Interfacial properties of pulmonary surfactant layers

Advances in Colloid and Interface Science 117 (2005) 33 – 58 www.elsevier.com/locate/cis

Interfacial properties of pulmonary surfactant layers R. Wu¨stneck a,*, J. Perez-Gil b, N. Wu¨stneck a, A. Cruz b, V.B. Fainerman c, U. Pison a a

Charite´, Campus Virchow-Klinikum, Universita¨tsmedizin Berlin, Klinik fu¨r Ana¨sthesiologie und operative Intensivmedizin, Spandauer Damm 130, 14050 Berlin, Germany b Departamento de Bioquimica, Facultad de Biologia, Universidad Complutense, 28040 Madrid, Spain c Medical Physicochemical Centre, Medical University Donetsk, Prospekt Ilischa 1, 83003 Donetsk, Ukraine Available online 24 August 2005

Abstract The composition of the pulmonary surfactant and the border conditions of normal human breathing are relevant to characterize the interfacial behavior of pulmonary layers. Based on experimental data methods are reviewed to investigate interfacial properties of artificial pulmonary layers and to explain the behavior and interfacial structures of the main components during compression and expansion of the layers observed by epifluorescence and scanning force microscopy. Terms like over-compression, collapse, and formation of the surfactant reservoir are discussed. Consequences for the viscoelastic surface rheological behavior of such layers are elucidated by surface pressure relaxation and harmonic oscillation experiments. Based on a generalized Volmer isotherm the interfacial phase transition is discussed for the hydrophobic surfactant proteins, SP-B and SP-C, as well as for the mixtures of dipalmitoylphosphatidylcholine (DPPC) with these proteins. The behavior of the layers depends on both the oligomerisation state and the secondary structure of the hydrophobic surfactant proteins, which are controlled by the preparation of the proteins. An example for the surface properties of bronchoalveolar porcine lung washings of uninjured, injured, and Curosurf treated lavage is discussed in the light of surface behavior. An outlook summarizes the present knowledge and the main future development in this field of surface science. D 2005 Elsevier B.V. All rights reserved. Keywords: Pulmonary surfactant; Surface behavior; Phospholipids; Surfactant protein

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The composition of the pulmonary surfactant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological and interfacial border conditions of breathing . . . . . . . . . . . . . . . . . . . . . . Methods of surface characterization for pulmonary surfactant films . . . . . . . . . . . . . . . . . . Surface behavior of films containing the main hydrophobic components of the pulmonary surfactant . Interfacial properties of spread DPPC layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interfacial properties of spread lipid/protein layers . . . . . . . . . . . . . . . . . . . . . . . . . . . Influence of the surfactant protein SP-A on the surface properties . . . . . . . . . . . . . . . . . . . Surface dilatational rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stress relaxation of spread pulmonary layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viscoelastic properties of spread pulmonary layers . . . . . . . . . . . . . . . . . . . . . . . . . . . Oligomerisation and secondary structure of the hydrophobic pulmonary surfactant proteins . . . . . . Influence of oligomerisation and secondary structure on the surface behavior of SP-B and SP-C . . . Modeling of mixed surfactant protein/DPPC layers . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. E-mail address: [email protected] (R. Wu¨stneck). 0001-8686/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cis.2005.05.001

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15. Influence of h-SP-C on surface behavior of mixed pulmonary layers 16. An example of surface behavior of lung washings . . . . . . . . . . 17. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The properties of surfactant mixtures may differ significantly from those of their containing individual components. One native surfactant mixture is the pulmonary surfactant, which covers the alveolar surfaces of the lung and which was optimized by evolution to fulfill several functions [1]. Pulmonary surfactant is one of the first barriers participating in innate immune defense of the lung. Therefore it should interact with different viruses, bacteria and fungi [2– 5], and with inhaled glycoconjugated allergens [6,7]. It should maintain the gas exchange of the lung, which is an active process governed by tissue and interfacial forces. To stabilize the lung and to hold it open the pulmonary surfactant film should be mechanically stable to guarantee a large exchange area [8] and it should decrease the surface tension thus reducing the interfacial work to support the gas exchange under dynamic conditions. In the following we will focus on the interfacial behavior of the alveolar lining film and the role of the different surface active components of the pulmonary surfactant, which determine this behavior. We will review the way of discovering the composition of the pulmonary surfactant, the border conditions of the breathing process, and how the interfacial behavior of this surfactant was characterized. Three main aspects will be the surface rheological behavior, the modeling of the surface film, and its structure. Furthermore we will show some examples in order to understand the function of the different pulmonary surfactant components and review further fields of investigation.

2. The composition of the pulmonary surfactant Von Neergaard [9] was the first who investigated the static repulsive force of excised lungs of different species, including human lungs. He stated that the pressure required maintaining the lung inflated with air was greater than that required keeping the lung open when it was filled with isotonic solutions. He concluded that the pressure difference was caused by the surface tension forces and that lung washings contain surface active substances. Pattle firstly investigated lung washings and found that they contain a nearly insoluble material which could reduce the surface tension to 0 mN/m [10]. In fact this is a really surprising but thermodynamically absurd result. Actually any interface disappears at zero surface tension, which happens only in crucial situations, i.e. at the critical pressure

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and temperature of the liquid phase or in turn of the formation of bicontinuous phases [11] such discussed for microemulsion formation. What Pattle really found was the formation of a new (solid) phase at the liquid –gas interface. A possible explanation for measuring such strange effects was given by Bangham [12]. Despite the different views concerning surface activity, lung washings strongly reduce surface tension and there is no single surfactant, which could reduce surface tension in a similar way. Strong reduction of surface tension, however, can be achieved by mixing different surface active substances. This was the starting point to find out in more detail which substances are present in lung washings. By treating lung extracts with protease Pattle and Thomas [13] found lipoprotein to be present. Brown [14] found that the pulmonary surfactant contained different phospholipids with dipalmitoylphosphatidylcholine (DPPC) being the main component. King and Clements [15] isolated a large protein from lung washings, which was later named surfactant protein A (SP-A) [16], and suggested that this protein would play an important role in reducing the surface tension. In 1980 Metcalfe et al. [17] reported chloroform – methanol organic extracts of pulmonary surfactant retained the ability of natural surfactant to achieve surface tensions near 0 mN/m. At that time it was assumed that the films formed by lung washings do not contain any other proteins, but further investigations showed that two small, low molecular weight hydrophobic proteins, which dissolved in organic solvents, were retained in the lipid extracts [18 – 20]. These proteins are now known as SP-B and SP-C, and these proteins have been implicated as important contributors to the surface activity of pulmonary surfactant [16]. Later on another surfactant associated protein was discovered, SP-D, which belong to the family of collectins. SP-D is hydrophilic. Therefore it does not influence the surface behavior directly [21]. The lipid composition of pulmonary surfactant of different species was reviewed by Veldhuizen et al. [22]. In all species, phosphatidylcholine comprised approx. 80%, about half of which is DPPC. In rat and ovine lungs, only 25% and 40%, respectively, of the DPPC is surfactant associated [23]. The remaining PC in surfactant was composed primarily of molecular species containing monoand dienoic fatty acids, with only minor amounts of short chains or polyunsaturated alkyl groups. Very recently, the discovery has been made that some heterothermic mammals, which spends a significant period of time in torpor, where body temperature is only ¨ 15 -C

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

[24], have only around 15% DPPC, whereas the dominant PC molecular species is PC(16:0/16:1) at 30% (Sandra Orgeig and Chris Daniels, personal communication). However, surfactant from this species still contains a total of 46% di-saturated PC, as it contains a significant proportion (17%) of highly unusual PC molecular species, i.e. PC(16: 0/14: 0), as well as some highly unusual alkyl species. Remarkably, heterothermic mammals do not appear to alter their PC molecular species composition during torpor, indicating that the unique phospholipid composition of their surfactant may already be adapted to the variable living temperature that is characteristic of the heterotherm lifestyle. The acidic phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), accounted for 8 – 15% of the total surfactant phospholipid pool with most species. The remaining phospholipids were present in small amounts although some importance has been proposed for the role of minor amounts of alkyl-ether lipids in surfactant activities [25]. Cholesterol, the major neutral lipid in the pulmonary system, is an important component for surfactant structural and functional properties under dynamic conditions. Cholesterol may account for up to 8 –10% of surfactant by weight in human lungs [26], meaning in the order of 20% cholesterol-to-phospholipid as a molar basis. The amount of cholesterol in surfactant seems to be optimized both from an ontogenetic and an evolutionary point of view [27] suggesting a major role to define optimal surfactant performance.

3. Physiological and interfacial border conditions of breathing Clements first started to investigate the surface tension of lung washings by using a Langmuir film balance [28 – 30]. He found that films formed by adsorption of the lung washing components achieved a surface pressure of about 45 mN/m and that these films could be compressed up to a surface pressure of about 60 mN/m without collapsing. The surface pressure P is the difference between the surface tension of the solvent (water or saline, respectively) c 0, and that of a surfactant film covered surface c, i.e. P = c 0
c. Therefore the highest possible value for P should be c 0 for an insoluble monolayer, which however is unrealistic for the same reason as c cannot become zero for an existing interface [12]. This equation was introduced into surface science to connect two different concepts, i.e. adsorption phenomena and monolayer behavior, and it was pointed out by Gaines [31] that it is valid only under appropriate circumstances. An important point in order to understand the breathing process was the determination of the surface tension of the alveolar surface in vivo. First attempts of in situ determination were made by Schu¨rch [32,33]. He was then able to determine in cat lung that alveolar tension

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decreased to about 1 mN/m upon deflation to 40% total lung capacity. There was no difference in surface tension on alveolar size [34]. In order to determine this tension Bachofen used the spreading behavior of an oil droplet placed onto excised rabbit lungs to obtain the alveolar surface tension at different stages of inflation and deflation [34]. While Bachofen did the animal work and perfused the lung, Schu¨rch performed the micropuncture of subpleural alveoli [35]. A maximal surface tension of approximately 30 mN/m was measured at total lung capacity (TLC), which decreased during lung deflation to about 1 mN/m at 40% TLC (equivalent to the functional residual lung capacity). This work was still in situ, but it was the closest to the in vivo situation, as the lungs were perfused. Although the measurement is a complicated task other attempts with anaesthetized sheep and not anaesthetized horses [36 –38] confirmed this finding. A surface tension of approximately 32 mN/m was found at the air – mucus interface in these animals. The measurements in the trachea and large airways were in line with the measurements in alveoli at 100% TLC, although they did not confirm the results in alveoli on deflation. Considering the delicate measurement procedure one can assume that the surface tension measured at the alveolar interface in situ approximately agrees with the equilibrium surface tension in vitro at surface saturation. The equilibrium surface tension at surface saturation is the lowest surface tension of pure or mixed phospholipid films that can be achieved spontaneously, giving sufficient lipids, i.e. approximately 50 Ag/ml of total phospholipid and a long enough time to adsorb at the air –liquid interface [38]. Using morphological methods it was determined that there is a connection between the expansion of the lung (fraction of TLC) and the increase of the surface area of the alveolar lining layer [39 – 41]. Tschumperlin and Margulies [41] assumed that a strain alveolar lining layer occurs above 40% of TLC. In the case of low tidal breathing (40 –70% of TLC) the alveolar lining layer is expanded in the order of 7 –15%. At inflation of the lung to 100% of TLC the increase of the surface area ranges up to 30 – 35%. Recently the inflation of individual alveoli during the generation of a pressure/volume curve was visualized by direct in vivo microscopy [42,43]. It was found that the normal uninjured lung does not increase significantly in volume by simple isotropic (balloon-like) expansion of alveoli. Cochrane [44] stated that there are only small changes in alveolar size during the respiratory cycle. As stated previously by Schu¨rch et al. [45] normal tidal breathing might not require extensive over-compression leading to near zero surface tension, whereas it may be a different matter when the surfactant is compromised by inhibitory agents, e.g. blood and serum proteins, or when the lung is mechanically ventilated. Pulmonary surfactant proteins increased adsorption rate, but led to the same equilibrium surface tension at surface saturation. This value is in good agreement with a plateau or a kink-point in the P/A isotherms observed for

