Study of water vapor and surfactant absorption by lipid model systems using the quartz crystal microbalance

Skin is constantly exposed to surfactants which compromise the essential barrier function of normal healthy skin. To model the interactions of surfactants with the barrier lipids of the stratum corneum (SC), it is essential to develop in vitro and in vivo quantitative measurement methods to predict, evaluate, and demonstrate the effect of the different surfactant chemistries and technologies on skin. In the current work, in vitro water vapor uptake and surfactant absorption onto skin lipid model films were quantitatively studied using a technique based on the piezoelectric effect, the quartz crystal microbalance (QCM). This approach is straightforward and reliable in providing subtle surface/interface related mass change information with high resolution and sensitivity. The results show that barrier properties of the lipid model system may be damaged by surfactant absorption, as well as by long-term water exposure due to alterations to the lipid film structure. Surfactant absorption is found to be concentration dependent even beyond its critical micelle concentration (CMC). QCM results for different surfactant systems are consistent with reported clinical data in showing that clinically milder surfactants (SLES) do not perturb the film as much as clinically harsh surfactants (SDS).     

Exposure to Polymers Reverses Inhibition of Pulmonary Surfactant by Serum,Meconium, or Cholesterol in the Captive Bubble Surfactometer

Dysfunction of pulmonary surfactant in the lungs is associated with respiratory pathologies such as acute respiratory distress syndrome or meconium aspiration syndrome. Serum, cholesterol, and meconium have been described as inhibitory agents of surfactant’s interfacial activity once these substances appear in alveolar spaces during lung injury and inflammation. The deleterious action of these agents has been only partly evaluated under physiologically relevant conditions. We have optimized a protocol to assess surfactant inhibition by serum, cholesterol, or meconium in the captive bubble surfactometer. Specific measures of surface activity before and after native surfactant was exposed to inhibitors included i), film formation, ii), readsorption of material from surface-associated reservoirs, and iii), interfacial film dynamics during compression-expansion cycling. Results show that serum creates a steric barrier that impedes surfactant reaching the interface. A mechanical perturbation of this barrier allows native surfactant to compete efficiently with serum to form a highly surface-active film. Exposure of native surfactant to cholesterol or meconium, on the other hand, modifies the compressibility of surfactant films though optimal compressibility properties recover on repetitive compression-expansion cycling. Addition of polymers like dextran or hyaluronic acid to surfactant fully reverses inhibition by serum. These polymers also prevent surfactant inhibition by cholesterol or meconium, suggesting that the protective action of polymers goes beyond the mere enhancement of interfacial adsorption as described by depletion force theories.     

Neuromediator action on surfactant system activity and the functioning area of the lung

Experiments on rats have demonstrated the effect of neurotransmitter function on the activity of the surfactant system and functioning area of the lung (FAL). Excess transmitters gave rise to activation of the lung surfactant system and increase in the FAL, whereas the deficiency of transmitters lowered surfactant activity and the area of the air-blood barrier.     

Enhanced surfactant adsorption via polymer depletion forces: a simple model for reversing surfactant inhibition in acute respiratory distress syndrome

Lung surfactant adsorption to an air-water interface is strongly inhibited by an energy barrier imposed by the competitive adsorption of albumin and other surface-active serum proteins that are present in the lung during acute respiratory distress syndrome. This reduction in surfactant adsorption results in an increased surface tension in the lung and an increase in the work of breathing. The reduction in surfactant adsorption is quantitatively described using a variation of the classical Smolukowski analysis of colloid stability. Albumin adsorbed to the interface induces an energy barrier to surfactant diffusion of order 5 k(B)T, leading to a reduction in adsorption equivalent to reducing the surfactant concentration by a factor of 100. Adding hydrophilic, nonadsorbing polymers such as polyethylene glycol to the subphase provides a depletion attraction between the surfactant aggregates and the interface that eliminates the energy barrier. Surfactant adsorption increases exponentially with polymer concentration as predicted by the simple Asakura and Oosawa model of depletion attraction. Depletion forces can likely be used to overcome barriers to adsorption at a variety of liquid-vapor and solid-liquid interfaces.     

