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A self-assembled phospholipid monolayer at an air–water interface is a well-defined model system for studying surface thermodynamics, membrane biophysics, thin-film materials, and colloidal soft matter. Here we report a study of two-dimensional phase transitions in the dipalmitoylphosphatidylcholine (DPPC) monolayer at the air–water interface using a newly developed methodology called constrained drop surfactometry (CDS). CDS is superior to the classical Langmuir balance in its capacity for rigorous temperature control and leak-proof environments, thus making it an ideal alternative to the Langmuir balance for studying lipid polymorphism. In addition, we have developed a novel Langmuir–Blodgett (LB) transfer technique that allows the direct transfer of lipid monolayers from the droplet surface under well-controlled conditions. This LB transfer technique permits the direct visualization of phase coexistence in the DPPC monolayer. With these technological advances, we found that the two-dimensional phase behavior of the DPPC monolayer is analogous to the three-dimensional phase transition of a pure substance. This study has implications in the fundamental understanding of surface thermodynamics as well as applications such as self-assembled monolayers and pulmonary surfactant biophysics.

Drop shape techniques are extensively used for surface tension measurement. The accuracy of surface tension measurements using a pendant drop setup depends mainly on the drop shape. As the pendant drop shape becomes close to spherical, the performance of drop shape techniques deteriorates. It is important to consider how to optimize the pendant drop design and how to quantify the accuracy of the surface tension measurements. In this paper, the design and accuracy of pendant drop methods is considered in detail. Dimensional analysis is used to describe similarity in pendant drop shapes and to express the problem using appropriate dimensionless groups. A quantitative criterion called shape parameter is then used to express quantitatively the difference in shape between a given experimental drop and a spherical shape. The shape parameter is found to depend only on two dimensionless groups: the dimensionless volume and the Bond number. A critical shape parameter (minimum value of the shape parameter that guarantees a specified accuracy) is shown to depend only on Bond number. A set of experiments were performed with pure liquids to illustrate the change of the critical shape parameter with the Bond number. Results show that high accuracy is indeed reachable independent of the algorithms used.

Drop shape techniques are widely used for surface tension measurement. As the shape becomes close to spherical, the performance of drop shape techniques deteriorates. A quantitative criterion called shape parameter was previously introduced to quantify the meaning of "well-deformed" drops and "close to spherical" drops. In this paper, a modified definition of the shape parameter that does not depend on the radius of curvature at the drop apex is proposed. Such definition is applicable to different constellations of pendant and sessile drops/bubbles. Dimensional analysis is used to describe similarity in constrained sessile drop shapes and to express the problem using appropriate dimensionless groups. The proposed shape parameter is found to depend only on two dimensionless groups: the dimensionless volume (drop volume normalized by the cube of the holder radius) and the Bond number (using the drop holder's radius as the length scale). A critical shape parameter (minimum value of the shape parameter that guarantees a specified accuracy) is shown to depend only on Bond number. A set of experiments were performed with pure liquids to illustrate the change of the critical shape parameter with the Bond number.

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.

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.

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.

The film adsorption of bovine lipid extract surfactant (BLES) onto the air–liquid interface was examined using axisymmetric drop shape analysis. In combination with a pendant drop constellation, BLES concentrations as high as 10 mg/ml were studied, i.e. concentrations far higher than those accessible with the captive bubble set-ups. ‘Adsorption clicks’, i.e. dynamic processes in which the interfacial tension of surfactant films decreases quickly in a stepwise fashion, were studied at concentrations below 1 mg/ml. Adsorption clicks with high magnitudes up to approximately 35 mJ/m2 (within 0.2 s) were observed. The rate of adsorption was investigated as a function of surfactant concentration. At concentrations below 1 mg/ml, the rate of adsorption is highly concentration dependent. Surfactant films formed on 1 mg/ml BLES solutions reached a surface tension of about 25 mJ/m2 in approximately 10 s, while 0.1 mg/ml BLES required more than 100 s to reach a similar value.

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.

