Interaction between chitosan and bovine lung extract surfactants

The interaction between a cationic polyelectrolyte, chitosan, and an exogenous bovine lung extract surfactant (BLES) was studied using dynamic compression/expansion cycles of dilute BLES preparations in a Constrained Sessile Drop (CSD) device equipped with an environmental chamber conditioned at 37 °C and 100% R.H. air. Under these conditions, dilute BLES preparations tend to produce variable and relatively high minimum surface tensions. Upon addition of “low” chitosan to BLES ratios, the minimum surface tension of BLES-chitosan preparations were consistently low (i.e. < 5 mJ/m²), and the resulting surfactant monolayers (adsorbed at the air-water interface) were highly elastic and stable. However, the use of “high” chitosan to BLES ratios induced the collapse of the surfactant monolayer at high minimum surface tensions (i.e. > 15 mJ/m²). The zeta potential of the lung surfactant aggregates in the subphase suggests that chitosan binds to the anionic lipids (phosphatidyl glycerols) in BLES, and that this binding is ultimately responsible for the changes in the surface activity (elasticity and stability) of these surfactant-polyelectrolyte mixtures. Furthermore the transition from “low” to “high” chitosan to BLES ratios correlates with the flocculation and de-flocculation of surfactant aggregates in the subphase. It is proposed that the aggregation/segregation of “patches” of anionic lipids in the surfactant monolayer produced at different chitosan to BLES ratios explains the enhancing/inhibitory effects of chitosan. These observations highlight the importance of electrostatic interactions in lung surfactant systems.     

Restoring the activity of serum-inhibited bovine lung extract surfactant (BLES) using cationic additives

In this work four cationic additives were used to improve the surface activity of lung surfactants, particularly in the presence of bovine serum that was used as a model surfactant inhibitor. Two of those additives were chitosan in its soluble hydrochloride form with average molecular weights of 113 kDa and 213 kDa. The other two additives were cationic peptides, polylysine 50 kDa and polymyxin B. These additives were added to bovine lipid extract surfactant (BLES) and the optimal additive-surfactant ratio was determined based on the minimum surface tension upon dynamic compression, carried out in a constrained sessile drop (CSD) device in the presence of 50 μl/ml serum. At the optimal ratio all the BLES-additive mixtures were able to achieve desirable minimum surface tensions. The optimal additive-surfactant ratios for the chitosan chlorides are consistent with a previously proposed patch model for the binding of the anionic lipids in BLES to the positive charges in chitosan. For the peptides, the optimal binding ratios were consistent with ratios established previously for the binding of these peptides to monolayers of anionic lipids. The optimal formulation containing these peptides were able to reach low minimum surface tension in systems containing 500 μl/ml of serum, matching the effectiveness of a lung surfactant extract that had not undergone post-separation processes and therefore contained all its proteins and lipids (complete lung surfactant).     

Restoring the charge and surface activity of bovine lung extract surfactants with cationic and anionic polysaccharides

Chitosan, a cationic polysaccharide, has been found to improve the surface activity of lung surfactant extracts in the presence of various inhibitors. It has been proposed that chitosan binds to anionic lipids (e.g. phosphatidyl glycerols) in lung surfactants, producing stable lipid films at the air-water interface. This binding also reverses the net charge of the surfactant aggregates, from negative to positive. Unfortunately, positively charged aggregates may adsorb or interact with the negatively charged epithelial tissue, leading to poor surfactant performance. To address this issue an anionic polysaccharide, dextran sulfate (dexS), was used as a secondary coating to reverse the charge of chitosan-lung surfactant extracts without affecting the surface activity of the preparation. The dynamic surface tension and zeta potential of bovine lipid extract surfactant (BLES) containing chitosan chloride (chiCl) and dexS were evaluated as a function of dexS concentration. These studies were conducted in the absence and presence of sodium bicarbonate buffer, and in the absence and presence of bovine serum used as model inhibitor. It was determined that using an appropriate concentration of dexS, especially at physiological pH, it is possible to restore the negative charge of the surfactant aggregates, and retain their surface activity, even in the presence of bovine serum. High concentrations of dexS affect the binding of chiCl to BLES, and the surface activity of the preparation.     

