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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.

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.

A drop shape technique using a constrained sessile drop constellation (ADSA-CSD) has been introduced as a superior technique for studying spread films specially at high collapse pressures [Saad et al. Langmuir 2008, 24, 10843-10850]. It has been shown that ADSA-CSD has certain advantages including the need only for small quantities of liquid and insoluble surfactants, the ability to measure very low surface tension values, easier deposition procedure, and leak-proof design. Here, this technique was applied to investigate mixed DPPC/DPPG monolayers to characterize the role of such molecules in maintaining stable film properties and surface activity of lung surfactant preparations. Results of compression isotherms were obtained for different DPPC/DPPG mixture ratios: 90/10, 80/20, 70/30, 60/40, and 50/50 in addition to pure DPPC and pure DPPG at room temperature of 24 degrees C. The ultimate collapse pressure of DPPC/DPPG mixtures was found to be 70.5 mJ/m2 (similar to pure DPPC) for the cases of low DPPG content (up to 20%). Increasing the DPPG content in the mixture (up to 40%) caused a slight decrease in the ultimate collapse pressure. However, further increase of DPPG in the mixture (50% or more) caused a sharp decrease in the ultimate collapse pressure to a value of 59.9 mJ/m2 (similar to pure DPPG). The change in film elasticity was also tracked for the range of mixture ratios studied. The physical reasons for such changes and the interaction between DPPC and DPPG molecules are discussed. The results also show a change in the film hysteresis upon successive compression and expansion cycles for different mixture ratios.

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. 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.

The surface activity of bovine lipid extracted surfactant (BLES) preparations used in surfactant replacement therapy is studied in dynamic film compression/expansion cycles as a function of relative humidity, surfactant concentration, compression rate, and compression periodicity. BLES droplets were formed in a constrained sessile droplet configuration (CSD). Images obtained during cycling were analyzed using axisymmetric drop shape analysis (ADSA) to yield surface tension, surface area, and drop volume data. The experiments were conducted in a chamber that allowed both humid (100% RH), and "dry" air (i.e. less than 20% RH) environments. It was observed that in humid environments BLES films are not stable and tend to have poor surface activity compared to BLES films exposed to dry air. Further analysis of the data reveal that if BLES films are compressed fast enough (i.e. at physiological conditions) to avoid film hydration, lower minimum surface tensions are achieved. A film hydration-relaxation mechanism is proposed to explain these observations.

The in vitro adsorption kinetics of lung surfactant at air-water interfaces is affected by both the composition of the surfactant preparations and the conditions under which the assessment is conducted. Relevant experimental conditions are surfactant concentration, temperature, subphase pH, electrolyte concentration, humidity, and gas composition of the atmosphere exposed to the interface. The effect of humidity on the adsorption kinetics of a therapeutic lung surfactant preparation, bovine lipid extract surfactant (BLES), was studied by measuring the dynamic surface tension (DST). Axisymmetric drop shape analysis (ADSA) was used in conjunction with three different experimental methodologies, i.e., captive bubble (CB), pendant drop (PD), and constrained sessile drop (CSD), to measure the DST. The experimental results obtained from these three methodologies show that for 100% relative humidity (RH) at 37 degrees C the rate of adsorption of BLES at an air-water interface is substantially slower than for low humidity. It is also found that there is a difference in the rate of surface tension decrease measured from the PD and CB/CSD methods. These experimental results agree well with an adsorption model that considers the combined effects of entropic force, electrostatic interaction, and gravity. These findings have implications for the development and evaluation of new formulations for surfactant replacement therapy.

Pulmonary surfactant is crucial to maintain the proper functioning of the respiration system. Certain types of blood proteins (e.g. albumin) were found to inhibit the activity of pulmonary surfactant. Axisymmetric Drop Shape Analysis (ADSA) was used to study the effect of dextran to restore the activity of an albumin-inhibited pulmonary surfactant. It was found that dextran could effectively restore surface tension properties of the inhibited surfactant in vitro. Furthermore, dextran improved the performance of pulmonary surfactants when albumin was absent. It was found that when a surfactant film was under high compression (e.g. above 70% surface area reduction), the presence of dextran increased film stability, so that the film could sustain high surface pressures without being collapsing.