Gastric infection with Helicobacter pylori results in chronic active gastritis and in some individuals is associated with complications such as peptic ulceration and gastric cancers. A balance between bacterial factors and host responses may determine disease outcome. The mouse-adapted H. pylori strain SS1 has been utilized as a model to study disease pathogenesis. Although chronic gastritis is observed in this murine model of H. pylori infection, other complications of disease seen in the human host (such as peptic ulceration) are not identified. The objectives of this study were to characterize virulence factors of the mouse-adapted H. pylori strain SS1 and determine host responses to infection. Vacuolating cytotoxin activity of H. pylori strain SS1 was determined after incubation of HEp-2 cells with culture supernatant for 24 hr. Polymerase chain reaction was performed to detect the presence of the cagA and cagE genes. Chemokine responses from human gastric epithelial cells infected with H. pylori SS1 were assessed by measurement of the concentration of interleukin-8 in cell-free supernatants. C57BL/6 and gld mice were infected with strain SS1 or sham-infected. Eight weeks following infection, gastric tissues were obtained for histological analysis and surface hydrophobicity was measured by axisymmetric drop-shape analysis. H. pylori strain SS1 was cytotoxin negative, cagA positive, and cagE positive, but induced only a modest interleukin-8 response (684 ± 140 pg/ml) from AGS gastric epithelial cells in comparison to a clinical isolate (4170 ± 410 pg/ml, P < 0.0005). Increased inflammation was observed in the stomachs of H. pylori strain SS1-infected animals compared to uninfected controls. Gastritis was not associated with any disease complications. Despite mucosal inflammation, infected mice did not demonstrate alterations in gastric surface hydrophobicity (42.2° ± 2.2° and 41.4° ± 3.2° for C57BL/6 and gld, respectively) compared to uninfected mice (43.2° ± 2.3° and 39.5° ± 1.6°, respectively). In conclusion, murine infection with H. pylori SS1, which contains putative bacterial virulence factors, results in gastric inflammation. However, the mucosal changes are not associated with alterations in surface hydrophobicity. Therefore, the mouse model of infection with H. pylori, strain SS1 may not serve as an entirely appropriate model to study host factors associated with disease complications.

Molecular interactions between small molecules and proteins, such as binding of lipids to proteins, are of fundamental importance in various biological processes. A recently-developed method based on dynamic surface tension measurement is efficient and versatile in detecting such molecular interactions: Axisymmetric Drop Shape Analysis (ADSA) provides a tool for measuring the surface tension (γ) response to surface area changes. Through the analysis of the γ response pattern, surface competitive adsorption between small organic molecules and protein molecules can be detected. Surface squeeze-out of small molecules by proteins can also be observed. Molecular binding of lipids to proteins manifests itself in a modification of the γ response which is not compatible with a simple superposition of the two individual patterns. The specific binding can be studied in terms of dose effects and specificity.

Helicobacter pylori is a recently recognized bacteria which colonizes the stomach and causes gastritis, gastric cancers and peptic ulceration. However gastric and duodenal ulcers occur in only a minority of H. pylori -infected individuals. Surface hydrophobicity is an important virulence factor of many pathogenic bacteria since it is known to be involved in mediating mucosal adherence. In this study, surface hydrophobicity of H. pylori strains originally isolated from children and adolescents was correlated with gastroduodenal pathology including both endoscopic evidence of duodenal ulceration and histologic evidence of duodenitis. Contact angle measurements on 11 H. pylori isolates were used to characterize surface hydrophobicity. Axisymetric drop-shape analysis revealed contact angles ranging from 7.5 to 15.3°. Contact angles of the strains isolated from patients with duodenal ulcers (11.9±2.6°) were not different from values in isolates obtained from children with gastritis alone (8.9±5.2°; P =0.16). Contact angles of H. pylori isolated from patients with and without duodenitis were also comparable (10.9±2.8° and 10.0±9.6°; P =0.69). These findings indicate that additional virulence properties likely are required for H. pylori to induce peptic ulceration in infected humans.

The surface tension of aqueous solutions of Polymer JR400, the chloride salt of a trimethylammonium derivative of hydroxyethyl cellulose, has been measured as a function of polymer concentration. Axisymmetric drop shape analysis profile has been employed to record the dynamic surface tension response of the solutions to a sawtooth-shaped variation in the surface area. Dilute solutions of the polymer (concentration

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.

Of the many methodologies used in surface thermodynamic experimentation, Axisymmetric Drop Shape Analysis (ADSA) has proven to be one of the most accurate and versatile. Until recently, however, ADSA has had two main limitations: (1) the inability to analyse drops with near-zero curvature at the apex; and (2) problems with drop edge detection when the contrast between the drop and the background is not adequately sharp. In this paper, ‘second-generation’ ADSA algorithms and new image analysis schemes are introduced as solutions to these shortcomings. To illustrate these improvements, an inverted sessile air bubble in a pulmonary surfactant suspension is used. This ‘captive bubble’ geometry was chosen to overcome the problems of film leakage at low surface tensions. The modified experimental apparatus, called ADSA-captive bubble (CB), in conjunction with the new software, proves to be a powerful technique to investigate these types of systems. ADSA-CB is fully automated, requiring less user intervention, enabling the collection of large amounts of data, and thereby providing intricate details of surface behaviour. The so-called ‘squeeze-out’ and film collapse phenomena are examined closely. To demonstrate the problems of film leakage, pendant (hanging) drop experimental results are also presented.