Electrostatic barrier to recovery of dipalmitoylphosphatidylglycerol monolayers after collapse

The reincorporation of lipids into monolayers at the air-water interface after collapse is important to the maintenance of low surface tensions on subsequent expansion and compression cycles. For single component, anionic dipalmitoylphosphatidylglycerol monolayers, the fraction of recovered lipid is proportional to the subphase ionic strength. The collapse mechanism and structure of the collapsed materials appear unchanged with ionic strength. A simple electrostatic barrier model shows that the fractional recovery depends exponentially on the Debye length; this is verified by experiment. This simple model suggests possible catalytic roles for the cationic lung surfactant specific proteins SP-B and SP-C that induce structural changes in the monolayer that may act as charge-neutralizing docking sites for surfactant in the subphase, leading to faster and more efficient recovery.     

Effect of the hydrophobic surfactant proteins on the surface activity of spread films in the captive bubble surfactometer

The main function of pulmonary surfactant, a mixture of lipids and proteins, is to reduce the surface tension at the air/liquid interface of the lung. The hydrophobic surfactant proteins SP-B and SP-C are required for this process. When testing their activity in spread films in a captive bubble surfactometer, both SP-B and SP-C showed concentration dependence for lipid insertion as well as for lipid film refinement. Higher activity in DPPC refinement of the monolayer was observed for SP-B compared with SP-C. Further differences between both proteins were found, when subphase phospholipid vesicles, able to create a monolayer-attached lipid reservoir, were omitted. SP-C containing monolayers showed gradually increasing minimum surface tensions upon cycling, indicating that a lipid reservoir is required to prevent loss of material from the monolayer. Despite reversible cycling dynamics, SP-B containing monolayers failed to reach near-zero minimum surface tensions, indicating that the reservoir is required for stable films.     

The molecular mechanism of monolayer-bilayer transformations of lung surfactant from molecular dynamics simulations

The aqueous lining of the lung surface exposed to the air is covered by lung surfactant, a film consisting of lipid and protein components. The main function of lung surfactant is to reduce the surface tension of the air-water interface to the low values necessary for breathing. This function requires the exchange of material between the lipid monolayer at the interface and lipid reservoirs under dynamic compression and expansion of the interface during the breathing cycle. We simulated the reversible exchange of material between the monolayer and lipid reservoirs under compression and expansion of the interface. We used a mixture of dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol, cholesterol, and surfactant-associated protein C as a functional analog of mammalian lung surfactant. In our simulations, the monolayer collapses into the water subphase on compression and forms bilayer folds. On monolayer reexpansion, the material is transferred from the folds back to the interface. The simulations indicate that the connectivity of the bilayer aggregates to the monolayer is necessary for the reversibility of the monolayer-bilayer transformation. The simulations also show that bilayer aggregates are unstable in the air subphase and stable in the water subphase.     

Surface respreading after collapse of monolayers containing major lipids of pulmonary surfactant

Isotherms have been obtained near 37 degrees C for a series of repetitive compressions and expansions of monolayers that contain major components of lung surfactant. The minimum surface tension or maximum surface pressure which could be achieved under conditions of dynamic compression, and the rate of return of lipid from excluded phase to the monolayers were measured. Monolayers of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or of DPPC plus 10 or 30 mol% of the calcium salt of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG) (POPG-Ca) achieved very high surface pressures or low surface tensions (near 0 mN m-1), but they showed no return of material from the collapse phases under the test conditions. Monolayers of POPG-Ca alone collapsed at relatively low surface pressures (high surface tensions), but showed good return of material from the collapse phase into the monolayer. Monolayers containing more complex mixtures of lipids (DPPC, phosphatidylglycerol (PG), unsaturated phosphatidylcholine (PC), cholesterol (chol] in ratios similar to those found in surfactant achieved minimum surface tensions intermediate between those of monolayers with less complex compositions. These more complex mixtures showed a better rate of return of lipids from the collapse phases to the monolayer than did simple DPPC-POPG mixtures. 31P-NMR and differential scanning calorimetric investigations of the mixture DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POP G/DPPG/chol (10:4:2:1:3) showed that in the bulk phase at 37 degrees C, it was in bilayers in the liquid-crystalline state.     