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spread phospholipid/protein films under equilibrium conditions. As it was shown by Putz et al. [46] adsorbed or spread films of pulmonary surfactant components led to comparable values in this respect. In contrast to adsorption experiments spreading techniques had the advantage, that the amount of surface active material is exactly known. In addition, the amount of pulmonary surfactant material to reach equilibrium is considerably smaller. Surfactant adsorbed from the aqueous subphase forms not simple monolayers but multilayered films. These films are likely composed of an interfacial monolayer plus several associated bilayers [47]. Study of surface dynamics of solvent spread films may therefore oversimplify the behavior of surfactant interfacial structures as they may exist in vivo. To maintain the gas exchange area in lungs under the dynamic conditions of breathing pulmonary surfactant need to reach mechanically stable films of low surface tensions under high compression [8]. To investigate such physiological relevant films for a more profound understanding of breathing it is necessary to study a range of surface pressure from approximately 35 up to 65 mN/m. When taking into account extreme conditions like coughing or when human nearly dies laughing, a surface pressure up to 70 mN/m may occur and need to be studied, although Schu¨rch found surface pressure of nearly 70 mN/m upon deflation even at approximately 40% TLC [35]. An alternative view on pulmonary films was introduced by Scarpelli [48 – 50]. He introduced the concept of the alveolar surface network, which supposes a totally fluid intraacinar conformation of the alveolar surface liquid continuum circulating, both in series and in parallel, through ultrathin (to < 7 nm) molecular conduits formed by appositions of unit bubbles of alveolar gas. Appositions of unit bubble films include bubble-to-bubble at the alveolar entrance, across alveolar ducts, and at pores of Kohn, bubble-to-epithelial cell surface, and bubble-to-open surface liquid layer of the terminal conducting airways. These appositions of monolayer bubble films could create macrochannels that would potentially modulate alveolar surface layer transfers, volume and flow throughout the acinus and between acinar surface and both the interstitium and the terminal conducting airways surfaces, and microchannels along the broadest surfaces of the appositions. These bubbles were virtualized [49]. As the bubble shrinks, surfactants of its film are compressed and the surface tension of the film falls rapidly until the surfactants are maximally compressed, at which point the surface tension is virtually zero. It should be pointed out here, that also the surface tension of foam lamellas could not become zero even when Newton black films are formed. Therefore the author used the term ‘‘virtually zero’’. Interesting is the thickness of the foam films, which is actually twice the monolayer thickness. The implications of this model to understand surfactant performance under normal as compared to pathological situations still have to be evaluated.

4. Methods of surface characterization for pulmonary surfactant films The most important characteristic of the surface behavior of pulmonary surfactant is the surface tension or the surface pressure. It was first determined by using the Langmuir balance. There are, however, some experimental shortcomings of the Langmuir film balance when used for pulmonary layers. The first is that the surface area is large and therefore a relatively large amount of pulmonary surfactant is needed for such measurements. Special care has to be taken for the contact angle at the Wilhelmy plate. This should be mentioned here because surface pressures higher than that of the subphase were measured by using this device [51], which would yield negative surface tensions. Serious problems arise from the leakage of such equipment, especially when the films are highly compressed. Leakage problems also limit the application of the pulsating bubble surfactometer [52] or the pendant drop technique [53] to a level of film pressure < 65 mN/m. The captive bubble surfactometer introduced by Schu¨rch et al. [45,54 – 56] offers a leak-proof system because the surface film is not interrupted by plastic walls, barriers or outlets. In this apparatus, an air bubble floats against a hydrophilic roof of a 1% agarose gel by buoyancy. The water layer between the bubble and the air –agarose interface is thin but still prevent adhesion of the bubble to the gel itself. Bubble volume is controlled by varying the pressure in the measuring chamber. Nevertheless a disadvantage of this device is that surface active material can penetrate into the agarose gel. It was found, however, that also a glass or a stainless steel roof can be used [57 – 60], which also is sufficiently hydrophilic to prevent the adhesion of pulmonary surfactant material and which can be easily cleaned. This device can be used for both investigations of adsorbed and spread films. Surface tension, bubble surface, and volume can be determined by using the axisymmetric bubble shape analysis. Even turbid lipid/protein suspensions can be characterized [61,62]. Beside surface tension measurements this device can be also used to characterize the surface rheological behavior of pulmonary systems. Usually only the surface compressibility, i.e. the reversed elasticity modulus derived from P/A isotherms is reported in literature. Surface rheological characterization, however, requires considering the dependence of surface rheological parameters on the kind of stress and deformation, because these parameters depend on surface stress and strain and their velocities. For the captive bubble surfactometer two surface dilatational methods were applied, the harmonic bubble oscillation [63,64] and transient surface stress relaxation experiments [65], which yield surface dilatational elasticity, viscosity, and corresponding relaxation times of surface stress or strain. One shortcoming of the classical captive bubble device was that it could not be used for surface spectroscopy, i.e.

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

Brewster angle microscopy, IR, UV, CD and others. Here the Langmuir film balance was preferred, to observe and analyze the films either in situ or upon transference onto Langmuir-Blodgett (LB) supports. Films have also been spread on quartz for CD or KBr plates for FTIR measurements [66]. Fluorescence light microscopy (FLM) was used firstly by Perez-Gil et al. [67] to investigate the lateral distribution of pulmonary surfactant components by using a Langmuir film balance, and scanning force microscopy (SFM) for resolving the topography of pulmonary layers [68,69] on a molecular level. Meanwhile there is some development to combine the captive bubble with additional equipment, which would extend the application of the captive bubble device. Galla et al. successfully introduced a new attempt. They used small air bubbles, diameter 50 –500 Am, formed in a solution at hydrophobic patches on mica when the hydrostatic pressure was reduced within a closed chamber. Varying the pressure within the chamber changed the area of the droplets. Using adsorbed pulmonary films Knebel et al. investigated these films at the air –liquid interface by using FLM and SFM [70,71]. A different approach to investigate pulmonary surfactants is based on the ideas of Scarpelli [49], who showed that pulmonary surfactant forms foam bubbles in vivo, thus forming an alveolar surface network. Exerowa et al. used techniques for characterization of foam film stability [72] to evaluate pulmonary films. She started her investigations in the middle of the 70th, i.e. before foam lamellas in the lung were virtualized. Studies of human amniotic fluid, which contains pulmonary surfactant, showed a direct correlation between the rapid formation of stable black films and the concentration of phosphatidylcholine in amniotic fluid [73,74]. The technique is easy to handle and has the advantage of high sensitivity; evaluation of film dynamics and the disjoining pressure, and the direct visualization of surface film architecture [75 – 77]. The probability of black film formation is concentration dependent and failure to obtain stable black films was correlated with high risk of respiratory distress syndrome at birth [77]. It is plausible that deficits in pulmonary surfactant proteins facilitate the film drainage and film rupture. Such measurements might be helpful to characterize interfacial behavior of pulmonary films and for practical use.

5. Surface behavior of films containing the main hydrophobic components of the pulmonary surfactant Pulmonary surfactant is a mixture of components with different surface activity. The most hydrophobic lipid component is DPPC. Two of the pulmonary surfactant proteins, SP-B, and SP-C, are extremely hydrophobic and consequently surface-active. Therefore mixtures of these components were often used to simulate the behavior of pulmonary surfactant. For some aspects mixtures containing DPPG have been frequently used [78]. This is justified in

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some aspects, although not all features of the natural pulmonary surfactant can be adequately covered by such model systems. In the following we will summarize some of the particularities of the pulmonary surfactant, which may be clearly explained by the behavior of simple model systems. We will also discuss terms like ‘‘squeeze-out’’, ‘‘overcompression’’, and ‘‘collapse’’. These terms have been used by different authors with different meanings. The adsorption rate of a system that contains a mixture of components of different surface activity to form a surface film depends on the concentration of the components and on their diffusion coefficients when diffusion controlled mechanism is assumed. When such a layer is compressed with avoiding film collapse (destruction of a monolayer) the most hydrophilic components can be removed from the surface and should desorb, provided that there is no interaction between the different components causing a desorption barrier. In pulmonary surfactant, there are some unsaturated lipid components less surface active than DPPC, which can be assumed to be removed from the interface during compression, but there is no systematic knowledge of all aspects of the interaction of these different lipid components. When using only monolayers spread from organic solutions of the most hydrophobic components some features are neglected, but the films resulted still show main features, which seem to be relevant for the pulmonary surfactant performance. Substantial work has been carried out during the last years to characterize surface behavior of simple model systems and obviously the behavior of the pulmonary surfactant and its components in comparison to such simple systems is unusual in many aspects.

6. Interfacial properties of spread DPPC layers In the case of high compression (deep expiration) the classical model of pulmonary surfactant films assumes that a DPPC enriched film is formed, which can be compressed to a surface pressure of å 70 mN/m, which is formally a nearly zero surface tension. At those pressures the DPPC monolayer collapses and the surface pressure remains constant at further compression. Formation of a new phase has then to be assumed and as it was pointed out this solid phase should be highly stable mechanically to prevent alveolar collapse [12]. This demand and the fact that a DPPC layer under such extreme conditions collapses is inconsistent, because collapsed structures are usually not stable mechanically and cannot resist further increase of pressure. In the case of surface expansion (inspiration) the interfacial layer should be restored fast enough to form a complete pulmonary layer again. Adsorption, rearrangement, and a subsequent re-spreading of removed molecules would be a relatively slow process. Vesicles of pure DPPC for instance form surface layers only at extremely slow rates [47].

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Fig. 1 shows a typical P/A isotherm of a spread DPPC layer determined by using a captive bubble device [58] in combination with an Axisymmetric Bubble Shape Analysis program, ADSA-CB [57]. It can be seen that the spread DPPC monolayer can be compressed up to very high surface pressure. The cross section of vertically orientated DPPC molecule is about 0.40 nm2/molecule. Therefore it is surprising that a surface pressure of about 70 mN/m is achieved at a surface coverage that is definitely less than the cross section of a DPPC molecule. In contrast the corresponding surface coverage is already established at a surface pressure of about 50 mN/m where the slope of the P/A isotherm only slightly decreases. Therefore in the range 50 –70 mN/m the layer is ‘‘over-compressed’’. Some authors [22,79] ignore the molecular cross section and uses the term ‘‘over-compressed’’ as synonym of a collapsed layer, i.e. for a layer that realizes a film pressure of about 70 mN/m. At this pressure the surface coverage, however, is 0.27 nm2/ molecule, which is about 70% of the calculated minimum area at which all DPPC molecules should be vertically orientated in a densely packed monolayer. Furthermore, there is usually an abrupt break down of film pressure when a collapsed insoluble monolayer is expanded. In contrast, the over-compressed DPPC layer can be expanded whereas the expansion wing of the isotherm exactly follows that of compression in the range of high film pressure. There is only a slight hysteresis when the DPPC layer is not deeply compressed into the collapse region at about 70 mN/m. Even the LE – LC plateau, which is observed at about 10 mN/m, is repetitive. A cycling (repeatedly compression and expansion) without hysteresis is possible within the range 0.75 – 0.27 nm2/molecule, which was also reported by Veldhuizen et al. [22]. This is a unique property of the

Fig. 1. Quasi-equilibrium P/A isotherms determined by using the captive bubble surfactometer. Red curve, curve 1: DPPC for compression and expansion. Green curve, curve 2: DPPC + 0.25 wt.% SP-B + 3 wt.% SP-C (SP-C in a conformation) for compression. Layers spread on water at 23 -C using a chloroform – methanol (3/1) mixture. Black solid line: curves for the best fit by using Eqs. (4) – (8). The LE – LC plateau of the DPPC layer is found at 11 mN/m. There is a small hysteresis. The green curve shows a well-pronounced plateau at 51 mN/m for the mixture.

