Mode of Interaction of Amphiphilic α-Helical Peptide with

Dec 24, 2005 - and DPPC/egg-PC (1:1) and their combinations by the Wilhelmy ... system of DPPC/egg-PC, the collapse pressure (πc; ∼42 mN m-1) was ...
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Langmuir 2006, 22, 1182-1192

Mode of Interaction of Amphiphilic r-Helical Peptide with Phosphatidylcholines at the Air-Water Interface Hiromichi Nakahara,† Shohei Nakamura,† Takato Hiranita,† Hideya Kawasaki,‡ Sannamu Lee,§ Gohsuke Sugihara,§ 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, Graduate School of Science, Kyushu UniVersity, 6-1-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; and Department of Chemistry, Faculty of Science, Fukuoka UniVersity, 8-19-1 Nanakuma, Johnan-ku, Fukuoka 814-0180, Japan ReceiVed September 12, 2005. In Final Form: NoVember 13, 2005 Surface pressure (π)-, surface potential (∆V)-, and dipole moment (µ⊥)-area (A) isotherms and morphological behavior were examined for monolayers of a newly designed 18-mer amphiphilic R-helical peptide (Hel 13-5), DPPC, and DPPC/egg-PC (1:1) and their combinations by the Wilhelmy method, ionizing electrode method, fluorescence microscopy (FM), and atomic force microscopy (AFM). The newly designed Hel 13-5 showed rapid adsorption into the air-liquid interface to form interfacial films such as a SP-B function. Regardless of the composition and constituents in their multicomponent system of DPPC/egg-PC, the collapse pressure (πc; ∼42 mN m-1) was constant, implying that Hel 13-5 with the fluid composition of egg-PC is squeezed out of Hel 13-5/DPPC/egg-PC monolayers accompanying a two- to three-dimensional phase transformation. FM showed that adding a small amount of Hel 13-5 to DPPC induced a dispersed pattern of ordered domains with a “moth-eaten” appearnce, whereas shrinkage of ordered domains in size occurred for the DPPC/egg-PC mixture with Hel 13-5. Furthermore, AFM indicated that (i) the intermediate phase was formed in pure Hel 13-5 systems between monolayer states and excluded nanoparticles, (ii) protrusions necessarily located on DPPC monolayers, and (iii) beyond the collapse pressure of Hel 13-5, Hel 13-5 was squeezed out of the system into the aqueous subphase. Furthermore, hysteresis curves of these systems nicely resemble those of the DPPC/SP-B and DPPC/SP-C mixtures reported before.

Introduction Langmuir monolayers at the air-water interface induce experimentally simple, convenient, and useful model systems for biophysical studies in cell biology and physiology. In fact, Langmuir monolayer behavior of a mixture of proteins and phospholipids is converted to pulmonary surfactant (PS) behavior in vivo to treat respiratory distress syndrome (RDS), acute respiratory distress syndrome (ARDS), etc. in the clinical field. These days, some researchers have clarified the correlations from in vitro to in vivo behavior.1,2 PS is a complex mixture of multiple lipids (∼90 wt %) and four surfactant proteins (SP-A, -B, -C, and -D, ∼10 wt %). The lipid and protein composition of human PS mainly includes phosphatidylcholines (PC; ∼50% DPPC) and the two amphiphilic surfactant proteins, SP-B and SP-C.3 Many studies4-6 have shown that two hydrophobic pulmonary surfactant proteins (SP-B and SP-C) triggered the reversible exclusion of materials from the * Author to whom correspondence should be addressed [telephone/fax +81-92(642) 6669; e-mail [email protected]; Website http:// 210.233.60.66/∼kaimen/]. † Division of Biointerfacial Science, Kyushu University. ‡ Department of Chemistry, Kyushu University. § Department of Chemistry, Fukuoka University. (1) Goerke, J. Biochim. Biophys. Acta 1998, 1408, 79-89. (2) Lipp, M. M.; Lee, K. Y. C.; Waring, A.; Zasadzinski, J. A. Biophys. J. 1997, 72, 2783-2804. (3) Veldhuizen, R.; Nag, K.; Orgeig, S.; Possmayer, F. Biochim. Biophys. Acta 1998, 1408, 90-108. (4) Ding, J.; Takamoto, D. Y.; Nahmen, A. V.; Lipp, M. M.; Lee, K. Y. C.; Waring, A. J.; Zasadzinski, J. A. Biophys. J. 2001, 80, 2262-2272. (5) Nag, K.; Perez-Gil, J.; Ruano, M. L. F.; Worthman, L. A. D.; Stewart, J. Biophys. J. 1998, 74, 2983-2995. (6) Wang, Z.; Gurel, O.; Baatz, J. E.; Notter, R. H. J. Biol. Chem. 1996, 271, 19104-19109.

