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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., (2010 Elsevier B.V. All rights reserved.)

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 113kDa and 213kDa. The other two additives were cationic peptides, polylysine 50kDa 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 microl/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 microl/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)., (Copyright 2010 Elsevier B.V. All rights reserved.)

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 degrees 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.

Contact angle hysteresis of liquids with different molecular and geometrical properties on high quality films of four fluoropolymers was studied. A number of different causes are identified for hysteresis. With n-alkanes as probe liquids, contact angle hysteresis is found to be strongly related to the configuration of polymer chains. The largest hysteresis is obtained with amorphous polymers whereas the smallest hysteresis occurs for polymers with ordered molecular chains. This is explained in terms of sorption of liquid by the solid and penetration of liquid into the polymer film. Correlation of contact angle hysteresis with the size of n-alkane molecules supports this conclusion. On the films of two amorphous fluoropolymers with different molecular configurations, contact angle hysteresis of one and the same liquid with "bulky" molecules is shown to be quite different. On the surfaces of Teflon AF 1600, with stiff molecular chains, the receding angles of the probe liquids are independent of contact time between solid and liquid and similar hysteresis is obtained for all the liquids. Retention of liquid molecules on the solid surface is proposed as the most likely cause of hysteresis in these systems. On the other hand, with EGC-1700 films that consist of flexible chains, the receding angles are strongly time-dependent and the hysteresis is large. Contact angle hysteresis increases even further when liquids with strong dipolar intermolecular forces are used. In this case, major reorganization of EGC-1700 chains due to contact with the test liquids is suggested as the cause. The effect of rate of motion of the three-phase line on the advancing and receding contact angles, and therefore contact angle hysteresis, is investigated. For low viscous liquids, contact angles are independent of the drop front velocity up to approximately 10 mm/min. This agrees with the results of an earlier study that showed that the rate-dependence of the contact angles is an issue only for liquids with high viscosity.

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 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.

Chitosan is a natural, cationic polysaccharide derived from fully or partially deacetylated chitin. Chitosan is capable of inducing large phospholipid aggregates, closely resembling the function of nonionic polymers tested previously as additives to therapeutic lung surfactants. The effects of chitosan on improving the surface activity of a dilute lung surfactant preparation, bovine lipid extract surfactant (BLES), and on resisting albumin-induced inactivation were studied using a constrained sessile drop (CSD) method. Also studied in parallel were the effects of polyethylene glycol (PEG, 10 kD) and hyaluronan (HA, 1240 kD). Both adsorption and dynamic cycling studies showed that chitosan is able to significantly enhance the surface activity of 0.5 mg/mL BLES and to resist albumin-induced inactivation at an extremely low concentration of 0.05 mg/mL, 1000 times smaller than the usual concentration of PEG and 20 times smaller than HA. Optical microscopy found that chitosan induced large surfactant aggregates even in the presence of albumin. Cytotoxicity tests confirmed that chitosan has no deleterious effect on the viability of lung epithelial cells. The experimental results suggest that chitosan may be a more effective polymeric additive to lung surfactant than the other polymers tested so far.

Contact angles of a homologous series of naphthalene compounds on films of a fluorinated acrylate polymer (EGC-1700) deviate from an ideal pattern of contact angles. The deviations increase with the electronegativity of the constituent atoms of the liquid molecules. The results suggest that an uneven distribution of electrostatic charges over the molecules creates strong dipole moments, giving rise to fairly strong dipole-dipole and dipole-induced dipole interactions between liquid molecules and the EGC-1700 chains, which have large dipole moments. In comparison, contact angles of the same probe liquids on the films of Teflon AF 1600, which have small dipole moments, fall on a smooth curve representing the surface tension of the polymer film.

Contact angles of a series of n-alkanes (i.e., n-heptane to n-hexadecane) are studied on two functionalized maleimide copolymers (i.e., poly(ethene-alt-N-(4-(perfluoroheptylcarbonyl)aminobutyl)maleimide) (ETMF) and poly(octadecene-alt-N-(4-(perfluoroheptylcarbonyl)aminobutyl)maleimide) (ODMF)). On the homogeneous ETMF films, all liquids show a smooth motion of the three-phase line. In contrast, on ODMF surfaces that are found to consist of mainly fluorocarbons and small patches of hydrocarbons, short-chain n-alkanes show a stick-slip pattern. By increasing the chain length of the probe liquids, stick-slip is reduced significantly. The phenomenon is discussed in the framework of the Cassie equation. It is found that the upper limit of contact angles in the stick-slip pattern is given by the advancing angle that would be obtained on the pure fluorocarbon surface, whereas the lower limit of the stick-slip pattern is given by the Cassie angle.

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.

