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Langmuir Monolayer of Artificial Pulmonary Surfactant Mixtures with an Amphiphilic Peptide at the Air/Water Interface: Comparison of New Preparations with Surfacten (Surfactant TA) Hiromichi Nakahara,† Sannamu Lee,‡ Gohsuke Sugihara,‡ Chien-Hsiang Chang,| and Osamu Shibata*,†,§ DiVision of Biointerfacial Science, Graduate School of Pharmaceutical Sciences, Kyushu UniVersity, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, Department of Chemistry, Faculty of Science, Fukuoka UniVersity, 8-19-1 Nanakuma, Johnan-ku, Fukuoka 814-0180, Japan, Department of Chemical Engineering, National Cheng Kung UniVersity, Tainan 701, Taiwan, and Department of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Nagasaki International UniVersity, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan ReceiVed October 13, 2007. In Final Form: January 11, 2008 Interfacial behavior was studied on the pulmonary lipid mixture containing a newly designed amphiphilic R-helical peptide (Hel 13-5) that consists of 13 hydrophobic and 5 hydrophilic amino acid residues. Moreover, the data obtained were compared with those of commercially available Surfacten (Surfactant TA) which has been clinically used for neonatal respiratory distress syndrome (NRDS) in Japan. Surface pressure (π)-A and surface potential (∆V)-area (A) isotherms were measured for our synthetic preparations and Surfacten. Herein, a mixture of dipalmitoylphosphatidylcholine (DPPC)/egg-phosphatidylglycerol (PG)/palmitic acid (PA) (68:22:9 by weight) was used as the constituent of basic preparations. Monolayers were spread on 0.02 M Tris buffer (pH 7.4) with 0.13 M NaCl at the air/liquid interface, and the surface behavior was investigated by employing the Wilhelmy method, an ionizing electrode method, and fluorescence microscopy (FM). Cyclic compression and expansion isotherms of the prepared materials (or products) (DPPC/PG/PA/Hel 13-5) were examined to confirm the spreading and respreading ability. For the prepared products, a plateau region exists on π-A and ∆V-A isotherms at ∼42 mN m-1, indicating that Hel 13-5 is squeezed out of surface monolayers together with fluid components (PG) upon lateral compression. That is, the squeeze-out phenomenon induces a 2D-3D phase transformation. In particular, the inclination of the π-A isotherms at XHel 13-5 ) 0.1 in the plateau region was almost zero irrespective of the molecular area. As proposed in the earlier report (Nakahara, H.; Lee, S.; Sugihara, G.; Shibata, O. Langmuir 2006, 22, 5792-5803), an observed refluorescence phenomenon was discussed for FM measurements. This phenomenon provides evidence of the squeeze-out motion with fluid molecules. Furthermore, the cyclic π-A and ∆V-A isotherms show larger hysteresis areas and better respreading abilities in comparison with the previous ternary systems (DPPC/PG/Hel 13-5 and DPPC/PA/Hel 13-5) that are very important properties in pulmonary functions. FM photographs and the temperature dependence of π-A and ∆V-A isotherms suggest that the phase behavior of the present preparation product is very similar to that of Surfacten in terms of the domain size and in parameters such as collapse pressures, maximum ∆V values, and so on. These results demonstrate that PG and PA even in the present preparations work well for compression-expansion cycling as is the case in the previous ternary systems, and the present preparations show comparable properties to Surfacten in vitro.
