Langmuir 2006, 22, 3243-3250
3243
Lung-Surfactant-Meconium Interaction: In Vitro Study in Bulk and at the Air-Solution Interface T. Gross,† E. Zmora,‡ Y. Levi-Kalisman,§ O. Regev,*,§,⊥,| and A. Berman*,†,§,| Department of Biotechnology Engineering, Neonatal IntensiVe Care Unit, Soroka Medical Center and Department of Biomedical Engineering, Department of Chemical Engineering, The Ilse Katz Center for Meso- and Nanoscale Science and Technology, and the R. Stadler MinerVa Center for Mesoscale Macromolecular Engineering, Ben-Gurion UniVersity, Beer-SheVa, 84105, Israel ReceiVed August 3, 2005. In Final Form: NoVember 29, 2005 Lung surfactants (LSs) form a monolayer at the lung’s alveoli air-solution interface and play a crucial role in making normal breathing possible by reducing the surface tension. LS are affected by various agents that hamper their normal functioning. Tobacco smoke [Bringezu, F.; Pinkerton, K. E.; Zasadzinski, J. A. Langmuir 2003, 19, 29002907] and meconium, the first excrement of the newborn, are examples for such LS poison. In neonates, intrauterine aspiration of meconium is a known cause for morbidity and mortality. We studied in vitro the interactions between modified porcine LSs (Curosurf), used as LS replacement, and meconium, as well as between their artificial analogues, phospholipids mixture, and taurocholic acid (TA), respectively. The interactions were examined both in the bulk solution and at the air-water interface, representing the pre- and postnatal situations. It was found that the artificial analogues represent the natural system reliably and exhibit similar effects. TA, a principle component of bile, is an amphiphilic sterol compound in which the hydrophilic and hydrophobic moieties are presented at different faces of the sterol plane. Here we found that TA affects the structure of both monolayers at the interface and surfactant aggregates in solution. A likely poisoning mechanism is by stereoselective penetration of TA into the lamellar or monolayer structures, thus disrupting the contiguous structure of the intact monolayer or the bilayer vesicle structure.
Introduction Gas exchange in the lungs takes place by diffusion through a thin water layer that coats the alveoli surface. The air-water interface in the lungs is covered by a monolayer of lung surfactant (LS) that is composed of phospholipids and hydrophobic proteins that reduce the surface tension. The dynamic performance of the LS monolayer is essential for its operation. As air is cyclically inhaled and exhaled, the alveoli surface area varies correspondingly. To maintain the alveoli surface permanently wetted, it is imperative that the lung surfactant monolayer be continuous, would not be dewetted, and as little as possible LS material would be lost into the aqueous phase during the breathing cycle due to collapse of the monolayer. Several agents are known to poison the lung surfactant monolayer, thus obstructing its vital role. This situation is particularly abundant in newborn infants, where aspiration of meconium (the first excrement of the newborn)-contaminated amniotic fluid is a common pathological condition in case of fetal stress. LS is a complex mixture of approximately 90 wt % lipids, mainly phospholipids, and 10 wt % proteins. The phospholipids comprise approximately 70% phosphatidylcholine (mainly the disaturated dipalmitoylphosphatidylcholine (DPPC)), approximately 10% phosphatidylglycerol (mainly dipalmitoylphosphatidylglycerol (DPPG)), and trace amounts of other phospho* Corresponding authors. † Department of Biotechnology Engineering. ‡ Neonatal Intensive Care Unit, Soroka Medical Center, and Department of Biomedical Engineering. § The Ilse Katz Center for Meso- and Nanoscale Science and Technology. ⊥ Department of Chemical Engineering. | R. Stadler Minerva Center for Mesoscale Macromolecular Engineering. (1) Bringezu, F.; Pinkerton, K. E.; Zasadzinski, J. A. Langmuir 2003, 19, 2900-2907.
lipids.2-4 While the phosphatidylcholine fraction is mostly saturated, the phosphatidylglycerol lipid fraction usually contains mono-unsaturated acyl chains. In contrast to LS composition in humans, mammalian laboratory animals LSs feature a significant fraction (up to 30%) of unsaturated acyl chains.5 The LS protein fraction includes four surfactant proteins denoted as SP-A, SPB, SP-C, and SP-D. The main function of the hydrophobic SP-B and SP-C proteins is to promote adsorption of lipids to the airwater interface, while SP-A is associated with the formation of tubular myelin (TM), a special extracellular supramolecular lipid structure which serves as a reservoir for LS spreading from the bulk. Both SP-A and SP-D are hydrophilic proteins that are related to the regulation of the immune defense of the lungs.3,6,7 LS produced in type II pneumocyte cells is organized in multilamellar aggregates, lamellar bodies (LB), and is then secreted into the alveolar space, where it undergoes structural reorganization into TM before it spreads and covers the airliquid interface of the alveoli as a thin film.8 LS can be inhibited in vivo by a variety of substances such as blood, plasma, serum proteins,9-11 tobbaco smoke,1 lipids,12 (2) Morgenroth, K.; Newhouse, M. The Surfactant System of the Lungs: Morphology and Clinical Significance; De Gruyer: Berlin, New York, 1988. (3) Goerke, J. Biochim. Biophys. Acta 1998, 1408, 79-89. (4) Krol, S.; Ross, M.; Sieber, M.; Kunneke, S.; Galla, H. J.; Janshoff, A. Biophys. J. 2000, 79, 904-918. (5) Postle, A. D.; Heeley, E. L.; Wilton, D. C. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2001, 129, 65-73. (6) Perez-Gil, J.; Keough, K. M. W. Biochim. Biophys. Acta 1998, 1408, 203217. (7) Vaandrager, A. B.; van Golde, L. M. Biol. Neonate 2000, 77, 9-13. (8) McCabe, A. J.; Wilcox, D. T.; Holm, B. A.; Glick, P. L. J. Pediatr. Surg. 2000, 35, 1687-1700. (9) Warriner, H. E.; Ding, J.; Waring, A. J.; Zasadzinski, J. A. Biophys. J. 2002, 82, 835-842. (10) Zasadzinski, J. A.; Alig, T. F.; Alonso, C.; de la Serna, J. B.; Perez-Gil, J.; Taeusch, H. W. Biophys. J. 2005, 89, 1621-1629. (11) Taeusch, H. W.; de la Serna, J. B.; Perez-Gil, J.; Alonso, C.; Zasadzinski, J. A. Biophys. J. 2005, 89, 1769-1779.