DPPC layer, which cannot be explained in the framework of the traditional monolayer concept [53]. Structural studies were first approached on traditional models of surfactant films consisting of interfacial monolayers prepared by spreading small volumes of organic solutions containing simple lipid or lipid/protein mixtures at the air – liquid interface of a surface balance. Inclusion into such organic solution of a trace of fluorescently-labeled lipid probes made possible to obtain epifluorescence microscopic images from the interface [67,80,81]. Detailed observation of changes in the distribution of the probe at the interface while it is continuously compressed provided information on the microscopic structure of the monolayers at the different segments of the compression isotherms. Fig. 2 shows a scheme of the potential interfacial organization of phospholipid molecules at different pressures in the compression isotherm of a DPPC monolayer, as seen by epifluorescence microscopy. The P/A isotherm given in Fig. 2 was measured using the Langmuir film balance. The data only negligibly differ from that determined by using the captive bubble surfactometer. Changes in the distribution of the fluorescent probe NBD-PC are restricted to the liquidexpanded (LE) – liquid-condensed (LC) coexistence plateau, observed between 8 and 13 mN/m in the DPPC isotherm, at 24 -C. Ordered lipid molecules at the liquid-condensed phase exclude the bulky fluorescent probe, resulting in fluorescence-depleted dark areas contrasting against a fluorescent background of liquid-expanded phase. Observation of the progressive condensation of the lipid film as the monolayer is compressed along the plateau has provided detailed information on the effects of variables such as temperature, lipid composition, or compression speed on the nucleation and dynamics of film condensation [80,82,83]. Above 13 mN/m at 24 -C or about 40 mN/m at 37 -C [58], pure DPPC films consist of a single condensed phase that completely segregates most fluorescent probes. DPPC is the only component in pulmonary surfactant that can be condensed upon compression of the corresponding interfacial monolayer, at physiological temperatures, and this is related with its ability to reach the highest surface pressures. Images of mixed DPPC/POPG (palmitoyloleoylphosphatidylglycerol) films were also obtained using fluorescence microscopy (FLS) [51]. At different stages of compression bright streaks crossed this lipid layer at a surface pressure of nearly 68 mN/m. These streaks were interpreted as layer folding. Furthermore, dark domains on bright background showed liquid-condensed phase coexisting with liquidexpanded phase at extremely high surface pressures. The formation of the 3D structures at high surface pressure was reversible and temperature dependent [51]. Similar results were also observed for lipid mixtures with surfactant proteins [84,85]. Gopal and Lee [51] stated that folding or vesiculation in biphasic monolayers originates favorably at the boundary between the liquid-expanded phase and condensed domains and that any monolayer that undergoes folding is able to sustain high surface pressures. And indeed,

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Fig. 2. P/A isotherm of a DPPC monolayer at 24 -C. Addition of 1% molar of the fluorescently-labeled lipid NBD-PC allows observation of compressiondriven lateral transitions in the DPPC film as changes in the distribution of the label at the surface corresponding with interfacial organization of the lipid at the different surface pressures. (LE: liquid-expanded phase, LC: liquid-condensed phase, LE – LC: liquid-condensed – liquid-expanded coexistence, images width is 100 Am).

the reversible formation of 3D structures could explain all these unusual features observed for spread DPPC layers, including the over-compression, the weak hysteresis, and the fast reformation of the surface structure, provided the unfolding is sufficiently fast and nearly complete. It is also suited to resolve the apparent contradiction between mechanical stability and collapse.

7. Interfacial properties of spread lipid/protein layers Some studies have analyzed the effect of the presence of the hydrophobic proteins SP-B and SP-C on the compression-driven condensation of DPPC films [67,86 –88]. Curve 2 in Fig. 1 shows a quasi-equilibrium P/A isotherm for compression of a mixture of DPPC + SP-B + SP-C. The contents where chosen based on the assumption that the SPB to SP-C ratio in pulmonary surfactant is 1:3 (by weight) [89] and that the content of protein in the alveolar film contains 8 –10 wt.% protein [90]. The dimension of x-axis is nm 2/molecule. For these mixtures the molecule dimension at the surface is arbitrary because it is composed by several individual components. However, since the cross section of DPPC at the surface is dominating the cross section of proteins the latter is negligible. In contrast to pure DPPC isotherms, lipid/protein layers have a slightly pronounced LE –LC plateau at about 10 mN/ m and a large extended surface pressure plateau in the range of 51 mN/m. The appearance of the latter was initially interpreted as a kind of refining of surface active material from the interface, which is usually found for mixtures of DPPC and less surface active materials [53,58]. The surface pressure remains constant until this process is completed

and all less surface active material is removed into the surrounding phases (gas or liquid). The surface pressure of this plateau is similar to the equilibrium surface tension reported for adsorption layers of pulmonary surfactant, and also to the values found by Nag et al. [88] for DPPC + SP-B and DPPC + SP-C. The appearance of the plateau in the range of about 45– 52 mN/m in the quasi-equilibrium isotherm (depending on the mixture and temperature) was controversially discussed. Some authors argued that under equilibrium conditions this plateau indicates a film collapse, and a higher surface pressure cannot be realized [91 –93]. Other authors showed, however, that the film pressure very well may exceed the plateau value [47,94,95]. We could show that under dynamic conditions the plateau value can be exceeded, whereas the film is compressed to some metastable states [96]. For a dynamical process the plateau is deformed into a range of changing slope of P/A. Fig. 3 shows an example of P/A dependencies of the mixed layer DPPC + SP-B + SP-C at different compression speeds. These isotherms were successively recorded for the same DPPC layer shown in Fig. 1. Although at these compression rates a hysteresis occurs the next following compression cycle exactly repeats the former one because all structural changes are intermediately restored. These curves show that under dynamic conditions surface pressures higher than 51 mN/m can be easily achieved. Similar results were achieved by Hall et al. [92,97] with different pulmonary surfactant mixtures that could be compressed to a metastable state and which were mechanical stable for a prolonged period at pressures exceeding the equilibrium pressure, thus confirming the findings of Schu¨rch et al. [45]. In some cases during compression a ‘‘clicking’’ was reported, which is a sudden

40

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

70

∏ [mN/m]

60 50 40 30 20 10 0 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

A [nm2/molecule] Fig. 3. P/A isotherms of spread layers of DPPC + 0.25 wt.% SP-B + 3 wt.% SP-C (in a conformation) at different speeds of compression/expansion in 10
3 nm2/s: green 0.875, red 2.2, blue 8.75; 23 -C. The plateau observed at slow compression rates degenerates at higher compression rates.

reduction of the surface pressure that is caused by structural changes at the interface. The extension of this plateau was shown to depend on the content of protein in the mixture [64]. The actual structure and location of 3D structures in the plateau region was contrary discussed. Recent investigation showed different discoid structures [91,97], vesicles [98] associated with the interface, or buckles coexisting with flat layers [93, 99]. These results confirm the assumption of sectors of a multilamellar phase surrounded by monolayer areas. Diamant et al. [100] showed that differing elastic properties of two coexisting phases (LE and LC) generally form non-flat topography, where domains of one phase are elevated with respect to the other phase. Crane et al. [101] found that mixed monolayers of lung surfactant phospholipids tend to remain phases separated at high compression, which is usually a tendency for monolayers that contain components with different hydrophilic charged head groups and different apolar alkyl chains [100,102 –104]. Also the existing liquidexpanded domains in highly compressed pulmonary layers were confirmed by others [91,105]. A profound compression of solvent-spread DPPC layers into the collapse region was shown to form even some visible 3D structures, which partially were detached from the bubble surface and sank slowly down into the surrounding liquid phase in a captive bubble device [59]. Based on the isotherms of the pure proteins roughly a cross section of SP-B at the interface was determined of about 6.4 [106] and about 2.4 nm2/molecule for SP-C [58]. Although this is not the minimum cross section for a highly compressed protein film the surface pressure start to increase after the squeeze-out plateau at about 0.5 nm2/molecule, which is nearly confirmed by curve 2 in Fig. 1. If the whole protein amount would be excluded from the interface a further increase of the surface pressure would be expected to occur at about 0.4 nm2/molecule. Actually the increase of P starts at about 0.26 nm2/molecule. This confirms the data by Taneva

et al. that a part of DPPC molecules is removed by the surfactant proteins and squeezed out from the interface [107 – 109]. The exact determination of the amount of lipid molecules per protein molecule has important uncertainties because the area occupied by remaining loops and tails of the proteins at the interface cannot be established especially when taking into account the formation of 3D structures and the co-existence of LE and LC phases even at higher surface pressures. Therefore under dynamic conditions starting from the plateau region the layer is over-compressed and may finally collapse at about 68 to 70 mN/m depending on temperature for strong over-compression. Originally the formation of a DPPC enriched layer was assumed, which is quite reasonable when taking into consideration, however, that the DPPC layer relaxes the surface pressure by forming different 3D structures and that therefore not exclusively DPPC remains at the interface. Presence of either of the two hydrophobic proteins is enough to promote rapid formation of interfacial films from pure DPPC bilayers, which are extraordinarily reluctant to adsorb in the absence of the proteins [86,110,111]. Both, SP-B and SP-C, perturb the condensation of DPPC at the LE –LC coexistence pressures, presumably by inhibiting nucleation of condensed domains at the early stages. As a result of this perturbation, condensation of protein-containing monolayers produces much more numerous but smaller condensed microdomains (see Fig. 4) [67,88]. It has been proposed that such alteration of the microscopic morphology could be related with substantial changes induced by the proteins in the rheological properties of the films that would still be able to sustain high pressures while having substantial flexibility (inverse viscosity). A maximal flexibility of the films would be important to facilitate rapid respreading during expansion [90]. In the recent years, the application of scanning force microscopy (SFM) has allowed to obtain details of the structure of these films at much higher resolution, even in the absence of perturbing exogenous probes. Using SFM, the observation of the effect of SP-B to perturb the condensation of DPPC has been extended to the nanometer scale (see Fig. 5), and a clear correlation has been found between the effect of SP-B on the morphology of the monolayers and a significant increase in the stability of the films to mechanical rupture [69]. Nucleation of condensed domains of DPPC during compression seems also to produce an uneven distribution of the surfactant proteins that can be important for surfactant protein-associated activities (Fig. 6). Lateral distribution in specific regions of the films can be studied using fluorescent labeled proteins. Excess of SP-B at the interface induces formation of clusters that also preferentially associate with the boundaries of condensed microdomains (Fig. 6A) [69], and we have recently found that SP-C also accumulates close to the condensed phase borders (Fig. 6B). Concentration of proteins at the contact borders between disordered/expanded regions and ordered/

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

41

Fig. 4. P/A isotherms of DPPC monolayers in the absence or in the presence of surfactant protein SP-B or SP-C (10% w/w). Presence of surfactant proteins alters the compression-driven condensation of DPPC monolayers as observed on the isotherms and by epifluorescence microscopy of transferred films at the indicated surface pressures (mN/m). Observation of monolayers is allowed by the presence of the fluorescent probe NBD-PC (0.5% molar); image width is 350 Am.

condensed areas could be the expression of a general effect that thermodynamically favors exclusion of film impurities into packing discontinuities that would act as a sort of twodimensional sinks. Alternatively, preferential accumulation of surfactant proteins at the domain boundaries could be part of their specific properties, providing additional stabilizing effects to phase coexistence and therefore promoting

Fig. 5. SFM topographic images of a DPPC monolayer in the absence and in the presence of SP-B (5% w/w) transferred onto mica substrates at 11 mN/m. The perturbing effect of the protein on the packing structure of DPPC is patent both on the micro- and nano-structure of the film. The width of the images is indicated (courtesy of A. Cruz, L. Vazquez, and M. Velez J. Perez-Gil).

simultaneously suitable stability and flexibility. The occurrence of some other special activities of the proteins once accumulated at specific regions of the surface layers cannot be discarded. Pulmonary surfactant is more than just DPPC, and seminal studies showed that films formed by the full complex mixture of lipids and proteins in surfactant organic extract also showed lateral phase separation under compression (Fig. 7A) [112– 114]. Elegant calculations by the group of Steve Hall demonstrated that segregated condensed areas in surfactant films are enriched in DPPC [114] suggesting that the relative high proportion of this saturated phospholipid in surfactant may be required for structural reasons. The intrinsic tendency of DPPC molecules to selforganize in condensed domains (only at temperatures below 41 -C, the gel-to-liquid crystalline melting temperature for this phospholipid) triggers a compression-driven lateral reorganization of surfactant lipid and protein molecules in the interfacial film. Such structural reorganization is

Fig. 6. Nucleation of surfactant proteins is observed on films compressed to 11 mN/m at the boundaries of condensed phases. (A) SP-B clusters of ˚ height are observed by AFM microscopy on DPPC approximately 4 A transferred films containing the protein (10% w/w; scale bar, 2 Am). (B) Texas Red labeled SP-C (5% w/w) is observed as fluorescent clusters (red) on the boundaries of DPPC domains. The monolayer contained also NBDPC (green) to allow simultaneous observation of the lipid distribution (scale bar is 20 Am).