Introduction A pulmonary surfactant (PS) containing the lung alveoli is a complex of ∼90% lipids and ∼10% surfactant proteins.1 Dipalmitoylphosphatidylcholine (DPPC) is the most abundant component of PS lipids and plays an important role in lowering surface tension down to near zero in the multilayer state. The lipids contain a smaller but significant amount of phosphatidylglycerol (PG) and palmitic acid (PA) besides DPPC.1-4 Four surfactant proteins (SP) are commonly known: the hydrophilic SP-A and SP-D and the hydrophobic SP-B and SP-C. Of the four proteins, SP-B and SP-C are essential components to the adjustment of surface activity as a squeeze-out phenomenon, proposing that unsaturated lipids and proteins in PS are selectively * Corresponding author. E-mail:
[email protected]. URL: http:// www.niu.ac.jp/∼pharm1/lab/physchem/indexenglish.html. † Kyushu University. ‡ Fukuoka University. § Nagasaki International University. | National Cheng Kung University. (1) Veldhuizen, R.; Nag, K.; Orgeig, S.; Possmayer, F. Biochim. Biophys. Acta 1998, 1408, 90-108. (2) Kruger, P.; Baatz, J. E.; Dluhy, R. A.; Losche, M. Biophys. Chem. 2002, 99, 209-228. (3) Postle, A. D.; Heeley, E. L.; Wilton, D. C. Comp. Biochem. Physiol. A 2001, 129, 65-73. (4) Yu, S.-H.; Possmayer, F. J. Lipid Res. 2003, 44, 621-629.
and reversibly removed from the air/alveolar fluid interface.5-7 However, its mechanism has not been made clear yet. Premature infants lacking an abundant amount of PS are suffered from neonatal respiratory distress syndrome (NRDS). Exogenous surfactant preparations extracted from bovine or porcine lungs are commonly administered to NRDS patients for their therapy. For example, Curosurf (Chiesi Pharmaceutici, Parma, Italy), Survanta (Ross Laboratories, Columbus, OH), and Surfacten (Surfactant TA; Mitsubishi Pharma Corporation, Osaka, Japan) are analogue to native pulmonary surfactant, and they have been clinically used in each country. Although the preparations above are dramatically effective for the patients, they involve the risk of animal infections such as bovine spongiform encephalopathy (BSE), potential viral contamination, and inherent immunity. Other drawbacks include a costly purification procedure and the difficulty of producing batchto-batch uniformity. Therefore, the preparations fully made of synthetic surfactants with or without proteins (or peptides) would be desired in the clinical surfactant replacement therapy for NRDS. Recently, in addition, basic clinical research on the expanded application of these preparations to acute respiratory distress (5) Taneva, S.; Keough, K. M. Biophys. J. 1994, 66, 1137-1148. (6) Taneva, S.; Keough, K. M. Biophys. J. 1994, 66, 1149-1157. (7) Taneva, S.; Keough, K. M. Biophys. J. 1994, 66, 1158-1166.
10.1021/la703180x CCC: $40.75 © 2008 American Chemical Society Published on Web 03/04/2008
Monolayer of Artificial Pulmonary Surfactants
syndrome (ARDS) has been actively carried out.8 As a most effective lipid replacement, Tanaka et al. have originally developed a DPPC/PG/PA (68:22:9 by weight) mixture.9 The composition of this mixture mimics that of the lipids existing in the amnion liquid. It has been reported that the surface properties of this mixture are enhanced by adding a small amount of the lipid-binding protein.9 Therefore, the DPPC/PG/PA mixture has been used by many researchers to develop new pulmonary preparations and to clarify pulmonary function.10-12 Recently, Surfaxin, a synthetic PS preparation with a KL4 peptide (consisting of novel 