Samples of pulmonary surfactant were obtained from rabbits by repeated lung lavage. Surface tension measurements of the samples were made in such a way as to avoid the possibility of film leakage. It was confirmed that the relative magnitudes of the surface tension values are strongly correlated to the amount of phospholipids in the samples. Furthermore, the surface tension measurements are more sensitive means to detect the difference between the lavage samples than the total phospholipid content.

Axisymmetric Drop Shape Analysis has been employed to measure the dynamic surface tension response to a sawtooth-shaped variation in the surface area. Four systems were studied: (a) bovine serum albumin (BSA) aqueous solution; (b) dimethyl sulfoxide (DMSO) added to the BSA aqueous solution; (c) ethyl alcohol (ethanol) added to the BSA aqueous solution; and (d) hepoxilin methyl ester, a lipidic hydroxy epoxide derived from arachidonic acid, dissolved in DMSO and then added to the BSA aqueous solution. In the mixtures of the solutions, competitive adsorption to the surface is revealed between the smaller molecules and the protein molecules, through the change of the pattern in the dynamic surface tension response to the surface area change. In the initial stage of the adsorption, the smaller organic molecules (DMSO, ethanol) diffuse to the surface at a faster rate than the protein molecules, impeding protein adsorption. However, in the cases of DMSO/BSA and ethanol/BSA solutions, the experiment indicates that the small organic molecules are squeezed out of the surface because of the compression/dilation process. After steady state is established, the surface tension pattern shows a skewed shape suggesting a conformational change in the protein. When hepoxilin, a naturally occurring biologically active C20 fatty acid containing a hydroxyl epoxide functionality, is present in the BSA solution, a rather symmetric shape is found in the dynamic surface tension changes, different from the skewed shape found in that of the pure BSA solution. This suggests that hepoxilin molecules interact with the protein, leading to a stabilization of one conformation of the protein molecules at the surface.

A pendant drop arrangement is employed in conjunction with the axisymmetric drop shape analysis-profile (ADSA-P) technique to study the effect of concentration and pH on the surface tension of insulin layers adsorbed at the solution-air interface. Three different insulin preparations are used: human zinc-free insulin and two of its analogs, B9AspB27Glu and B10Asp, at three concentrations, 0.01, 0.1 and 1.0 mg ml −1 , and two pH levels, 3 and 7.5. ADSA-P is shown to permit high resolution between the insulin preparations which differ only by one or two amino acids in their primary structures. Furthermore, the effects of pH and concentration on the surface tensions of insulin solutions are examined.

This paper reports dynamic surface tension experiments of a lung surfactant preparation, BLES, for a wide range of concentrations, compression ratios and compression rates. These experiments were performed using Axisymmetric Drop Shape Analysis–Constrained Sessile Drop (ADSA–CSD). The main purpose of the paper is to interpret the results in terms of physical parameters using the recently developed Compression–Relaxation Model (CRM). In the past, only the minimum surface tension was used generally for the characterization of lung surfactant films; however, this minimum value is not a physical parameter and depends on the compression protocol. CRM is based on the assumption that the dynamic surface tension response is governed by surface elasticities, adsorption and desorption of components of the lung surfactant. The ability of CRM to fit the surface tension response closely for a wide variety of parameters (compression ratio, compression rate and surfactant concentration) and produce sensible values for the elastic and kinetic parameters supports the validity of CRM.

