Lung Surfactant Adsorption Research Articles

Properties of mixed monolayers of clinical lung surfactant, serum albumin and hydrophilic polymers

It is now established that the surface activity of the clinically used lung surfactant is reduced by serum proteins and can be restored by adding the hydrophilic polymers. The mechanisms of lung surfactant inactivation by serum proteins and restoring effect by the hydrophilic polymers remain not completely understood.In this paper the state and rheological dilatational properties of surface films formed from clinical lung surfactant Exosurf, Survanta, Curosurf and Alveofact in the presence of serum albumin (BSA) and hydrophilic polymers polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) and Dextran were studied. The obtained results suggest that the lung surfactant and BSA mixtures spread at air–water interface form a DPPC/BSA mixed monolayers with lower content of DPPC. The presence of hydrophilic polymers PVP, PEG and Dextran restore the DPPC content in the surface film. The effectiveness of the DPPC spreading and formation of better compacted film increases in order Exosurf, Survanta, Curosurf, Alveofact.The obtained results are in accordance with the generally admitted ideas about the mechanisms of serum protein inactivation and restoring effect of hydrophilic polymers based on the previously studies of the lung surfactant adsorption rate.

Adsorption of lung surfactant by particles studied in an ex vivo model: effects of quartz and amorphous silica

AbstractParticles adsorb biomolecules such as proteins and lipids from biological fluids. Accordingly, respirable particles (aerodynamic diameter <3 μm) inside lung alveolae bind molecules from lung surfactant (LS) which is composed of lipids (90%) and surfactant proteins (10%). In this paper we show that particles impair lung function by adsorbing LS. To this aim, we suspended particles characterized by different surface sizes such as quartz DQ12 and amorphous silica HDKN20 in 0.9% NaCl and tested the effect of a particle containing fluid bolus on the compliance of the isolated rat lung in the presence and absence of surfactant. A method was deviced in which the left lung of the rat was ventilated ex vivo by periodic negative pressure pulses. As an indirect measure of lung compliance the maximal expiratory volume flow of the isolated lung (MEF‐IL) was measured. Experiments were carried out for 90 min during which MEF‐IL showed a slight run down by about 20%. Instillation of 0.9% NaCl further reduced MEF‐IL; this effect was attenuated if lung lavage fluid was used and was completely reversed by commercially available surfactant. Instillation of quartz DQ12 (5 mg) and HDK N20 (2 mg) suspended in 0.9% NaCl lowered MEF‐IL within 10 min by 50.3% and 58.2%, respectively. In both cases pre‐coating with or subsequent addition of surfactant abrogated the inhibitory effect of particles on MEF‐IL. Experiments show that suspended particles affect lung compliance by adsorbing surfactant and that this effect can be quantified by an ex vivo lung model.Partikel in biologischen Flüssigkeiten können Biomoleküle wie Proteine und Lipide adsorbieren. Dementsprechend binden lungengängige Partikel (aerodynamischer Durchmesser <3 μm) in den Alveolen Komponenten des Lungensurfactants, das aus Lipiden (90%) und Proteinen besteht (10%). In dieser Arbeit wird gezeigt, dass Partikel die Lungenfunktion über eine Bindung von Lungensurfactant beeinträchtigen können. Dazu wurden Partikel mit verschieden großer Oberfläche (Quarz DQ12, amorphe Kieselsäure HDKN20) in 0,9%iger NaCl‐Lösung suspendiert und der Effekt eines partikelhaltigen Flüssigkeitsbolus auf die Lungencompliance mit und ohne Zusatz von Lungensurfactant getestet. Für die Experimente wurde eine ex vivo‐Methode entwickelt, bei der die linke Lunge kontinuierlich durch zyklisch angelegte Unterdruckpulse ventiliert wurde. Als indirekte Messgröße der Lungencompliance diente der maximale exspiratorische Volumenstrom der isolierten Lunge (MEF‐IL). Die Messungen erstreckten sich über 90 min, innerhalb derer ein Abfall des MEF‐IL um ca. 20% stattfand. Instillation von 0,9%iger NaCl‐Lösung reduzierte den MEF‐IL zusätzlich. Dieser Effekt wurde durch Zusatz von Lungenspülflüssigkeit abgemildert, durch kommerziell erhältliches Lungensurfactant vollständig aufgehoben. Die Instillation von Quarz DQ12 (5 mg, n = 7) oder HDK N20 (2 mg, n = 7), suspendiert in 0,9%iger NaCl‐Lösung, senkte den MEF‐IL innerhalb von 10 min auf 50,3% bzw. 58,2%. In beiden Fällen hob eine Vorbehandlung der Partikel mit Surfactant oder eine nachträgliche Surfactant‐Zugabe den Effekt wieder auf. Die Experimente zeigen, dass Partikel die Compliance der Lungen beeinträchtigen und dass dieser Effekt mit einem ex vivo Lungenmodell gemessen werden kann.