21-amino acid residues), has been approved for clinical use.11,13,14 Moreover, the preparation containing recombinant SP-C (rSP-C; 34 amino acid residues) has been under active investigation for clinical efficacy.15-17 Both preparations are composed of the DPPC/PG/PA (68:22:9 by weight) mixture with the exception of minor difference in the PG species. The authors have verified the potential use of Hel 13-5 of an 18-mer amphiphilic R-helical peptide possessing 13 hydrophobic and 5 hydrophilic amino acid residues as a PS protein analogue.18-21 Hel 13-5 can form a stable monolayer at the air/ liquid interface near physiological temperature and can be squeezed out of a surface monolayer together with fluid components upon lateral compression.22,23 Furthermore, the rapid adsorption of DPPC toward the interface is induced by the addition of a small amount of Hel 13-5.23 These surface properties are essential to maintaining pulmonary functions during breathing. In previous work, each role of PG and PA components in the ternary DPPC/PG (68:22 by weight)/Hel 13-5 and the DPPC/PA (90:9 by weight)/Hel 13-5 systems has been proven.24 PG interacts selectively with Hel 13-5 as a result of electrostatic attraction, and the interaction facilitates the squeeze out of Hel 13-5 together with PG upon lateral compression. PA, however, forms rigid mixed monolayers with DPPC at the interface, promoting the squeeze out of Hel 13-5 from surface monolayers. Notice that the squeeze-out action is recognized as a “refluorescent phenomenon” in the DPPC/PA/Hel 13-5 systems by fluorescence microscopy measurements. That is, the earlier report suggested (8) Notter, R. H. Lung Surfactants: Basic Science and Clinical Applications; Marcel Dekker: New York, 2000; Vol. 149, pp 1-444. (9) Tanaka, Y.; Takei, T.; Aiba, T.; Masuda, K.; Kiuchi, A.; Fujiwara, T. J. Lipid Res. 1986, 27, 475-485. (10) Gustafsson, M.; Vandenbussche, G.; Curstedt, T.; Ruysschaert, J. M.; Johansson, J. FEBS Lett. 1996, 384, 185-188. (11) Ma, J.; Koppenol, S.; Yu, H.; Zografi, G. Biophys. J. 1998, 74, 18991907. (12) Gustafsson, M.; Palmblad, M.; Curstedt, T.; Johansson, J.; Schurch, S. Biochim. Biophys. Acta 2000, 1466, 169-178. (13) Revak, S. D.; Merritt, T. A.; Cochrane, C. G.; Heldt, G. P.; Alberts, M. S.; Anderson, D. W.; Kheiter, A. Pediatr. Res. 1996, 39, 715-724. (14) Cai, P.; Flach, C. R.; Mendelsohn, R. Biochemistry 2003, 42, 94469452. (15) Amrein, M.; von Nahmen, A.; Sieber, M. Eur. Biophys. J. 1997, 26, 349-357. (16) Krol, S.; Ross, M.; Sieber, M.; Kunneke, S.; Galla, H.-J.; Janshoff, A. Biophys. J. 2000, 79, 904-918. (17) Grigoriev, D. O.; Kragel, J.; Akentiev, A. V.; Noskov, B. A.; Miller, R.; Pison, U. Biophys. Chem. 2003, 104, 633-642. (18) Kiyota, T.; Lee, S.; Sugihara, G. Biochemistry 1996, 35, 13196-13204. (19) Kitamura, A.; Kiyota, T.; Tomohiro, M.; Umeda, A.; Lee, S.; Inoue, T.; Sugihara, G. Biophys. J. 1999, 76, 1457-1468. (20) Lee, S.; Furuya, T.; Kiyota, T.; Takami, N.; Murata, K.; Niidome, Y.; Bredesen, D. E.; Ellerby, H. M.; Sugihara, G. J. Biol. Chem. 2001, 276, 4122441228. (21) Furuya, T.; Kiyota, T.; Lee, S.; Inoue, T.; Sugihara, G.; Logvinova, A.; Goldsmith, P.; Ellerby, H. M. Biophys. J. 2003, 84, 1950-1959. (22) Nakahara, H.; Nakamura, S.; Lee, S.; Sugihara, G.; Shibata, O. Colloids Surf., A 2005, 270-271, 52-60. (23) Nakahara, H.; Nakamura, S.; Hiranita, T.; Kawasaki, H.; Lee, S.; Sugihara, G.; Shibata, O. Langmuir 2006, 22, 1182-1192. (24) Nakahara, H.; Lee, S.; Sugihara, G.; Shibata, O. Langmuir 2006, 22, 5792-5803.