10.1021/la0521241 CCC: $33.50 © 2006 American Chemical Society Published on Web 03/07/2006
3244 Langmuir, Vol. 22, No. 7, 2006
antibodies, and oxidants, etc. Meconium is also an effective LS poison in the case in which it is aspirated into the lungs around birth. Meconium aspiration syndrome (MAS) occurs mainly in term and postterm newborn infants. As a result of intrauterine asphyxia, the fetus may excrete meconium to the amniotic fluid. In some of these events, deep fetal breathing movements follow, which may result in meconium aspiration into the bronchial tree. A recently published paper suggests that damage to the LS can occur even before birth when the LS is in the bulk phase.13 The clinical effects of MAS can be obstruction of airways at severe cases, chemical pneumonitis, and inhibition of LS both structurally and by reducing its capabilities of lowering surface tension.14 According to Laplace law, ∆P ) 4γ/r, the pressure difference between the alveoli air space and the alveolar liquid lining (∆P) depends on the surface tension (γ) and the radius of the alveolus (r). Hence, reduction of the alveolar radius during expiration increases the pressure difference and can cause collapse of the alveolus. Therefore, the major role of the LS film at the airliquid interface of the alveoli is to reduce the surface tension and thus to decrease the tendency for alveolar collapse at the end of the expiration.8 Lipid monolayers undergo several phase transitions during surface compression: from a “gas” phase to a “liquid-expanded” (LE), “liquid-condense” (LC), and “solid” (S) phases. These are analogous to 3-D phases. At the gas phase, molecules are diluted and only rarely collide. At the LE phase, hydrophobic tails of the LS come into occasional contact with each other but are orientationally uncorrelated. The surface pressure of this phase is measurable. In the LC phase the hydrophilic headgroups of the LS are closer and the tails are fluid, yet form a continuous layer and orient roughly perpendicular to the interface. The solid phase is characterized by denser packing of the aliphatic tails with limited motion.15,16 Upon surface pressure increase, the monolayer collapses in either of three collapsed forms: fracture, buckling, or material loss into the subphase. The nature of the collapse depends on the elasticity and solubility properties of the monolayer.15,16 Lung surfactant monolayers in vivo experience surface pressures between 40 and 70 mN/m during the inhalationexpiration cycle and undergo SP-A, -B, and -C mediated organized transformation into surface associated three-dimensional lipid constructs while maintaining the LE-LC coexistence.17,18 As the expiration progresses and the surface area is reduced, gradual refinement process takes place where the unsaturated lipids are phase-separated and subsequently extruded from the monolayer. At the peak of expiration the intact monolayer is composed almost entirely of DPPC, which withstands high surface pressure.19 The condensed 2-D phases, (LC, S) can be imaged with a Brewster angle microscope (BAM). In BAM, p-polarized laser (12) Simberg, D.; Weisman, S.; Talmon, Y.; Faerman, A.; Shoshani, T.; Barenholz, Y. J. Biol. Chem. 2003, 278, 39858-39865. (13) Fraser, W. D.; Hofmeyr, J.; Lede, R.; Faron, G.; Alexander, S.; Goffinet, F.; Ohlsson, A.; Goulet, C.; Turcot-Lemay, L.; Prendiville, W.; Marcoux, S.; Laperriere, L.; Roy, C.; Petrou, S.; Xu, H.-R.; Wei, B.; the Amnioinfusion Trial Group N. Engl. J. Med. 2005, 353, 909-917. (14) Woods, R.; Glantz, J. C. In Maternal Fetal Medicine, Principle and Practice; 3rd ed.; Creasy, R. K., Resnik, R., Eds.; W. B. Saunders & Co.: Philadelphia, 1994; pp 413-414. (15) Lipp, M. M.; Lee, K. Y. C.; Takamoto, D. Y.; Zasadzinski, J. A.; Waring, A. J. Phys. ReV. Lett. 1998, 81, 1650-1653. (16) Zasadzinski, J. A.; Ding, J.; Warriner, H. E.; Bringezu, F.; Waring, A. J. Curr. Opin. Colloid Interface Sci. 2001, 6, 506-513. (17) Piknova, B.; Schief, W. R.; Vogel, V.; Discher, B. M.; Hall, S. B. Biophys. J. 2001, 81, 2172-2180. (18) Diemel, R. V.; Snel, M. M. E.; Waring, A. J.; Walther, F. J.; van Golde, L. M. G.; Putz, G.; Haagsman, H. P.; Batenburg, J. J. J. Biol. Chem. 2002, 277, 21179-21188. (19) Alonso, C.; Alig, T.; Yoon, J.; Bringezu, F.; Warriner, H.; Zasadzinski, J. A. Biophys. J. 2004, 87, 4188-4202.