Confocal Microscopy and Competitive Adsorption: A New Look At Polymer-Enhanced Lung Surfactant Adsorption

Lung surfactant (LS) is a mixture of lipids and proteins that lines the air-liquid interface of the alveolar walls and modulates the surface tension in the lungs. It therefore greatly reduces the mechanical work of breathing as well as prevents alveolar collapse upon expiration. Blood serum leaking into the alveoli as a result of trauma can lead to LS inhibition, which is one characteristic of acute respiratory distress syndrome (ARDS). The competitive adsorption of serum proteins, such as albumin, to the air-liquid interface of the alveoli blocks LS from forming a functional monolayer during ARDS. The addition of hydrophilic polymers, such as polyethylene glycol and chitosan, to the liquid subphase has been shown to enhance interfacial LS adsorption in vitro. Optimal amounts of polymer allow LS to form a functional monolayer in the presence of albumin, thus reversing inhibition. Albumin must be displaced from the air-liquid interface in order for a functional monolayer of LS to form. Imaging of the competitive adsorption process with confocal microscopy has allowed us to better understand the mechanisms behind forming an interfacial LS monolayer under inhibitory conditions. We can simultaneously track LS, polymer, and albumin, as well as separately visualize phenomena occurring at the interface from those occurring in the bulk. As a result of these capabilities, we have studied how various parameters affect the transport of LS to the interface and the displacement of albumin in order to form a functional surfactant monolayer.

Overcoming rapid inactivation of lung surfactant: analogies between competitive adsorption and colloid stability.

Lung surfactant (LS) is a mixture of lipids and proteins that line the alveolar air-liquid interface, lowering the interfacial tension to levels that make breathing possible. In acute respiratory distress syndrome (ARDS), inactivation of LS is believed to play an important role in the development and severity of the disease. This review examines the competitive adsorption of LS and surface-active contaminants, such as serum proteins, present in the alveolar fluids of ARDS patients, and how this competitive adsorption can cause normal amounts of otherwise normal LS to be ineffective in lowering the interfacial tension. LS and serum proteins compete for the air-water interface when both are present in solution either in the alveolar fluids or in a Langmuir trough. Equilibrium favors LS as it has the lower equilibrium surface pressure, but the smaller proteins are kinetically favored over multi-micron LS bilayer aggregates by faster diffusion. If albumin reaches the interface, it creates an energy barrier to subsequent LS adsorption that slows or prevents the adsorption of the necessary amounts of LS required to lower surface tension. This process can be understood in terms of classic colloid stability theory in which an energy barrier to diffusion stabilizes colloidal suspensions against aggregation. This analogy provides qualitative and quantitative predictions regarding the origin of surfactant inactivation. An important corollary is that any additive that promotes colloid coagulation, such as increased electrolyte concentration, multivalent ions, hydrophilic non-adsorbing polymers such as PEG, dextran, etc. added to LS, or polyelectrolytes such as chitosan, also promotes LS adsorption in the presence of serum proteins and helps reverse surfactant inactivation. The theory provides quantitative tools to determine the optimal concentration of these additives and suggests that multiple additives may have a synergistic effect. A variety of physical and chemical techniques including isotherms, fluorescence microscopy, electron microscopy and X-ray diffraction show that LS adsorption is enhanced by this mechanism without substantially altering the structure or properties of the LS monolayer.

