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1 Chemistry and Physics of Lipids 161 (2009) Contents lists available at ScienceDirect Chemistry and Physics of Lipids journal homepage: Miscibility and phase behavior of DPPG and perfluorocarboxylic acids at the air water interface Hiroki Yokoyama a, Hiromichi Nakahara b, Osamu Shibata a,b, a Division of Biointerfacial Science, Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi-ku, Fukuoka , Japan b Department of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Nagasaki International University, Nagasaki , Japan article info abstract Article history: Received 18 May 2009 Received in revised form 17 June 2009 Accepted 18 June 2009 Available online 26 June 2009 Keywords: Langmuir monolayer Perfluorocarboxylic acid DPPG Surface pressure Fluorescence microscopy Atomic force microscopy The miscibility and phase behavior of two components of phospholipids and perfluorocarboxylic acids [FCn; perfluorododecanoic acid (FC12), perfluorotetradecanoic acid (FC14), perfluorohexadecanoic acid (FC16), and perfluorooctadecanoic acid (FC18)] have been systematically investigated using Langmuir monolayer technique. Dipalmitoylphosphatidylglycerol (DPPG) is utilized as a phospholipid component in biomembranes. Surface pressure () molecular area (A) and surface potential ( V) A isotherms have been measured for the DPPG/FCn systems on 0.15 M NaCl (ph 2.0) at K. From the isotherm results, two-dimensional phase diagrams are constructed and classified into miscible and immiscible patterns. Furthermore, the phase behavior of the DPPG/FCn systems has been morphologically examined using fluorescence microscopy (FM) and atomic force microscopy (AFM). These images indicate different phases among the four systems. In particular, specific phase morphology is observed in the middle molar fraction range for the DPPG/FC14 system; FC14 is selectively excluded from mixed DPPG FC14 monolayers to be concentrated in the phase boundary as surface pressure increases. Then DPPG is refined as a patched film. Moreover, the data obtained here are compared to those in the previous systems in which different kinds of phospholipids were treated. Through a series of the miscibility investigations, it is proposed that combinations of hydrophobic chain lengths and of polar headgroups contribute to the monolayer miscibility between phospholipids and perfluorocarboxylic acids Elsevier Ireland Ltd. All rights reserved. 1. Introduction Highly fluorinated surfactants have been widely used in material sciences as well as in the biomedical field (Krafft and Riess, 1998; Krafft, 2001; Riess, 2001). The fluorinated surfactants take on both hydrophobicity and lipophobicity and thus their high possibility is also being expected in an industrial field. In fact, perfluorinated surfactants have the possibility for use and application as blood substitutes (Riess and Krafft, 1998) and lung surfactants (Gerber et al., 2006, 2007). However, the residual effect of the perfluorinated surfactants (particularly, perfluorocarboxylic acids) on the human body and environment is commonly recognized as the significant problem. From the environmental point of view, the environmental fate of the perfluorocarboxylic acids has been exclusively associated with their anionic species. The anionic perfluorocarboxylates Corresponding author at: Department of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Nagasaki International University, Huis Ten Bosch, Sasebo, Nagasaki , Japan. Tel.: ; fax: address: wosamu@niu.ac.jp (O. Shibata). URL: pharm1/lab/physchem/indexenglish.html (O. Shibata). are not expected to partition into the air phase at all, and sorption by soils and sediments, which usually carry a net negative charge, is expected to be quite smaller compared to the corresponding undissociated acids (Higgins and Luthy, 2006). Therefore, the distribution of the perfluorocarboxylic acids in the global environment has been recently investigated in terms of an equilibrium acid dissociation constant (Burns et al., 2008; Goss, 2008). In another sense, the importance of fluorinated amphiphiles in advanced clinical trials has been advocated (Krafft and Riess, 1998; Riess and Krafft, 1998; Riess, 2001, 2002). However, it is still unknown how the fluorinated chemicals affect the human body and interact with biomembranes. Langmuir monolayers have been intensely adopted as experimental paradigms of biomembranes for elucidating the interfacial properties and mechanisms of surfactants (Nakahara et al., 2005b; Broniatowski and Dynarowicz-Latka, 2006; Broniatowski et al., 2007; Yukitake et al., 2008). The monolayer technique has an advantage in simplifying physicochemical behavior of biomembranes and can easily visualize the phase variation using Brewster angle microscopy (BAM) (Hénon and Meunier, 1991; Hönig and Möbius, 1991), fluorescence microscopy (FM) (McConnell, 1991; Matsumoto et al., 2007), and atomic force microscopy (AFM) (Josefowicz et al., 1993; Nakahara et al., 2006b). Thus, the monolayer miscibility of perfluorocarboxylic acids with several components of biomem /$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi: /j.chemphyslip