Gross et al.
beam is incident at the liquid surface at an angle (53° from the normal for water) at which total refraction takes place. The presence of condensed monolayer changes the refractive index; thus, the Brewster condition no longer holds, and reflection from the condensed domains takes place. In contrast, the LE and 2-D gas phases are sparse and therefore appear darker in BAM images because of the similarity between their effective refractive indices to that of exposed water. The LE phase has a low contrast, and it can hardly be imaged due to surface flows. The LC/LE phase ratio depends on the surface pressure, average molecular area, temperature, and the ionic species present in the subphase.4,15,16,20,21 The in-vitro effect of meconium on the physical properties and morphology of LS have been studied.22-26 It has been shown that the presence of meconium can significantly interfere with the surface properties by mixing with LS, thus causing transient increase in surface tension, the viscosity of LS, and the surface spreading rate (SSR), which expresses the time required to reach the equilibrium surface tension. The presence of meconium decreases the adsorption rate of LS from solution and changes its morphology.22,23 It has also been shown that the effects of meconium on surfactant depend on the LS concentration.24-26 In addition, both the hydrophobic and the hydrophilic fractions of meconium have inhibitory effects on LS.24,26 It has been speculated that meconium may inhibit LS activity by competing with surfactant molecules for area at the air-liquid interface during adsorption or intercalation into the LS film during dynamic compression.9,24 Similar inactivation mechanism by serum proteins probably originate from electrostatic and/or steric barriers, as was recently suggested by Zasadzinski.10 In this study we examined the supramolecular changes in LS after exposure to meconium, in two in-vitro environments: in bulk and at the air-liquid interface, representing pre- and postnatal situations, respectively. The bulk mimics the alveoli filled with lung fluid before birth. The second setup, at the air-liquid interface, represents the postnatal situation, where the alveoli are inflated with air. In each setup the surfactant-meconium interactions were investigated in two in-vitro systems, termed “artificial” and “natural”, that mimic the in-vivo conditions. The rationale for studying artificial model systems originates from the complexity of the natural living biological system. The latter consists of a complex mixture of lipids and proteins on the LS part, and a mixture of bile salts, lipids, proteins, cell debris, and other excretions in the meconium counterpart. To simplify the study of the interactions between two such complicated mixtures, we devised an “artificial” setup in which only the primary components of each system participate. Thus, in the artificial system the LS is represented by DPPC or a 4:1DPPC/DPPG mixture27 and the meconium is embodied by taurocholic acid (TA), a principal component of bile and meconium. The rationale for the choice of fully saturated lipids for the artificial setup stemmed from the fact that DPPC monolayers represent the peak of expiration in LS. At this situation the monolayer is in its most condensed state but, due to the high surface pressure, is also the (20) Zhao, J.; Vollhardt, D.; Brezesinski, G.; Siegel, S.; Wu, J.; Li, J. B.; Miller, R. Colloids Surf., A 2000, 171, 175-184. (21) Vollhardt, D.; Fainerman, V. B.; Siegel, S. J. Phys. Chem. B 2000, 104, 4115-4121. (22) Park, K. H.; Bea, C. W.; Chung, S. J. J. Korean Med. Sci 1996, 11, 429-436. (23) Bea, C. W.; Takahashi, A.; Chida, S.; Sasaki, M. Pediatr. Res 1998, 44, 187-191. (24) Moses, D.; Holm, B. A.; Spitale, P.; Liu, M.; Enhorning, G. Am. J. Obstet. Gynecol. 1991, 164, 477-481. (25) Oh, M. H.; Bea; C. W. Eur. J. Pediatr. 2000, 159, 770-774. (26) Sun, B.; Curstedt, T.; Song, G.-W.; Robertson, B. Biol. Neonate 1993, 63, 96-104. (27) Bourdos, N.; Kollmer, F.; Benninghoven, A.; Ross, M.; Sieber, M.; Galla, H. J. Biophys. J 2000, 79, 357-369.