monolayers by the compression beyond the collapse pressure of surfactant proteins. RDS commonly has often fatal complications that follow a variety of illnesses, trauma, sepsis, and shock. The typical treatment uses replacement pulmonary surfactants derived mainly from animals. For example, one of the most commonly used clinical replacement surfactants in the United States is Survanta, a natural bovine pulmonary extract supplied with DPPC, PA, and triglycerides.7,8 However, it has serious problems: zoonotic infections such as bovine spongiform encephalopathy (BSE); a zoonotic viral disease that infects domestic and wild animals; and the huge cost of surfactant production. For these defects, it is quite necessary that the effective and low-cost drug with no potential of animal infection should be developed immediately. Recently, Surfaxin has reached endpoint results of phase 3 clinical trials for RDS in premature infants. Surfaxin consists of a novel 21-amino acid peptide (leucine and lysine repeated units), KL4, as a substitute for SP-B.9-12 The primary sequence of SP-B suggests that it may contain several amphipathic helices, which has led to speculation that such structures might play an important role in the function of that protein. It could be pointed out that another synthetic peptide, which was synthesized to mimic SPB’s amphipathic helices and has been studied extensively, should (7) Ding, J.; Doudevski, I.; Warriner, H. E.; Alig, T.; Zasadzinski, J. A. Langmuir 2003, 19, 1539-1550. (8) Park, S. Y.; Hannemann, R. E.; Franses, E. I. Colloids Surf. B 1999, 15, 325-338. (9) Cai, P.; Flach, C. R.; Mendelsohn, R. Biochemistry 2003, 42, 9446-9452. (10) Cochrane, C. G.; Revak, S. D.; Merritt, T. A.; Heldt, G. P.; Hallman, M.; Cunningham, M. D.; Easa, D.; Pramanik, A.; Edwards, D. K.; Alberts, M. S. Am. J. Respir. Crit. Care Med. 1996, 153, 404-410. (11) Ma, J.; Koppenol, S.; Yu, H.; Zografi, G. Biophys. J. 1998, 74, 18991907. (12) 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.

10.1021/la0524925 CCC: $33.50 © 2006 American Chemical Society Published on Web 12/24/2005

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pulmonary surfactant, and is used best for RDS patients in Japan.19 We present here a study of Langmuir monolayer behavior of a mixture of Hel 13-5 and pure DPPC and the DPPC/egg-PC (1:1 mol/mol) as a model system of replacement pulmonary surfactants using a homemade Langmuir trough (see Materials and Methods). It has been found that fluid materials are squeezed out of the multicomponent monolayers to form surface-associated protrusions.20 However, it is not very clear toward which the protrusions are located, the air or the subphase. Therefore, the main aims of the present study are (i) to investigate the surface properties (surface pressure and surface potential as well as surface topography) as functions of film compositions and (ii) to elucidate which side the excluded material goes into, the air or the subphase, judging from two kinds of atomic force microscopy (AFM) images (topography and phase contrast). This will be very useful for investigations of the interfacial phenomena for synthetic peptides as mimicking native surfactant proteins and of replacement surfactant preparations for treatment of RDS. Materials and Methods

Figure 1. (a) Ribbon diagram (indicating a secondary structure, R-helix) of Hel 13-5 and helical wheel representations of (b) Hel 13-5 and (c) KL4. L (leucine) and W (tryptophan) are hydrophobic amino acids, and K (lysine) is a hydrophilic amino acid. (d) π-t isotherms of pure DPPC and the representative DPPC/Hel 13-5 mixture system (XHel 13-5 ) 0.1) adsorbing from vesicles in 0.15 M NaCl (0.9% NaCl solution) at 298.2 ( 1.0 K.