2 104 H. Yokoyama et al. / Chemistry and Physics of Lipids 161 (2009) branes has been studied as a model system in advanced clinical trials (Shibata et al., 1996; Lehmler and Bummer, 2004; Nakahara et al., 2005a; Rontu and Vaida, 2007; Yokoyama et al., 2009). The fundamental and systematic study is very important and desired in the industrial and biomedical fields, where fluorochemicals are treated and applied. Herein, we utilize dipalmitoylphosphatidylglycerol (DPPG) as a component of biomembranes. Phosphatidylglycerols are secondly main components next to phosphatidylcholines in lavaged lung surfactants (Notter, 2000). In the present study, the monolayer miscibility of DPPG and the perfluorocarboxylic acids (FCn, n = 12, 14, 16, 18) has been systematically investigated employing the monolayer technique. Surface pressure () molecular area (A) and surface potential ( V) A isotherms are measured and then the interaction between DPPG and FCn is analyzed in terms of the additivity rule for mean molecular areas and of the interaction parameter. Furthermore, the two-dimensional (2D) phase diagrams are constructed by plotting transition and collapse pressures against molar fraction of FCn. The phase behavior of the binary systems upon compression is morphologically observed as a function of surface pressure using fluorescence microscopy (FM) and atomic force microscopy (AFM). Moreover, these data are compared to those obtained in the previous studies (Nakahara et al., 2005a; Yokoyama et al., 2009) to clarify the effect of hydrophobic chain combinations and phospholipids polar headgroups on the monolayer miscibility behavior. 2. Materials and methods 2.1. Materials Perfluorocarboxylic acids [FCn; perfluorododecanoic (n = 12), perfluorotetradecanoic (n = 14), perfluorohexadecanoic (n = 16), and perfluorooctadecanoic acids (n = 18)] were purchased from Fluorochem (Derbyshire, United Kingdom). They were purified by repeated recrystallizations from n-hexane/acetone mixed solvents. The purity of these amphiphiles (>99%) was checked by GC MS (QP-1000, Shimadzu, Kyoto) (Nakahara et al., 2005a; Tsuji et al., 2008). l- -Dipalmitoylphosphatidylglycerol (DPPG; purity > 99%) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). DPPG was supplied as its sodium salt. 3,6-Bis(diethylamino)-9-(2- octadecyloxycarbonyl) phenyl chloride (R18) came from Molecular Probes Inc. (Eugene, OR) as a fluorescent probe. They were used without further purification. n-hexane, methanol, and chloroform with especially prepared grade (99.5%; Kanto Chemical Co., Inc., Tokyo, Japan) were used as spreading solvents. These amphiphiles were dissolved in chloroform:methanol = 2:1 (v/v) for DPPG and in n-hexane:methanol:chloroform = 10:5:1 (v/v/v) for FCn. A 0.15 M NaCl subphase was prepared using thrice distilled water (surface tension = mn m 1 at K; resistivity = 18 M cm). Sodium chloride (nacalai tesque, Kyoto, Japan) was roasted at 1023 K for 24 h to remove any surface-active organic impurities. The ph of the subphase was kept at 2.0 with hydrochloric acid (HCl; ultra fine grade, nacalai tesque) throughout the experiment to prevent FCn from dissolving in the subphase (Nakahara et al., 2005a). 3. Methods 3.1. Surface pressure area isotherms The surface pressure () of monolayers was measured using an automated homemade Wilhelmy balance, which was the same as that used in the previous studies (Nakahara et al., 2005b; Matsumoto et al., 2007; Nakahara et al., 2008). The surfacepressure balance (AG-245; Mettler Toledo, Greifensee, Switzerland) had a resolution of 0.01 mn m 1. The pressure-measuring system was equipped with filter paper (Whatman 541, periphery = 4.0 cm, Whatman International Ltd., Maidstone, England). The trough was made from Teflon-coated brass (area = 750 cm 2 ), and Teflon-made barriers (both hydrophobic and lipophobic) were used in this study. More details of the trough performance were described in the previous papers (Nakahara et al., 2006a,b; Nakamura et al., 2007). The A isotherms were recorded at ± 0.1 K. The spreading solvents of DPPG (0.5 mm) and FCn (1.0 mm) were allowed to evaporate for 15 min prior to compression. The monolayer was compressed at a speed of 0.12 nm 2 molecule 1 min 1. The standard deviations for molecular surface area and surface pressure were 0.01 nm 2 and 0.1mNm 