Lung-Surfactant-Meconium Interaction
Langmuir, Vol. 22, No. 7, 2006 3245
most sensitive for various perturbations. We chose to examine the effect of TA on a monolayer of condensed phase saturated lipids in order to emulate the situation of LS at its extreme state. Furthermore, the temperature (25 °C) of the artificial setup was chosen to be well below the gel transition temperature at moderate surface pressure in order to certify the LC phase predominates. The natural setup was examined at 37 °C in order to provide LS proteins their native ambience. The selected temperatures for the artificial and natural setups were intended to direct the investigated systems into the desired state of mostly LC phase. Moreover, it has been shown that the monolayer and phase behavior of a similar model system, DPPC/POPG (POPG: Palmitoyloleylphosphatidylcholine) isotherm, is independent of temperature over the 25-37 °C range.9,11 The effects of meconium or TA on the surfactant aggregation forms in the bulk were examined by cryogenic transmission electron microscope (cryo-TEM) imaging. The experiments at the air-liquid interface were performed by compressing the surfactant on a Langmuir trough (LT) and simultaneously imaging it using BAM. Materials and Methods Materials. First-pass meconium collected from healthy term human neonates was diluted in distilled water (Direct-Q, Millipore S.A., France), homogenized (TPC-25, Telsonic Ultrasonic, Switzerland), centrifuged in 4500 rpm for 30 min (Labofuge-400R, Heraeus Instrument, Germany) and then lyophilized (Beta 1-8, CHRIST Ltd., Switzerland), and kept at 4 °C. Before the experiments the meconium extract was resuspended to the desirable concentration. Modified porcine lung surfactant (Curosurf, Chiesi Pharmaceutici, Parma, Italy) was donated by Teva Medical, Israel. Information on Curosurf composition is from the manufacturer. Accordingly, the total lipid content is 99% and 1% hydrophobic proteins (SP-B, SPC).26 For the interface experiments the lung surfactant was dried and resuspended in chloroform/methanol (1:1) to make 2.78 mg/mL spreading solution concentration (approximately 3.75 mM). Chloroform and methanol were purchased from Frutarom, Israel. In the bulk experiments Curosurf was used as received. Dipalmitoyl l-R-phosphatidylcholine (DPPC), dipalmitoyl l-Rphosphatidyl-DL-glycerol, (DPPG), taurocholic acid (TA) (sodium salt), sodium chloride, Trizma base, and calcium chloride were purchased from Sigma Chemical Co. (St Louis, MO). Experimental Methods. Interface Methods. Compression isotherms were performed on a Langmuir film balance (Nima Technology, Model 611), using filter paper as a Wilhelmy plate. Monolayers were prepared by slowly applying 20 to 50 µL of LS spreading solution or 2 mM lipid mixture, spreading solutions in chloroform/methanol on the subphase interface, at 37 °C for Curosurf and 25 °C for the the DPPC/DPPG system. After 15 min for equilibration, the monolayers were compressed at a rate of 15 cm2/ min. The area per molecule a, was calculated according to a)
AM cNaV
(1)
A, surface area; c, mass concentration of the spreading solution; M, molecular weight; Na, Avogadro number; V, volume of the injected spreading solution. The phase transitions in the monolayers were visualized with a Brewster angle microscope (BAM2+, Nanofilm Technology Gmbh, Go¨ttigen, Germany) coupled to the Langmuir film balance. Experiments in which pure saline subphase was replaced by meconium solution were performed by simultaneously pumping in and out solutions with a two-channel peristaltic pump. One trough volume of target subphase solution (e.g. 0.5 mg/mL meconium) was slowly (15 mL/min) pumped in while the original subphase was pumped out at the same rate, to keep the water level unchanged.
Three compression-expansion cycles were performed on water before the onset of solution exchange. Four cycles took place during subphase exchange, and finally seven cycles on the newly replaced meconium subphase solution after the exchange process was terminated. Each cycle was run to 80% of compression area before collapse. Bulk Methods. Dilute solutions of LS and the lipids were used for cryo-TEM. Curosurf was used as received. A 4:1 DPPC/DPPG mixture was dissolved in water to the desired concentration and observed without further steps. The surfactant mixtures were reacted with the respective “contaminants”, TA or meconium extract, and were visualized employing cryo-TEM technique. This observation technique preserves the structure by rapid vitrification of the samples in their original aqueous solution. Samples were prepared in a controlled environment vitrification system (CEVS).28 In this method, a 3 µL droplet of the solution is deposited on a TEM grid, coated with a holey carbon film (Lacey substrate, Ted Pella, Ltd.). The grid is blotted to remove excess fluid, resulting in a thin film (20-300 nm) of the solution suspended over the grid’s holes. A temperature of 37 °C and 98-100% humidity were kept constant during the sample preparation. Vitrification was done by rapid plunging of the sample into liquid ethane at its freezing point. The vitrified specimens were transferred under liquid nitrogen to transmission electron microscope (JEOL 1200 EXII TEM) equipped with a cold stage (model 626, Gatan Inc., Pleasanton, CA) and a CCD camera (Gatan, Bioscan). Minimal dose techniques (using the microscope MDS software) were applied to ensure minimal beam damage. Turbidity of bulk samples was measured on a HP 8452A diode array UV-vis spectrophotometer, using 1 cm optical path length quartz cuvettes.
Results and Discussion The experimental setups used in this study were based on comparisons between surfactant-contaminant pairs in the artificial and natural systems. The artificial system includes DPPC and DPPG in 4:1 molar ratio to mimic the lung surfactant and TA, a major component of bile in meconium,29 to mimic meconium. The natural system includes components from natural sources: Curosurf and meconium. Each system was investigated both at the interface and in the bulk. Characterization of Monolayers at the Air-Liquid Interface: Mimicry of Postnatal Situation. (1) “Artificial” System. The compression isotherms of DPPC/ DPPG (4:1) on saline (0.9% NaCl) in the presence or absence of 10-5 M TA solution are shown in Figure 1, together with respective BAM images. The 2-D, gas-LE phase transition of DPPC/DPPG (4:1) is clearly depicted in the isotherm on saline (solid line). The pressure offset indicates the transition is at 116 Å2/molecule. Further compression leads to nucleation of LC islands in equilibrium with the LE phase. This takes place above the surface pressure of π ∼ 10 mN/m and is manifested in a bend in the isotherm followed by a small plateau region. BAM images (Figure 1 A-D) indicate that LC islands, which are ∼10 µm in diameter, are being compressed but do not completely merge: even at the high surface pressure, isolated patches of darker, LE regions are still clearly observed. The monolayer’s collapse pressure is ∼58 mN/m, and the limiting area is 60 Å2/molecule. The isotherm of DPPC/DPPG (4:1) on the TA solution (Figure 1, dashed line) is different from the one on the saline solution. The pressure offset on the TA solution is greater than 240 Å2/ molecule, suggesting that a considerable amount of subphase material penetrated into the film. The LE-LC phase transition is less sharp compared to the isotherm in the presence of TA. The transition cannot be discerned on the compression isotherm (28) Talmon, Y. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 364-372. (29) Murai, T.; Mahara, R.; Kurosawa, T.; Kimura, A.; Tohma, M. J. Chromatogr., B: Biomed. Sci. Appl. 1997, 691, 13-22.