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.

Axisymmetric drop shape analysis-profile (ADSA-P) was employed to obtain accurate measurements of the concentration dependence of the film pressure of human serum albumin at a water/decane interface. Fifteen aqueous human albumin solutions ranging in concentration from 0.0001 to 5 mg ml−1 were used. Three domains were identified in the effect of the bulk protein concentration, C, on the interfacial tension, γ: (a) a slow change of γ at low bulk concentrations (C 10−2 mg ml−1). At the two lowest bulk albumin concentrations, 0.0001 and 0.001 mg ml−1, negative interfacial pressures were observed that may be attributed to repulsive electrostatic interactions and the formation of a surface depletion layer. At all other concentrations, a positive surface pressure was measured. Moreover, based on our data on the variation of γ with C, Gibbs' adsorption equation was used to provide an account of the dependence of the surface concentration on C. The possibility of the applicability of the Gibbs' adsorption equation exists for low bulk protein concentrations (C < 10−3 mg ml−1).

The adsorption kinetics of human albumin (HA) at a 0.02 mg ml−1 aqueous solution/decane interface and the temperature dependence of the interfacial tension from 20 to 60°C were studied using Axisymmetric Drop Shape Analysis. This is a non-perturbation method which allows us to study the interfacial tension for a long period of time, without a significant change in the interfacial area. Two criteria were applied to obtain “experimental equilibrium” values of the interfacial tension and interfacial pressure. The rate of change of interfacial pressure with temperature was found to be about 0.1 mJm−2°C−1 from 20 to 45°C. The interfacial pressures were found to be approximately 10 mJ m−2 larger than those reported elsewhere for the HA solution/air interface at room temperature. For the concentration used, a plateau in the interfacial pressure was obtained and can be interpreted on the basis of saturation at temperatures above 45°C.

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.

Contact angle data, measured by using a sessile drop arrangement in conjunction with Axisymmetric Drop Shape Analysis-contact Diameter (ADSA-CD), were used to quantify the effects of ammonia gas plasma treatment on the surface properties of previously untreated polystyrene surfaces. The surface tension of treated polystyrene samples is considerably higher than that of untreated samples. The increase in surface tension following plasma treatment is attributed to the addition of amine groups to the surface.

A micropipette aspiration technique was used to study the extent of adhesion of live and glutaraldehyde-fixed endothelial cells to four solid fibres with different surface tensions, γSV: glass (γSV > 72mj m−2); nylon (γSV = 41 mJ m−2); polyester (γSV = 30 mJ m−2); FC721-coated glass (γSV = 13 mJ m−2). The cells and the fibres were immersed in phosphatebuffered saline (PBS), distilled water (for fixed cells), and complete culture medium. The contact angles of PBS on layers of cells were measured by the Axisymmetric Drop Shape Analysis-Contact Diameter (ADSA-CD) technique. Subsequently, the contact angle data were used in conjunction with the equation of state approach to determine the cell surface tensions, γCV. For a live cell layer, the contact angle was 3.1 ° corresponding to a γCV value of approximately 72 mJ m−2, and for fixed cells, an average contact angle of 36.6° was measured corresponding to a γCV of approximately 60 mJ m−2. A small positive pressure of 1 Pa was applied to deposit the cells onto the fibres before the initiation of aspiration. The force of detachment is reported as a function of the duration of pressure application for the four test fibres in each medium. The results are interpreted by using a thermodynamic model for cell adhesion based on the relevant interfacial tensions. For both live and fixed cells, the forces of detachment increase with increasing duration of pressure application. The measured forces ranged from (3 ± 0) × 10−11N (live cells in culture medium on glass) to (3709 ± 374) × 10−11 N (fixed cells in PBS on FC721), where the errors are the 95% confidence limits. For fixed cells with PBS as a medium, the measured forces of detachment increased with decreasing solid surface tension. This corresponds to a trend predicted by the thermodynamic adhesion model for the case of surface tension of the liquid γLV > γCV and confirmed by the contact angle measurements. In water, although the magnitude of the forces is smaller than that in PBS, the trend is the same as that in PBS and is again predicted by the model. In complete culture medium, the extent of adhesion decreases greatly and the forces of detachment for all test surfaces are indistinguishable from one another. This can be attributed to the prior adsorption of proteins from the medium onto the fibres, resulting in an alteration of their surface properties. For live endothelial cells in PBS, the trend is opposite to that for the fixed cells. According to the thermodynamic model, this trend corresponds to the case of γLV