1, respectively Surface potential measurements The surface potential ( V) was simultaneously recorded with surface pressure while the monolayer was compressed. It was monitored by using an ionizing 241 Am electrode 1 2 mm above the interface while a reference electrode was dipped in the subphase. The standard deviation for the surface potential was 5 mv. The other experimental conditions were the same as those described in the previous papers (Shibata et al., 1996; Nakamura et al., 2007; Nakahara et al., 2008) Fluorescence microscopy The fluorescence microscope (BM-1000, U.S.I. System, Fukuoka, Japan) observations and the compression isotherm measurements were carried out simultaneously. A spreading solution of the surfactants was prepared as a mixed solution doped with 1 mol% of a fluorescence probe (R18). A 300 W xenon lamp (XL 300, Pneum) was used for the excitation of the FM probes. Excitation and emission wavelengths were selected by an appropriate beam splitter/filter combination (Mitutoyo band path filter of 546 nm, Olympus cutoff filter of 590 nm). The monolayer was observed by using 20-fold magnification of a long-distance objective lens (Mitutoyo, f = 200/focal length = 20 mm). The other experimental conditions were the same as those in the previous study (Nakahara et al., 2006a,b, 2008). Image processing and analysis were carried out by using Scion Image Beta 4.02 for Windows (Scion Corp., Frederick, MD). The total area of ordered domains (or liquid-condensed (LC) domains) was calculated and expressed as a percentage per frame area Atomic force microscopy Langmuir Blodgett (LB) film preparations were carried out with the KSV Minitrough (KSV Instruments Ltd., Helsinki, Finland). Freshly cleaved mica (Okenshoji Co., Tokyo, Japan) was used as a supporting solid substrate for film deposition (a vertical dipping method). At selected surface pressures, a transfer velocity of 5mmmin 1 was used for film-forming materials on a 0.15 M NaCl (ph 2.0) at K. The transfer occurs such that the hydrophilic part of the monolayer is juxtaposed to mica while the hydrophobic part is exposed to air. LB films with deposition ratio of 1 were used in the experiments. AFM images were obtained using an SPA 400 instrument (Seiko Instruments Inc., Chiba, Japan) at room temperature in the tapping mode, which provided both a topographical image and a phase contrast image. The tapping mode images (256 or 512 points per line) were collected with scan rates of Hz, using silicon tips (Olympus Co., Japan) with a nominal spring constant of 1.5 or 16 N m 1 under normal atmosphere (Nakahara et al., 2006b, 2009a; Nakamura et al., 2007). The lateral and vertical resolutions were 0.2 and 0.1 nm, respectively. The transferred samples

3 H. Yokoyama et al. / Chemistry and Physics of Lipids 161 (2009) were checked for possible tip-induced deformation by zooming out after a region had been scanned. 4. Results and discussion 4.1. Compression isotherms The perfluorocarboxylic acids (FCn) used here are able to form a stable Langmuir monolayer on the acidic subphase of less than ph 2.0 (Nakahara et al., 2005a; Yokoyama et al., 2009). Thus, the subphase ph of 0.15 M NaCl aqueous solution, which mimics a saline concentration, was kept at 2.0 throughout the experiments to avoid dissolution of the film-forming materials into the subphase. The surface pressure () molecular area (A) and surface potential ( V) A isotherms of two-component monolayers composed of DPPG and FCn (n = 12, 14, 16, 18) at K are shown in Fig. 1. The compression isotherms of single FCn monolayers have been already reported previously, where detail analyses on monolayer state, transition pressure, and limiting molecular area were performed (Nakahara et al., 2005a; Yokoyama et al., 2009). As for pure DPPG, the A isotherm is strongly affected by subphase temperature and ionization state of the DPPG polar headgroup (Sacré and Tocanne, 1977; Maltseva et al., 2006). In particular, these factors have an influence on a first-order liquid-expanded (LE)/liquidcondensed (LC) transition pressure. Under the fixed temperature, the transition pressure decreases with a decrease in ionization degree of the DPPG polar headgroup. Correspondingly, the limiting molecular area of DPPG monolayers shifts to smaller values, which Fig. 1. Surface pressure () molecular area (A) and surface potential ( V) A isotherms of the binary (A) DPPG/FC12, (B) DPPG/FC14, (C) DPPG/FC16, and (D) DPPG/FC18 systems on 0.15 M NaCl (ph 2.0) at K for different molar fractions (X FCn). (Inset) Enlarged A isotherms in the transition regions (A and B), where the transition pressures are indicated by dashed arrows. The representative transition pressures are also indicated by dashed arrows in (C) and (D).