X-Ray Diffraction and Reflectivity Validation of the Depletion Attraction in the Competitive Adsorption of Lung Surfactant and Albumin

Lung surfactant (LS) and albumin compete for the air-water interface when both are present in solution. Equilibrium favors LS because it has a lower equilibrium surface pressure, but the smaller albumin is kinetically favored by faster diffusion. Albumin at the interface creates an energy barrier to subsequent LS adsorption that can be overcome by the depletion attraction induced by polyethylene glycol (PEG) in solution. A combination of grazing incidence x-ray diffraction (GIXD), x-ray reflectivity (XR), and pressure-area isotherms provides molecular-resolution information on the location and configuration of LS, albumin, and polymer. XR shows an average electron density similar to that of albumin at low surface pressures, whereas GIXD shows a heterogeneous interface with coexisting LS and albumin domains at higher surface pressures. Albumin induces a slightly larger lattice spacing and greater molecular tilt, similar in effect to a small decrease in the surface pressure. XR shows that adding PEG to the LS-albumin subphase restores the characteristic LS electron density profile at the interface, and confirms that PEG is depleted near the interface. GIXD shows the same LS Bragg peaks and Bragg rods as on a pristine interface, but with a more compact lattice corresponding to a small increase in the surface pressure. These results confirm that albumin adsorption creates a physical barrier that inhibits LS adsorption, and that PEG in the subphase generates a depletion attraction between the LS aggregates and the interface that enhances LS adsorption without substantially altering the structure or properties of the LS monolayer.

Competitive Adsorption: A Physical Model for Lung Surfactant Inactivation

Charged, surface-active serum proteins can severely reduce or eliminate the adsorption of lung surfactant from the subphase to the alveolar air-liquid interface via a kinetically controlled competitive adsorption process. The decreased surfactant concentration at the interface leads to higher surface tensions during the compression of the interface during breathing. The correspondence between the factors governing colloid stability and competitive adsorption is validated via a new method of measuring surfactant and serum protein adsorption rates to the air-water interface, using quantitative Brewster angle microscopy (BAM). Competitive adsorption from a 10 mg/mL albumin subphase prevents the adsorption of lung surfactant from even high subphase concentrations due to the fast diffusion of the water-soluble proteins to the interface. The formation of an albumin film causes an electrostatic and steric barrier to subsequent surfactant adsorption, which can destroy the necessary properties of functional lung surfactant: low surface tension during compression and rapid respreading after film collapse. Surfactant inactivation is at least partially due to decreased surfactant adsorption; such decreased adsorption due to the presence of serum proteins may play a role in the development and severity of acute respiratory distress syndrome.

Studying the Effects of Protein and Lipid Composition on Lung Surfactant Adsorption Through Confocal Microscopy

Lung surfactant (LS) is a mixture of lipids and proteins that lines the air-water interface of the alveolar walls. It modulates the surface tension in the lungs which greatly reduces the mechanical work of breathing and also prevents the collapse of the alveoli upon expiration. Although lipids are the major constituent of LS, the hydrophobic surfactant proteins SP-B and SP-C play an integral role in proper adsorption of LS to the air-water interface. Understanding the role of these proteins will allow for better design of exogenous LS therapies for the treatment of respiratory distress syndrome. Confocal microscopy's abilities to optically section a sample and simultaneously image multiple dyes provide an ideal tool to study LS adsorption in vitro. Combining three-dimensional, multi-component imaging with surface tension measurements allows us to determine whether a particular surfactant mixture can successfully transition from bilayer aggregates in the bulk to a functional monolayer on the interface. SP-B and SP-C are believed to play an integral role in both the transport of aggregates to the interface and the unfolding of surfactant bilayers into a monolayer. Confocal microscopy has allowed us to study the importance of lipid and protein composition on the transport and unfolding of LS.

