Surface activity and film formation from the surface associated material of artificial surfactant preparations

AbstractSurfactant proteins B and C (SP-B and SP-C) are present in natural derived surfactant preparations used for treatment of respiratory distress syndrome. Herein the surface activity of an SP-C analogue (SP-C(LKS)), a hybrid peptide between SP-C and bacteriorhodopsin (SP-C/BR) and a model peptide (KL4) was studied with a captive bubble surfactometer (CBS). The peptides were mixed with either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol (PG) (7:3, by weight) or DPPC/PG/palmitic acid (68:22:9, by weight) at a concentration of 1 mg/ml in HEPES buffer, pH 6.9 and a polypeptide/lipid weight ratio of 0.02–0.03. In some lipid/peptide preparations also 2% of SP-B was included. Adsorption, monitored as surface tension vs. time for 10 min after bubble formation did not show discernible differences for the whole set of preparations. Equilibrium surface tensions of approximately 25 mN/m were reached after 5–10 min for all preparations, although those with SP-C/BR appeared not to reach end point of adsorption within 10 min. Area compression needed to reach minimum surface tension of 0.5–2.0 mN/m was least for the KL4 preparation, about 13% in the first cycle. 3% SP-C(LKS) in DPPC:PG (7:3, by weight) reached minimum surface tension upon 27% compression in the first cycle. If DPPC:PG:PA (68:22:9, by weight) was used instead only 16% area compression was needed and 14% if also 2% SP-B was included. 3% SP-C(LKS) in DPPC:PG (7:3, by weight)+2% SP-B needed 34% compression to reach minimum surface tension. The replenishment of material from a surface associated surfactant reservoir was estimated with subphase depletion experiments. With the 2% KL4 preparation incorporation of excess material took place at a surface tension of 25–35 mN/m during stepwise bubble expansion and excess material equivalent to 4.3 monolayers was found. When 2% SP-B was added to 3% SP-C(LKS) in DPPC:PG (7:3, by weight) the number of excess monolayers increased from 1.5 to 3.6 and the incorporation took place at 30–40 mN/m. When SP-B was added to 3% SP-C(LKS) in DPPC:PG:PA (68:22:9, by weight) the number of excess monolayers increased from 0.5 to 3.4 and incorporation took place at 40–50 mN/m. With 2% SP-C/BR incorporation took place at 40–45 mN/m, frequent instability clicks were observed and excess material of approximately 1.1 monolayer was estimated

A comparative study of mechanisms of surfactant inhibition

AbstractPulmonary surfactant spreads to the hydrated air–lung interface and reduces the surface tension to a very small value. This function fails in acute respiratory distress syndrome (ARDS) and the surface tension stays high. Dysfunction has been attributed to competition for the air–lung interface between plasma proteins and surfactant or, alternatively, to ARDS-specific alterations of the molecular profile of surfactant. Here, we compared the two mechanisms in vitro, to assess their potential role in causing respiratory distress. Albumin and fibrinogen exposure at or above blood level concentrations served as the models for testing competitive adsorption. An elevated level of cholesterol was chosen as a known adverse change in the molecular profile of surfactant in ARDS. Bovine lipid extract surfactant (BLES) was spread from a small bolus of a concentrated suspension (27 mg/ml) to the air–water interface in a captive bubble surfactometer (CBS) and the bubble volume was cyclically reduced and increased to assess surface activity of the spread material. Concentrations of inhibitors and the concentration and spreading method of pulmonary surfactant were chosen in an attempt to reproduce the exposure of surfactant to inhibitors in the lung. Under these conditions, neither serum albumin nor fibrinogen was persistently inhibitory and normal near-zero minimum surface tension values were obtained after a small number of cycles. In contrast, inhibition by an increased level of cholesterol persisted even after extensive cycling. These results suggest that in ARDS, competitive adsorption may not sufficiently explain high surface tension, and that disruption of the surfactant film needs to be given causal consideration

Translocation and potential neurological effects of fine and ultrafine particles a critical update