3246 Langmuir, Vol. 22, No. 7, 2006
Gross et al.
Figure 1. BAM images and the corresponding compression isotherms (bottom right) of DPPC/DPPG (4:1) on saline (A-D, solid line) and on TA (E-G, dashed line). Letters on the compression isotherms indicate the positions where the corresponding images were taken from. Note that the diagonal pattern is interference from the BAM optics that cannot be removed. BAM images are 450 µm on a side.
Figure 2. BAM images and the corresponding compression isotherms of Curosurf on water (A-C, solid line) and on meconium (0.25 mg/mL) containing the subphase (D-F, dashed line). Meconium subphase alone also forms a compressible surface layer that was imaged with BAM (G, dotted line). The compression isotherms are depicted in the lower left panel. The letters A-G indicate the position on the isotherms at which the BAM image was acquired. BAM images are 450 µm on a side.
but takes place around point E, where LC domains appear. BAM images taken in the presence of TA (Figure 1E-G) indicate that the LC domains become progressively more abundant until their complete merge at 30 mN/m. (2) “Natural” System. The natural model includes Curosurf monolayer and meconium solubilized in the subphase. Figure 2 depicts isotherms of Curosurf on water and on meconium solutions with their corresponding BAM images, as well as a compression isotherm of meconium alone. The isotherm of Curosurf on water (Figure 2, solid line) is smooth without any apparent features. The pressure offset is at 120 Å2/molecule, and the collapse pressure is ∼43 mN/m. The transition from LE to LC/LE coexistence is visualized by BAM images (Figure 2A-C) and is taking place at a surface pressure below 10 mN/m. However, the onset of LC domains is not accompanied by any change in the π-A isotherm. The discrete form of the LC islands is retained during the whole compression course: the domains,
having circular shape, are uniform, ca. 10 µm in diameter and do not coalesce even at high surface pressure or after the collapse point. This is in contrast to the behavior of the lipid monolayer in the artificial system (Figure 1C and D), where partial coalescence is observed. The differences between the two systems may stem not only from the different lipid composition but also from the presence of surfactant proteins in Curosurf and the different temperature regime used for each system, as explained before. The π-A isotherms of Curosurf on saline solution (not shown) are similar to those on water. Small differences are observed in the area per molecule (this value is slightly higher for saline). This can be attributed to attraction forces between zwitterionic choline moieties and Na+ and Cl- ions, thus forming an effective larger headgroup. A comparison of the compression isotherms of Curosurf and of DPPC/DPPG (4:1) with and without the inhibiting agents,
Lung-Surfactant-Meconium Interaction
Langmuir, Vol. 22, No. 7, 2006 3247
Table 1. Summary of Isotherms Parameters of Synthetic and Natural Source Systems Used for Comparison between Pure and Poisoned Systems DPPC/DPPG (4:1) 2/molecule)
pressure offset (Å surface pressure of nucleation of LC islands (mN/m) diameter of LC islands (µm) and their shape surface pressure of a uniform monolayer (mN/m) surface pressure of collapse (mN/m) limiting area per molecule (Å2/molecule) n, replicates
Curosurf
on saline
on TA
on water
on meconium
108 ( 2.12 ∼10 10 (round) >30 57 ( 1.41 61.5 ( 2.12 2
na 13 5-10 (round) 30 52 ( 1.73 85 ( 0.58 3
110 ( 17.7 10 10 (round) na 43 ( 1.16 86 ( 12.3 8
na 0 20-50 (“flowers”) 38 29 ( 0.82 170 ( 8.2 4
meconium and TA, respectively, points at some differences. These originate from the different molecular composition of the film and the contaminants. Table 1 presents the parameters characterizing the isotherms. Curosurf monolayer includes amphiphilic proteins (SP-B, SP-C) and several other phospholipids that are absent in the DPPC/DPPG (4:1) monolayer. The isotherm of Curosurf is smooth without any plateau on it, whereas in that of the DPPC/DPPG (4:1) monolayer a plateau is evident. This plateau is possibly associated with enthalpy change of a first-order transition.30 We speculate that this difference arises from the simplicity of the binaric DPPC-DPPG system compared to the multicomponent Curosurf, where intricate phase transitions are superimposed. Another difference can be visualized with BAM images. At the end of the Curosurf compression, the monolayer still consists of individual LC islands (Figure 2C), similarly to domains in the DPPC/DPPG (4:1) monolayer, where individual domains are still visible (Figure 1D). The fast exchange between the two phases is essential for normal breathing as it allows effective response of the LS coverage to the constantly changing alveoli surface area.17 (Only at the expiration end, the monolayer is almost pure LC phase, made primarily of the DPPC fraction.) For the Curosurf, this is likely to take place through a fast LCLE phase transition across the long contact line between these phases (vide infra). Meconium alone, in concentration of 0.25 mg/ml in the subphase, is amphiphilic and forms a surface layer. This is observed by BAM as a perforated continuous layer throughout the compression isotherm (for example, Figure 2G). Curosurf monolayer on subphase containing meconium appears as 20-50 µm wide flower-shaped aggregates at a low surface compression with considerable surface activity already at a high molecular area (Figure 2D-F). The limiting area per molecule is ∼170 Å2/molecule (dotted line in Figure 2; it is difficult to estimate the exact value due to the moderate slope of the isotherm, Figure 2, dashed isotherm). The high value of the limiting area per molecule implies that substances originating from the meconium solution penetrate from the bulk to the interface. The increased size of the LC domains in the presence of meconium and their eventual partial merging result in reduction in the LCLE contact line (Figure 2F). Hence, equilibration of the phases is expected to be slower. Further compression of the monolayer leads to a denser monolayer that collapses at relatively low surface pressure, 30 mN/m. This behavior is qualitatively similar to that of the DPPC/DPPG (4:1) isotherm on TA solution (Figure 1G), as shown and discussed above. The measured variables of the compression isotherms and BAM images of the LS/meconium (natural) and lipids/TA (artificial) experimental systems are summarized in Table 1, where comparisons are made between the pure and poisoned systems, but not between the Curosurf and lipid mixture systems.