Influence of Lipid Saturation Grade and Headgroup Charge: A Refined Lung Surfactant Adsorption Model

Rapid adsorption of surfactant material to the air/liquid interface of the lung is essential for maintaining normal lung function. The detailed mechanism of this process, however, remains unclear. In this study, we elucidate the influence of lipid saturation grade and headgroup charge of surface layer lipids on surfactant protein (SP)-induced vesicle insertion into monolayers spread at the air/water interface of a film balance. We used dipalmitoylphosphatidlycholine (DPPC),1,2-dipalmitoyl- sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol (POPG) as monolayer lipids doped with either hydrophobic surfactant-specific protein SP-B or SP-C (0.2 and 0.4 mol %, respectively). Vesicles consisting of DPPC/DPPG (4:1, mol ratio) were injected into a stirred subphase to quantify adsorption kinetics. Based on kinetic film balance and fluorescence measurements, a refined model describing distinct steps of vesicle adsorption to surfactant monolayers is presented. First, in a protein-independent step, lipids from vesicles bridged to the interfacial film by Ca 2+ ions are inserted into defects of a disordered monolayer at low surface pressures. Second, in a SP-facilitated step, active material insertion involving an SP-B- or SP-C-induced flip-flop of lipids occurs at higher surface pressures. Negatively charged lipids obviously influence the threshold pressures at which this second protein-mediated adsorption mechanism takes place.

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.

Competitive Adsorption of Lung Surfactant and Serum Proteins at the Air-Liquid Interface: A Grazing Incidence X-Ray Diffraction Study

AbstractThe competitive adsorption of lung surfactant (LS) and albumin at the air-liquid interface and the ability of polyethylene glycol (PEG) to mediate LS adsorption are analyzed using pressure area isotherms and grazing incidence x-ray diffraction (GIXD). The addition of albumin drastically reduces the amount of LS on the interface and slightly increases the LS lattice spacing. The addition of PEG restores the characteristic LS peaks, yielding a slightly more compact lattice. The scattering results are consistent with recent work which proposed that albumin creates a physical barrier which eliminates LS adsorption and that PEG enhances LS adsorption but does not significantly change LS surface ordering.

The Mechanism of Chitosan Enhanced Lung Surfactant Adsorption at the Air-Liquid Interface in the Presence of Serum Proteins

ABSTRACTPressure-area isotherms and fluorescence microscopy were used to investigate the impact of chitosan on the competitive adsorption between lung surfactant (LS) and serum proteins at the air-liquid interface. Isotherms demonstrate an optimum chitosan concentration to mediate LS adsorption; higher concentrations actually reduce the amount of LS which can adsorb. Fluorescence microscopy images show the transition from a serum protein to LS-covered interface for the optimum chitosan concentration; this transition goes through a sharply phase separated coexistence region. The results suggest that the cationic chitosan molecules mediate adsorption of the negatively charged LS aggregates by reducing the electrostatic barrier imposed by negatively charged interfacial serum proteins.

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.