4 106 H. Yokoyama et al. / Chemistry and Physics of Lipids 161 (2009) Fig. 2. Mean molecular areas of the binary DPPG/FC12, DPPG/FC14, DPPG/FC16, and DPPG/FC18 monolayers as a function of X FCn at the representative surface pressures (5 and 30 mn m 1 ) on 0.15 M NaCl (ph 2) at K. The dashed lines were drawn assuming additivity, and the solid circles and open circles represent experimental values. The error bars are included within the markers. indicates formation of more packed monolayers. In the present condition (ph 2.0), the A isotherm of DPPG (curve 1) has the transition pressure ( eq )at 3mNm 1 and the collapse pressure ( c )at 54 mn m 1, which are determined from an inclination change of the corresponding V A isotherm (Matsumoto et al., 2007; Nakamura et al., 2007; Nakahara et al., 2009b; Yokoyama et al., 2009). For the V A isotherm, the V value reaches 408 mv at the close-packed state. Although these values are different from those obtained in the previous study (Nakahara et al., 2009a), the deviation is attributed to the above-mentioned effect of the ionization degree, which is induced by different ph of the subphase (Maltseva et al., 2006). The A and V A isotherms of the binary DPPG/FCn systems have been measured as a function of molar fraction of FCn (X FCn ) to elucidate the two-component miscibility in the monolayer state. For the DPPG/FC12 system (Fig. 1A), the A isotherms shift successively to smaller area at high surface pressures as X FC12 increases. The transition pressure originated from pure DPPG and FC12 monolayers varies with X FC12, indicated by dashed arrows typically for X FC12 = 0, 0.1, and 0.3 in the inset of Fig. 1A. The change implies that the two components are miscible within a monolayer state. The V A isotherms also shift to negative values over the whole molecular area with an increase in X FC12. Particularly, the shift of the V A isotherms against X FC12 becomes larger in magnitude than that of the A isotherms due to the better sensitivity of surface potential measurements (Matsumoto et al., 2007; Nakamura et al., 2007; Nakahara et al., 2008). In the case of the DPPG/FC14 system (Fig. 1B), the transition pressure increases with increasing X FC14, as seen in the inset. In addition, the collapse pressure of the two-component monolayers becomes larger compared with that of single DPPG and FC14 monolayers. These variations suggest the better monolayer miscibility between DPPG and FC14. The DPPG/FC16 (Fig. 1C) and DPPG/FC18 (Fig. 1D) systems show the constant transition pressure of 3mNm 1, which is the same as eq of DPPG, regardless of a variation in X FCn (indicated by dashed arrows). The constant value provides evidence that DPPG is immiscible with FC16 and FC18 in the monolayer state Additivity rule A mutual interaction between DPPG and FCn monolayers is analyzed by examining whether the variation of the mean molec- Fig. 3. Two-dimensional phase diagrams based on the variation in phase transition pressure ( eq ) and collapse pressure ( c ) on 0.15 M NaCl (ph 2.0) at K, as a function of X FCn: (a) DPPG/FC12, (b) DPPG/FC14, (c) DPPG/FC16, and (d) DPPG/FC18 systems. The dashed lines were calculated according to Eq. (1) for = 0. The open circles and solid circles represent experimental eq and c values, respectively. M indicates a mixed monolayer formed by DPPG and FCn species, while Bulk denotes a solid phase of DPPG and FCn ( bulk phase may be called solid phase ). The error bars are included within the markers.

5 H. Yokoyama et al. / Chemistry and Physics of Lipids 161 (2009) ular areas (A) versus X FCn satisfies the additivity rule (Marsden and Schulman, 1938; Shah and Schulman, 1967). A comparison between the experimental area (A) and the estimated area based on ideal mixing (drawn by dashed lines) is shown in Fig. 2 at selected surface pressures (5 and 30 mn m 1 ). For the DPPG/FC12 and DPPG/FC14 systems, the experimental values indicated a large positive deviation from the ideal line at 5 mn m 1. This implies existence of a repulsive interaction between DPPG and FCn at low surface pressures. Judging from the fact that the interaction in the bulk between hydrocarbon and fluorocarbon chains is smaller than that between hydrocarbons, the interaction between the polar headgroups of PG and COOH may not contribute to the positive deviation. At 30 mn m 1, the experimental values show a good agreement with the ideal line, which indicates no repulsive and attractive interaction between the two components at high surface pressures. In the cases of n = 16 and 18, the degree of the positive deviation at 5 mn m 1 is smaller than that for n = 12 and 14. Accordingly, the molecular area comes to deviate negatively with an increase in fluorocarbon chain length in the region of larger X FCn. The fact suggests that the increase of the chain length results in independent and respective change for hydrocarbon and fluorocarbon chain at low surface pressures. In addition, the systems (n = 16 and 18) become similar to the systems (n = 12 and 14) in the additivity at high surface pressures Two-dimensional phase diagrams Shown in Fig. 3 are two-dimensional phase diagrams for the binary DPPG/FCn systems. The phase diagrams are constructed on the basis of the disordered/ordered transition pressure ( eq ) and the monolayer collapse pressure ( c ) versus X FCn. Being focused on the transition pressure, the DPPG/FC12 and DPPG/FC14 systems indicate a linear eq variation with X FCn. The change provides evidence of the miscibility between the two components at low surface pressures. In contrast, the other systems (n = 16 and 18) indicate the constant pressure of 3mNm 1 ( eq of pure DPPG monolayers) regardless of the X FCn variation, which implies that the DPPG/FC16 and DPPG/FC18 systems form immiscible monolayers. For the collapse pressure, the systems of n = 12 and 14 show that the c values Fig. 4. Typical FM images of the DPPG/FC12 monolayers for (a) X FC12 = 0 (DPPG), (b) X FC12 = 0.1, (c) X FC12 = 0.2, and (d) X FC12 = 0.3 at 5, 7, and 10 mn m 1. In the coexistent phases, the percentage (%) in lower-right corner refers to the ordered domains in the micrograph. The monolayer contained 1 mol% fluorescent probe. The scale bar represents 100 m.