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that the pulmonary preparations with Hel 13-5 peptides needed to contain both the components to develop the more effective medicines. In the present study, we have investigated the interfacial behavior of spread monolayers of the DPPC/PG/PA (68:22:9 by weight) mixture, Hel 13-5, and Surfacten (Surfactant TA). The aims of this work are to enhance the PS functions of our synthetic preparations (DPPC/PG/PA/Hel 13-5) by extending the previous investigations22-24 and to confirm the potential use of our preparations in comparison with Surfacten from the viewpoint of the basic study of their interfacial behavior. For in vivo administration, Surfacten is commonly suspended in sterile saline. In this study, however, it was dissolved in the organic solvent and was spread onto the subphase to verify the in vitro behavior of spread monolayers of our preparations and Surfacten. These examinations will provide information on the clarification of PS functional mechanisms through the air/alveolar and into the development of synthetic PS preparations with proteins or peptides. Materials and Methods Materials. Hel 13-5 (MW ) 2203 Da) was synthesized as described previously.18 Dipalmitoylphosphatidylcholine (DPPC, purity >99%) and egg-phosphatidylglycerol (PG, purity >99%) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). PG was supplied as its sodium salt. Palmitic acid (PA, purity >99%) was purchased from Sigma (St. Louis, MO). Surfacten (Surfactant TA) is a bovine lung extract produced for clinical use by Mitsubishi Pharma Corporation (Osaka, Japan). Surfacten contains mainly phospholipids and two surfactant-specific proteins of SP-B and SPC. The extract is supplemented with DPPC, PA, and tripalmitin.9 3,6-Bis(diethylamino)-9-(2-octadecyloxycarbonyl) phenyl chloride (R18) was obtained from Molecular Probes Inc. (Eugene, OR) as a fluorescent probe. These lipids were used without further purification or characterization. n-Hexane (>99.5%), ethanol (>99.5%), chloroform (>99.7%), and methanol (>99.7%) were used as spreading solvents and were obtained from Cica-Merck (Uvasol, Tokyo, Japan), nacalai tesque (Koto, Japan), Kanto Chemical Co., Inc. (Tokyo, Japan), and Merck (Uvasol), respectively. Tris(hydroxymethyl)aminomethane (Tris) and acetic acid (HAc) of guaranteed reagent grade for the preparation of a subphase were purchased from nacalai tesque. Sodium chloride (nacalai tesque) was roasted at 1023 K for 24 h to remove all surface-active organic impurities. The lipid mixture with a fixed ratio, DPPC/PG/PA (68:22:9 by weight), was used in the present study. The subphase was kept at 0.02 M Tris buffer (pH 7.4) with 0.13 M NaCl solution throughout the experiment to approach the conditions in a living body as supported by many researchers.11,25-27 The substrate solution was prepared using thrice-distilled water (surface tension ) 71.96 mN m-1 at 298.2 K and electrical resistivity ) 18 MΩ cm). Methods. Surface Pressure-Area Isotherms. The surface pressure (π) of monolayers was measured using an automated homemade Wilhelmy balance, which was the same as that used in the previous studies.23,24,28 The surface-pressure balance (Mettler Toledo, AG64) had a resolution of 0.01 mN m-1. The pressure-measuring system was equipped with filter paper (Whatman 541, periphery ) 4 cm). The trough was made from Teflon-coated brass (area ) 750 cm2), and Teflon-made barriers (both hydrophobic and lipophobic) were used in this study. More details of the trough performance were described in the previous paper.24 (25) Flanders, B. N.; Vickery, S. A.; Dunn, R. C. J. Phys. Chem. B 2002, 106, 3530-3533. (26) Koppenol, S.; Tsao, F. H. C.; Yu, H.; Zografi, G. Biochim. Biophys. Acta 1998, 1369, 221-232. (27) Lipp, M. M.; Lee, K. Y.; Waring, A.; Zasadzinski, J. A. Biophys. J. 1997, 72, 2783-2804. (28) Nakahara, H.; Nakamura, S.; Kawasaki, H.; Shibata, O. Colloids Surf., B 2005, 41, 285-298.
3372 Langmuir, Vol. 24, No. 7, 2008 The π-A isotherms were recorded over the temperature range from 298.2 to 310.2 K to within (0.1 K. The subphase and the ambient air temperature were precisely controlled by a thermostat and a clean-room-grade ribbon heater, respectively.22 Stock solutions of DPPC (1.35 mM), PG (0.68 mM), and PA (1.35 mM) were prepared in n-hexane/ethanol (9:1 v/v), and that of Hel 13-5 was made in n-hexane/ethanol (4.5:5.5 v/v). For our experiments, Surfacten (0.47 mg/mL) was dissolved in chloroform/methanol (2:1 v/v) to verify the in vitro behavior of spread monolayers of our preparations and Surfacten. The spreading solvents were allowed to evaporate for 15 min prior to compression. The monolayer was compressed at a speed of