Ultrastructure of the blood-air barrier in comparison with the indices of surfactant surface activity

Ultrastructure of the air-blood barrier and surface surfactant activity were studied at different time periods of nonspecific inflammation of the lungs in guinea pigs. The animals were sacrificed 3 days, 2 weeks and 1, 2 and 4 months after beginning of the experiment. It has been demonstrated that in early periods of lung inflammation there was edema of all components of the air-blood barrier. Subsequent development of inflammation is accompanied by surface activity decrease associated with dystrophic changes in the epithelial cells of alveoli. At the same time there are compensatory changes in the lungs, directed to eliminate deficiency of surfactant.     

Changes in surface activity of surfactant and ultrastructure of the air-blood barrier in alcoholic intoxication in experimental animals

In the results of complex investigation of the lungs of 26 white rats, it was established, that there is the suppression of surface active properties of surfactant under influence of ethanol. In acute poisoning this suppression is associated with direct injury of surfactant with ethanol and inactivation of surfactant with serum proteins, which appear in the alveolar space because of the edema of air-haematic barrier. In prolonged influence the suppression of the surface activity of surfactant is due to the increase of its catabolism with alveolar macrophages.     

Pulmonary surfactant system of newborn rats in alcoholic intoxication of pregnant rats

Data, received in investigation of the lungs of 45 newborn rats, show, that there is the suppression of the surface active properties of surfactant in animals, born from female rats with simulated alcoholic intoxication in pregnancy period. The decrease of the surface activity of surfactant may be connected with direct injury influence of alcohol on surfactant as well as with inactivation of surfactant with serum proteins, which appear in the alveolar space because of the increase of the permeability of components of air-haematic barrier. The suppression of the surface active properties of surfactant is accompanied by reinforcement of the functional activity of the 2nd type pneumocytes and appearance of the hypertrophic forms of these cells.     

Molecular weight dependence of the depletion attraction and its effects on the competitive adsorption of lung surfactant

Albumin competes with lung surfactant for the air-water interface, resulting in decreased surfactant adsorption and increased surface tension. Polyethylene glycol (PEG) and other hydrophilic polymers restore the normal rate of surfactant adsorption to the interface, which re-establishes low surface tensions on compression. PEG does so by generating an entropic depletion attraction between the surfactant aggregates and interface, reducing the energy barrier to adsorption imposed by the albumin. For a fixed composition of 10 g/L (1% wt.), surfactant adsorption increases with the 0.1 power of PEG molecular weight from 6 kDa-35 kDa as predicted by simple excluded volume models of the depletion attraction. The range of the depletion attraction for PEG with a molecular weight below 6 kDa is less than the dimensions of albumin and there is no effect on surfactant adsorption. PEG greater than 35 kDa reaches the overlap concentration at 1% wt. resulting in both decreased depletion attraction and decreased surfactant adsorption. Fluorescence images reveal that the depletion attraction causes the surfactant to break through the albumin film at the air-water interface to spread as a monolayer. During this transition, there is a coexistence of immiscible albumin and surfactant domains. Surface pressures well above the normal equilibrium surface pressure of albumin are necessary to force the albumin from the interface during film compression.     

Ultrastructural morphology of the air-blood barrier and pulmonary surfactant in pulmonary inflammation in experimental alcoholic intoxication

Combined investigation of ultrastructure of components of air-haematic barrier and surface-active properties of surfactant in 21 guinea pigs' lungs with simulated pneumonia against a background of alcoholic intoxication was carried out. It was established, that alcoholic intoxication aggravates a deficiency of pulmonary surfactant occurred in pneumonia because of its high phagocytosis with alveolar macrophages. The increase of mobilization of alveolar macrophages in alcoholic intoxication may be connected with the rise of surfactant secretion by hyperfunctional pneumocytes of the 2nd type. Stopping of alcoholic intoxication may lead to normalization of qualitative composition of surfactant phospholipids.     

