Zdenka Policova

Chitosan Enhances the In Vitro Surface Activity of Dilute Lung Surfactant Preparations and Resists Albumin-Induced Inactivation

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.

Semiautomatic measurement of contact angles on cell layers by a modified axisymmetric drop shape analysis

Abstract 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 effect of dextran to restore the activity of pulmonary surfactant inhibited by albumin.

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.

A study of captive bubbles with axisymmetric drop shape analysis

Abstract 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.

Measurement of contact angles from the maximum diameter of non-wetting drops by means of a modified axisymmetric drop shape analysis

Abstract 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) applied to protein solutions

Abstract 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 Youngs 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 Youngs equation to albumin and fibrinogen solutions is discussed in terms of the solid-protein solution interfacial tensions.

The effect of concentration on the bulk adsorption of bovine lipid extract surfactant

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.

Mixed DPPC/DPPG monolayers at very high film compression.

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.

Axisymmetric Drop Shape Analysis−Constrained Sessile Drop (ADSA-CSD): A Film Balance Technique for High Collapse Pressures

Collapse pressure of insoluble monolayers is a property determined from surface pressure/area isotherms. Such isotherms are commonly measured by a Langmuir film balance or a drop shape technique using a pendant drop constellation (ADSA-PD). Here, a different embodiment of a drop shape analysis, called axisymmetric drop shape analysis-constrained sessile drop (ADSA-CSD) is used as a film balance. It is shown that ADSA-CSD has certain advantages over conventional methods. The ability to measure very low surface tension values (e.g., <2 mJ/m2), an easier deposition procedure than in a pendant drop setup, and leak-proof design make the constrained sessile drop constellation a better choice than the pendant drop constellation in many situations. Results of compression isotherms are obtained on three different monolayers: octadecanol, dipalmitoyl-phosphatidyl-choline (DPPC), and dipalmitoyl-phosphatidyl-glycerol (DPPG). The collapse pressures are found to be reproducible and in agreement with previous methods. For example, the collapse pressure of DPPC is found to be 70.2 mJ/m2. Such values are not achievable with a pendant drop. The collapse pressure of octadecanol is found to be 61.3 mJ/m2, while that of DPPG is 59.0 mJ/m2. The physical reasons for these differences are discussed. The results also show a distinctive difference between the onset of collapse and the ultimate collapse pressure (ultimate strength) of these films. ADSA-CSD allows detailed study of this collapse region.

The temperature dependence of the interfacial tension of aqueous human albumin solution/decane

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.