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 adsorption kinetics of human albumin (HA) at the aqueous solution/air interface and the surface relaxation behaviour of HA adsorption layers after transient area disturbances are studied using an axisymmetric drop shape technique (ADSA). Transient relaxation measurements were performed with the ADSA technique by changing the volume of the pendent drop by definite increments. In the concentration interval studied, the relaxation behaviour of HA is not diffusion controlled. Relaxation phenomena observed during the induction time show the presence of structured adsorption layers. The relaxation behaviour of a quasi-equilibrium HA adsorption layer is independent of the HA concentration. The calculated apparent diffusion coefficients represent the order of magnitude of the rate constant of the real mechanism. A comparison of results calculated from adsorption kinetics and relaxation data shows good agreement with an apparent diffusion coefficient in the order of 0.001 cm2s−1.

The surface tensions of human albumin, immunoglobulin G, and fibrinogen were determined by using two different techniques: (1) the freezing front technique and (2) the pendant drop arrangement in conjunction with axisymmetric drop shape analysis-profile (ADSA-P). The changes in surface tension, resulting from the adsorption of proteins from solution, at various concentrations, onto polymer particles are reported by using the freezing front technique. Furthermore, the adsorption of proteins onto the solution—air interface is also reported by using the pendant drop arrangement. The large differences between the two types of measurements are discussed in terms of the different extents of conformational changes in response to different interfacial tensions driving these changes. A further qualitative difference between the two methods as used here is that the ADSA surface tension measurement registers only the properties of the top few angstroms of the layer whereas the solidification front technique registers the presence of much thicker layers.

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 (

A modified axisymmetric drop shape analysis approach, ADSA-MD (maximum diameter) was developed to measure the contact angles of non-wetting drops front top-view images of the drop. The approach numerically solves the Laplace equation of capillarity given the following input parameters: maximum diameter and volume of the drop, liquid surface tension, density difference between the two fluid phases and the gravity constant. The computed contact angles are in good agreement with those from ADSA-P, an approach which uses the profile of the drop to determine the contact angle. This new technique is particularly suited for systems where the quality of the solid substrate is poor, such as in the case of biological systems. For these situations contact angle determination from the profile is difficult, if not impossible, due to the difficulty in locating the three-phase contact line. The ADSA-MD approach was used to determine the contact angle of water sessile drops on colon sections of New zealand white rabbits.

Axisymmetric drop shape analysis (ADSA) is applied to study time evolution of profiles of pendant and sessile insulin and protein solution drops. Human, bovine and porcine insulin preparations are studied with respect to surface tension kinetics (pendant drop) and contact angle kinetics on hydrophobic FC-721 (fluorocarbon) surfaces. ADSA permits high resolution between the different insulin preparations, which can be distinguished by slight but reproducible differences in surface tensions and contact angles. Application of Young's equation, however, results in nearly identical solid-protein solution interfacial tensions. The time dependence of contact angles of albumin and fibrinogen solution drops on Acetal and FC-721 is presented. Scatter of data is more pronounced on the hydrophilic Acetal surface than on the hydrophobic FC-721 one. Further, an irregular motion of the three-phase contact line is observed. The applicability of Young's equation to albumin and fibrinogen solutions is discussed in terms of the solid-protein solution interfacial tensions.

The surface properties of selected drugs and liposomes (drug delivery vesicles) were characterized by two independent techniques: contact angle and sedimentation volume methods. Firstly, advancing contact angles θA, of different liquids were measured on compressed powders of drugs (tablets) and on layers of liposomes. The contact angles were determined by means of the sessile drop method, and in some cases by the axisymmetric drop shape analysis — contact diameter technique (ADSA-CD). The surface tensions, γSV, were then calculated from the contact angles measured on the different surfaces. Secondly, another method, the sedimentation volume, Vsed, technique was applied to estimate directly the solid/vapour surface tensions of drug powders and those of the liposomes originally suspended in binary liquid mixtures. The experiments were performed with six different drugs used both in powdered form (Vsed) and as powder compacts (contact angle). Multilamellar (MLV) liposomes were prepared from phospholipid (PL) and cholesterol (Chol) in different molar ratios. Both empty liposomes (without drug) and drug loaded vesicles were investigated as dispersions for the sedimentation experiments and as deposited liposomal layers for the contact angle measurements. The surface tensions obtained from Vsed and calculated from θA for the drugs showed an agreement. The order of the hydrophobicity of the drugs can be established from these results. It was also found that the surface tensions of the empty liposomes with different PL/Chol ratios depend on their composition: the hydrophilic character of liposomes decreases with the increasing cholesterol content. Sedimentation results indicated that the encapsulated drug might affect the surface properties of liposomes. The effect of drug depends on the PL/Chol molar ratio as well as on the properties and the concentration of the encapsulated drug. It was found that the change in surface properties of liposomal layers due to drug encapsulation cannot be detected by the traditional (sessile drop) contact angle measuring method. However, the ADSA-CD technique provided higher sensitivity. The surface tensions obtained for the deposited layers of drug loaded vesicles by this technique were similar to those obtained from the Vsed studies performed with liposome dispersions.