Mechanisms of polyelectrolyte enhanced surfactant adsorption at the air-water interface

Chitosan, a naturally occurring cationic polyelectrolyte, restores the adsorption of the clinical lung surfactant Survanta to the air-water interface in the presence of albumin at much lower concentrations than uncharged polymers such as polyethylene glycol. This is consistent with the positively charged chitosan forming ion pairs with negative charges on the albumin and lung surfactant particles, reducing the net charge in the double-layer, and decreasing the electrostatic energy barrier to adsorption to the air-water interface. However, chitosan, like other polyelectrolytes, cannot perfectly match the charge distribution on the surfactant, which leads to patches of positive and negative charge at net neutrality. Increasing the chitosan concentration further leads to a reduction in the rate of surfactant adsorption consistent with an over-compensation of the negative charge on the surfactant and albumin surfaces, which creates a new repulsive electrostatic potential between the now cationic surfaces. This charge neutralization followed by charge inversion explains the window of polyelectrolyte concentration that enhances surfactant adsorption; the same physical mechanism is observed in flocculation and re-stabilization of anionic colloids by chitosan and in alternate layer deposition of anionic and cationic polyelectrolytes on charged colloids.     

Inactivation of pulmonary surfactant due to serum-inhibited adsorption and reversal by hydrophilic polymers: experimental

The rate of change of surface pressure, pi, in a Langmuir trough following the deposition of surfactant suspensions on subphases containing serum, with or without polymers, is used to model a likely cause of surfactant inactivation in vivo: inhibition of surfactant adsorption due to competitive adsorption of surface active serum proteins. Aqueous suspensions of native porcine surfactant, organic extracts of native surfactant, and the clinical surfactants Curosurf, Infasurf, and Survanta spread on buffered subphases increase the surface pressure, pi, to approximately 40 mN/m within 2 min. The variation with concentration, temperature, and mode of spreading confirmed Brewster angle microscopy observations that subphase to surface adsorption of surfactant is the dominant form of surfactant transport to the interface. However (with the exception of native porcine surfactant), similar rapid increases in pi did not occur when surfactants were applied to subphases containing serum. Components of serum are surface active and adsorb reversibly to the interface increasing pi up to a concentration-dependent saturation value, pi(max). When surfactants were applied to subphases containing serum, the increase in pi was significantly slowed or eliminated. Therefore, serum at the interface presents a barrier to surfactant adsorption. Addition of either hyaluronan (normally found in alveolar fluid) or polyethylene glycol to subphases containing serum reversed inhibition by restoring the rate of surfactant adsorption to that of the clean interface, thereby allowing surfactant to overcome the serum-induced barrier to adsorption.     

Ultrastructural and morphometric study of the lung of the European salamander,Salamandra salamandra L.

Ultrastructural and morphometric investigations were performed on the lung of the European salamander, Salamandra salamandra L. Folds of first and second order are covered with a ciliated epithelium containing goblet cells. The respiratory surface of the lung is lined by a single type of cell which, in amphibians, combines features of type I and type II alveolar cells of the mammalian lung. In the salamander the respiratory and ciliated epithelial cells as well as goblet cells possess electron dense and lucent vesicles in their cytoplasm as well as lamellar bodies. A small amount of surfactant, composed most probably of phospholipids and mucopolysaccharides, was observed covering the entire inner surface of the lung. Morphometric methods were used to determine the dimensions of the perinuclear region of pneumocytes, the thickness of the air-blood barrier and lung wall, and also the diameter of capillaries. The thickness of the respiratory air-blood barrier was found to be considerably higher than that of the corresponding barrier in mammals.     