The adhesion of a hydrophilic Escherichia coli and a hydrophobic Staphylococcus epidermidis was significantly higher to silicone-coated latex than to a hydrophilic lubricious-coated catheter after 24 h incubation. Time-course studies showed a steady increase in viable E. coli adhesion to the hydrophilic catheter over 24 h. However, in contrast to thermodynamic modelling predictions, S. epidermidis adhered well to the hydrophilic catheter within 30–60 min. By 18 h the adherent S. epidermidis were non-viable, apparently demonstrating the presence of an unidentified antibacterial factor on this catheter. A range of creatinine concentrations had some effect on the adhesion. Bacterial binding was significantly higher in low urea concentrations (

It has been reported in the literature that sugars such as dextrose and sucrose increase the surface tension of water. The effect was interpreted as a depletion of the solute molecules from the water-air interface. This paper presents accurate measurements of the surface tension of different concentrations of dextrose solution as well as its polymer (i.e., dextran). An automated drop shape technique called axisymmetric drop shape analysis (ADSA) was used for the surface tension determination. The surface tension measurement is presented as a function of a shape parameter, P(s), which has been used to quantify the range of the applicability of ADSA. The results of the above study show that dextrose solutions decrease the surface tension of water in contradiction to the results obtained from the weight drop method in the literature. The surface tension decreases continuously with increasing concentration. A similar effect was observed for the dextran solutions. To verify that the setup and the methodology are capable of accurately measuring increases in surface tension, a similar experiment was conducted with a sodium chloride solution with a concentration of 1 M. It is well-known that electrolyte solutions, e.g., sodium chloride, increase the surface tension of water. The results obtained from ADSA verify that the sodium chloride increases the surface tension of water by 1.6 mJ/m(2). It is concluded that dextrose and dextran decrease the surface tension of water. Thus, there is no evidence of depletion. To identify the sources of discrepancy between the results of ADSA and those reported in the literature, the experiments were repeated for different concentrations and the rate of drop formation using the drop weight method. It was found that the rate of drop formation is most likely the source of error in the results reported in the literature.

Shortage or malfunction of pulmonary surfactant in alveolar space leads to a critical condition termed respiratory distress syndrome (RDS). Surfactant replacement therapy, the major method to treat RDS, is an expensive treatment. In this paper, the effect of poly(ethylene glycol) (PEG) to improve dynamic surface activity of a bovine lipid extract surfactant (BLES) was studied by axisymmetric drop shape analysis (ADSA) and a captive bubble method. The activity of BLES + PEG mixtures was compared to that of a natural surfactant containing surfactant proteins A and D. When PEG was added into BLES mixtures, the surface tension hysteresis of BLES films was minimized when the films were compressed by more than 50%. PEG also helps to quickly restore surfactant films after film collapse. Thus, as far as surface tension effects go, the findings suggest that PEG might be used as a substitute for surfactant-associated protein SP-A in therapeutic surfactant products, and might also be used to reduce the amount of BLES required in clinical applications.

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 primary role of lung surfactant is to reduce surface tension at the air-liquid interface of alveoli during respiration. Axisymmetric drop shape analysis (ADSA) was used to study the effect of poly(ethylene glycol) (PEG) on the rate of surface film formation of a bovine lipid extract surfactant (BLES), a therapeutic lung surfactant preparation. PEG of molecular weights 3,350; 8,000; 10,000; 35,000; and 300,000 in combination with a BLES mixture of 0.5 mg/mL was studied. The adsorption rate of BLES alone at 0.5 mg/mL was much slower than that of a natural lung surfactant at the same concentration; more than 200 s are required to reach the equilibrium surface tension of 25 mJ/m(2). PEG, while not surface active itself, enhances the adsorption of BLES to an extent depending on its concentration and molecular weight. These findings suggest that depletion attraction induced by higher molecular weight PEG (in the range of 8,000 to 35,000) may be responsible for increasing the adsorption rate of BLES at low concentration. The results provide a basis for using PEG as an additive to BLES to reduce its required concentration in clinical treatment, thus reducing the cost for surfactant replacement therapy.

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).

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.