Influence of surface chemistry and topography of particles on their immersion into the lung's surface-lining layer.

Inhaled and deposited spherical particles, 1-6 micrometer in diameter and of differing surface chemistry and topography, were studied in hamster intrapulmonary conducting airways and alveoli by electron microscopy. Polystyrene and Teflon particles, as well as puffball spores, were found submersed in the aqueous lining layer and adjacent to epithelial cells. The extent of particle immersion promoted by a surfactant film was assessed in a "floating-drop-surface balance" by light microscopy. Teflon and polystyrene spheres were immersed into the subphase by 50-60% at film surface tensions of 25 and 30 mJ/m(2), respectively, and totally submersed at 15 and 25 mJ/m(2), respectively. Puffball spores were immersed by approximately 50% at 22 mJ/m(2) and totally submersed at film surface tensions of 相似文献   

Effect of budesonide and salbutamol on surfactant properties.

The objective of this study was to evaluate the in vitro effect of budesonide and salbutamol on the surfactant biophysical properties. The surface-tension properties of two bovine lipid extracts [bovine lipid extract surfactant (BLES) and Survanta] and a rat lung lavage natural surfactant were evaluated in vitro by the captive bubble surfactometer. Measurements were obtained before and after the addition of a low and high concentration of budesonide and salbutamol. Whereas salbutamol had no significant effect, budesonide markedly reduced the surface-tension-lowering properties of all surfactant preparations. Surfactant adsorption (decrease in surface tension vs. time) was significantly reduced (P < 0.01) at a high budesonide concentration with BLES, both concentrations with Survanta, and a low concentration with natural surfactant. At both concentrations, budesonide reduced (P < 0.01) Survanta film stability (minimal surface vs. time at minimum bubble volume), whereas no changes were seen with BLES. The minimal surface tension obtained for all surfactant preparations was significantly higher (P < 0.01), and the percentage of film area compression required to reach minimum surface tension was significantly lower after the addition of budesonide. In conclusion, budesonide, at concentrations used therapeutically, adversely affects the surface-tension-lowering properties of surfactant. We speculate that it may have the same adverse effect on the human surfactant.     

Interaction of lung surfactant proteins with anionic phospholipids.

Langmuir isotherms, fluorescence microscopy, and atomic force microscopy were used to study lung surfactant specific proteins SP-B and SP-C in monolayers of dipalmitoylphosphatidylglycerol (DPPG) and palmitoyloleoylphosphatidylglycerol (POPG), which are representative of the anionic lipids in native and replacement lung surfactants. Both SP-B and SP-C eliminate squeeze-out of POPG from mixed DPPG/POPG monolayers by inducing a two- to three-dimensional transformation of the fluid-phase fraction of the monolayer. SP-B induces a reversible folding transition at monolayer collapse, allowing all components of surfactant to remain at the interface during respreading. The folds remain attached to the monolayer, are identical in composition and morphology to the unfolded monolayer, and are reincorporated reversibly into the monolayer upon expansion. In the absence of SP-B or SP-C, the unsaturated lipids are irreversibly lost at high surface pressures. These morphological transitions are identical to those in other lipid mixtures and hence appear to be independent of the detailed lipid composition of the monolayer. Instead they depend on the more general phenomena of coexistence between a liquid-expanded and liquid-condensed phase. These three-dimensional monolayer transitions reconcile how lung surfactant can achieve both low surface tensions upon compression and rapid respreading upon expansion and may have important implications toward the optimal design of replacement surfactants. The overlap of function between SP-B and SP-C helps explain why replacement surfactants lacking in one or the other proteins often have beneficial effects.     