42

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

Fig. 7. (A) Film formed from whole native purified surfactant obtained by spreading of a small amount of the surfactant suspension containing NBDPC (1% molar) on top of a pure water interface. The film was transferred onto a glass support after compression to 36 mN/m, the scale bar is 20 Am. (B) A giant unilamellar vesicle (GUV) formed from native surfactant shows coexistence of fluid ordered (red) and fluid disordered (green) phases when observed by confocal microscopy. Surfactant contained 0.5% molar each of the fluorescent probes DiI C18 (red) and Bodipy-PC (green), the scale bar is 5 Am. Image courtesy of J. Bernardino de la Serna, J. Perez-Gil and L.A. Bagatolli.

probably required to support highly compressed states while producing arrangements that re-spread efficiently during expansion. Interestingly, very recent studies have been able to determine that native pulmonary surfactant membranes as obtained from lung lavages do present from their assembly a well-defined lateral structure, including coexistence of fluid ordered and fluid disordered phases (Fig. 7B) [115]. Such lateral structure imposes a defined lateral sorting of proteins and lipid species that is probably important for surfactant function, and that is transmitted to the interfacial films. Undisputed is the function of those surface-associated structures, which seem to justify the denotation ‘‘surfactant reservoir’’[47]. Fluorescence microscopic investigations confirmed that small amounts of surfactant proteins remain associated with surfactant films even in the case of strong compression, suggesting that these proteins are involved in restoration of the original pulmonary layer [88]. Scanning force microscopy of supported surfactant layers has also shown that proteins promote compression-driven formation of membrane structures that remain associated to the surface film. Thus the main hydrophobic components of the pulmonary surfactant are stored during compression in 3D structures in the vicinity of the surface and therefore facilitate fast reformation of the pulmonary layer during interfacial expansion. Experimental evidence that opened a different interpretation of the structure of pulmonary surfactant films was obtained by Schu¨rch et al. [47]. They were able to visualize the ultrastructure of surface films fixed and preserved in lung tissue preparations. Interfacial films were seen in those experiments as multilayered structures, composed of several surface-associated membranes. Many evidences have been obtained since then suggesting that pulmonary surfactant proteins SP-B and SP-C facilitate formation of what is called a surfactant surface reservoir [94,116,117]. This reservoir is composed of several tightly packed surfactant membranes closely associated with the interfacial monolayer. The present working model suggests that there exist a

continuous and dynamic flow of surface active lipid species between the monolayer directly located at the interface and the associated bilayered structures. Surfactant structures adsorbing to the interface from the bulk phase join the reservoir as a previous step to the real interfacial adsorption [118]. During compression, a proportion of lipid (and perhaps also protein) molecules leave out the interface in a process that has been called squeeze-out [119, 120]. In the traditional model, a selective molecular squeeze-out process would be responsible to refine the composition of the interfacial film to end in a DPPC-enriched state that would be able to support the highest surface pressures at the end of compression. Our present view is that certain regions of the laterally organized surface film initiate, when compressed beyond a threshold pressure, three-dimensional folds or transitions that move, probably in a cooperative way, massive amounts of lipids from the interface down to the associated reservoir [97,118,121]. These two-dimension to three-dimension structural transitions would be responsible for the typical ‘‘squeeze-out plateaus’’ observed when compressing pulmonary surfactant films. Pure lipid model films producing coexistence of condensed and expanded regions during compression have also been shown to produce this kind of three-dimensional transitions [93]. Hydrophobic surfactant proteins would modulate and optimize these compression-driven structural transformations of the films, to maintain the squeezed structures closely associated to the surface film [118,121]. Several techniques, specially SFM, have clearly demonstrated how both SP-B [68,122] and SP-C [68,123] do initiate formation of three-dimensional structures during compression of the film beyond equilibrium pressures. Using FLS and SFM, Knebel et al. [70,71] not only principally confirmed the data found with transferred LB layers [68], but definitely showed that the multilayer phase in the plateau range is at least in part orientated towards the aqueous phase. Using fluorescent microscopy and quenching experiments, Smith et al. [92,97] confirmed a model with discoid structures orientated to the gas phase. The hydrophobic a-helical part of SP-C is assumed to span the lipid bilayer, thus enhancing the lipid release [70] during film expansion, which verifies the principal scheme of squeeze out depicted in [90] and [118]. Palmitoylation of the protein seems to be particularly important to sustain association of SP-C/lipid membrane structures, and it has been proposed that SP-C could act as a bridge between the interfacial monolayer and a neighbor bilayer or between two bilayers, in the reservoir [68,118,123]. Participation of the proteins in formation of the reservoir during compression could also ensure a competent structure to promote reinsertion of lipids backwards during expansion, a process that is necessary to respread the surface film with almost no hysteresis. Results of cycling experiments (repeated compression and expansion of the pulmonary layer) are often reported in literature. The results of such experiments depend on both the speed of cycling and the range of pressure or surface

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

coverage, respectively. Following the traditional concept most of these experiments were started at the equilibrium surface tension at surface saturation, or at the plateau value of the P/A isotherm. Subsequently the layer was compressed to maximum accessible surface pressure, followed by an expansion, thus simulating breathing processes. This procedure does not really correspond to normal breathing as explained before. The differences between the P/A isotherms are rather small as long as the films are not deeply compressed into the collapse region (surface tension å 1 – 3 mN/m). It is not surprising that the incipiently reversible squeeze out procedure is changed when the film is repeatedly over-compressed into the collapse region. Such procedure yields irreversible collapsed structures, which causes an effectively loss of surface active material. This consequently shifts the P/A isotherms and yield to results suggesting that extremely high surface pressure is easily accessible after repeated cycling. This observation was designated as refinement. Veldhuizen et al. [124] concluded: ‘‘These findings (observations of Crane and Hall [125]) indicate that compression rates must be standardized and care must be taken when conclusions are drawn from c min (minimum surface tension) values in captive bubble surfactometer studies’’. A representative example for the formation of irreversible 3D structures at the interface was produced by repeatedly over-compressing a spread DPPC/SP-C/SP-B layer. These structures are visible [59] (an analogous picture is given in Fig. 16). There are some patches modifying the contour of the bubble. From the physiological point of view the formation of irreversible collapsed structures is not critical as a loss of surface active material can be compensated by adsorption from the bulk phase. The collected over-compressed patches are sporadically separated from the surface accompanied by ‘‘clicking’’, which is a temporary jump of the surface pressure. All other phospholipids are less surface active than DPPC. They interact with other components of the pulmonary surfactant and were assumed to be partially removed from the interface by compression. Nevertheless even at surface pressures just before collapse palmitoyloleoylphosphatidylglycerol was observed in mixed films with DPPC by fluorescence microscopy at the interface thus forming separate phases [51]. Furthermore the surface behavior is different when Ca2+ is present. DPPG was shown to interact specifically with SP-B [20,126 –128]. SP-B was found to interact more strongly with PG than SP-C in DPPC/DPPG mixtures which could facilitate the removal of PG-enriched regions from the interface [127,129]. Cholesterol also enhances adsorption of DPPC vesicles which has been traditionally interpreted as a consequence of the increase of fluidity and film flexibility (inverse viscosity), and which could also improve re-spreading [130,131]. Cholesterol is a critical actor in producing that lateral separation and in maintaining the liquid character of

43

the segregated two-dimensional phases. The role of cholesterol in the structure and functional properties of surfactant films has been very much underestimated in spite that cholesterol can reach proportions in surfactant as high as 20% molar ratio with respect to phospholipids [115]. Cholesterol is also responsible for a dramatic miscibility of phases that occurs in surfactant films when compressed above a critical pressure [132,133]. A clear conclusion is that the structure of fully compositionally complex surfactant films is very dynamic, and that the composition of surfactant has been probably optimized during evolution to provide such a dynamic behavior under the exigent conditions imposed by the lung mechanics. The study of the structure of pulmonary surfactant films subjected to dynamic compression is particularly important at the pressure regime that goes from the equilibrium surface pressure to the collapse. Structures formed at those pressures are responsible to sustain stable films collapsing close to 70 mN/m, but also to re-spread efficiently during expansion. Sterol, however, cannot be squeezed out easily from DPPC – cholesterol spread films [131,134,135] thus preventing extreme surface tension decrease. However, very recent results suggest that the presence of cholesterol in pulmonary surfactant may still be required for surfactant bilayers to adopt a well-defined in-plane structure including lateral separation of cholesterol and DPPC-enriched fluidordered regions from cholesterol depleted liquid-disordered phase. Such structures would be competent for rapid interfacial adsorption and could provide substantial mechanical stability when associated to the surface films subjected to dynamic compression [115].

8. Influence of the surfactant protein SP-A on the surface properties Although SP-A is hydrophilic and therefore only weakly surface active, it has been proposed that it influences the adsorption and film formation, re-spreading, and cycling behavior of pulmonary surfactant films. Early Langmuir balance studies seemed to demonstrate that SP-A promotes interfacial phospholipid adsorption, although only in the presence of SP-B [47,56,94,136,137], whereas this cooperative effect between the surfactant proteins was shown to be calcium dependent [138 –140]. Using fluorescent labeled SP-A it was shown that SP-A accumulates at the boundaries of condensed DPPC domains [141 – 143] and several evidences point that from that location, SP-A is able to attract SP-B to form SP-A/SP-B complexes [144]. This is in agreement with data of Taneva and Keough [145] who found that the hydrophobic surfactant proteins, SP-B or SPC, may enhance the incorporation of SP-A into mixed lipid monolayers to a similar extent at low surface pressures. The results revealed that SP-B and SP-C in their pure monolayers had similar abilities in promoting the adsorption of SP-A, whereas SP-B, when present in lipid films in the LE –

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R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

LC coexistence state, displayed a higher capacity to attract SP-A from the subphase than SP-C. Furthermore SP-A had a positive effect on refinement when it was present in the subphase. However, this effect was only observed when SPA was combined with SP-B and incubated with subphase vesicles [124]. Comprehensively Rodriguez-Capote et al. recently showed that the adsorption kinetics in mixed lipid systems and in the presence of SP-B or SP-C was not significantly affected by SP-A [79]. They found, however, an influence in the maximum depression of surface tension1 during dynamic cycling, which was strongly reduced in the simultaneous presence of SP-B and SP-A. Furthermore these authors compared the interaction between the surfactant proteins, DPPC, and acidic POPG (palmitoyloleoylglycerophosphoglycerol), or zwitterionic POPC (palmitoyloleoylglycerophosphocholine). They found that SP-A enhanced the ability of the zwitterionic PL samples containing SP-B to reduce the surface tension, whereas this effect was more pronounced in the presence of acidic lipids. The results suggest specific interactions between SPB and phosphatidylglycerol, and between SP-B and SP-A, which finally influences the surface behavior of the whole system. In summary, SP-A influences the surface behavior mainly indirectly and especially in the range of low surface pressures. In spite of this, SP-A seems to be not essential for breathing, because SP-A knock-out mice could breathe normally but were extremely susceptible to infections [146]. Our present view is that SP-A may have evolved as a major element in lung defense, while maintaining proper surface properties not to disturb the surfactant dynamics mainly developed at the alveolar interface by the other components.