in fact not be amphipathic. However, it has recently been reported that KL4 peptide-containing surfactant and a natural pulmonary extraction were similar in terms of efficacy and safety when used for the prevention and treatment of RDS in preterm infants.13,14 Recently, we synthesized a de novo-designed 18-mer amphiphilic R-helical peptide, Hel 13-5, consisting of 13 hydrophobic and 5 hydrophilic amino acid residues, which is 3 residues less than KL4.15 When it takes R-helical structure, the hydrophobic part and the hydrophilic part are completely separated in R-helical structure (Figure 1b). Therefore, it is expected to be able to mimic the biophysical functions of SP-B and be safe for RDS patients. In addition, we have previously reported that Hel 13-5 could induce neutral liposomes to adopt long nanotubular structures and that the interaction of specific peptides with specific phospholipid mixtures could induce the formation of membrane structures resembling cellular organelles such as the Golgi apparatus.16-18 Our preliminary monolayer experiments using a modified Wilhelmy surface balance showed that a mixture of Hel 13-5 and phospholipids spread and adsorbed quickly, comparable with Surfacten, which is a modified natural bovine (13) Moya, F. R.; Gadzinowski, J.; Bancalari, E.; Salinas, V.; Kopelman, B.; Bancalari, A.; Kornacka, M. K.; Merritt, T. A.; Segal, R.; Schaber, C. J.; Tsai, H.; Massaro, J.; d’Agostino, R. Pediatrics 2005, 115, 1018-1029. (14) Sinha, S. K.; Lacaze-Masmonteil, T.; i Soler, A. V.; Wiswell, T. E.; Gadzinowski, J.; Hajdu, J.; Bernstein, G.; Sanchez-Luna, M.; Segal, R.; Schaber, C. J.; Massaro, J.; d’Agostino, R. Pediatrics 2005, 115, 1030-1038. (15) Kiyota, T.; Lee, S.; Sugihara, G. Biochemistry 1996, 35, 13196-13204. (16) Kitamura, A.; Kiyota, T.; Tomohiro, M.; Umeda, A.; Lee, S.; Inoue, T.; Sugihara, G. Biophys. J. 1999, 76, 1457-1468. (17) 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. (18) Furuya, T.; Kiyota, T.; Lee, S.; Inoue, T.; Sugihara, G.; Logvinova, A.; Goldsmith, P.; Ellerby, H. M. Biophys. J. 2003, 84, 1950-1959.