DPPC/DPPG monolayers, prepared on meconium containing subphase (0.25 mg/mL), exhibited compression isotherms and BAM appearance that did not differ from the behavior of meconium alone upon surface compression (results not shown). Hence it can be concluded that the meconium dominated the surface when artificial lipids are spread at the interface. Compression of Curosurf on the subphase containing a higher concentration of meconium (0.5 mg/mL) also shows behavior similar to the meconium alone (not shown). We therefore conclude that the inhibiting effect of meconium on Curosurf monolayer takes place at twice as high concentration as that needed for a similar effect in the artificial lipid system, suggesting better stability of the natural LS product against higher meconium concentrations. The inhibiting effects of meconium on lung surfactant surface tension reduction and on monolayer elasticity have been shown before.31 Two possible mechanisms were suggested.31 The first, lipids from meconium origin, compete with LS for area at the interface. These lipids form a less stable monolayer that collapses at low surface pressure. Second, meconium induces the aggregation of LS (lipids and proteins) into clusters; thus, the formation of a stable and functioning monolayer at the interface is inhibited. Compression-expansion cycles of Curosurf at the air-water interface are shown in Figure 3. This experiment mimics the expiration-inspiration cycles in the lung during breathing (though at a much slower rate due to experimental limitations). The aim of these experiments was to observe changes that take place in the film upon intercalation of foreign substances. For the Curosurf over saline subphase, the isotherms were repeatable without noticeable changes in surface tension reduction ability. The traces of the compression and expansion are nearly identical. During the experiment meconium extract was added to the subphase to make a final concentration in the trough of 0.5 mg/mL. Following the meconium addition, isotherms gradually became flatter and an increase of the area of the pressure offset was observed. Hysteresis between compression and expansion of the film became apparent in the presence of meconium, indicating the inferior dynamic performance due to meconium. Moreover, a significant area expansion, from 80 to 180 Å2/molecule with time is observed in the compression isotherms when meconium is present in the subphase. This indicates continuous penetration of meconium surface-active components into the LS monolayer. Characterization of LS in the Bulk: Mimicry of the Prenatal Situation. (1) “Artificial” System. Lipid mixtures of DPPC/DPPG (4:1; 4.0 mg/mL) form unilamellar vesicles (ULV) in solution with diameters ranging from 100 to 550 nm (Figure 4A). Upon addition of 2.8 mM TA the observed unilamellar vesicles appear to be rougher and almost perforated (Figure 4C). In addition, fragments and threadlike structures are observed,
(30) Petty, M. C. Langmuir Blodgett Films-An Introduction; Cambridge University Press: Cambridge, England, 1996.
(31) Taeusch, H. W.; Lu, K. W.; Goerke, J.; Clements, J. A. Am. J. Respir. Crit. Care. Med. 1999, 159, 1391-1395.
3248 Langmuir, Vol. 22, No. 7, 2006
Gross et al.
Figure 3. Subphase replacement experiment. Compressionexpansion cycles of Curosurf on the water subphase (empty circles). Transition state followed the subphase substitution with meconium solution (filled diamonds). Compression-expansion cycles on meconium-containing subphase (solid thin lines) exhibit continuous area expansion due to the penetration of surface-active substances from the subphase. The hysteresis between expansion and compression traces indicates the deteriorated dynamic response of the LS film in the presence of meconium.