ABSTRACT: Particulate air pollution has been associated with respiratory and cardiovascular disease. Evidence for cardiovascular and neurodegenerative effects of ambient particles was reviewed as part of a workshop. The purpose of this critical update is to summarize the evidence presented for the mechanisms involved in the translocation of particles from the lung to other organs and to highlight the potential of particles to cause neurodegenerative effects.Fine and ultrafine particles, after deposition on the surfactant film at the air-liquid interface, are displaced by surface forces exerted on them by surfactant film and may then interact with primary target cells upon this displacement. Ultrafine and fine particles can then penetrate through the different tissue compartments of the lungs and eventually reach the capillaries and circulating cells or constituents, e.g. erythrocytes. These particles are then translocated by the circulation to other organs including the liver, the spleen, the kidneys, the heart and the brain, where they may be deposited. It remains to be shown by which mechanisms ultrafine particles penetrate through pulmonary tissue and enter capillaries. In addition to translocation of ultrafine particles through the tissue, fine and coarse particles may be phagocytized by macrophages and dendritic cells which may carry the particles to lymph nodes in the lung or to those closely associated with the lungs. There is the potential for neurodegenerative consequence of particle entry to the brain. Histological evidence of neurodegeneration has been reported in both canine and human brains exposed to high ambient PM levels, suggesting the potential for neurotoxic consequences of PM-CNS entry. PM mediated damage may be caused by the oxidative stress pathway. Thus, oxidative stress due to nutrition, age, genetics among others may increase the susceptibility for neurodegenerative diseases. The relationship between PM exposure and CNS degeneration can also be detected under controlled experimental conditions. Transgenic mice (Apo E -/-), known to have high base line levels of oxidative stress, were exposed by inhalation to well characterized, concentrated ambient air pollution. Morphometric analysis of the CNS indicated unequivocally that the brain is a critical target for PM exposure and implicated oxidative stress as a predisposing factor that links PM exposure and susceptibility to neurodegeneration.Together, these data present evidence for potential translocation of ambient particles on organs distant from the lung and the neurodegenerative consequences of exposure to air pollutants

Ultrafine Particles Cross Cellular Membranes by Nonphagocytic Mechanisms in Lungs and in Cultured Cells

High concentrations of airborne particles have been associated with increased pulmonary and cardiovascular mortality, with indications of a specific toxicologic role for ultrafine particles (UFPs; particles < 0.1 μm). Within hours after the respiratory system is exposed to UFPs, the UFPs may appear in many compartments of the body, including the liver, heart, and nervous system. To date, the mechanisms by which UFPs penetrate boundary membranes and the distribution of UFPs within tissue compartments of their primary and secondary target organs are largely unknown. We combined different experimental approaches to study the distribution of UFPs in lungs and their uptake by cells. In the in vivo experiments, rats inhaled an ultrafine titanium dioxide aerosol of 22 nm count median diameter. The intrapulmonary distribution of particles was analyzed 1 hr or 24 hr after the end of exposure, using energy-filtering transmission electron microscopy for elemental microanalysis of individual particles. In an in vitro study, we exposed pulmonary macrophages and red blood cells to fluorescent polystyrene microspheres (1, 0.2, and 0.078 μm) and assessed particle uptake by confocal laser scanning microscopy. Inhaled ultrafine titanium dioxide particles were found on the luminal side of airways and alveoli, in all major lung tissue compartments and cells, and within capillaries. Particle uptake in vitro into cells did not occur by any of the expected endocytic processes, but rather by diffusion or adhesive interactions. Particles within cells are not membrane bound and hence have direct access to intracellular proteins, organelles, and DNA, which may greatly enhance their toxic potential

Formation and structure of surface films: captive bubble surfactometry

AbstractThe adsorption model for soluble surfactants has been modified for suspensions of pulmonary surfactant. The dynamic adsorption behavior may be governed by a two-step process: (1) the transfer of molecules between the surface layer and the subsurface layer, which has a thickness of a few molecular diameters only; (2) the exchange of molecules between the subsurface and the bulk solution. The first step is an adsorption process and the second step is a mass transfer process. Between the subsurface and the bulk solution is an undisturbed boundary layer where mass transport occurs by diffusion only. The thickness of this boundary layer may be reduced by stirring. Rapid film formation by adsorption bursts from lipid extract surfactants, as observed in the captive bubble system, suggests that the adsorption process as defined above is accompanied by a relatively large negative change in the free energy. This reduction in the free energy is provided by a configurational change in the association of the specific surfactant proteins and the surfactant lipids during adsorption. The negative change in the free energy during film formation more than compensates for the energy barrier related to the film surface pressure. In the traditional view, the extracellular alveolar lining layer is composed of two parts, an aqueous subphase and a surfactant film, believed to be a monolayer, at the air–water interface. The existence and continuity of the aqueous subphase has recently been demonstrated by Bastacky and coworkers, and a continuous polymorphous film has recently been shown by Bachofen and his associates, using perfusion fixation of rabbit lungs with slight edema. In the present chapter, we have described a fixation technique using a non-aqueous fixation medium of perfluorocarbon and osmium tetroxide to fix the peripheral airspaces of guinea pig lungs. A continuous osmiophilic film which covers the entire alveolar surface, including the pores of Kohn, is demonstrated. By transmission electron microscopy, the surface film frequently appears multilaminated, not only in the alveolar corners or crevices, but also at the thin air–blood barrier above the capillaries. Disk-like structures or multilamellar vesicles appear partially integrated into the planar multilayered film. In corners and crevices, tubular myelin appears closely associated with the surface film. Tubular myelin, however, is not necessary for the generation of a multilaminated film. This is demonstrated in vitro by the fixation for electron microscopy of a film formed from lipid extract surfactant on a captive bubble. Films formed from relatively high surfactant concentration (1 mg/ml of phospholipid) are of variable thickness and frequent multilayers are seen. In contrast, at 0.3 mg/ml, only an amorphous film can be visualized. Although near zero minimum surface tensions can be obtained for both surfactant concentrations, film compressibility and mechanical stability are substantially better at the higher concentrations. This appears to be related to the multilaminated structure of the film formed at the higher concentration