9. Surface dilatational rheology The rheology of the liquid bulk phase is based usually on isochoric deformation, because liquids are almost incompressible. The rheology of the bulk phase is therefore mainly a shear rheology. In contrast the interface can be sheared but also deformed by compression and expansion. For the latter the breathing process is a classical natural example. Therefore the dilatational parameters are of interest for the surface behavior of pulmonary surfactant films, because the surface area of the lung is dynamically changed. Consequently one strictly has to distinguish between shear and dilatational rheology for the interface. Since the lung is full of converging and diverging passages it is also possible that for some

aspects surface shear rheology becomes important. As we will show below those effects, however, are very small. The fundamentals of surface rheology, including the 2D conception of surface rheology and the connection between shear and dilatational properties, were described in detail elsewhere [147]. There are different experimental approaches to determine surface dilatational parameters. One option is to record the surface stress decay in the case of a transient surface pressure jump [65,148]. This experiment can be used to determine relaxation times, i.e. s = g d / e d, with g d being the dilatational viscosity and e d the dilatational modulus. Another experimental approach is using periodical oscillations of the surface area and corresponding surface pressure [63] that yield anisotropic effects. The dilatational modulus contains not only the storage elasticity, but also the dissipative component, which is affected by the retarded flow. The complex modulus then is given by ed ¼ jed jcosh þ ijed jsinh ¼ er þ iei

ð1Þ

with e r and e i being the real and the imaginary part of the complex modulus, respectively, and h the loss angle. The dilatational elasticity becomes jed j ¼


dc : dlnA

ð2Þ

From the phase angle the dilatational viscosity g d can be calculated as [149] gd ¼

jejsinh x

ð3Þ

x = 2pf is the circular frequency of the area oscillation. The physical meaning of the dilatational viscosity is complex. It contains any relaxation process from the surface layer as well as the intrinsic viscosity [150]. For an interfacial pulmonary layer stressed in the frequency range of breathing it contains conformational changes of the components at the interface, and rearrangement processes of surface structures. Extreme slow rearrangement processes in the time scale of minutes or hours can be neglected for breathing processes. On the other hand any influence of adsorption/desorption processes is usually too fast to influence the dilatational parameters in the frequency range of breathing. An exception is the adsorption of surface material released by liposomes, which is too slow to influence the dilatation behavior [111].

10. Stress relaxation of spread pulmonary layers 1

Rodriguez-Capote et al. [79] uses the term surface activity as a synonym of the maximum depression of surface tension. This is a common way to evaluate and compare functional performance of pulmonary surfactant preparations. In contrast, in the context of surface science, a substance is more surface active than another when it reduces the surface tension of the solvent at a lower bulk concentration.

Studies of the stress relaxation of monolayers yield information about the viscoelastic properties of the interface, whereas in the time scale of the experiment a series of conformational changes and rearrangement processes is covered. Starting from an equilibrium state the surface layer is transiently compressed or expanded. To establish a new

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

Table 1 Main relaxation times determined after transient layer compression or expansion starting at P å 50 mN/m using the decay of the surface pressure main relaxation time s max [s], (a – a-helical structure, h – h-sheet) System

DPPC DPPG DPPG DPPC + 2 mol% DPPC + 2 mol% DPPC + 3 mol% mol% SP-B DPPC + 3 mol% mol% SP-B

70 60

Π [mN/m]

equilibrium some molecular reorientation, adsorption/ desorption, re-spreading, and structural rearrangement processes are necessary, which are not completed instantaneously. This process is accompanied by a time dependent increase or decrease of surface pressure, respectively. From the decay of the surface pressure a relaxation time can be determined. The sense of this characteristic time was analyzed and discussed by Maxwell [151]. All processes much faster than this time can be described mainly by elasticity, all processes much slower by viscosity. In the range of the relaxation time the viscosity retards the elastic behavior. A realistic assumption to explain viscoelastic processes is a series of relaxing elements, which are distributed around a main relaxation time, s. The procedure of evaluation is given elsewhere [65,148], whereas for the limited time range of breathing the assumption of a single relaxation process is sufficient. Practically the transient deformation should be small to avoid destruction and non-linear behavior of the surface layer. For the special case of the pulmonary surfactant it is interesting to start such stress relaxation experiments from the equilibrium surface tension, i.e. a surface pressure of about 50 mN/m. Taking into account the results shown in Fig. 6 it becomes clear that even starting from the plateau value determined by the P/A isotherms a change of surface pressure can be achieved when the transient change of bubble volume is executed sufficiently fast. Table 1 gives some relaxation times determined for different spread layers. Clearly distinguished is the role of SP-B, which usually strongly decreases the relaxation time. This means in layers that contain SP-B every deformation of surface is fast equilibrated (the exception is the system with SP-C in h-sheet structure, which will be explained later). This, however, is not the only difference. After a transient jump of surface pressure the system may return to the former equilibrium state or realize a new steady state. Fig. 8 shows some measured decays of surface pressure. SPB remarkably accelerates the relaxation process. Nevertheless it was found that the only system that restores the

45

50 40 30 20 0

50

100

time [s] Fig. 8. P/t of different spread pulmonary layers after transient compression (black symbols) or expansion (white symbols). (h, g): DPPC + 2 wt.% aSP-C, (r, ‚): DPPC + 0.5 wt.% SP-B, ( , o): DPPC + 0.25 wt.% SP-B + 3 wt.% a-SP-C, squares: DPPC + 0.25 wt.% SP-B + 3 wt.% h-SP-C at 23 -C. In opposite to all other curves this one for the mixture DPPC + 0.25 wt.% SP-B + 3 wt.% a-SP-C a fast relaxation and an agreement of the curves after monolayer compression and expansion.

.

equilibrium state (P = 51 mN/m) is DPPC + 3 mol% a-SPC + 0.25 mol% SP-B, i.e. the mixture that contains both proteins where SP-C is in native conformation [96]. The relaxation behavior of SP-B and SP-C suggests that the presence of SP-B is essential for expiration and the presence of both proteins SP-B and SP-C is essential for inspiration process of the breathing cycle. For all other systems a steady state is established which differs remarkably from the previous state. In conclusion the stress relaxation experiments yield information that in normal cases re-spreading, molecular reorientation in the pulmonary layer, and molecular transfer processes for molecules not far away from the interface (reservoir), all completed within seconds. It was pointed out by Schu¨rch et al. [45] that the rapid re-establishment of equilibrium during inspiration to total lung capacity keeps the surface component of lung recoil pressure low, which helps to minimize the work of breathing [152]. The stress relaxation is sensitive to the correct physiological composition of the pulmonary surfactant. It is useful to mention that both, elasticity and viscosity depend on temperature. In contrast, the relaxation time depends on temperature only to a small degree, which was first mentioned by Maxwell [151]. Experimentally this could be confirmed also for spread DPPG layers [148].

Temperature Main relaxation time [s] [-C] Compression Dilatation 30 35 40 SP-B 23 SP-C 23 a-SP-C + 0.25 23

29 T 12 19.5 T 1.2 18.2 T 1.7 2.4 T 0.3 35 T 11.4 2.5 T 0.25

22 T 9.8 19.8 T 0.7 17.5 T 1.8 4.8 T 0.4 16 T 8.6 4.3 T 0.38

h-SP-C + 0.25 23

66.8 T 15.6

61.5 T 8.6

11. Viscoelastic properties of spread pulmonary layers A very impressive insight into the viscoelastic behavior yields the dependence of the dilatational elasticity or viscosity on both the surface pressure and the frequency. Such dependencies can be determined by harmonic drop or bubble oscillation at a constant surface pressure and for a given frequency [63]. The amplitudes of bubble oscillation

46

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A 250

ε d [mN/m]

200 150 100 50 0 10

50

-2

35 10

f [Hz]

Π [mN/m]

20

-1

5

B 400 300

ηd [mNs/m] 200 100 0 10

50

-2

35 20

f [Hz]

10-1

5

Π [mN/m]

In contrast to that the elasticity of a pure DPPC layer (Fig. 10) strongly increases up to a surface pressure of about 35 mN/m, passes a stationary minimum at the point where the over-compression starts, increases temporary and subsequently decreases continuously in the range of 55– 60 mN/ m. It breaks completely when the DPPC layer collapses [64]. The dilatational viscosity shows some maxima which corresponds to the main relaxation times determined elsewhere [58], which however do not influence the behavior in the frequency range of breathing. The compressibility is the inversed elasticity. Therefore for pulmonary surfactant the interfacial layers become extremely compressible in the squeeze out plateau, i.e. small changes of the surface area practically do not change the surface pressure. In fact in this range the interfacial work becomes minimum, which supports the breathing process. When assuming a widely DPPC enriched film existing at higher surface pressures the elasticity would approach to the behavior shown in Fig. 10A, which however should at surface pressure above the plateau initially reach a finite value > 0 mN/m. In comparison to the behavior of the DPPC layer the film containing also the surfactant associated proteins SP-B and SP-C is less elastic, i.e. more compressible, in the whole range of elasticity accessible for measurements.

A

Fig. 9. e d (A) and g d (B) via frequencies f and P for spread layers of DPPC + 0.25 wt.% SP-B + 3 wt.% a-SP-C at 23 -C. There is pronounced viscoelastic behavior in the frequency range of human breathing.

are usually small to avoid changes of the interfacial structure and non-linearity effects of viscoelastic behavior. This ideally meets the demands of alveolar deformation during normal tidal breathing [43]. The frequency of normal human breathing is in the range 0.04 – 0.2 Hz [153]. Both dilatational elasticities and viscosities, and their dependencies on frequencies, surface pressure, and temperature were reported for different films which contained the most hydrophobic components of pulmonary surfactant [64,106,154,155]. The surface dilatational elasticity of a mixture DPPC + 3 mol% a-SP-C + 0.25 mol% SP-B is given in Fig. 9. The elasticity increases with the surface pressure and gets maximum just before the squeeze out process starts. In the range of squeeze out plateau the elasticity strongly decreases, because the amplitudes of surface pressure oscillations become too small for evaluation. The elasticity is only weakly influenced by the frequency. The dilatational viscosity at the same time is considerably low and does not strongly increase even at pressures in the range of the squeeze out plateau. It only slightly increases at low frequencies. Therefore the main characteristic for the surface dilatational parameters is the elasticity and the elastic effects are only weakly retarded by the corresponding viscosity.

250 200

ε d [mN/m]

150 100 50 0 0.006

0.016

0.025

10

20

40 30

60 50

Π [mN/m]

f [Hz]

B 400

300

ηd [mNs/m]

200

100 0 0.006

30 0.016

f [Hz]

0.025

10

20

40

60 50

Π [mN/m]

Fig. 10. e d (A) and g d (B) via frequencies f and P for spread layers of DPPC at 23 -C. The dilatational elasticity increases up to 50 mN/m, but decreases at higher surface pressure.

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58 350 1

300

ε d [mN/m]

250

2

200 3

150 100 50 0 0

10

20

30

40

50

60

Π [mN/m] Fig. 12. e d via P for DPPC + 2 wt.% a-SP-C (1), DPPC + 2 wt.% SP-B (2), DPPC + 0.25 wt.% SP-B + 3 wt.% SP-C (3) at frequency 0.1 Hz and 20 -C. SP-C in a conformation. The dilatational elasticity is decreased when SP-C and SP-B are present.