Materials. Hel 13-5 (molecular mass ) 2203 Da) was synthesized by Fmoc strategy based on the solid-phase technique starting from Fmoc-Leu-PEG-PS resin (0.1 mmol scale) with a Perseptive 9050 automatic peptide synthesizer and purified by HPLC with a reversedphase column (20 × 250 mm, YMC C8) as described previously.15 Dipalmitoylphosphatidylcholine (DPPC; purity > 99%) and egg phosphatidylcholine (egg-PC; purity > 99%) were obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). 3,6-Bis(diethylamino)9-(2-octadecyloxycarbonyl) phenyl chloride (R18) came from Molecular Probes as a fluorescent probe. They were used without further purification or characterization. n-Hexane and ethanol (especially prepared reagent, 99.5%) used as spreading solvents came from Merck (Uvasol) and Nacalai Tesque, respectively. Tris(hydroxymethyl)aminomethane (Tris) and acetic acid (HAc) for the preparation of a subphase were purchased from Nacalai Tesque and supplied as guaranteed reagent. Sodium chloride (Nacalai Tesque) was roasted at 1023 K for 24 h to remove any surface-active organic impurities. We used two model surfactant lipids: pure DPPC21 and DPPC/ egg-PC22 (DPPC/egg-PC ) 1:1, by molar ratio). 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. This condition is widely used by many researchers.2,11,23,24 Methods. Interfacial Adsorption. Samples of DPPC and the representative DPPC/Hel 13-5 mixture (XHel 13-5 ) 0.1) were dissolved in chloroform/methanol (2:1 in volume ratio, Kanto Chemical Co., Inc., Tokyo, Japan). After the evaporation of the solvent under a stream of nitrogen, the dried mixtures were reconstituted in 0.15 M NaCl (∼2 mg/mL) by sonicating the mixtures on ice with a sonicator bath (Branson model 3510 ultrasonic cleaner; 100 W, 42 kHz output, Branson Cleaning Equipment Co., Shelton, CT) for 30 min to remove materials from an inner wall of the vial. A microprobe sonicator (1.5 min, pulse 50%, Branson model 250D sonifier, Branson Cleaning Equipment Co.) with 50% powered individual bursts lasting 1 s was used on ice to produce vesicles with minimal hydration. The kinetics of surfactant adsorption, via monomer in equilibrium with the surface film after the injection of surfactant vesicles below an air-liquid interface, were monitored using a commercially (19) Lee, S.; Sugihara, G.; Shibata, O.; Yukitake, H. JP Patent Appl. P 2004305006A, 2004. (20) Krol, S.; Ross, M.; Sieber, M.; Ku¨nneke, S.; Galla, H.-J.; Janshoff, A. Biophys. J. 2000, 79, 904-918. (21) Avery, M. E.; Mead, J. Am. J. Dis. Child. 1959, 97, 517-523. (22) Ohmori, N.; Niidome, T.; Kiyota, T.; Lee, S.; Sugihara, G.; Wada, A.; Hirayama, T.; Aoyagi, H. Biochem. Biophys. Res. Commun. 1998, 245, 259-265. (23) Flanders, B. N.; Vickery, S. A.; Dunn, R. C. J. Phys. Chem. B 2002, 106, 3530-3533. (24) Koppenol, S.; Tsao, F. H. C.; Yu, H.; Zografi, G. Biochim. Biophys. Acta 1998, 1369, 221-232.

1184 Langmuir, Vol. 22, No. 3, 2006 available surface balance system (MM 10060, KSV Instruments, KSV software ver. 2.46, Helsinki, Finland). Adsorption experiments were done at 298.2 ( 1.0 K in a dish with a 9-mL 0.15 M NaCl subphase, which was stirred (∼200 rpm) to minimize diffusion resistance as reported previously.25 One hundred and fifty microliters of vesicular suspensions was injected into the stirred subphase, and then the change in surface pressure due to the adsorption was measured as a function of time by the force on a partially submerged Wilhelmy plate composed of a filter paper (Whatman 541, periphery ) 2 cm). The final surfactant concentration was uniform at 0.033 mg/mL. Surface Pressure-Area Isotherms. The surface pressure (π) of the monolayer was measured by using an automated homemade Wilhelmy balance, which was the same as that used in the previous studies.26 The surface pressure balance (Mettler Toledo, AG-64) has a resolution of 0.01 mN m-1. The pressure-measuring system was equipped with a 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. The compression process of typical phospholipids such as DPPC could reach pressures close to 70 mN m-1 without leakage of materials by using hydrophilic barriers; however, an entire Teflon-made system was possible to prevent adsorption of materials to barriers and to elucidate quantitative analyses up to monolayer collapse states of phospholipids exactly. The surface potential measurement also supports this behavior (see Results and Discussion). The π-A isotherms were recorded at 298.2 ( 0.05 K. Stock solutions of DPPC (1.35 mM) and egg-PC (1.35 mM) were prepared in n-hexane/ethanol (9:1 v/v), and those of Hel 13-5 were made in n-hexane/ethanol (4.5:5.5 v/v). The spreading solvent was allowed to evaporate for 15 min prior to compression. The monolayer was compressed at a speed of 42 mN m-1, the ∆V-A isotherms of 0.05 e XHel 13-5 e 0.3 gradually increased, whereas the ∆V of the other mole fractions shows nearly constant value. Judging from the above phenomena, if the surface pressure goes beyond the collapse pressure, the ∆V-A isotherms become almost parallel to the area axis. Namely, the rising of ∆V beyond the second kink point means that the packing state or orientation of the molecule is still developing. At the least, these results support the squeeze-out phenomenon of Hel 13-5 from the monolayer surface. However, the value of ∆V became almost similar (∼500 mV) at ∼55 mN m-1, indicating that Hel 13-5 molecules do not squeeze out completely from the monolayer (to be stated later in more detail). (43) Piknova, B.; Schief, W. R.; Vogel, V.; Discher, B. M.; Hall, S. B. Biophys. J. 2001, 81, 2172-2180.