Effects of albumin and erythrocyte membranes on spread monolayers of lung surfactant lipids

Dipalmitoyl phosphatidylcholine (DPPC), one of the main constituents of lung surfactant is mainly responsible for reduction of surface tension to near 0 mN/m during expiration, resisting alveolar collapse. Other unsaturated phospholipids like palmitoyloleoyl phosphatidylglycerol (PG), palmitoyloleoyl phosphatidylcholine (POPC) and neutral lipids help in adsorption of lung surfactant to the air–aqueous interface. Lung surfactant lipids may interact with plasma proteins and hematological agents flooding the alveoli in diseased states. In this study, we evaluated the effects of albumin and erythrocyte membranes on spread films of DPPC alone and mixtures of DPPC with each of PG, POPC, palmitoyloleoyl phosphatidylethanolamine (PE), cholesterol (CHOL) and palmitic acid (PA) in 9:1 molar ratios. Surface tension–area isotherms were recorded using a Langmuir–Blodgett (LB) trough at 37 °C with 0.9% saline as the sub-phase. In the presence of erythrocyte membranes, DPPC and DPPC + PA monolayers reached minimum surface tensions of 7.3 ± 0.9 and 9.6 ± 1.4 mN/m, respectively. Other lipid combinations reached significantly higher minimum surface tensions >18 mN/m in presence of membranes (Newman Keul's test, p < 0.05). The relative susceptibility to membrane inhibition was [(DPPC + PG, 7:3) = (DPPC + PG, 9:1) = (DPPC + POPC) = (DPPC + PE) = (DPPC + CHOL)] > [(DPPC + PA) = (DPPC)]. The differential response was more pronounced in case of albumin with DPPC and DPPC + PA monolayers reaching minimum surface tensions less than 2.4 mN/m in presence of albumin, whereas DPPC + PG and DPPC + POPC reached minimum surface tensions of around 20 mN/m in presence of albumin. Descending order of susceptibility of the spread monolayers of lipid mixtures to albumin destabilization was as follows: [(DPPC + PG, 7:3) = (DPPC + PG, 9:1) = (DPPC + POPC)] > [(DPPC + PE) = (DPPC + CHOL)] > [(DPPC + PA) = (DPPC)] The increase in minimum surface tension in presence of albumin and erythrocyte membranes was accompanied by sudden increases in compressibility at surface tensions of 15–30 mN/m. This suggests a monolayer destabilization and could be indicative of phase transitions in the mixed lipid films due to the presence of the hydrophobic constituents of erythrocyte membranes.

Inhibition of Pulmonary Surfactant Adsorption by Serum and the Mechanisms of Reversal by Hydrophilic Polymers: Theory

A theory based on the Smolukowski analysis of colloid stability shows that the presence of charged, surface-active serum proteins at the alveolar air-liquid interface can severely reduce or eliminate the adsorption of lung surfactant from the subphase to the interface, consistent with the observations reported in the companion article (pages 1769–1779). Adding nonadsorbing, hydrophilic polymers to the subphase provides a depletion attraction between the surfactant aggregates and the interface, which can overcome the steric and electrostatic resistance to adsorption induced by serum. The depletion force increases with polymer concentration as well as with polymer molecular weight. Increasing the surfactant concentration has a much smaller effect than adding polymer, as is observed. Natural hydrophilic polymers, like the SP-A present in native surfactant, or hyaluronan, normally present in the alveolar fluids, can enhance adsorption in the presence of serum to eliminate inactivation.

Dipalmitoyl lecithin and lung surfactant adsorption at an air-liquid interface by respirable particles

The effects of kaolin and flyash on synthetic dipalmitoyl lecithin and lung surfactant from the lavage of excised rat lungs were studied using a Wilhelmy-type surface balance. After control studies with dipalmitoyl lecithin and rat lung surfactant, particles were sprinkled onto the surface at minimum surface area and the surface tensions vs surface areas were recorded. The results showed increased minimum surface tensions and decreased hysteresis areas when the particles were added. More particles were required to change the hysteresis area of lung surfactant than were required for synthetic dipalmitoyl lecithin. The effects of changes in surface areas of alumina and zinc oxide particles on dipalmitoyl lecithin were also studied. The amount of particles required to reduce surface tension-surface area hysteresis increased as the particles' surface area per gram decreased. This study indicates that respirable dusts may adsorb dipalmitoyl lecithin or lung surfactant in vivo and, therefore, may prevent surface tension from reaching the low values generally thought to occur in vivo.