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.

Gastric infection with Helicobacter pylori results in chronic active gastritis and in some individuals is associated with complications such as peptic ulceration and gastric cancers. A balance between bacterial factors and host responses may determine disease outcome. The mouse-adapted H. pylori strain SS1 has been utilized as a model to study disease pathogenesis. Although chronic gastritis is observed in this murine model of H. pylori infection, other complications of disease seen in the human host (such as peptic ulceration) are not identified. The objectives of this study were to characterize virulence factors of the mouse-adapted H. pylori strain SS1 and determine host responses to infection. Vacuolating cytotoxin activity of H. pylori strain SS1 was determined after incubation of HEp-2 cells with culture supernatant for 24 hr. Polymerase chain reaction was performed to detect the presence of the cagA and cagE genes. Chemokine responses from human gastric epithelial cells infected with H. pylori SS1 were assessed by measurement of the concentration of interleukin-8 in cell-free supernatants. C57BL/6 and gld mice were infected with strain SS1 or sham-infected. Eight weeks following infection, gastric tissues were obtained for histological analysis and surface hydrophobicity was measured by axisymmetric drop-shape analysis. H. pylori strain SS1 was cytotoxin negative, cagA positive, and cagE positive, but induced only a modest interleukin-8 response (684 +/- 140 pg/ml) from AGS gastric epithelial cells in comparison to a clinical isolate (4170 +/- 410 pg/ml, P0.0005). Increased inflammation was observed in the stomachs of H. pylori strain SS1-infected animals compared to uninfected controls. Gastritis was not associated with any disease complications. Despite mucosal inflammation, infected mice did not demonstrate alterations in gastric surface hydrophobicity (42.2 degrees +/- 2.2 degrees and 41.4 degrees +/- 3.2 degrees for C57BL/6 and gld, respectively) compared to uninfected mice (43.2 degrees +/- 2.3 degrees and 39.5 degrees +/- 1.6 degrees, respectively). In conclusion, murine infection with H. pylori SS1, which contains putative bacterial virulence factors, results in gastric inflammation. However, the mucosal changes are not associated with alterations in surface hydrophobicity. Therefore, the mouse model of infection with H. pylori, strain SS1 may not serve as an entirely appropriate model to study host factors associated with disease complications.

In a recent paper,(1) we demonstrated that molecular interactions between biopolymers and other smaller molecules can be detected by means of dynamic surface tension measurements. In the present paper, we demonstrate that the same methodology can be employed for investigating dose effects and specificity of molecular interactions. Three similar lipids were chosen for this study: 12(S)-hydroxy-5(Z), 8(Z),10(E),14(Z)-eicosatetraenoic acid (12(S)-HETE-free acid), methyl 12(S)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoate (12(S)-HETE-methyl ester), and 5(Z),8(Z),11(Z), 14(Z)-eicosatetraenoic acid (arachidonic acid-free acid). These substances were added to a fatty acid free bovine serum albumin (BSA) aqueous solution at different lipid concentrations. The characteristic tension response indicates that molecular interactions between 12(S)-HETE-free acid and BSA exist. The detected interactions are concentration dependent: at a molecular ratio of lipid to protein of 1:1, the binding of 12(S)-HETE-free acid to BSA is hydrophobic in nature; at the molecular ratio of lipid to protein of 10:1, a secondary binding occurs and is hydrophilic in nature. Similar molecular interactions were not detected between 12(S)-HETE-methyl ester or arachidonic acid-free acid and BSA, indicating that the interactions between 12(S)-HETE-free acid and BSA are specific. As an independent means, surface elasticity is used to probe the molecular interactions at the interface. In the case of 12(S)-HETE-free acid but not its methyl ester or arachidonic acid, distinct higher surface elasticities were observed at lipid concentrations in excess of a molecular ratio of lipid to protein of 1:1. This finding reinforces the above stipulations.