Mimic Pulmonary Surfactant Monolayers

Figure 4. Surface pressure (π)-area (A) isotherms, surface potential (∆V)-A isotherms, and surface dipole moment (µ⊥)-A isotherms of the DPPC/Hel 13-5 and DPPC/egg-PC (DPPC/egg-PC 1:1 mol/ mol)/Hel 13-5 mixtures on a 0.02 M Tris buffer solution (pH 7.4) with 0.13 M NaCl at 298.2 K: (A) DPPC/Hel 13-5 (A′, 0 e XHel 13-5 e 0.3); (B) DPPC/egg-PC (DPPC/egg-PC 1:1 mol/mol)/Hel 13-5 (B′, 0 e XHel 13-5 e 0.3); (inset, B′) frame size of 100 µm × 100 µm for XHel 13-5 ) 0.5 at its transition pressure (πeq).

In the case of the mixture of DPPC/egg-PC and Hel 13-5 (Figure 4B′), the π-A isotherm of XHel 13-5 ) 0.05 also had two small plateau regions at the surface pressure of ∼26 mN m-1 (shown by an arrow and supported by an inserted FM image), which is the same transition surface pressure as that of the DPPC/ egg-PC monolayer at ∼42 mN m-1. However, all other π-A isotherms had only the second plateau, indicating that the kink point of the disorder/order transition became unclear with increasing amount of Hel 13-5. Similarly to the DPPC/Hel 13-5 system, the second plateaus were widened as the amount of Hel 13-5 increased (Figure 4B,B′) and second kink points (∼42 mN

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m-1) were independent of the compositions. Interesting behavior was observed for the collapse, which means that the DPPC/ egg-PC monolayer formed a stable film up to ∼43 mN m-1, whereas the DPPC/egg-PC/Hel 13-5 mixture (0 < XHel 13-5 e 0.2) remained in a stable state up to ∼48 mN m-1. As egg-PC is composed of a mixture of various kinds of saturated and unsaturated acyl chain lengths, some acyl chains are fluid in monolayer surfaces. Thus, Hel 13-5 might be squeezed out from the DPPC/egg-PC/Hel 13-5 mixture with fluid components of egg-PC in the monolayer, and more rigid components (DPPC, saturated PC in egg-PC, and so on in the monolayer) show the second kink point. As the fluid components were eliminated from the interface, the surface pressure of stable states increased to ∼48 mN m-1. These results lead to a possible conclusion that a mixture of Hel 13-5 and fluid components of egg-PC are squeezed out of the DPPC/egg-PC/Hel 13-5 mixture at ∼42 mN m-1. The ∆V-A isotherms for 0 < XHel 13-5 e 0.2 gradually increased at pressures >42 mN m-1 as those of the DPPC/Hel 13-5 mixture. Such kinds of the behavior as the squeezing out, rising ∆V, and collapse pressure of the second kink points have been observed in the cases of SP-B and -C.44,45 Additivity Rule. A better understanding of the interactions between DPPC or DPPC/egg-PC and Hel 13-5 is provided by examining whether the mean molecular surface areas as a function of XHel 13-5 satisfy the additivity rule.46,47 A comparison between the experimental mean molecular areas (solid points) and the calculated mean molecular areas (dashed lines) of ideal mixing is shown in Figure 5 at four different surface pressures (5, 15, 25, and 35 mN m-1). For the DPPC/Hel 13-5 mixture, they showed good linearity at all surface pressures. This result is quite similar to that of the DPPC/SP-B and DPPC/SP-C systems.36,48 For the DPPC/egg-PC/Hel 13-5 system, they showed good linearity (0 < XHel 13-5 e 0.6) and a slight positive deviation (0.6 < XHel 3-5 e 1) at all surface pressures. The linearity generally indicates the complete phase separation or the ideal mixing pattern of two- or three-component monolayers. Judging from the fluorescent micrographs mentioned later (Figures 7-9), both of the monolayers support the former. The surface potential (∆V) of the monolayers was also analyzed in terms of the additivity rule at the four pressures. For the DPPC/ Hel 13-5 and DPPC/egg-PC/Hel 13-5 multicomponent systems, the experimental ∆V values are presented by the solid circles in Figure 6a,b, respectively, where the dashed lines show the ∆V values calculated by assuming the additivity rule. For the DPPC/ Hel 13-5 system, they show big deviations (0.005 e XHel 13-5 e 0.2) from and a close linearity (0.2 < XHel 13-5 e 0.9) to the calculated ones at 5 mN m-1. At 15-35 mN m-1, they wholly indicate the negative deviations but show the strange behavior at smaller molar fractions (0.005 e XHel 13-5 e 0.2). This is due to a response to the squeeze-out phenomenon of Hel 13-5 caused by morphological change. For the DPPC/egg-PC/Hel 13-5 system, on the other hand, they show greatly positive deviations at 5 mN m-1. Especially at 15-35 mN m-1, they show outstanding positive deviations (0.05 e XHel 13-5 e 0.3), whereas good linearity or slight positive deviation was observed 0.3 < XHel 13-5 e 0.8. Similar to the ∆V changes of the DPPC/Hel 13-5 system, those of smaller molar regions (0.05 e XHel 13-5 e 0.3) show the specific behavior. This might result from the above-mentioned squeeze(44) Diemel, R. V.; Snel, M. M. E.; Waring, A. J.; Walther, F. J.; Golde, L. M. G. V.; Putz, G.; Haagsman, H. P.; Batenburg, J. J. J. Biol. Chem. 2002, 277, 21179-21188. (45) Takamoto, D. Y.; Lipp, M. M.; von Nahmen, A.; Lee, K. Y. C.; Waring, A. J.; Zasadzinski, J. A. Biophys. J. 2001, 81, 153-169. (46) Marsden, J.; Schulman, J. H. Trans. Faraday Soc. 1938, 34, 748-758. (47) Shah, D. O.; Schulman, J. H. J. Lipid Res. 1967, 8, 215-226. (48) Taneva, S. G.; Keough, K. M. W. Biophys. J. 1994, 66, 1149-1157.