6 108 H. Yokoyama et al. / Chemistry and Physics of Lipids 161 (2009) of two-component monolayers are different from those of the single components depending upon the X FCn. On the other hand, the c values remain the same for the systems of n = 16 and 18, independently of X FCn variations. These results suggest that DPPG is miscible with FC12 and FC14, whereas it is immiscible with FC16 and FC18. The coexistence phase boundary between the ordered monolayer phase and the bulk phase can be theoretically simulated from the Joos equation (Joos and Demel, 1969) under the assumption of a regular surface mixture with a hexagonal lattice in the miscible DPPG/FCn systems (n = 12 and 14): { } ( c 1 = x s 1 exp m c 1 )ω 1 exp{(x s kt 2 )2 } { } ( c + x s 2 exp m c 2 )ω 2 exp{x(x s kt 1 )2 } (1) where x s 1 and xs denote the molar fractions in the two-component 2 monolayers of components 1 and 2, and c 1 and c are the col- 2 Fig. 5. Typical FM images of the DPPG/FC14 monolayers for (a) X FC14 = 0.1, (b) X FC14 = 0.2, (c) X FC14 = 0.3, (d) X FC14 = 0.4, and (e) X FC14 = 0.5 at 5, 7, and 10 mn m 1. In the coexistent phases, the percentage (%) in lower-right corner refers to the ordered domains in the micrograph. The monolayer contained 1 mol% fluorescent probe. The scale bar represents 100 m.

7 H. Yokoyama et al. / Chemistry and Physics of Lipids 161 (2009) Fig. 6. Typical FM images of the DPPG/FC16 monolayers for (a) X FC16 = 0.05, (b) X FC16 = 0.1, and (c) X FC16 = 0.2 at 5, 7, and 10 mn m 1. The monolayer contained 1 mol% fluorescent probe. The scale bar represents 100 m. lapse pressures of components 1 and 2, m c is the collapse pressure of the two-component monolayer at a given composition of x s 1 and x s 2, ω 1 and ω 2 are the corresponding limiting molecular area at the collapse points, is the interaction parameter, and kt is the product of the Boltzmann constant and the Kelvin temperature. The solid curve at high surface pressures is drawn by curve fitting to the experimental c values as a variable of the interaction parameter in the above equation (Eq. (1)). The interaction parameters of both the systems are negative values; = 0.40 for DPPG/FC12 and = 0.48 for DPPG/FC14. The negative value means that an interaction energy between different molecules is higher in magnitude than that among the same molecules. The interaction energies ( ε = RT/6) were calculated to be 165 J mol 1 for DPPG/FC12 and 198 J mol 1 for DPPG/FC14. This suggests that the interaction mode of the DPPG/FC12 system is almost the same as that of the DPPG/FC14 system, judging from the additivity of molecular areas (Fig. 2) and from the inclination of transition pressures ( eq / X FCn ). Accordingly, the resultant parameters ( and ε) show that the interaction between DPPG and FC12 molecules is also similar in magnitude to that between those of DPPG and FC14. The interaction parameters () calculated from the Joos equation (Eq. (1)) for the binary DPPG/FCn, dipalmitoylphosphatidylcholine (DPPC)/FCn (Nakahara et al., 2005a), and dimyristoylphosphatidylethanolamine (DMPE)/FCn (Yokoyama et al., 2009) systems are summarized in Table 1. All of the systems indicate that the phospholipids entirely tend to interact strongly with FC14 rather than FC12. Note that DPPG possesses the same hydrocarbon chain length (fully saturated) as DPPC. However, the DPPG/FCn systems indicate smaller values compared to the DPPC/FCn systems. This may be attributed to the following reasons. The first is the difference in spatial size (or bulkiness) of the polar headgroups between DPPG and DPPC (in fact, PG < PC). DPPG is possible to form a more ordered monolayer at high surface pressures. Thus, the magnitude of closepacked monolayer orientation is thought to be correlated with the interaction between phospholipids and FCn. The second is a charge effect of the headgroups. The pk a value of the phosphate group in phospholipids is estimated to be (Papahadjopoulos, 1968; Maltseva et al., 2006). Under the present condition of ph 2.0, the phosphate group of DPPG is fully protonated (98 100%, see Appendix A) and then DPPG monolayers have no charges. The phosphate group of DPPC is also almost protonated (98 100%) and DPPC monolayers are positively charged due to their choline group. As for FCn, it has been reported that the pk a of perfluorooctadecanoic acid (FC8) is 3.8 and the values of long-chain FCn (> 10) are expected to be similar or slightly lower than that of FC8 as they resemble structurally (Burns et al., 2008). Thus, the carboxyl group in FCn are Table 1 Interaction parameters () of the two-component DPPG/FCn, DPPC/FCn, and DMPE/FCn monolayers on 0.15 M NaCl (ph 2.0) at K. Systems Interaction parameter () n =12 n =14 n =16 DPPG/FCn DPPC/FCn a (0 < X FC16 0.3) DMPE/FCn b a Nakahara et al. (2005a). b Yokoyama et al. (2009).