Effect of humidity on the stability of lung surfactant films adsorbed at air-water interfaces

The effect of humidity on the film stability of Bovine Lipid Extract Surfactant (BLES) is studied using the captive bubble method. It is found that adsorbed BLES films show distinctly different stability patterns at two extreme relative humidities (RHs), i.e., bubbles formed by ambient air and by air prehumidified to 100% RH at 37 degrees C. The differences are illustrated by the ability to maintain low surface tensions at various compression ratios, the behavior of bubble clicks, and film compressibility. These results suggest that 100% RH at 37 degrees C tends to destabilize the BLES films. In turn, the experimental results indicate that the rapidly adsorbed BLES film on a captive bubble presents a barrier to water transport that retards full humidification of the bubble when ambient air is used for bubble formation. These findings necessitate careful evaluation and maintenance of environmental humidity for all in vitro assessment of lung surfactants. It is also found that the stability of adsorbed bovine natural lung surfactant (NLS) films is not as sensitive as BLES films to high humidity. This may indicate a physiological function of SP-A and/or cholesterol, which are absent in BLES, in maintaining the extraordinary film stability in vivo.     

More than a monolayer: relating lung surfactant structure and mechanics to composition

Survanta, a clinically used bovine lung surfactant extract, in contact with surfactant in the subphase, shows a coexistence of discrete monolayer islands of solid phase coexisting with continuous multilayer "reservoirs" of fluid phase adjacent to the air-water interface. Exchange between the monolayer, the multilayer reservoir, and the subphase determines surfactant mechanical properties such as the monolayer collapse pressure and surface viscosity by regulating solid-fluid coexistence. Grazing incidence x-ray diffraction shows that the solid phase domains consist of two-dimensional crystals similar to those formed by mixtures of dipalmitoylphosphatidylcholine and palmitic acid. The condensed domains grow as the surface pressure is increased until they coalesce, trapping protrusions of liquid matrix. At approximately 40 mN/m, a plateau exists in the isotherm at which the solid phase fraction increases from approximately 60 to 90%, at which the surface viscosity diverges. The viscosity is driven by the percolation of the solid phase domains, which depends on the solid phase area fraction of the monolayer. The high viscosity may lead to high monolayer collapse pressures, help prevent atelectasis, and minimize the flow of lung surfactant out of the alveoli due to surface tension gradients.     

Effect of humidity on the stability of lung surfactant films adsorbed at air-water interfaces

The effect of humidity on the film stability of Bovine Lipid Extract Surfactant (BLES) is studied using the captive bubble method. It is found that adsorbed BLES films show distinctly different stability patterns at two extreme relative humidities (RHs), i.e., bubbles formed by ambient air and by air prehumidified to 100% RH at 37 °C. The differences are illustrated by the ability to maintain low surface tensions at various compression ratios, the behavior of bubble clicks, and film compressibility. These results suggest that 100% RH at 37 °C tends to destabilize the BLES films. In turn, the experimental results indicate that the rapidly adsorbed BLES film on a captive bubble presents a barrier to water transport that retards full humidification of the bubble when ambient air is used for bubble formation. These findings necessitate careful evaluation and maintenance of environmental humidity for all in vitro assessment of lung surfactants. It is also found that the stability of adsorbed bovine natural lung surfactant (NLS) films is not as sensitive as BLES films to high humidity. This may indicate a physiological function of SP-A and/or cholesterol, which are absent in BLES, in maintaining the extraordinary film stability in vivo.     

Physiological and inflammatory response to instillation of an oxidized surfactant in a rat model of surfactant deficiency.