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Figure 5. Mean molecular area (A) of the (a) DPPC/Hel 13-5 and (b) DPPC/egg-PC (DPPC/egg-PC 1:1 mol/mol)/Hel 13-5 mixtures as a function of XHel 13-5 at four different pressures. The dashed lines were calculated by assuming the additivity rule; the solid points represent experimental values.

out phenomena of Hel 13-5. These results strongly suggest that the addition of just a small amount of Hel 13-5 induces an easy squeeze-out of Hel 13-5 from the monolayer phase. Fluorescence Microscopy. A series of FM images of two systems (DPPC/Hel 13-5 and DPPC/egg-PC/Hel 13-5) at surface pressures from 15 to 50 mN m-1 are presented in Figures 7-9. FM images of DPPC/Hel 13-5 are shown in Figure 7, where all of them showed the disorder/order coexistence states; that is, the dark domains reflect the ordered phase of DPPC, whereas the bright regions reflect the disordered phases of DPPC and Hel 13-5. The ordered domains of all molar fractions are growing in area with increasing surface pressure from 15 to 20 mN m-1. When the films were compressed further, they formed dark homogeneous images consisting of almost the ordered phase up to the collapse pressure except for XHel 13-5 ) 0.025. It should be noted that the addition of a small amount of Hel 13-5 to DPPC induced the “moth-eaten” aggregation (shown by an arrow) of LC domains made of pure DPPC. This moth-eaten aggregation occurred only in the specific case where a small amount of Hel 13-5 coexists with DPPC. In addition, its aggregation enlarged and expanded the regions of each LC domain. Therefore, the disordered phases of Hel 13-5 penetrated into the ordered domains of DPPC and promoted the nucleation of the DPPC ordered domain; the percentage of ordered domains in each total image increased at 15 mN m-1 (35% for pure DPPC, 53% for XHel 13-5 ) 0.005, and 50% for XHel 13-5 ) 0.025). At 50 mN m-1, FM images of DPPC and XHel 13-5 ) 0.005 showed a dark homogeneous image due to quenching of the FM probe. Two regions were observed at XHel 13-5 ) 0.025 as the same images at 50 mN m-1. Hall and co-workers43 indicated that beyond the second plateau, shining disklike particles corresponding to the formation of a surface-associated reservoir appeared on the FM image aside from disordered and ordered regions. In general,

Nakahara et al.

