vapour, Langmuir isotherms exhibited condensed character and a significant reduction in maximum surface pressure was noted in all cases. Langmuir isocycles, reflective of the human breathing cycle, demonstrated condensed character on smoke vapour delivery. A reduction in the maximum surface pressure was clear only in the case of cigarette vapour application. The components of cigarette vapour can cause oxidative damage to pulmonary surfactant and impair recycling. Neutral nicotine molecules can weaken the structure of the monolayer and cause destabilisation. A protective effect was evident in the case of repeated surfactant compression – relaxation cycles (i.e. the ability to reduce the surface tension term was impaired less), demonstrating a likely innate biological defensive mechanism of the lung. E‐cigarette vapour appeared to have a reduced impact on surfactant performance, which may hold value in harm reduction over the longer term. Copyright © 2017 John Wiley & Sons, Ltd.     

表面活性剂对硫醇单层膜修饰金电极电化学行为的影响

利用自组装方法在金电极表面制备成硫醇单层膜, 循环伏安和交流阻抗实验表明硫醇单层膜与表面活性剂十六烷基三甲基溴化胺作用后, 其电化学行为发生变化, 对于带有不同电荷的探针分子, 表现出一定的选择性响应. 即使是带有相同电荷的探针分子, 由于与表面活性剂的作用方式不同, 也使它们通过硫醇单层膜在电极表面产生电化学响应的程度完全不同.