8 110 H. Yokoyama et al. / Chemistry and Physics of Lipids 161 (2009) partially dissociated ( 87%) and FCn monolayers are in the anionic form. That is, electrostatic attraction makes DPPC polar headgroups interact more favorably with FCn ones. It is suggested that a certain intermolecular distance and mutual interaction force between polar headgroups are required as a miscibility factor between phospholipids and FCn Fluorescence microscopy The interfacial phase behavior of the binary DPPG/FCn monolayers has been systematically observed as a function of X FCn and surface pressure by the fluorescence microscopy (FM). Shown in Fig. 4 are FM images of the DPPG/FC12 monolayers. Pure DPPG monolayers indicate a homogeneously bright image corresponding to LE (disordered) phases below the transition pressure ( 3mNm 1 ). Beyond the transition pressure, the nucleation of LC (ordered) domains with a dark contrast occurs and then the domains becomes larger in size as surface pressure increases (Fig. 4(a)). Correspondingly, their occupied area in the images increases from 43 (at 5 mn m 1 ) to 70% (at 10 mn m 1 ). On further compression to the collapse pressure (at 54 mn m 1 ), the image becomes almost dark, corresponding to uniform LC or solid phases. Addition of small amount FC12 to DPPG undergoes a change in transition pressure of the ordered domain nucleation and in shape of the domains. The domain shape shifts somewhat to leaflike structures due to the variation in line tension of phase boundary between disordered and ordered domains. In the region of higher X FC12 (data not shown), the appearance of ordered domains shifts to higher surface pressure with decreasing X FC12 from 1 to 0.9, which is similar to the previous systems (Nakahara et al., 2005a; Yokoyama et al., 2009). These morphological phenomena provide complementary evidence of the miscibility between DPPG and FC12 in the monolayer state. As for the DPPG/FC14 monolayers in Fig. 5, FM images show a different morphological variation from those for the DPPG/FC12 systems. In the region of 0 X FC (Fig. 5(a)), the phase behavior (the domain shape in particular) is similar to that for the DPPG/FC12 system. With increasing molar fraction to X FC (Fig. 5(b) and (c)), however, the domain shape transforms into a circular domain. This behavior means that the contribution of line tension of the phase boundary to the domain shape considerably changes; that is, a dominative factor of the domain shape is expected to shift in line tension from negative (0 X FC14 0.1) to positive (0.1 < X FC14 0.3) as X FC14 increases. When FC14 is added further, an interesting phase morphology is observed in Fig. 5(d) and (e), which did not appear in the previous systems (Nakahara et al., 2005a; Yokoyama et al., 2009). The ringlike domains composed of bright contents and dark edges are formed and the organization becomes clearer in contrast with an increase in surface pressure. The bright contents and dark edges respectively consist of ordered DPPG and FC14 monolayers by atomic force microscopy. More detail analyses and discussions are made in the latter section. In this stage, the monolayer miscibility between DPPG and FC14 is at least supported morphologically. In the DPPG/FC16 system (Fig. 6), the phase morphologies are quite different from those of the DPPG/FC12 and DPPG/FC14 monolayers. The ordered domain nucleation occurs at the constant surface pressure of 3mNm 1 ( eq of DPPG alone) regardless of a variation in X FC16. In addition, each ordered domain is not separately Fig. 7. Typical FM images of the DPPG/FC18 monolayers for (a) X FC18 = 0.05, (b) X FC18 = 0.1, and (c) X FC18 = 0.2 at 5, 7, and 10 mn m 1. The monolayer contained 1 mol% fluorescent probe. The scale bar represents 100 m.