Helicobacter mustelae is a gastric pathogen in ferrets that adheres to epithelial cells both in vitro and in vivo. In this study, the authors examine the role of surface hydrophobic properties in the adhesion of these organisms to eukaryotic cell surfaces. The surface properties of six H. mustelae strains were characterized by hydrophobic interaction chromatography (HIC), salt aggregation testing (SAT) and contact-angle measurement by axisymmetric drop-shape analysis (ADSA). Contact angles in multiple regions of the gastrointestinal tract, obtained from infected and uninfected ferrets, were also measured. The cell surface of H. mustelae was found to be hydrophilic by SAT but relatively hydrophobic by HIC. Contact-angle measurements for H.mustelae (mean 22.5 degrees, 95% confidence interval [CI] 9.3 degrees to 35.7 degrees) were higher than values previously reported for Helicobacter pylori (mean 12.1 degrees, 95% CI 2.0 degrees to 22.2 degrees, p0.05). The body of the stomach was more hydrophilic in infected ferrets (mean contact angle 59.9 degrees, 95% CI 52.5 degrees to 67.3 degrees) than in uninfected animals (mean contact angle 94.2 degrees, 95% CI 84.4 degrees to 104.0 degrees, p0.05). Reductions in the surface hydrophobicity of the ferrets' stomachs were correlated with the degree of mucosal inflammation (p0.01). These findings demonstrate that H. mustelae has surface properties comparable to those of H. pylori strains. Like the human stomach infected by H. pylori, there is a reduction in surface hydrophobicity of the ferret antrum associated with H. mustelae infection and the resulting mucosal inflammatory cell response.

To quantitate surface hydrophobicity of the stomach, we measured contact angles formed with water droplets in different regions of rabbit stomach at varying ages (suckling, weanling, and adult). Contact angles were measured using novel methods: axisymmetric drop-shape analysis-contact diameter for contact angles90 degrees and axisymmetric drop-shape analysis-maximum diameter for contact angles90 degrees. To determine whether gastric mucus was responsible for the physical properties of the surface mucosa, the surface tension of mucus derived from the body of stomach was measured by axisymmetric drop-shape analysis on pendant drops. Contact angles of adult antrum 82.9 degrees +/- 5.5 degrees (mean +/- SEM) were greater than in the body of stomach (36.1 degrees +/- 2.6 degrees, p = 0.0001). Contact angles on mucosa obtained from the body of the stomach of both suckling rabbits (76.4 degrees +/- 2.7 degrees) and weanling rabbits (84.2 degrees +/- 2.9 degrees) were greater than in adult animals (ANOVA, p0.05). Pendant drop analysis of mucus derived from the body of stomach showed a high surface tension (57.72 +/- 0.06 mJ/m2, mean +/- SD). We conclude that there are maturational changes and regional differences in the surface hydrophobicity of the lapine stomach. These changes are likely caused by changes in the overlying mucus layer.

To quantitate surface hydrophobicity of the intestine, we measured contact angles formed with water droplets in multiple regions of rabbit intestine at varying ages (suckling, weanling, and adult) and after dinitrochlorobenzene-induced colitis. Contact angles were measured using novel methods: axisymmetric drop-shape analysis-contact diameter for contact angles less than 90 degrees and axisymmetric dropshape analysis-maximum diameter for contact angles greater than 90 degrees. To determine whether mucus was present on the surface of intestine used, indirect immunofluorescence was performed using antibody specific to goblet cell mucin. To confirm that intestinal mucus could be responsible for the physical properties of surface mucosa, surface tensions of mucus prepared from distal ileum, distal colon, and inflamed distal colon of adult rabbits were measured by axisymmetric drop-shape analysis on pendant drops. Contact angles of adult small intestine [duodenum, 38.0 +/- 11.2 degrees (SD); jejunum, 44.0 +/- 22.9 degrees; ileum, 56.4 +/- 23.3 degrees] were less than proximal colon (93.2 +/- 6.7 degrees; P less than 0.05) and distal colon (86.4 +/- 24.2 degrees; P less than 0.05). Contact angles on proximal colon from suckling rabbits (53.2 +/- 8.4 degrees) were less than both weanling (93.2 +/- 23.3 degrees; P less than 0.05) and adult rabbits (93.2 +/- 6.7 degrees; P less than 0.05). Contact angles on inflamed adult distal colon (54.7 +/- 20.6 degrees) were decreased from values on normal distal colons (86.4 +/- 24.2 degrees). Indirect immunofluorescence demonstrated that mucin was present in both vacuoles of goblet cells and on the colonic surface.(ABSTRACT TRUNCATED AT 250 WORDS)