Wetting and spreading of a surfactant film on solid particles: influence of sharp edges and surface irregularities

In addition to particle size and surface chemistry, the shape of particles plays an important role in their wetting and displacement by the surfactant film in the lung. The role of particle shape was the subject of our investigations using a model system consisting of a modified Langmuir-Wilhelmy surface balance. We measured the influence of sharp edges (lines) and other highly curved surfaces, including sharp corners or spikes, of different particles on the spreading of a dipalmitoylphosphatidyl (DPPC) film. The edges of cylindrical sapphire plates (circular curved edges, 1.65 mm radius) were wetted at a surface tension of 10.7 mJ/m2 (standard error (SE) = 0.45, n = 20) compared with that of 13.8 mJ/m2 (SE = 0.20, n = 20) for cubic sapphire plates (straight linear edges, edge length 3 mm) (p 0.05). The surfaces of the plates showed significantly higher resistance to spreading compared to that of the edges, as substantially lower surface tensions were required to initiate wetting (p 0.05), respectively, whereas the surfaces were wetted at 3.8 mJ/m2 (SE = 0.89, n = 20) and 5.8 mJ/m2 (SE = 0.52, n = 20) (p < 0.05), respectively. Further experiments with pollen of malvaceae and maize (spiky and fine knobbly surfaces) were wetted at 10.0 mJ/m2 (SE = 0.52, n = 10) and 22.75 mJ/m2 (SE = 0.81, n = 10), respectively (p < 0.05). These results show that resistance to spreading of a DPPC film on various surfaces is dependent on the extent these surfaces are curved. This is seen with cubic sapphire plates which have at their corners a radius of curvature of about 0.75 microm, spiky malvaceae pollen with an even smaller radius on top of their spikes, or talc with various highly curved surfaces. These highly curved surfaces resisted wetting by the DPPC film to a higher degree than more moderately curved surfaces such as those of cylindrical sapphire plates, maize pollens, or polystyrene spheres, which have a surface free energy similar to that of talc but a smooth surface. The macroscopic plane surfaces of the particles demonstrated the greatest resistance to spreading. This was explained by the extremely fine grooves in the nanometer range, as revealed by electron microscopy. In summary, to understand the effects of airborne particles retained on the surfaces of the respiratory tract, and ultimately their pathological potential, not only the particle size and surface chemistry but also the particle shape should be taken in consideration

Interaction of fine particles and nanoparticles with red blood cells visualized with advanced microscopic techniques

So far, little is known about the interaction of nanoparticles with lung cells, the entering of nanoparticles, and their transport through the blood stream to other organs. The entering and localization of different nanoparticles consisting of differing materials and of different charges were studied in human red blood cells. As these cells do not have any phagocytic receptors on their surface, and no actinmyosin system, we chose them as a model for nonphagocytic cells to study how nanoparticles penetrate cell membranes. We combined different microscopic techniques to visualize fine and nanoparticles in red blood cells: (I) fluorescent particles were analyzed by laser scanning microscopy combined with digital image restoration, (II) gold particles were analyzed by conventional transmission electron microscopy and energy filtering transmission electron microscopy, and (III) titanium dioxide particles were analyzed by energy filtering transmission electron microscopy. By using these differing microscopic techniques we were able to visualize and detect particles < or = 0.2 microm and nanoparticles in red blood cells. We found that the surface charge and the material of the particles did not influence their entering. These results suggest that particles may penetrate the red blood cell membrane by a still unknown mechanism different from phagocytosis and endocytosis