9 H. Yokoyama et al. / Chemistry and Physics of Lipids 161 (2009) dispersed but fuses one another at 5 and 7 mn m 1 (Fig. 6(b) and (c)). Then, further compression leads to formation of a successive network as seen at 10 mn m 1. The fusion of domains means a phase separation of two components (Nakahara et al., 2005a; Yokoyama et al., 2009). The FM images for the DPPG/FC18 system (Fig. 7) show the similar morphology to those for the DPPG/FC16 system. However, there is the following difference in FM morphology between the both systems. At X FCn = 0.05, the FM images of n =16(Fig. 6(a)) show the ordered domain of the circular shape. On the other hand, the successive network composed of ordered domains is formed in the system of n =18 (Fig. 7(a)). The difference suggests that the degree of immiscibility between DPPG and FC18 is more enhanced than that between DPPG and FC16. Nevertheless, the monolayer immiscibility for the systems of n = 16 and 18 is morphologically confirmed Atomic force microscopy The FM observations provide morphological evidence of the monolayer miscibility in terms of changes in transition pressure of the domain nucleation and in shape of the ordered domains against X FCn. However, there exist some molar fractions where ordered domains cannot be traced in the FM measurement due to its limitation of resolution and magnification. Therefore, AFM observations of Langmuir Blodgett (LB) films transferred onto mica have been performed to elucidate the miscibility between DPPG and FCn in the selected X FCn regions. Fig. 8 shows representative AFM height images for the DPPG/FCn systems (n = 12, 14, 16, 18) for X FCn = 0.7 (n = 12, 14) and 0.5 (n = 16, 18) at 5 and 30 mn m 1. Using the software (CS Chem3D Pro Ver. 5.0, CambridgeSoft Corp., MA), we can estimate hydrophobic chain lengths; 1.83 for DPPG, 1.18 for FC12, 1.41 for FC14, 1.65 for FC16, and 1.88 nm for FC18. The AFM image of X FC12 = 0.7 at 5 mn m 1, which is below the transition pressure (see Fig. 3(a)), exhibits two different phases; bright (higher) domain consists of disordered DPPG monolayers and the dark (lower) domain is made of disordered FC12 monolayers. This phase separation indicates weak interaction between DPPG and FC12 at lower surface pressures. On further compression to 30 mn m 1, which is beyond the transition pressure, both the domains are finely dispersed as seen in the corresponding crosssectional profile. The thickness in the cross-sectional images is almost the same as that of the estimated value (0.65 nm) under the assumption that monolayer components are in orientation perpendicular to the interface. For the DPPG/FC14 system (X FC14 = 0.7), the AFM image at 5 mn m 1 indicates the two contrasts similarly to the DPPG/FC12 system. However, the phase morphology is quite different from the DPPG/FC12 system. The bright region is composed of dispersed DPPG and FC12 (ordered) monolayers as seen in the cross-sectional image. Thus, judging from the estimated height difference between the two components (0.42 nm), dark regions are found to be disordered FC14 monolayers. Considering that FC14 alone forms an ordered monolayer without the transition pressure in the present condition, the interaction with DPPG liquefies the ordered monolayer (or disorder of FC14 monolayers). In fact, FC14 monolayers show the transition pressure of 7mNm 1 on different subphase condition of 0.15 M NaCl (ph 6) (data not shown). As surface pressure increases to 30 mn m 1, the size of the disordered FC14 domains becomes smaller and the finely dispersed DPPG and FC14 monolayer mainly occupy the image. As for the DPPG/FC16 system (X FC16 = 0.5), the AFM image at 5mNm 1 exhibits assemblies of dark dots (indicated by a straightline arrow), dark islands (indicated by a dashed arrow), and Fig. 8. Representative AFM topographic images of DPPG/FCn (n = 12, 14, 16, 18) for X FCn = 0.7 (n = 12, 14) and = 0.5 (n = 16, 18) at 5 and 30 mn m 1 on 0.15 M NaCl (ph 2.0) at K. The scan area is 5 m 5 m. The cross-sectional profiles along the scanning line (white line) are drawn below the each image. The height difference between the arrowheads is indicated in the cross sectional profile.