The focus of this study was to establish whether the advancing solidification front technique could be used to quantitate the surface hydrophoblcity of protein-coated polymer materials. The method has the advantage that it allows a study of this question without having to expose the protein-coated particle to an air interface which would denature the adsorbed protein layer. We have also used the technique to establish whether substrates with different surface tensions would induce a different extent of conformational change in the adsorbed protein molecules. Such changes are reflected by differences in the surface tension of different substrate materials coated by one and the same protein. The results with a low bulk protein concentration (< 0.1%) show a decreasing surface tension of the adsorbed protein layer with increasing substrate hydrophoblcity, suggesting more extensive conformational changes on the more hydrophobic surfaces. At high bulk protein concentrations (0.5% and above) the surface...

The contact angle between a sessile drop of liquid and a bed of biological particles such as bacterial cells provides a measure of the hydrophobicity or, more precisely, the surface tension of the cells. While this surface characteristic plays an important role in diverse processes such as cell adhesion and phagocytosis, it is difficult to obtain contact angles which are meaningful in a thermodynamic sense using traditional methods of measurement (e.g., goniometric techniques) because: (1) the contact angles, which are inherently small on biological substrates in their native hydrated state, are difficult to measure with precision; (2) the contact angle of the sessile drop invariably decreases as the liquid is absorbed into the layer of cells, and; (3) the heterogeneous and often rough surfaces produced by any method of cell deposition give rise to sessile drops which do not possess a perfectly circular perimeter. A modified axisymmetric drop shape analysis (ADSA-CD) permits the contact angle of a sessile drop to be calculated from the average diameter of the drop as viewed from above, and provides the means to circumvent these problems. The ADSA-CD procedure was used to measure the contact angle of water on layers of three different species of bacteria. The average contact angles for T. thiooxidans and Staph. epidermidis were 16.9 and 20.6°, respectively. The contact angles for two different strains of T. ferrooxidans were 11.7 and 10.5° and were not statistically different. The 95% confidence intervals for these means, obtained from fewer than 17 independent measurements were less than or equal to ± 1.0°. We show that this novel strategy provides: (1) increased objectivity with respect to the goniometric method; (2) a precise estimate of the contact angle in spite of the fact that the contact angle is time dependent; (3) a simple means of assessing surface quality and the circularity of the drop periphery on biological surfaces.

The role of hydrophobicity in the attachment of enteropathogens to gastrointestinal mucosa is controversial. In vitro binding of Escherichia coli RDEC-1 to rabbit intestine is dependent on the expression of pili. We examined in vitro adherence of piliated RDEC-1 after altering either the hydrophobicity of the organisms, the hydrophobicity of the substrate for attachment, or the surface tension of the suspending liquid. Hydrophobicity of RDEC-1 was determined using four complementary methods. In each assay piliated RDEC-1 demonstrated relatively more hydrophobic properties compared with both organisms grown to suppress pilus expression and a mutant that cannot express mannose-resistant pili. When piliated RDEC-1 were pretreated with tetramethyl urea to disrupt hydrophobic bonds surface hydrophobicity decreased. Concurrently, bacterial adherence to rabbit ileal microvillus membranes, mucus and mucin was reduced. Binding of piliated organisms to hydrophobic surfaces was significantly higher compared to both nonpiliated bacteria and the adherence of piliated RDEC-1 to relatively hydrophilic surfaces. Addition of propanol reduced the surface tension of the suspending liquid, and decreased adhesion of piliated RDEC-1 to polystyrene by 80%. Conversely, adherence of piliated organisms to a hydrophilic surface increased 12-fold after lowering the surface tension of the suspending liquid. We conclude that hydrophobic properties have a role in mediating in vitro adherence of this E. coli enteric pathogen.