Figure 6. Surface potential (∆V) of the (a) DPPC/Hel 13-5 and (b) DPPC/egg-PC (DPPC/egg-PC 1:1 mol/mol)/Hel 13-5 mixtures as a function of XHel 13-5 at four different pressures. The dashed lines were calculated by assuming the additivity rule; the solid points represent experimental values.

Figure 7. FM images of the DPPC/Hel 13-5 mixture system at 298.2 K for surface pressures of 15, 20, and 50 mN m-1: (a) pure DPPC; (b) XHel 13-5 ) 0.005; (c) XHel 13-5 ) 0.025. In the coexistence phase, percentage refers to the ordered domains in the micrograph. The monolayers contain 1 mol % fluorescent probe (R18). The scale bar in the lower right represents 100 µm.

when the monolayers build toward the air above the collapse pressure, the resultant particles shine due to scattering of the light. Such a shining spot did not appear in our images, implying that the Hel 13-5 was squeezed out of the binary monolayers into the aqueous subphase. In the next section, we discuss this in more detail. We could not compare and analyze FM images of the DPPC/ egg-PC/Hel 13-5 system due to quite small ordered domains of

Mimic Pulmonary Surfactant Monolayers

Figure 8. FM images of the system I (DPPC/egg-PC 0.75:0.25 mol/mol)/Hel 13-5 mixture at 298.2 K for surface pressures of 20 and 30 mN m-1: (a) system I; (b) XHel 13-5 ) 0.005; (c) XHel 13-5 ) 0.025. In the coexistence phases, the percentage refers to the ordered domains in the micrograph. The monolayers contain 1 mol % fluorescent probe (R18). The scale bar in the lower right represents 100 µm.

Figure 9. FM images of the system II (DPPC/egg-PC 0.85:0.15 mol/mol)/Hel 13-5 mixture at 298.2 K for surface pressures of 20 and 30 mN m-1: (a) system II; (b) XHel 13-5 ) 0.005; (c) XHel 13-5 ) 0.025. In the coexistence phases, percentage refers to the ordered domains in the micrograph. The monolayers contain 1 mol % fluorescent probe (R18). The scale bar in the lower right represents 100 µm.

DPPC/egg-PC. Consequently, to confirm the behavior of this system, we added more DPPC to it. Herein, we need to develop a protocol that would define, at least to some extent, the new model surfactant lipids: system I (DPPC/egg-PC ) 0.75:0.25 mol/mol) and system II (DPPC/egg-PC ) 0.85:0.15 mol/mol). Figures 8 and 9 show FM images of systems I and II at 20 and 30 mN m-1, respectively. In Figure 8, system I indicates the disorder/order coexistence states, where the dark domains reflect the ordered domains of DPPC and the bright ones the disordered phases of DPPC, egg-PC, and Hel 13-5. Ordered domains of all molar fractions are growing in size with increasing surface pressure. In addition, the disordered/ordered states continued to exist beyond 30 mN m-1 up to collapse pressures (the data are not shown). It is quite remarkable that the addition of a small amount of Hel 13-5 to system I induced such interaction that ordered domains shrank in size contrary to the DPPC/Hel 13-5 mixture. For this behavior, the regions of the disordered phase increased with the amount of Hel 13-5; the percentage of ordered domains in the total image decreased both at 20 mN m-1 (16% for system I and 10% for XHel 13-5 ) 0.005) and at 30 mN m-1 (42% for system I and 39% for XHel 13-5 ) 0.005). Images of the system II/Hel 13-5 system are shown in Figure 9, indicating the disordered/ordered coexistence states. Ordered domains are growing with increasing surface pressure in all molar fractions, and the disordered/ordered coexistence states continued