10 112 H. Yokoyama et al. / Chemistry and Physics of Lipids 161 (2009) Fig. 9. FM and AFM images of DPPG/FC14 for X FC14 = 0.5 at 5, 20, and 40 mn m 1 on 0.15 M NaCl (ph 2.0) at K. The scale bar in the FM image at 40 mn m 1 represents 100 m. The scan area of AFM images is 30 m 30 m. The cross-sectional profiles along the scanning line (white line) are drawn below the each image. The height difference between the arrowheads is indicated in the cross sectional profile. homogeneous bright regions. When the monolayer is further compressed up to 30 mn m 1, the dark dots and islands become smaller in size and number. Considering the reduction in size and number upon compression and the estimated height difference (FC16 is lower by 0.18 nm), the dark dots and islands are made mainly of FC16 monolayers. Unfortunately, the image does not provide information how DPPG interacts with FC16 at high surface pressures due to almost the same hydrophobic chain lengths of the two components. In the case of the DPPG/FC18 system (X FC18 = 0.5), the AFM images at 5 mn m 1 show dark circular domains with thickness of 1.5 nm. The circular domains are composed of FC18 and considerably grow in vertical thickness with increasing surface pressure to 30 mn m 1. This implies that FC18 forms three or four layers in the circular domain. The phenomenon was also observed in the previous DMPE/FC18 system (Yokoyama et al., 2009) and was induced by a characteristic property of the longer fluorinated chain of FC18 as reported for fluorinated alkanes (Krafft et al., 2001). The present AFM images ensure the complete immiscibility between DPPG and FC18 in the monolayer state. FM images and the corresponding AFM images of the DPPG/FC14 system at X FC14 = 0.5 are shown in Fig. 9. The images indicate a remarkable phase behavior that has never been reported, as far as the authors know. The FM images at each surface pressure resemble the corresponding AFM images, although there are some differences between FM and AFM in the experimental conditions (e.g., Langmuir or LB films, FM probe addition, magnification, and so on). This similarity bears out a good reproduction of Langmuir monolayers at the air water interface onto the solid support. The AFM image at 5 mn m 1 shows circular domains (bright) with a thickness of 2.2 nm. Considering the occupied molecular areas of the two components (X FC14 = 0.5) in the image and their difference in hydrophobic chain length, the circular domains and dark segments are composed of ordered DPPG FC14 monolayers and disordered FC14 monolayers, respectively. With increasing surface pressure to 20 mn m 1, the dark segments fuse one another to form a circular dark patch (indicated by straight-line arrows in FM and AFM images). The circular bright domains become smaller in size and are surrounded by a dark edge, which is indicated by dashed arrows. The edges are lower than the surroundings by 0.4 nm. This value is almost the same as the estimated height difference (0.42 nm) between DPPG and FC14. This means that ordered FC14 monolayers are concentrated in the circular edges. Upon further compression, the size of the circular (bright) domains become smaller and the dark patches of disordered FC14 monolayers also disappear at 40 mn m 1. It is suggested that the FC14 (ordered) components are extruded from the inside of circular domains into the edges on lateral compression and then the component in the circular domains are refined to ordered DPPG monolayers. That is, the appearance of the circular domains with dark edges means the limitation of miscibility between the two components. In the system of n =14, the gradual exclusion against surface pressure reveals the immiscibility of the two components in the middle X FC14 range in spite of the majority of mixed DPPG FC14 monolayers. These observations demonstrate that the binary DPPG/FC14 mixture can be assigned to a partially miscible system in the morphological aspect. 5. Conclusions The two-component monolayer miscibility for the DPPG/FCn (n = 12, 14, 16, 18) systems has been systematically investigated in the isothermal (thermodynamic) and morphological aspect. From the former analyses (macroscale), the four systems can be clearly

11 H. Yokoyama et al. / Chemistry and Physics of Lipids 161 (2009) classified into two patterns in terms of the variations in transition and collapse pressure with a molar fraction of FCn; miscible systems (n = 12 and 14) and immiscible systems (n = 16 and 18). On the other hand, FM and AFM images (submicro nanoscale) indicate specific phase behavior in the middle X FC14 range for the DPPG/FC14 system. The behavior reveals the existence of small patches of DPPG monolayers, which means the specific phase separation between DPPG and FC14. This phenomenon demonstrates that the DPPG and FC14 monolayer is assigned to a partially miscible system differently from the isothermal results. Considering a series of the two-component miscibility behavior of phospholipids and perfluorocarboxylic acids, a combination of hydrophobic chain lengths and a property of phospholipids polar headgroups are found to become significant miscibility factors. Namely, in the former case, FCn can be miscible with the phospholipids with the same or shorter hydrophobic chains. On the other hand, in the latter case, a certain intermolecular distance and mutual interaction force between polar headgroups of phospholipids and FCn are required to be miscible each other. In particular, the species of phospholipid polar headgroups (PG, PC, and PE) contributes to a variation in phase morphology of the binary monolayers. These results will become quite useful for industrial areas, biomedical fields, environmental sciences, technologies, and so on. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS). This work was also supported by a Grant-in-Aid for Young Scientists from JSPS (H.N.). Appendix A. The monolayer ionization degree ( ) is determined by the proton concentration at the surface ([H + ] s ) and the surface equilibrium constant for the acid group dissociation (K s ): 1 = K s [H + (2) ] s If the proton concentration in the bulk ([H + ] b ) is known, the surface proton concentration can be calculated using the Boltzmann equation: ( [H + ] s = [H + ] b exp zeϕ 0 kt ) whsere z denotes the charge number of monolayers, e is the elementary electric charge, and ϕ 0 is the potential difference of the ionic layer. Furthermore, the Gouy Chapman approach (Gaines, 1966; Maltseva et al., 2006) is used to relate ϕ 0 to the charge density ( e/a) under the condition of NaCl solution at K: ϕ 0 = 2kT ( 1.37 ) e sinh 1 A (4) c where c (in mol/l) is the 1 1 electrolyte concentration. The following equation is derived from combining Eqs. 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