Pulmonary surfactant is a mixture of phospholipids ( approximately 90%) and surfactant-associated proteins (SPs) ( approximately 10%) that stabilize the lung by reducing the surface tension. One proposed mechanism by which surfactant is altered during acute lung injury is via direct oxidative damage to surfactant. In vitro studies have revealed that the surface activity of oxidized surfactant was impaired and that this effect could be overcome by adding SP-A. On the basis of this information, we hypothesized that animals receiving oxidized surfactant preparations would exhibit an inferior physiological and inflammatory response and that the addition of SP-A to the oxidized preparations would ameliorate this response. To test this hypothesis, mechanically ventilated, surfactant-deficient rats were administered either bovine lipid extract surfactant (BLES) or in vitro oxidized BLES of three doses: 10 mg/kg, 50 mg/kg, or 10 mg/kg + SP-A. When instilled with 10 mg/kg normal surfactant, the rats had a significantly superior arterial Po2 responses compared with the rats receiving oxidized surfactant. Interestingly, increasing the dose five times mitigated this physiological effect, and the addition of SP-A to the surfactant preparation had little impact on improving oxygenation. There were no differences in alveolar surfactant pools and the indexes of pulmonary inflammation between the 10 mg/kg dose groups, nor was there any differences observed between either of the groups supplemented with SP-A. However, there was significantly more surfactant and more inflammatory cytokines in the 50 mg/kg oxidized BLES group compared with the 50 mg/kg BLES group. We conclude that instillation of an in vitro oxidized surfactant causes an inferior physiological response in a surfactant-deficient rat.     

Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability

Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.     

A modified squeeze-out mechanism for generating high surface pressures with pulmonary surfactant

The exact mechanism by which pulmonary surfactant films reach the very low surface tensions required to stabilize the alveoli at end expiration remains uncertain. We utilized the nanoscale sensitivity of atomic force microscopy (AFM) to examine phospholipid (PL) phase transition and multilayer formation for two Langmuir-Blodgett (LB) systems: a simple 3 PL surfactant-like mixture and the more complex bovine lipid extract surfactant (BLES). AFM height images demonstrated that both systems develop two types of liquid condensed (LC) domains (micro- and nano-sized) within a liquid expanded phase (LE). The 3 PL mixture failed to form significant multilayers at high surface pressure (π while BLES forms an extensive network of multilayer structures containing up to three bilayers. A close examination of the progression of multilayer formation reveals that multilayers start to form at the edge of the solid-like LC domains and also in the fluid-like LE phase. We used the elemental analysis capability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to show that multilayer structures are enriched in unsaturated PLs while the saturated PLs are concentrated in the remaining interfacial monolayer. This supports a modified squeeze-out model where film compression results in the hydrophobic surfactant protein-dependent formation of unsaturated PL-rich multilayers which remain functionally associated with a monolayer enriched in disaturated PL species. This allows the surface film to attain low surface tensions during compression and maintain values near equilibrium during expansion.     

Constrained sessile drop as a new configuration to measure low surface tension in lung surfactant systems.

Existing methodology for surface tension measurements based on drop shapes suffers from the shortcoming that it is not capable to function at very low surface tension if the liquid dispersion is opaque, such as therapeutic lung surfactants at clinically relevant concentrations. The novel configuration proposed here removes the two big restrictions, i.e., the film leakage problem that is encountered with such methods as the pulsating bubble surfactometer as well as the pendant drop arrangement, and the problem of the opaqueness of the liquid, as in the original captive bubble arrangement. A sharp knife edge is the key design feature in the constrained sessile drop that avoids film leakage at low surface tension. The use of the constrained sessile drop configuration in conjunction with axisymmetric drop shape analysis to measure surface tension allows complete automation of the setup. Dynamic studies with lung surfactant can be performed readily by changing the volume of a sessile drop, and thus the surface area, by means of a motor-driven syringe. To illustrate the validity of using this configuration, experiments were performed using an exogenous lung surfactant preparation, bovine lipid extract surfactant (BLES) at 5.0 mg/ml. A comparison of results obtained for BLES at low concentration between the constrained sessile drop and captive bubble arrangement shows excellent agreement between the two approaches. When the surface area of the BLES film (0.5 mg/ml) was compressed by about the same amount in both systems, the minimum surface tensions attained were identical within the 95% confidence limits.     