Surface shear rheology has been reported not often for pulmonary surfactant components. The effects, however, are small in comparison to those of dilatation. An example of the shear viscosity via surface pressure is given in Fig. 13. The curve was obtained for a spread film containing recombinant SP-C at the surface TRIZMA buffer/air. The measurements were performed using a torsion pendulum device [156]. Basically, a freely oscillating pendant titanium ring that touches the surface produced a small shear deformation of the surface layer. A small twocompartment Langmuir trough was used to vary the surface pressure [157]. The film between the disk and the wall of the dish was sheared. The transferred torque was correlated with the shear viscosity. As seen by the graph the shear viscosity starts to increase at a surface pressure of about 30 mN/m and steeply increases in the plateau range of surface tension. At 40 mN/m it increases up to 0.8 mNs/m, which is nearly 2 orders of magnitude less than the dilatational viscosity for DPPC + 0.25 800

600

ηs [µNs/m]

The dependence of the elasticity on temperature is shown for a mixed layer DPPC + 2 mol% SP-C at a constant frequency of 0.1 Hz (Fig. 11). With increasing surface pressure the elasticity increased simultaneously and becomes maximum at about 46 mN/m. Thereafter, when the film pressure passes the plateau in the P/A isotherms, the elasticity is drastically decreased. The surface pressure decreased at about 42 mN/m with some irregularities, which indicates that this rigid film became unstable at 20 -C. With increasing temperature the elasticity is depressed, i.e. the compressibility of the layer strongly increased. Notice, however, the overall shape of the curve does not change depending on the temperature. There was a rapid decrease of elasticity when the temperature was increased from 20 to 30 -C. Within the range 30 –40 -C the elasticity also drops but considerably less. The elasticity of different DPPC/surfactant protein mixtures at 20 -C and 0.1 Hz is compared in Fig. 12. The temperature 20 -C was chosen, because there are to be expected the most pronounced effects. The elasticity also increases with the surface pressure but less when SP-B is present. For the mixed layer DPPC + 3 mol% SP-C + 0.25 mol% SP-B a very smooth curve results which indicates that the flexibility (inversed viscosity) of these films increases, which corresponds with the low relaxation times for this mixture. When the film pressure passes the plateau range the elasticities of all films decreased. For the film DPPC + 2 mol% SP-B a stationary maximum of elasticity was observed in the range slightly above the LE – LC plateau. The most compressible films were found for DPPC + 3 mol% SP-C + 0.25 mol% SP-B. The maximum elasticity of about 300 mN/m shown in curve 1 (Fig. 12) is in quite good agreement with the dependencies of the real part of complex dynamic elasticity of spread recombinant SP-C monolayer reported by Grigoriev et al., which were obtained from longitudinal wave measurements on a Langmuir trough and frequencies of 0.2 and 0.8 Hz [155]. These results also confirmed the low dilatational viscosity and the increasing elasticity during compression up to the squeeze out plateau.

47

400

200 0 0

10

20

30

40

50

Π [mN/m]

Fig. 11. The dependence of e d on temperature T for a spread mixed layer of DPPC + 2 mol% SP-C at frequency 0.1 Hz. SP-C in a conformation.

Fig. 13. Surface shear viscosity g s vs. surface pressure for a spread rSP-C film (2% recombinant SP-C, mixture of DPPC and POPG in a ratio of 70:30, and 5 wt.% palmitic acid), 20 -C. Using a tungsten wire of 100 Am diameter the oscillation frequency was of the order of 0.1 Hz. All rheological experiments were performed with a deflection angle of 2- to minimize distortions of the monolayer. The measuring geometry yields an initial relative deformation of the surface of 8.7% (with kind permission of J. Kra¨gel).

48

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

SP-B + 3 wt.% SP-C. Recently these data were principally confirmed by Alonso and Zasadzinski [158] who used a different measuring device and reported data for DPPC layers. The order of magnitude of these data is very close to the data given here. As mentioned above other lipid components in pulmonary surfactant like unsaturated lipids or cholesterol may modulate the viscoelastic behavior of surfactant layers. There are no studies so far about this influence. This, however, is especially important in the context of the development of new surfactant preparations for therapeutic application. Many artificial surfactants used today incorporate additives such as palmitic acid, which should affect only vague known suitable surface viscoelastic properties of surfactant. The main point to optimize properties of artificial compositions today seems to be only the ability of the pulmonary layers to sustain extremely high surface pressure, which is important for infant therapeutic application. A better understanding of the whole breathing process and the different viscoelastic effects caused by the interplay of all surfactant components should help for further optimization and improvement of respiratory therapies.

12. Oligomerisation and secondary structure of the hydrophobic pulmonary surfactant proteins There are always differences when comparing experimental data obtained by different groups for lung surfactants, which cannot be finely explained. Such differences are normal for biological systems, but thoroughly isolating the components and restoring the system artificially should extensively reduce them. Several scientific groups chose this way, nevertheless after all the differences did not vanish satisfactorily. This was the reason to study the influence of different isolation and purification procedures on the results of surface behavior. Native SP-B and SP-C have a-helical segments. Furthermore SP-B and SP-C are known to form oligomers. Nothing is known about surfactant protein conformation at the interface and the influence of protein conformation on interfacial behavior. SP-B consists of 79 amino acids and has a molecular weight of 8690 Da. The content of hydrophobic amino acids

is 40.5% (11 Val, 13.9%; 15 Leu, 19%; and 6 Ile, 7.6%). Natural SP-B is commonly a homodimer in all known species, with two monomeric units linked by disulfide bounds at Cys 48. Bovine SP-B occurs also as covalent trimer [159,160], and was found to form oligomers [89]. SPB is remarkably thermally stable, and its a-helical domains are not much influenced by reduction of the disulfide bounds [160,161]. SP-C consists of 35 amino acids and has a molecular weight of 4200 Da. The content of hydrophobic amino acids is 65.7% (12 Val, 34.3%; 7 Leu, 20%; and 4 Ile, 11.4%). Despite its low molecular weight, SP-C has several structural features. The a-helical valyl-rich domain consists of amino acids in positions 11 –34 and contains 25 amino acids. It was found that this a-helix is transformed into h-sheet by incubation in solution [162]. Furthermore palmitoylation of the cysteines at positions 5 and 6 increases the hydrophobicity of SP-C [163 – 165]. The pulmonary surfactant proteins used were obtained from cell-free sheep lung lavage. The hydrophobic components of pulmonary surfactant were extracted from butanol or chloroform – methanol and isolated using gel exclusion or high performance liquid chromatography. The properties of hydrophobic surfactant proteins obtained by different isolation and purification procedures were investigated by circular dichroism (CD), Fourier transform infrared (FTIR), and matrix-assisted laser desorption/ ionization time-of-flight mass (MALDI-TOF) spectroscopy [66,166]. CD measurements were executed by using spread films at quartz plates. FTIR was measured also for spread films at KBr plates. In both cases a and h structures of the surfactant proteins could be distinguished, which gives an evident hint, that these conformations are sufficiently stable at interfaces and that these structures can be also obtained at the air –water interface. Table 2 shows the oligomerisation and the secondary structure of SP-B and SP-C samples obtained by different procedures of isolation and purification. The results given in Table 2 clearly show that SP-B in all samples occur with an a-helical structure and usually forms oligomers. SP-C forms only monomers although depending on the procedures of preparation its secondary structure was different. In the case of using butanol as extraction solvent

Table 2 The oligomerisation and the secondary structure of SP-B and SP-C samples obtained by different procedures of isolation and purification [167] Protein sample

Extraction solvent/isolation method/volume of lipid extracts

Protein storage concentration [mg/ml]

Oligomerisation state

Secondary structure at the interface

SP-B-1 SP-B-2 SP-B-3 SP-C-1 SP-C-2 SP-C-3

Butanol/LH 60 column/5 ml Chloroform – methanol/HPLC Chloroform – methanol/HPLC Butanol/LH 60 column/5 ml Chloroform – methanol/HPLC Chloroform – methanol/HPLC

0.04 – 0.06 0.8 – 1.0 0.8 – 1.0 0.04 – 0.06 0.8 – 1.0 0.8 – 1.0

Dimers Oligomers Oligomers Monomers Monomers Monomers

a-Helix a-Helix a-Helix a-Helix a-Helix + h-sheet Antiparallel h-sheet

column/5 ml column /1.5 ml column/5 ml column/1.5 ml

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

SP-C showed a typical a-helical structure. In the case of chloroform – methanol extraction, however, there was always a part of SP-C found in h-sheet conformation. For the SP-C-3 sample when the volume of lipid extract was increased to 1.5 ml CD and FTIR spectra strongly corresponded to secondary structure of antiparallel h-sheet. These results definitely emphasize that care is necessary comparing surface behavior of identical proteins without knowing their conformation. Taken together, these observations described in detail in [66] suggest that a-helix to h-sheet transition may originate as a simple consequence of increase of the summary protein/ lipid concentration. The mechanism may be common in the development of several human diseases of different etiology characterized by the extracellular deposition of amyloid and may influence lung diseases such as alveolar proteinosis, acute respiratory distress syndrome, and lung fibrosis.

13. Influence of oligomerisation and secondary structure on the surface behavior of SP-B and SP-C To quantify the influence of oligomerisation and secondary structure of proteins we used an thermodynamic approach based on a generalized Volmer isotherm [168]. This approach has the advantage that it can be used to describe complex adsorption processes with isotherms that show several kink points and plateaus [169,170], which is also characteristic for the isotherms of spread SP-B and SP-C layers [66]. Actually there is no thermodynamic concept taking into account the compression of a monolayer to a surface coverage below the minimum area demand of a molecule, which would be necessary for pulmonary layers at high surface pressure. Therefore present approaches are restricted to describe P/A isotherms up to pressures where formation of 3D structures occurs. The equation of state for monolayers in the fluid state was represented by Volmer’s equation, i.e. for the gaseous and the liquid-expanded (LE) state [64,169,170]: P¼

mkT
Pcoh : A
x

ð4Þ

Here, P is the surface pressure, k is the Boltzmann constant, T is the temperature, x is the partial molecular area for monomers (or the limiting area of molecule in the gaseous state), A is the area per molecule, and m is the number of kinetically independent units, i.e. fragments or ions of a molecule [64]. P coh is the cohesion pressure, which accounts for the intermolecular interaction. For the coexistence region of LE and LC phases the generalized equation leads to: P¼

mkT ab
Pcoh A
x½1 þ eðab
1Þ

ð5Þ

49

a and b are given by the relation between the aggregation constant and P, and by the fraction of the monolayer free from aggregates, respectively:   A P
Pc x ð6Þ exp
e a¼ Ac kT b ¼ 1 þ xð1
eÞða
1Þ=A:

ð7Þ

A c the molecular area, which corresponds to the onset of the phase transition at P =P c expressed by a kink point in the isotherm, e = 1
x cl / x, with x cl being the area per one monomer in a cluster. The area per molecule in the cluster can differ from the limiting area per molecule in the gaseous state. This fact was taken into account by the parameter e = e 0 + gP: xcl ¼ xð1
eÞ ¼ xð1
e0
gPÞ

ð8Þ

where e 0 is the relative jump of the area per molecule that occurs during condensation and which is connected with some changes of the molecular orientation at the interface. g is a relative two-dimensional compressibility of the condensed monolayer [170]. Using this model the experimental P/A isotherms of different samples of SP-B and SP-C were satisfactorily described for SP-B up to a surface coverage of 5 and for SPC up to 1.5 nm2/molecule [167] although the model takes into account only 6 independent parameters. Two of them are the surface pressure and the surface coverage of phase transition points, which are directly derived from critical points of the experimental isotherms. As this is a molecular approach SP-B dimers were treated as molecules. The parameters are given in Table 3. Table 3 gives the best fit parameters according to Eq. (4). The different SP-C samples show different phase transitions other than LE –LC, indicated by several kink points in the isotherm. Here for different ranges of the isotherm different parameters are characteristic. The results show that differences in the oligomerisation state (dimers or oligomers) of SP-B are reflected mainly by the number of kinetically independent units m (fragments) in the molecule. In contrast, differences in the secondary structure of SP-C are reflected by the cohesion pressure P coh, and the compressibility parameter g. The reason for the different phase transitions observed for SP-C-1 as well as that for SP-C-2 seems to be a decrease of the area per molecule in the condensed state and the corresponding coefficient of the two-dimensional compressibility of the protein molecules, respectively. With increasing surface coverage this compressibility parameter increases stepwise for the different states of the monolayer. In contrast to the samples SP-C-1 and SP-C-2 the sample with SP-C in h conformation (SP-C-3) shows no plateau in the P/A isotherm. The shape of the isotherm is typical for a coexistence of LE – LC phases in a wide range of surface coverage. Only one phase transition and a high monolayer compressibility coefficient g (0.01 m/mN) is characteristic

50

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

Table 3 Characteristic values determined by Eqs. (4) – (8) for surface pressure/area isotherms of SP-B-(1,2,3), SP-C-(1,2,3) monolayers (see Table 2) and DPPC Parameters 2