representing disintegrated membrane fractions (Figure 4B). Edgeon disks are also attributed to vesicles fragments32 (Figure 4B,C). Similar transition from vesicles to flat configuration has been observed in studies on the effects of addition of sodium cholate to phosphatidylcholine (PC) membranes.33-35 It was found that addition of low levels of TA (6-7.25 mM) to PC (9 mM) resulted in vesicle growth and coexisting small unilamellar vesicles and fragments of bilayers or sheets. These were interpreted as fragments with “stabilized” edges by sodium cholate. Increasing the sodium cholate concentration (to 8 mM) results in further disintegration of the flat sheets and formation of cylindrical mixed micelles.36 (2) “Semiartificial” System: Curosurf/TA. Cryo-TEM images of Curosurf (10 mg/mL) in the bulk are presented in Figure 5A. As expected, the main structures that can be seen are multilamellar and unilamellar vesicles with diameters ranging from 200 to 800 nm. Addition of 1.9 mM TA resulted in formation of elongated vesicles. The membranes appear to be open, broken, or rippled (Figure 5B). With increasing TA concentration to 5.6 mM, membrane debris and disklike fragments are imaged both edgeon and face-on (Figure 5C). This indicates that TA damages the Curosurf vesicles, in agreement with the artificial system findings. (2.1) Turbidity Measurements. Vesicular suspension of Curosurf (4.0 mg/mL in aqueous buffer) was examined for its light transmittance using a visible light spectrophotometer. At this concentration the suspensions are optically opaque, suggesting the existence of vesicles with average size larger than the visible light range. The change in the optical density (turbidity) is presented as a function of TA concentration (0.0-9.0 mM), at 400 nm (Figure 6). Decrease in the optical density (OD) due to disintegration of (32) Marques, E.; Regev, O.; Khan, A.; Miguel, M. D.; Lindman, B. J. Phys. Chem. 1998, 102, 6746-6758. (33) Walter, A.; Vinson, P. K.; Kaplun, A.; Talmon, Y. Biophys. J 1991, 60, 1315-1325. (34) Edwards, K.; Gustafsson, J.; Almgren, M.; G., K. J. Colloid Interface Sci. 1993, 161, 299-309. (35) Egelhaaf, S. U.; Schurtenberger, P.; Muller, M. J. Microsc. (Oxford) 2000, 200, 128-139. (36) Edwards, K.; Gustafsson, J.; Almgren, M.; Karlsson, G. J. Colloid Interface Sci. 1993, 161, 299-309.
Figure 4. Cryo-TEM micrographs of 5.33 mM DPPC/DPPG (4:1), (A) pure depicting spherical ULV’s and (B, C) after addition of 2.8 mM TA solution. In B, black arrows indicate edge-on disks and the circled regions show the remains of fractured vesicles. In C, white arrows show the rough surface of a vesicle and black arrows indicate fractured fragments. Scale bar: 200 nm.
the LS aggregate is the combined effect of both the decrease in aggregate size and the increase in the average distance between neighboring aggregates. Both effects cause the vesicular suspensions to become less turbid and reflect the shift in aggregation phase from vesicles to micelles. Since there is a strong dependence of the scatterer size on the wavelength, according to Rayleigh-
Lung-Surfactant-Meconium Interaction
Langmuir, Vol. 22, No. 7, 2006 3249
Figure 6. Optical density (OD) changes as a function of added TA to Curosurf vesicle suspension (5.33 mM). Turbidity was measured on a spectrophotometer in a wavelength of 400 nm. The midpoint of the OD drop is related to the scattering particles size. (The line is a guide for the eyes.)
Figure 7. Cryo-TEM micrographs of (A) a low concentration of Curosurf (0.67 mM) in the presence of meconium (0.25 mg/mL) and (B) a higher concentration of Curosurf (13.3 mM) in the presence of meconium (10 mg/mL): M, micelles; OV, open vesicle; ULV, unilamellar vesicle. Scale bars: 300 nm.
Figure 5. Cryo-TEM micrographs of Curosurf vesicles: (A) 13.3 mM solution (unilamellar and multilamellar vesicles were observed (white arrows)); (B) Curosurf with TA 1 mg/mL (1.9 mM), depicting deformed vesicle with rough surface; (C) Curosurf with 5.6 mM TA. Membrane fragments seen edge-on (white arrows) and face-on (circled) were observed in addition to ruptured vesicle membrane (black arrow).
Gans-Debye (RGD) theory,37 it can be concluded that within the TA concentration range that corresponds to the decrease in absorbance, the particles are degraded. In previous studies on the change in OD of phosphatidilcholine vesicles upon addition of cholate, similar behavior has been reported.33,34
(3) “Natural” System. Figure 7 shows images of Curosurf after exposure to meconium at 37 °C (0.5 mg/mL Curosurf with 0.25 mg/mL meconium (Figure 7A) and 10 mg/mL Curosurf with 10 mg/mL meconium (Figure 7B)). Unilamellar vesicles appear together with open vesicle and micelles. Observation of open vesicles and micelles suggests that meconium components penetrated into the Curosurf vesicles. These components obstruct the continuous lamellar organization of Curosurf vesicles; thus the vesicles loose their typical spherical shape and deform into structures with nonuniform curvatures. In the lungs, LS is produced and excreted by type II pneumocyte cells. As can be seen from Figures 4B,C, 5B,C and 7, meconium, or its TA analogue, penetrates into the vesicles, reduces their curvature, or eventually breaks them. In vivo, such structural changes could hinder the essential transition from LB to TM and reduce its effective spreading as a monolayer. As a consequence, the in-vivo spreading of the LS at the alveoli interface is inhomogeneous and its capability to reduce surface tension, and further, to sustain the repeated compression-expansion cycles (Figure 3) becomes ineffective.
Discussion and Conclusions In this study we present an artificial model as a simplified analogue system for the complex natural biological case of lung (37) Matsuzaki, K.; Murase, O.; Sugishita, K.; Yoneyama, S.; Akada, K.; Ueha, M.; Nakamura, A.; Kobayashi, S. Biochim. Biophys. Acta 2000, 1467, 219-226.