The role of the gel <=> liquid-crystalline phase transition in the lung surfactant cycle

Lipid polymorphism plays an important role in the lung surfactant cycle. A better understanding of the influence of phase transitions on the formation of a lipid film from dispersions of vesicles will help to describe the mechanism of action of lung surfactant. The surface pressure (or tension) of dispersions of DPPC, DMPC, and DPPE unilamellar vesicles was studied as a function of temperature. These aggregates rapidly fuse with a clean air-water interface when the system is at their phase transition temperature (Tm), showing a direct correlation between phase transition and film formation. Based on these results, an explanation on how fluid aggregates in the alveolar subphase can form a rigid monolayer at the alveolar interface is proposed.     

Lung surfactant protein SP-B promotes formation of bilayer reservoirs from monolayer and lipid transfer between the interface and subphase

We investigated the possible role of SP-B proteins in the function of lung surfactant. To this end, lipid monolayers at the air/water interface, bilayers in water, and transformations between them in the presence of SP-B were simulated. The proteins attached bilayers to monolayers, providing close proximity of the reservoirs with the interface. In the attached aggregates, SP-B mediated establishment of the lipid-lined connection similar to the hemifusion stalk. Via this connection, a lipid flow was initiated between the monolayer at the interface and the bilayer in water in a surface-tension-dependent manner. On interface expansion, the flow of lipids to the monolayer restored the surface tension to the equilibrium spreading value. SP-B induced formation of bilayer folds from the monolayer at positive surface tensions below the equilibrium. In the absence of proteins, lipid monolayers were stable at these conditions. Fold nucleation was initiated by SP-B from the liquid-expanded monolayer phase by local bending, and the proteins lined the curved perimeter of the growing fold. No effect on the liquid-condensed phase was observed. Covalently linked dimers resulted in faster kinetics for monolayer folding. The simulation results are in line with existing hypotheses on SP-B activity in lung surfactant and explain its molecular mechanism.     

Kinetics of phospholipid insertion into monolayers containing the lung surfactant proteins SP-B or SP-C

The lung surfactant proteins SP-B and SP-C are pivotal for fast and reversible lipid insertion at the air/liquid interface, a prerequisite for functional lung activity. We used a model system consisting of a preformed monolayer at the air/liquid interface supplemented with surfactant protein SP-B or SP-C and unilamellar vesicles injected into the subphase of a film balance. The content of SP-B or SP-C was similar to that found in lung lavage. In order to elucidate distinct steps of lipid insertion, we measured the time-dependent pressure increase as a function of the initial surface pressure, the temperature and the phosphatidylglycerol content by means of surface tension measurements and scanning force microscopy (SFM). The results of the film balance study are indicative of a two-step mechanism in which initial adsorption of vesicles to the protein-containing monolayer is followed by rupture and integration of lipid material. Furthermore, we found that vesicle adsorption on a preformed monolayer supplemented with SP-B or SP-C is strongly enhanced by negatively charged lipids as provided by DPPG and the presence of Ca2+ ions in the subphase. Hence, long-range electrostatic interactions are thought to play an important role in attracting vesicles to the surface, being the initial step in replenishment of lipid material. While insertion into the monolayer is independent of the type of protein SP-B or SP-C, initial adsorption is faster in the presence of SP-B than SP-C. We propose that the preferential interaction between SP-B and negatively charged DPPG leads to accumulation of negative charges in particular regions, causing strong adhesion between DPPG-containing vesicles and the monolayer mediated by Ca2+ ions, which eventually causes flattening and rupture of attached liposomes as observed by in situ SFM.