Contact angle measurements with a large number of liquids on the semi-fluorinated acryl polymer EGC-1700 films are reported. The surface tension was determined to be gammasv=13.84 mJ/m2 from contact angles of octamethylcyclotetrasiloxane (OMCTS) and decamethylcyclopentasiloxane (DMCPS). Inertness of these two liquids makes them ideal for determination of surface tension of low-energy fluoropolymers. On the other hand, contact angles of many other liquids deviated somewhat from a smooth contact angle pattern that represents the EGC-1700 surface tension. It is argued that noninertness of the molecules of these liquids gives rise to specific interactions with the polymer film, causing the deviations. Furthermore, contact angles of a series of n-alkanes (n-hexane to n-hexadecane) showed systematic deviations from this curve, similar to the trend observed for n-alkanes/Teflon AF 1600 systems studied earlier. Adsorption of vapor of short-chain liquids onto the polymer film caused their contact angles to fall above the gammasv=13.84 mJ/m2 curve, and a parallel alignment of molecules of the long-chain n-alkanes in the vicinity of the solid was the explanation for the deviation of their contact angles below it. It is found that vapor adsorption effect is more significant in the case of Teflon AF 1600, while the alignment of liquid molecules close to the surface is more pronounced for EGC-1700.

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.

Well-measured contact angles with different solid-liquid systems fall approximately on smooth patterns when plotted versus liquid surface tension. However, there are deviations of 1 degrees -3 degrees , which are outside the error limits. It is the purpose of this paper to elucidate the reasons for such deviations. Two types of liquids were selected for advancing contact angle measurements on Teflon AF 1600 coated surfaces: a series of n-alkanes ranging from n-hexane to n-hexadecane and five liquids consisting of bulky molecules, octamethylcyclotetrasiloxane (OMCTS), methyl salicylate, tetralin, cis-decalin, and octamethyltrisiloxane (OMTS). It was found that contact angles of the liquids with bulky molecules fall on a perfectly smooth curve corresponding to a solid surface tension of 13.64 +/- 0.1 mJ/m2. However, contact angles of n-alkanes deviated from this curve by up to 3 degrees in a complicated fashion. The observed trend suggests that more than one mechanism is responsible for the deviations. Substrate-induced rearrangement of liquid molecules in the close vicinity of the surface in the case of long-chain n-alkanes and adsorption of vapor onto the solid surface in the case of short-chain n-alkanes are the most likely explanations. The results suggest that liquids with bulky molecules appear to be suitable for contact angle measurements to characterize energetics of polymeric surfaces.

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.

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.

Two types of experiments were used to study the behavior of both advancing and receding contact angles, namely the dynamic one-cycle contact angle (DOCA) and the dynamic cycling contact angle (DCCA) experiments. For the preliminary study, DOCA measurements of different liquids on different solids were performed using an automated axisymmetric drop shape analysis-profile (ADSA-P). From these experimental results, four patterns of receding contact angle were observed: (1) time-dependent receding contact angle; (2) constant receding contact angle; (3) 'stick/slip'; (4) no receding contact angle. For the purpose of illustration, results from four different solid surfaces are shown. These solids are: FC-732-coated surface; poly(methyl methacrylate/n-butyl methacrylate) [P(MMA/nBMA)]; poly(lactic acid) (DL-PLA); and poly(lactic/glycolic acid) 50/50 (DL-PLGA 50/50). Since most of the surfaces in our studies exhibit time dependence in the receding contact angle, a more extended study was conducted using only FC-732-coated surfaces to better understand the possible causes of decreasing receding contact angle and contact angle hysteresis. Contact angle measurements of 21 liquids from two homologous series (i.e. n-alkanes and 1-alcohols) and octamethylcyclotetrasiloxane (OCMTS) on FC-732-coated surfaces were performed. It is apparent that the contact angle hysteresis decreases with the chain length of the liquid. It was found that the receding contact angle equals the advancing angle when the alkane molecules are infinitely large. These results strongly suggest that the chain length and size of the liquid molecule could contribute to contact angle hysteresis phenomena. Furthermore, DCCA measurements of six liquids from the two homologous series on FC-732-coated surfaces were performed. With these experimental results, one can construe that the time dependence of contact angle hysteresis on relatively smooth and homogeneous surfaces is mainly caused by liquid retention/sorption. The results also suggested that the contact angle hysteresis will eventually approach a steady state, where the rate of liquid retention-evaporation or sorption process would balance out each other. If the existence of contact angle hysteresis can be attributed to liquid sorption/retention, one should only use the advancing contact angles (measured on a dry surface) in conjunction with Young's equation for surface energetic calculations.

The application of spin trapping to the detection of transient radicals generated in heterogeneous systems is discussed. The major part of this review focuses on the interaction of light with particles in dispersion and the resultant radicals which are produced. This includes photoactive pigments such as CdS, TiO2, ZnO and phthalocyanine in a variety of solvents as well as micelles, vesicles and microemulsions containing molecules such as phthalocyanine or chlorophyll. The utility of spin trapping in heterogeneous systems which are not a function of illumination (i.e. electrochemical reactions, catalysis and biological systems) is also discussed. It is demonstrated that reaction mechanisms can be better understood by the indirect detection of the radical intermediates which are involved in the various processes. This impacts studies in solar energy utilization, electrophotography, catalysis, electrochemistry, photosynthesis and the disease process.