3250 Langmuir, Vol. 22, No. 7, 2006
Figure 8. Proposed schematic view of the possible TA penetration to the lipid monolayer at the air-water interface (A) and to the bilayer in bulk (B). The gray spheres denote the hydrophilic part of the lipid molecules, and the “zigzag” lines represent their hydrophobic part.
surfactant poisoning by meconium. This system enables one to assess the interactions that take place between key elements in the complex natural system. Using this approach, the penetration of meconium to the monolayer at the interface or to vesicles in the bulk was demonstrated. This was found to be detrimental to LS supramolecular order and function. A comparison between the two media (bulk and interface) points at similarities in damage characteristics: at the interface, small round LC domains become larger with convoluted shapes in the presence of meconium (Figure 2). This suggests a reduction in the line tension between the ordered LC and the disordered LE domains, hence reducing efficient response to compression-expansion events in the lung. Similarly, in the bulk, spherical vesicles become deformed, ruptured, and flattened aggregates, due to reduction of the lamellar structure ability to withstand surface tension. These results indicate that penetration of meconium changes the contact line or the surface tension of the aggregates. This could also affect the dynamic transition between monolayer to multilayer and reverse during the breathing process.38 In the absence of meconium, the existence of isolated domains at high surface pressure for both LS and their analogues (Figures 2C and 1D, respectively) indicates films that are more suitable for the breathing process. Such films have better flexibility since individual islands form a long contact line (a two-dimensional analogy to a high surface area of small particles in the bulk) between the LC and LE phases, thus allowing rapid equilibration between the two phases. The cryo-TEM observations on the destruction of the vesicles by addition of TA or meconium are corroborated by turbidity measurements. This indicates that the LS aggregate breakdown takes place at a TA concentration of ca. 2.5 mM, for LS concentration of 5.33 mM (app. 4.0 mg/mL). This yields a preliminary stoichiometry evaluation of the LS/TA interaction of roughly 2. The penetration of TA to surfactant aggregates can be interpreted by examination of our artificial model at the molecular level: TA is made of a sterol skeleton with a short, 6-C alkyl tail, consisting of sulfonate and amide groups, which give the tail hydrophilic character (Figure 8). In TA, all three hydroxyl groups on the sterol skeleton are located at the same side of its planar skeleton, while the hydrophobic methyl moieties are at (38) Wang, L.; Cai, P.; Galla, H.-J.; He, H.; Flach, C. R.; Mendelsohn, R. Eur. Biophys. J. 2005, 34, 243-254.
Gross et al.
the opposite face of the molecule (Figure 8). This gives the TA molecule amphiphilic character between its two faces (in contrast to cholesterol, where the hydrophilic and hydrophobic regions are arranged along the long molecule axis). Taurocholate and cholate molecules have been shown to cause solubilization, induction of vesicle leakage, and micellization of various phosphatydilcholine membranes and vesicles.39-41 On the basis of these and our wealth of supramolecular data, a stereochemical, molecular interaction is proposed. We assume that TA molecules penetrate into the phospholipid mono- or bilayers in an orientation parallel to the interface, so that its hydroxyl groups face the aqueous phase and the hydrophobic groups face the hydrophobic phase, as shown in Figure 8A. TA is moderately water soluble, and due to its “flat” molecular shape, it does not create stable monolayers at the air-water interface alone, since the molecules lying “flat” on the interface create much less lateral van der Waals interactions compared with “upright” oriented molecules. This makes TA a partially water soluble amphiphile. Hence TA spontaneously forms a Gibbstype monolayer at the air-solution interface. In contrast, LS lipids are water insoluble, thus forming a more stable Langmuirtype monolayer. It follows that the coexistence of these two monolayer types gives rise to competition, where the spread insoluble lipids displace the more soluble amphiphile from the interface, or vice-versa, depending on their respective concentrations, surface pressure, and temperature.42 The soluble amphiphile can be either a low molecular weight species (in this study) or a protein (e.g., serum albumin inactivation of LS10,11). Furthermore, penetration of TA into a DPPC/DPPG (4:1) monolayer can change its elasticity and stability at high pressure (Figure 1). Those changes could be critical during expiration in vivo. The dual face nature of TA molecules can similarly be recruited to rationalize the break-up of the lipid vesicles in the bulk, as evidenced by our cryo-TEM (Figures 4 and 5) and turbidity (Figure 6) measurements. The observed gradual deterioration of the lamellar structures, from small kinks to the formation of stable flat disks, can be accounted for by the stereoselective interaction of the TA hydrophobic face with exposed alkyl chains at lamellar edges, while the other, hydrophilic face interacts with the aqueous solution, as depicted schematically in Figure 8B. In summary, we have studied in vitro the effect of meconium on LS behavior at the interface and in bulk. A few combinations of natural and synthetic analogues were tested for both the LS and the meconium. TA or meconium cause similar damage to the supramolecular aggregation changes in the monolayer of Curosurf, or its synthetic analogue lipids, at the interface and damage their aggregate structures, in the bulk. It is therefore plausible to assume that the main poisoning agent for LS structure and consequently its function are bile salts from meconium. A possible mechanism of action is demonstrated in a simplified experimental system. Acknowledgment. We thank Teva Medical, Israel, for the generous gift of Curosurf for this study. LA0521241 (39) Chakrabarty, D.; Chakraborty, A.; Seth, D.; Hazra, P.; Sarkar, N. Chem. Phys. Lett. 2005, 412, 255-262. (40) Gantz, D. L.; Wang, D. Q.-H.; Carey, M. C.; Small, D. M. Biophys. J. 1999, 76, 1436-1451. (41) Forte, L.; Andrieux, K.; Keller, G.; Grabielle-Madelmont, C.; Lesieur, S.; Paternostre, M.; Ollivon, M.; Bourgaux, C.; Lesieur, P. J. Therm. Anal. Calorim. 1998, 51, 773-782. (42) Volinsky, R.; Kolusheva, S.; Berman, A.; Jelinek, R. Langmuir 2004, 20, 11084-11091.