The insulinogenic response to a standard i.v. dose of a sulfonylurea can be markedly augmented in normal, conscious dogs if they are given 30 min earlier a single i.v. dose of dihydroergotamine (DHE). Since the parent substance ergotamine posssed no such amplifying properties, further experiments were conducted to clarify the essential structural requirements that have to be fulfilled for an ergot alkaloid to act as an amplifier of sulfonylurea-stimulated insulin secretion. Amine alkaloids ergonovine, dihydroergonovone and dihydromethylergonovine had no amplifying potency, but the hydrogenated amino acid alkaloids dihydroergocornine, dihydroergocristine and dihydroergokryptine (Hydergine) were almost as potent amplifiers as was DHE. The data indicate that (a) DHE, Hydergine and by inference all hydrogenated amino acid alkaloids are potent amplifiers of sulfonylurea-stimulated insulin secretion; (b) saturation of the double bond at C9 and C10 of the lysergic acid moiety and the pressence of an amino acid side chain are essential structual requirements for an ergot alkaloid to function as an amplifier of the action of sulfonylureas; and (c) it appears that these compounds are acting chiefly by mechanisms other than alpha-adrenergic and serotonergic receptor blockade, perhaps as regulatory molecules inducing positive cooperative changes in integral proteins of the plasma membrane of beta cells.

The adhesion of five strains of bacteria, i.e., Staphylococcus aureus (strain 049), Staphylococcus epidermidis (strain 047), Escherichia coli (strains 055 and 2627), and Listeria monocytogenes, to various polymeric surfaces was studied. The design of the experimental protocol was dictated by thermodynamic considerations. From the thermodynamic model for the adhesion of small particles from a suspension onto a solid substratum, it follows that the extent of adhesion is determined by the surface properties of all three phases involved, i.e., the surface tensions of the adhering particles, of the substrate, and of the suspending liquid medium. In essence, adhesion is more extensive to hydrophilic substrata (i.e., substrata of relatively high surface tension) than to hydrophobic substrata, when the surface tension of the bacteria is larger than that of the suspending medium. When the surface tension of the suspending liquid is larger than that of the bacteria, the opposite pattern of behavior prevails. Suspensions of bacteria at a concentration of 10(8) microorganisms per ml were brought into contact with several polymeric surfaces (Teflon, polyethylene, polystyrene, and acetal and sulfonated polystyrene) for 30 min at 20 degrees C. After rinsing, the number of bacteria adhering per unit surface area was determined by image analysis. The surface tension of the suspending medium. Hanks balanced salt solution, was modified through the addition of various amounts of dimethyl sulfoxide. It was found that the number of bacteria adhering per unit surface area correlates well with the thermodynamic predictions and that these data may be used to determine the surface tension of the different bacterial species. The surface tensions of the bacteria obtained in this fashion are in excellent agreement with those obtained by other methods.

The sedimentation volume technique was employed to characterize the surface properties of proteincoated polymers. The sedimentation volume, V sed , of polymer particles suspended in aqueous solutions at 23°C was determined. The suspending binary liquid mixtures were chosen such that the surface tension,γ PV , of the protein-coated polymers fell between the surface tensions,γ LV , of the two liquids. A constant mass of a given polymer sample was suspended in constant volumes of the liquid mixtures of different concentrations. It was found that the sedimentation volume changed with varying compositions of the suspending liquid mixtures, as did the liquid surface tension. A maximum in V sed occurred when the surface tension of the suspending liquid was equal to that of the particles, i.e., whenγ LV = γ PV . The position of the V sed maxima, and hence the surface tension,γ PV , of the particles, was found to depend on polymer surface tension as well as on the type and bulk concentration of the coating protein solution. At high bulk concentrations the nature of the underlying substrate materials are entirely masked. Under these conditions V sed maxima, and hence the particle surface tensions, are independent of the nature of the substrate surface properties and are reflective only of the adsorbed protein. At low bulk concentrations a range in V sed maxima are observed, suggestive of substrate-induced conformational changes in the adsorbed protein molecules.

Thermodynamic model considerations suggest that the adhesion of biological cells to polymeric surfaces depends on the relative magnitude of the surface tension γSV of the substrates, the surface tension γCV of the cells, and the surface tension γLV of the suspending liquid medium. Glutaraldehyde-fixed erythrocytes are identified as suitable model particles for such studies. Experimental results of the extent of erythrocyte adhesion from suspension of mixtures of buffer and varying amounts of dimethyl sulfoxide (DMSO) confirm qualitatively and in certain aspects quantitatively the thermodynamic predictions. Specifically, the prediction that the extent of cell adhesion should become independent of substrate surface tension when γLV = γCV is confirmed by experiment. On the other hand for this case the free energy of adhesion predicting zero cell adhesion is not experimentally confirmed completely. However, performing these experiments with distilled water rather than buffer reduces erythrocyte adhesion to a negligible level. It emerges that van der Waals interactions are, by a large margin, the most important forces involved in cell adhesion to polymer surfaces. Nevertheless, not only divalent cations (through bridging effects), but also monovalent cations play a certain, though limited, role.

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.