The electron microscopic characteristics of stress lung

The influence of long-term (6 hours) immobilization stress on morphofunctional state of lung air-blood barrier was studied in experiments of the rats. It was shown that stress provoked the marked ultrastructural changes in the lungs, which were as follows: lung tissue oedema, pronounced thickening of lung air-blood barrier and its separate layers, edema-hemorrhagic syndrome, alveolar epithelial injury, disturbance of lung surfactant systems. Such a pathological complex may be designated as a "stress lung".     

Effect of exogenous pulmonary surfactant preparations on the structure of pulmonary alveoli in newborn rats

Treatment of pre-term newborns with exogenous surfactant preparation is a well established part of the therapy for respiratory distress syndrome of the newborns (RDS). Since the introduction of surfactant into clinical practice in 1980, hundreds of studies have been published describing beneficial effects of such treatment. There is only limited number of morphological publications reporting adverse effects of surfactant administration. The aim of the present study is to describe morphological changes in the lung after surfactant administration to healthy newborn rats. Two types of surfactant were used: Exosurf (Glaxo Wellcome, England) and Survanta (Abbott Laboratories, USA). Surfactant preparation were given intratracheally in single dose (bolus) (100 mg of lipids per kg b.w.). Animals from control group received 0.9% saline in equivalent volume. Lung specimens were taken 15, 20, 25 and 30 minutes after drug administration and evaluated by light and electron microscopy. There was no damage in lungs from the control group. Tissue specimens from the Exosurf group revealed severe pathological changes: foci of atelectasis, frank edema in the parenchyma, focal disruption of air-blood barrier, hemorrhages in many alveoli, surfactant particles in many alveolar capillaries, and strongly activated alveolar macrophages. In this group changes appeared as early as 15 min after surfactant administration and intensity of lung injury increased with time. Also, Survanta administration caused damage to the lung tissue. However, the changes were less intense and appeared later (20-25 minutes after Survanta treatment). In conclusion, the presented morphological findings proved that exogenous surfactant administration to healthy rat newborns caused lung damage. Comparing two different surfactant preparation, Exosurf and Survanta, it was shown that the former one produced stronger and faster damage to lung alveoli than the latter one.     

Effects of modified cellulose nanocrystals on the barrier and migration properties of PLA nano-biocomposites

The aim of this paper is to report the impact of the addition of cellulose nanocrystals on the barrier properties and on the migration behaviour of poly(lactic acid), PLA, based nano-biocomposites prepared by the solvent casting method. Their microstructure, crystallinity, barrier and overall migration properties were investigated. Pristine (CNC) and surfactant-modified cellulose nanocrystals (s-CNC) were used, and the effect of the cellulose modification and content in the nano-biocomposites was investigated. The presence of surfactant on the nanocrystal surface favours the dispersion of CNC in the PLA matrix. Electron microscopy analysis shows the good dispersion of s-CNC in the nanoscale with well-defined single crystals indicating that the surfactant allowed a better interaction between the cellulose structures and the PLA matrix. Reductions of 34% in water permeability were obtained for the cast films containing 1wt.% of s-CNC while good oxygen barrier properties were detected for nano-biocomposites with both 1wt.% and 5wt.% of modified and un-modified cellulose nanocrystals, underlining the improvement provided by cellulose on the PLA films. Moreover, the migration level of the studied nano-biocomposites was below the overall migration limits required by the current normative for food packaging materials in both non-polar and polar simulants.     

Electron microscopic demonstration of pulmonary surfactant proteins using procion brilliant blue H5GS

A new electron microscopy technique is described for detection of lung surfactant proteins with the copper-containing phthalocyanine dye, procion brilliant blue H5GS. The protein structures were stained concurrently with the fixation during perfusion through the pulmonary artery of a fixative-staining mixture containing glutaric aldehyde and a dye in the kakodilate buffer, pH 5.6-6, and in the course of a subsequent immersion of lung tissue pieces into the same mixture. Then the material was treated with thiosemicarbazide and post-fixed with OsO4. The dye did not penetrate intact cells. The electron-dense products of the histochemical reaction were located inside and on the surface of the surfactant membrane, in the hypophase of the surfactant complex, on the plasmalemma of air-blood barrier cells and in its micropinocytosis vesicles, as well as on the membranes of osmophilic plate-like bodies as their contents egressed into the alveolar lumen.     