SP-B-1

x, nm A c, nm2 P c, mN/m g, m/mN e0 m P coh, mN/m a b

5.0 13.2 21.0 0.00 0 60 8.26

SP-B-2 5.3 12.5 40.8 0.00 0 90 10.3

SP-B-3

SP-C-1 a

5.0 12.0 43.2 0.00 0 89 8.35

2.9/1.7 5.53/4.62b/3.66a 12.1/28.2b/38.9a 0.0/0.002b/0.0023a 0 18 14.8/18.3a

SP-C-2

SP-C-3

DPPC

2.0 3.62/3.22b 22.2/36.6b 0.0004/0.0025b 0 18 21.6

2.5 4.0 – 5.0 0.0 – 1.0 0.01 0 16 26.2 – 42.0

0.453 0.684 9.0 0.0009 0.1 1.0 8.45

Parameters of the third (structural) phase transition. Parameters of the second (structural) phase transition.

for the condensed SP-C-3. The cohesion pressure of SP-C-3 in h-sheet conformation is higher than the cohesion pressure of SP-C-1 in a-helical conformation, which is consistent with the higher tendency of this protein to aggregate. It must be pointed out here that this compressibility parameter g is a molecular value (compressibility of a protein molecule), which does not necessarily influence directly the rheological monolayer properties. Nevertheless there is a direct connection between these parameters [171] and the higher surface elasticities of SP-B films compared with those of SP-C. The last column in Table 3 shows the parameters used to describe the isotherm of DPPC for compression. The line shown in Fig. 1 confirms that the calculated curve is in good agreement with the experimental one (red curve) and agree well with data given in [172] at 22 -C. The result m = 1 is not unusual for small molecules. It means the whole molecule has to be seen as one kinetically independent unit and behaves extensively rigid. The physical content of the two-dimensional compressibility parameter correspond with a molecular orientation, where different orientated molecules occupy different area per molecule and where the layer thickness becomes maximum at vertically orientated molecules. Using GIXD measurements Vollhardt and Fainerman were able to determine this value independently for different spread monolayers [170,173]. A satisfactory approach is achieved up to a surface coverage of about 0.42 nm2/molecule corresponding to a surface pressure of about 50 mN/m, i.e. for the whole range of isotherm up to the minimum area per molecule for a vertically orientated DPPC molecule. Therefore both isotherms of the pulmonary surfactant proteins and DPPC can be described within the same theoretical frame, which open the possibility to treat the protein/DPPC mixtures.

14. Modeling of mixed surfactant protein/DPPC layers Taking into account Eq. (4) for a mixture of several insoluble components in the range of liquid-expanded state the surface pressure is P ¼ kT

~ni mi Ci
Pcoh 1
~ni Ci xi

ð9Þ

where C i is the surface concentration of ith component, P coh is the integral constant. Considering Eq. (4) for the individual components and for a strong diluted monolayer from Eq. (8) follows P ¼ ~Pi ;

ð10Þ

where P coh = const is assumed for all components. Here P i is the surface pressure of the individual components in the mixture for all values of C i . In the case of additive behavior n i is not included in Eq. (8). x can be calculated when taking the values for DPPC given in Table 2 (0.453 nm2) and considering the real molar ratio for the components. To evaluate the ratio of area occupied by the different species of the mixture DPPC + 2 mol% SP-C x can be determined by x = 0.98 * 0.453 + 0.02 * 2.9 = 0.502 nm2 and for DPPC + 0.5 mol% SP-B by x = 0.995 * 0.453 + 0.005 * 5 = 0.476 nm2. Therefore the ratio of area occupied are (0.98 I 0.453/0.502):(0.02 I 2.9/0.502) = 0.884:0.116 for DPPC + 2 mol% SP-C and (0.995 I 0.453/0.476):(0.005 I 5/ 0.476) = 0.947:0.053 for DPPC + 0.5 mol% SP-B, respectively. When we assume an area of 1 nm2 for DPPC, i.e. 88.4% in the mixture DPPC + SP-C, and the same value of C DPPC as in the mixture we will get an area of 1/0.884 = 1.13 nm2. For the mixture with 0.5 mol% SP-B we will get 1/ 0.947 = 1.056 nm2. Analogously the area per molecule of SPB and SP-C is 8.6 nm2 and 18.7 nm2, respectively. Fig. 14 shows the curves for the mixtures DPPC + 2 mol% SP-C and DPPC + 0.5 mol% SP-B. Curves 1 and 3 are calculated isotherms for these mixtures using Eq. (8). These curves (red) calculated for the additive cases definitely fail to describe the experimental data. It is presumable to suggest that the calculated results are determined not by the additive influence of SP-B and SP-C on the surface pressure of the mixed monolayer, but rather by the influence of the proteins on the state of DPPC, because DPPC occupies about 85 –95% of the surface. This influence, however, can be better discussed on the basis of results calculated by using Eqs. (4) – (8) and treating the mixture as individual systems. Table 4 gives the parameters calculated for the mixtures when taking into account Eqs. (4) – (8). The resulting isotherms for DPPC + 0.5 mol% SP-C and DPPC + 2 mol% a-SP-C (green curves Fig. 14) are in

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58 70

case of h-SP-C corresponds with the higher surface dilatational elasticity, e, determined by oscillation experiments, although the nature of these parameters is different as mentioned above.

60

4

Π [mN/m]

50

1

40

3

15. Influence of B-SP-C on surface behavior of mixed pulmonary layers

2

30

51

20 10 0 0.2

0.4

0.6

0.8

1

1.2

A [nm2/molecule] Fig. 14. Experimental surface pressure/area isotherms of DPPC + 0.5mol% SP-B (N), and DPPC + 2 mol% a-SP-C (g). Curve 1 calculated isotherm for DPPC + 0.5 mol% SP-B using Eq. (9), and curve 2 using Eqs. (4) – (8). Curve 3 calculated isotherm for DPPC + 2 mol% a-SP-C using Eq. (9), and curve 4 using Eqs. (4) – (8). T = 20 -C.

quite good agreement with the experimental isotherms. The presence of the proteins, SP-B and SP-C, in the DPPC layer decreases the cohesion pressure and increases x compared to the data given for DPPC in Table 3. The former corresponds with a decrease of the molecular interaction between the DPPC molecules within the layer in the presence of the protein. x is expected to increase negligibly in the additive case. In reality x is strongly increased. Both can explain this increase, a change of the molecular orientation of DPPC in the monolayer caused by the presence of the protein molecules, or by protein unfolding at the interface when DPPC is present in the layer. The values determined for x in the mixtures are larger than those determined for the individual components, i.e. the interaction between the DPPC molecules and pulmonary surfactant molecules are decreased. The compressibility parameter of the mixtures, g, is also increased in comparison to those determined for the individual components. This effect can be explained in the same way; as for x. g is maximum for the DPPC + SPB + a-SP-C. The smaller compressibility parameter in the

SP-C isolated from bronchoalveolar lavage (BAL) has mainly a-helical conformation [174,175]. As mentioned above transformation of pure SP-C in organic solution from a-helix into h-sheet was reported during the preparation of the protein at high protein concentration [66] and after 1 week incubation at a protein concentration of 4.6 mg/ml [174,176]. We have shown that both conformations of SP-C occur in surface layers [66,167] and influence their surface properties [167,177]. Recent studies demonstrated that amyloid-forming proteins with a-helical structure undergo a-helixYh-sheet transition [178]. Amyloid-like fibrils formed by SP-C were found in lung washings from patients with alveolar proteinosis [174]. This was the motivation to investigate changes of surface properties of model surfactant layers containing DPPC and the hydrophobic surfactant proteins SP-B, and SP-C with a and h conformation. Fig. 15 compares the P/A isotherms of DPPC + 0.25 mol% SP-B + 3 mol% SP-C with SP-C in a- and h conformation, respectively. The isotherms remarkably differ depending on the conformation of SP-C. There is no plateau for the system with SP-C in h conformation at about 51 mN/m, but a kink point instead at 0.45 –0.48 nm2/molecule. Further compression led to a rise up to a film pressure of about 70 mN/m. The isotherm wing for expansion shows that the compression wing is in principal repeated. A remarkable

Table 4 Characteristic values determined by Eqs. (4) – (8) for surface pressure/area isotherms of different mixtures of DPPC and pulmonary surfactant proteins Systems

DPPC + 0.5 mol% SP-B

DPPC + 2 mol% SP-C

DPPC + 0.25 mol% SP-B + 3 mol% a-SP-C

DPPC + 0.25 mol% SP-B + 3 mol% h-SP-C

x, nm2 A c, nm2 P c, mN/m g, m/mN e0 m P coh

0.54 0.75 12.31 0.0008 0.11 1 7.27

0.593 0.865 7.58 0.0015 0.06 1 7.34

0.734 0.973 11.3 0.004 0.07 1 5.62

0.667 0.918 9.5 0.0025 0.08 1 6.61

Fig. 15. Experimental surface pressure/area isotherms of DPPC + 0.25 mol% SP-B + 3 mol% SP-C: (‚) a-SP-C and (N) h-SP-C. Lines were calculated using Eqs. (4) – (8), green: DPPC + 0.25 mol% SP-B + 3 mol% a-SP-C, red: DPPC + 0.25 mol% SP-B + 3 mol% h-SP-C. T = 20 -C. No plateau is observed for the isotherm with h-SP-C in contrast to that with a-SP-C.

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

hysteresis, however, is found. This means that at least a part of collapsed structures had been formed. Taking into account the minimum area demand of a DPPC molecule the layer with h-SP-C is also overcompressed in a range of surface coverage 0.48 –0.27 nm2/molecule, which form of necessarily three-dimensional structures. Actually we have no data to specify which kind of 3D structures is formed by the system DPPC + SP-B + h-SP-C. Nevertheless when this film was repeatedly compressed deeply into this collapse range visible structures were detected by imaging the bubble in the captive bubble surfactometer. Fig. 16 shows a picture of the bubble with such a collapsed film. It can be seen that the contour of the bubble shows some irregularities. Furthermore some crumbs can be seen that are removed from the surface. This example shows the unusual properties of the h-SP-C films that definitely may form collapsed structures. Suggesting by the wing of the P/A isotherm for expansion the formation of these 3D structures is partially reversible, because there is no rapid break down of P. The nature of these 3D structures and their differences compared with those formed by a-SPC is still unknown. Remarkable differences are also revealed at the stress relaxation behavior. The main relaxation times, given in Table 1, exceed those of the same mixture with a-SP-C by more than one order of magnitude. Furthermore there is no complete return of the stress decays to the equilibrium state before transient change of the surface pressure (see Fig. 8). Similar differences were also observed with the surface dilatational characteristics (Fig. 17). The maximum values of elasticity at the surface pressure of the starting squeezeout process were higher in the systems containing SP-C in h conformation in comparison to those containing a-SP-C (Fig. 9). In the frequency range of human breathing (å 0.05 – 0.2 Hz) the surface dilatational viscosities were lower for the system containing SP-C in h conformation, whereas the elasticities were higher.

Fig. 16. 30 Al air bubble in the captive bubble device. At the surface a DPPC + 0.25 mol% SP-B + 3 mol% h-SP-C was spread, which was several times over-compressed. Three-dimensional structures were formed, which causes an irregular bubble contour.

A 250 200

ε d [mN/m]

150 100 50 0 10

-2

10

f [Hz]

-1

0 R1

10

20

40 30

50

Π [mN/m]

B 400 300

ηd 200 [mNs/m] 100 0

50 40 30 20 10

R1

52

10

-2

10

f [Hz]

-1

0

Π [mN/m]

Fig. 17. e d (A) and g d (B) via frequencies f and P for spread layers of DPPC + 0.25 wt.% SP-B + 3 wt.% h-SP-C at 23 -C. The dilatational elasticity becomes maximum at about 40 mN/m. It is slightly higher than that observed for the mixture containing a-SP-C (Fig. 9A). The behavior remains viscoelastic.