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beyond 30 mN m-1 to collapse pressures (the data are not shown). As is the case for the system I/Hel 13-5 system, the shrinkage of ordered domains was also observed by the addition of a small amount of Hel 13-5 to system II; that is, the percentage of ordered domains in the total image decreased at 20 mN m-1 (40% for system II, 37% for XHel 13-5 ) 0.005, and 33% for XHel 13-5 ) 0.025) and at 30 mN m-1 (52% for system II, 48% for XHel 13-5 ) 0.005, and 40% for XHel 13-5 ) 0.025). Considering the results in Figures 8 and 9, a similar interaction might occur in the case of the DPPC/egg-PC/Hel 13-5 system, too. These FM images (Figures 7-9) clearly show that the interactions between phosphatidylcholines and Hel 13-5 become quite different in the effect of Hel 13-5 addition between two rigid components and two coexistence substances in rigid or fluid state. In addition, the moth-eaten aggregation is assumed to be more unstable in terms of entropy and, therefore, we recognize that Hel 13-5 especially and preferentially interacts with fluid components (such as egg-PC). Atomic Force Microscopy. AFM for this study provided both topography and phase contrast images. The topography image reflects the sample topography, whereas the phase contrast image, which is originated from the energy loss of the oscillating AFM tip, shows the chemical structures of heterogeneous samples. Also, on surfaces with local variations of mechanical properties such as biological samples, the AFM phase image provides the best contrast of fine morphological and nanostructural features. Figure 10 shows AFM images of a pure Hel 13-5 monolayer transferred to mica substrates before collapse pressure at 35 mN m-1 and after the collapse at 45 mN m-1. AFM images of Hel 13-5 at below 35 mN m-1 were homogeneous (not shown). At 35 mN m-1, on the other hand, some bright domains appeared in the topography image (Figure 10a), which are slightly higher than the surrounding regions by ∼0.2 nm. The small protrusions suggest the existence of intermediate states toward monolayer collapses, because the height of the protrusions is much smaller than the diameter of R-helical Hel 13-5 (∼1 nm) estimated by a computer simulation (CS ChemOffice Ultra 5.0). After the collapse of Hel 13-5 at 45 mN m-1, many protrusions are observed over the whole range as shown in Figure 10c,d. They are located in a line, implying that the collapse of a Hel 13-5 molecule promotes another collapse beside it. Zasadzinski and co-workers4 reported that above the plateau pressure many protrusions appeared in the synthetic system of Survanta, which was a commercial RDS medicine containing a natural bovine pulmonary surfactant. The resultant patches were also observed in the DPPC/ POPG (palmitoyloleoyl-phosphatidylglycerol) films containing low amounts of protein analogues,44 indicating that they were induced by the squeeze-out of fluid compositions (one of PS functions). The zoomed images at 45 mN m-1 are shown in Figure 10e,f. In the topography image, the protrusions (bright) and monolayers (dark) of single-species Hel 13-5 molecules coexist. The height of these protrusions is ∼2.0-3.0 nm, suggesting that Hel 13-5 molecules are excluded from the monolayer after plateau regions on π-A isotherms and then a three-dimensional folding made of two or three Hel 13-5 molecules is formed. Notice that two kinds of domains are observed: the domain with the bright midpoint (indicated by an arrow) and that without the one (indicated by a dashed arrow). The former shows a central higher protrusion like an upheaval of mountains and like a hill, as shown in the phase contrast image of Figure 10f. These protrusions are found to be disklike in shape with a typical diameter of ∼33 nm, corresponding to aggregations containing ∼340 molecules of Hel 13-5. Scheme 1 represents the protrusion-forming model based on our results

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Figure 10. AFM images of the Hel 13-5 monolayer in a tapping mode: topography (a) and corresponding phase contrast images (b) transferred onto mica at 35 mN m-1 at the scan area of 400 × 400 nm; topography (c) and corresponding phase contrast image (d) transferred onto mica at 45 mN m-1 at the scan area of 5 × 5 µm. The zoomed-in images of the topography (c) and phase contrast image (d) are shown in (e) and (f) at the scan area of 400 × 400 nm, respectively. Scheme 1. Resultant Representation of Molecular Behavior for the Hel 13-5 Monolayer upon Compression: (a) at Low Surface pressure (