Pulmonary and gastric surfactants. A comparison of the effect of surface requirements on function and phospholipid composition

Surfactant is present in the alveoli and conductive airways of mammalian lungs. The presence of surface active agents was, moreover, demonstrated for avian tubular lungs and for the stomach and intestine. As the surface characteristics of these organs differ from each other, their surfactants possess distinct biochemical and functional characteristics. In the stomach so-called 'gastric surfactant' forms a hydrophobic barrier to protect the mucosa against acid back-diffusion. For this purpose gastric mucosal cells secrete unsaturated phosphatidylcholines (PC), but no dipalmitoyl-PC (PC16:0/16:0). By contrast, surfactant from conductive airways, lung alveoli and tubular avian lungs contain PC16:0/16:0 as their main component in similar concentrations. Hence, there is no biochemical relation between gastric and pulmonary surfactant. Alveolar surfactant, being designed for preventing alveolar collapse under the highly dynamic conditions of an oscillating alveolus, easily reaches values of <5 mN/m upon cyclic compression. Surfactants from tubular air-exposed structures, however, like the conductive airways of mammalian lungs and the exclusively tubular avian lung, display inferior compressibility as they only reach minimal surface tension values of approximately 20 mN/m. Hence, the highly dynamic properties of alveolar surfactant do not apply for surfactants designed for air-liquid interfaces of tubular lung structures.     

Surfactant-associated proteins: functions and structural variation

Pulmonary surfactant is a barrier material of the lungs and has a dual role: firstly, as a true surfactant, lowering the surface tension; and secondly, participating in innate immune defence of the lung and possibly other mucosal surfaces. Surfactant is composed of approximately 90% lipids and 10% proteins. There are four surfactant-specific proteins, designated surfactant protein A (SP-A), SP-B, SP-C and SP-D. Although the sequences and post-translational modifications of SP-B and SP-C are quite conserved between mammalian species, variations exist. The hydrophilic surfactant proteins SP-A and SP-D are members of a family of collagenous carbohydrate binding proteins, known as collectins, consisting of oligomers of trimeric subunits. In view of the different roles of surfactant proteins, studies determining the structure-function relationships of surfactant proteins across the animal kingdom will be very interesting. Such studies may reveal structural elements of the proteins required for surface film dynamics as well as those required for innate immune defence. Since SP-A and SP-D are also present in extrapulmonary tissues, the hydrophobic surfactant proteins SP-B and SP-C may be the most appropriate indicators for the evolutionary origin of surfactant. SP-B is essential for air-breathing in mammals and is therefore largely conserved. Yet, because of its unique structure and its localization in the lung but not in extrapulmonary tissues, SP-C may be the most important indicator for the evolutionary origin of surfactant.     

Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films, II: albumin-inhibited pulmonary surfactant films and the effect of SP-A

Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.     

Ultrastructure of the respiratory epithelium in the lungs of the tortoise,Testudo graeca

The respiratory epithelium in the lungs of the tortoise (Testudo graeca) has been studied by electron microscopy. The epithelium consists of a mosaic of two different cell types (here called "pneumonocytes"). Type I pneumonocytes are roughly squamous and possess attenuated flanges of cytoplasm which extend over the septal capillaries. Localized cytoplasmic expansions are often present near the periphery of these flanges. Most of the organelles are concentrated in the perinuclear region; the most prominent of these are the mitochondria and osmiophilic inclusions. In contrast, type II pneumonocytes are cuboidal and are richly endowed with organelles including large Golgi complexes, extensive endoplasmic reticulum and numerous inclusion bodies. The morphological evidence suggests that type I pneumonocytes are involved in the secretion of osmiophilic material (presumed to be pulmonary surfactant) and in maintaining the integrity of the air-blood barrier. Type II pneumonocytes appear to be concerned solely with the production of surfactant.