These results show that the films containing SP-C as hsheets are less compressible than those found for the mixed films with a-SP-C. Attempts to quantify this behavior show that the assumption of additive contribution of the individual components to the behavior of the layers fails in general for DPPC/surfactant protein mixtures. This is not surprising when considering that the films made from these mixtures contain mainly DPPC. The present mixture with DPPC + SP-B + SP-C = 1:0.0025:0.03 results in a molar ratio of 0.968:0.00242:0.0291. Because the area per molecule of the individual components strongly differ (0.4 nm2 for DPPC, 6 nm2 for SP-B (as dimer), and 2.4 nm2 for SP-C) the area per molecule expressed by the molar ratio becomes x = 0.986 I 0.4 + 0.00242 I 6 + 0.0291 I 2.4 = 0.479 nm2. Therefore the ratio of the individual components expressed by the area per molecule becomes (0.986 I 0.4/479):(0.00242 I 6/ 0.479):(0.0291 I1.5/0.479) = 0.82:0.03:0.15, i.e. 82% of the total area of the mixed layer is occupied by DPPC. Therefore it becomes more plausible to describe the behavior by treating the whole mixture like an individual component and to discuss changes of the parameters based on the influence of the proteins on the behavior of DPPC. The parameters determined by using Eqs. (4) –(8) are given

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

in Table 4 for the mixtures containing a-SP-C and h-SP-C, respectively. The calculated P/A isotherms are shown in Fig. 15 (red and green lines) and are in quite good agreement with the experimental data. As seen by the data given in Table 4 addition of both proteins decreases P coh of DPPC (compare Table 3), which corresponds with a decrease of the lipid interaction thus increasing the surface pressure in general, i.e. for SPB and SP-C, independent on the conformation. Furthermore the presence of the proteins in the layer increases the area per lipid molecule, x, in the LE range. If there would be no interaction between DPPC and SP-C, SP-B x would increase to 0.453 nm2/molecule. In fact, however, we see a much more pronounced increase of x. This could be caused by both changes of DPPC orientation in the presence of the proteins or protein unfolding in the presence of DPPC, as mentioned above for the influence of SP-B or SP-C on DPPC. Therefore also the compressibility coefficient, g, increases for the mixtures. An increase of g could also be explained by increasing the vertical orientation of DPPC within the clusters of the layer, or by protein unfolding, which would increase P. For instance, for the mixture DPPC + SPB + a-SP-C and P = 50 mN/m by using Eqs. (4) – (8) x would increase to 0.466 nm2/molecule when taking into account 82% of DPPC at the interface, instead of 0.734 I 0.87 = 0.64 nm2/molecule in the LE phase. A slightly increase of A c for the mixtures is also caused by some modification of the free energy for cluster formation [172]: DG0 ¼ RT lnðxcl =Ac Þ

ð11Þ

of DPPC by the influence of the proteins, because the increase of the area per molecule in the clusters must be compensated by an increase of A c. When comparing the influence of a-, and h-SP-C (see Table 4) P coh is decreased much stronger by a-SP-C than by h-SP-C, and also the increase of x is much more pronounced for a-SP-C. Therefore the interaction between DPPC molecules is decreased less by h-SP-C. Obviously, the molecular compressibility parameter is less increased by h-SP-C than by a-SP-C, which corresponds well with the higher elasticity of DPPC + SP-B + h-SP-C films found by the direct measurements shown in Fig. 17 in comparison to that of Fig. 9. Therefore the presence of h-SP-C changes the surface behavior of the model pulmonary layer such dramatically that the behavior definitely does not meet the demands of a natural alveolar layer [179], represented by DPPC + SPB + a-SP-C films. The mechanism may be common in the development of several human diseases of different etiology characterized by the extracellular deposition of amyloid [180] and may influence lung diseases such as alveolar proteinosis, acute respiratory distress syndrome, and lung fibrosis.

53

16. An example of surface behavior of lung washings One of the main directions for the investigations in the field of pulmonary surfactants has always been to get information about differences in the composition of pulmonary surfactant with regard to different lung diseases and to develop methods of clinical treatment [181 – 188]. Different trials have been attempted, most of all using the classical approaches and so-called cycling experiments [38,45,189 – 191]. The direct connection of experimental data to a pathological incident, however, is complicated, because of different manifestations of diseases. One example is shown demonstrating results obtained from porcine lung washings. The animals were anaesthetized and intubated with an endotracheal tube for mechanical ventilation. Unilateral lung contusion was induced on the right thorax using a standardized technique [192]. Unilateral lung contusion resulted in epithelial and endothelial barrier damage that increased permeability, leading to pulmonary edema: plasma components entered the alveoli and inhibited the function of pulmonary surfactant. Four hours after contusion the damaged lung was treated with a porcine natural surfactant preparation (Curosurf\, Serono, Switzerland). Curosurf is an organic solvent extract of porcine lung washings consisting of 99% polar lipids (mainly phospholipids) and 1% hydrophobic proteins, SPB and SP-C. For treatment 5 ml/kg Curosurf (80 mg phospholipids per ml) was instilled into the right, injured lung under visual control using a bronchoscope. Then bronchoalveolar lavages (BAL) were carried out through a bronchoscope that was placed through the endotracheal tube into selected lung areas. Ringer’s solution (8.6 g/l NaCl, 0.3 g/l KCl, 0.33 g/l CaCl2*2H2O) was instilled (7 10 ml) and gently withdrawn under negative pressure. Collected fluids were cooled on ice, filtered, and centrifuged to remove cells and debris (200 g, 10 min). The lavage procedure was performed in the left (normal) and the right (injured) lung. 4 h after the treatment the right lung was lavaged again. For adsorption measurements BAL samples were used without any further dilution or treatment. The total phospholipid concentrations of the BAL samples were 180 – 300 Ag/ml, which guaranteed saturation of the interface in any case. The pulmonary surfactant obtained from normal lungs adsorbs very fast and forms saturated interfacial layers with an equilibrium surface pressure of about 45 mN/m at 37 -C (Fig. 18). The extension of the plateau region is concentration dependent. As the exact surface concentration is unknown for the adsorption layer one can only state that the surface is saturated and the equilibrium surface pressure has been established. It is unknown, however, if the bulk concentration is high enough to reach extremely high surface pressures. Compression and expansion of these layers only slightly change the surface pressure because all surface deformation is equilibrated by fast re-entering or respreading of surface active material (expansion) or remov-

54

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58 70 60

γ [mN/m]

50 40 30 20 10 0 0

200

400

600

800

1000

1200

time [s] Fig. 18. Surface tension, c, vs. adsorption time for 3 different BAL, uninjured ( ), injured (0), and after Curosurf treatment (o). The surface tension immediately decreases during adsorption of BAL for the uninjured lung and remains constant. It decreases slowly for the BAL of the injured lung with remarkable data scattering. The Curosurf treatment accelerates the adsorption remarkably, but for small adsorption time still differences remain.

ing the material into a reservoir associated with the surface (compression). Because P only slightly changes during cycling in a wide range of surface coverage the surface films are highly compressible. The surface-active material in BAL from normal lungs adsorbed very fast thus depressing the surface tension strongly in a time scale < 1 s. After å 100 s the surface tension remained constant with a value that

differed from the initial one by only 1 –2 mN/m. In contrast the surface tension of the lavages from injured lung decreased slowly reaching a steady state only after 1100 s. Furthermore, there was some data scattering, which was never observed with BAL from normal lungs. In BAL samples obtained after Curosurf\ treatment the data scattering disappeared. However, the time to reach steady state remained long compared to BAL from normal lungs (120 s vs. 1 s). The equilibrium adsorption surface tension for all three BAL types was very similar and about 25– 27 mN/m, which corresponds to a surface pressure of 43– 45 mN/m at 37 -C. In Fig. 19 the P/A isotherms for all three native BAL types are shown after adsorption was completed. These curves were recorded by 10 successive cycling experiments, i.e. repeated linear compression and expansion. The adsorbed film from BAL samples of normal lungs had a steady state surface pressure of about 47 mN/m. There were practically no changes of surface pressure when such a film was compressed and expanded in the range 100– 50% of a relative area and by using a compression rate of 0.38 cm2/ min. In contrast, hysteresis occurred between the compression and expansion P/A isotherm when the adsorbed film was formed out of BAL from injured or treated lungs. Furthermore, there was remarkable data scattering of the isotherm from BAL of injured lungs, as was observed in the

A 60

Π [mN/m]

50 40 30 20 10 0 40

50

60

70

80

90

100

rel. area [%]

B

60

Π [mN/m]

50 40 30 20 10 0 40

50

60

70

80

90

100

rel. area [%] Fig. 19. P/A isotherms of adsorbed porcine BAL layers. Experimental results obtained from 10 compression and expansion cycles. The layer of the uninjured lungs (A, ) shows no hysteresis. The data for compression and expansion coincides. There is some data scattering in the case of the injured lung (B, r), as well as a pronounced hysteresis. After Curosurf treatment (o) the data scattering disappeared, the hysteresis however still remains.

.

R. Wu¨stneck et al. / Advances in Colloid and Interface Science 117 (2005) 33 – 58

adsorption measurements with this type of BAL. After treatment with Curosurf the hysteresis decreased but did not disappeared completely. In addition, data scattering was less obvious after treatment. Hysteresis implies non-reversible behavior and is never observed with lung during quiet tidal breathing [54]. In the case of a hysteresis the interfacial work of breathing is increased. These examples show that there are remarkable deviations between the normal lung lavage behavior and those after lung injury and after lung treatment of the injured lung. The results show that differences between the different samples can be established even when the initial bulk concentration is not exactly known, and that careful analysis of the surface behavior can provide important information to understand the nature of pathological alterations from a functional point of view. It was not the aim of these experiments to optimize treatment procedure, although an evaluation of such treatment could be possible.

17. Perspectives Pulmonary surfactant as a therapeutic option in clinical practice has been proven beneficial for the treatment of premature infants. For other lung diseases such option is under investigation. The use of exogenous surfactant preparation for acute lung injury in adults revealed conflicting results. The reason for these conflicting results is that the underlining mechanisms of acute lung injury is complex and could not be solved by one ‘‘magic’’ drug. However, new insights into the basic surface function of pulmonary surfactant will provide new strategies for the development of new concepts and probably new surfactant preparations with different compositional ingredients that might help to treat other patient populations than premature infants. There is no doubt that SP-B is crucial for pulmonary surfactant in vivo. Data of SP-B function from carefully done surface science experiments will help in the future to more consistently use or modify SP-B for treatment of pulmonary diseases. Total and partial liquid ventilation are liquid assisted ventilation techniques [193,194], which were not successful for the treatment of lung injury. A new technique, based on the concepts of liquid ventilation, is the perfluorocarbon vaporization in analogy to the application of inhalation anesthetic agents [195]. To our knowledge no systematic investigation has been reported concerning the surface behavior of model systems containing perfluorocarbons and a pulmonary surfactant layer up to now. Perfluorocarbons are supposed to displace at least partially the pulmonary surfactant layer, because they have a higher density than water and are known to interact weakly with hydrocarbons [8]. On the other hand an interaction between the fluorocarbon and pulmonary surfactant components were assumed, because of the low surface tension and the

55

improved compliance of the lung, which was reached by perfluorocarbons [193,195– 197]. These questions could be investigated by using the pendant drop technique where a fluorocarbon drop is placed into a chamber that contains a pulmonary surfactant solution. The captive bubble device allows the investigation of a pulmonary surfactant layer at the liquid – gas interface by the application of fluorocarbon from the gas phase. Such investigations would help to clarify the influence of perfluorocarbons on the interfacial behavior of a pulmonary surfactant layer. The influence of SP-C conformation on the interfacial behavior is important, because of the relevance of such structural changes for many other diseases that involve the formation of amyloid structures. Amyloid structure forming proteins other than SP-C should be involved in further investigations. Even when these proteins have no direct surface activity and are not functioning in vivo at interfaces, investigation of their adsorption characteristics to interfaces is useful, since it offers a fast and easy way to determine structure function correlation of a-helical and h-sheet configured proteins. Another promising approach is the investigation of the interaction of pulmonary surfactant layers with small inhaled particles [198,199]. Such investigations could help in better understanding common lung disease like asthma and chronic obstructive pulmonary disease (COPD). In addition, understanding the interaction of small particles with the pulmonary film and the tracheal surface will be useful to find new kind of treatments including nanotechnology derived drug carriers [200,201].

Acknowledgements The financial support by the Deutsche Forschungsgemeinschaft (Grant Pi 165/10-1/2, Pi 165/12-1) and Spanish Ministry of Science (BIO2003-0956) and Community of Madrid are gratefully acknowledged. The EU partially supported these studies (EFRE 2000-2006 2u¨/1, ‘‘Nano-Carrier’’).

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