Phase Behavior of an Equimolar Mixture of N-Palmitoyl-d-erythro

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Phase Behavior of an Equimolar Mixture of N-Palmitoyl-D-erythro-sphingosine, Cholesterol, and Palmitic Acid, a Mixture with Optimized Hydrophobic Matching Elana Brief,† Sungjong Kwak,§ John T. J. Cheng,‡ Neil Kitson, Jenifer Thewalt,*,†,‡ and Michel Lafleur*,§

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† Department of Physics and ‡Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6, §Department of Chemistry, Center for Self-Assembled Chemical Systems, Universit e de Montr eal, C.P. 6128, Succ. Centre Ville, Montr eal, Qu ebec, H3C 3J7 Canada, and Department of Dermatology and Skin Science, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4E8

Received January 28, 2009. Revised Manuscript Received March 17, 2009 The phase behavior and lipid mixing properties of an equimolar mixture of nonhydroxylated palmitoyl ceramide (Cer16), palmitic acid (PA), and cholesterol have been investigated using 2H NMR and vibrational spectroscopy. This mixture is formed by the three main classes of lipids found in the stratum corneum (SC), the top layer of the epidermis, and provides an optimized hydrophobic matching. Therefore, its behavior highlights the role played by hydrophobic matching on the phase behavior of SC lipids. We found that, below 45 °C, the mixture is essentially formed of coexisting crystalline domains with a small fraction of lipids (less than 20%) that forms a gel or fluid phase, likely ensuring cohesion between the solid domains. Upon heating, there is the formation of a liquid ordered phase mainly composed of PA and cholesterol, including a small fraction of Cer16. This finding is particularly highlighted by correlation vibrational microspectroscopy that indicates that domains enriched in cholesterol and PA include more disordered Cer16 than those found in the Cer16-rich domains. Solubilization of Cer16 in the fluid phase occurs progressively upon further heating, and this leads to the formation of a nonlamellar self-assembly where the motions are isotropic on the NMR time scale. It is found that the miscibility of Cer16 with cholesterol and PA is more limited than the one previously observed for ceramide III extracted from bovine brain, which is heterogeneous in chain composition and includes, in addition to Cer16, analogous ceramide with longer alkyl chains that are not hydrophobically matched with cholesterol and PA. Therefore, it is inferred that, in SC, the chain heterogeneity is a stronger criteria for lipid miscibility than chain hydrophobic matching.

Introduction The organization of the lipids present in the stratum corneum (SC), the top layer of the skin, has been shown to be largely responsible for the skin impermeability.1,2 This vital property is attributed to the significant proportion of crystalline or solid lipids. The presence of lipids in the crystalline phase with orthorhombic packing has been demonstrated using several techniques including X-ray diffraction,3-5 atomic force microscopy,6 and infrared spectroscopy.7,8 SC lipids form a complex mixture, and simplified models have been investigated in order to provide a detailed understanding of the rules dictating the formation of the solid phases. Most of the model mixtures included ceramides, free fatty acids, and sterol, as these are the *Corresponding authors. (M.L) Fax: (514) 343-7586. E-mail: michel. [email protected]. (J.T.) Fax: (778) 782-3592. E-mail: [email protected]. (1) Elias, P. M. J. Invest. Dermatol. 1983, 80, 44s. (2) Elias, P. M.; Menon, G. K. Adv. Lipid Res. 1991, 24, 1. (3) Bouwstra, J. A.; Gooris, G. S.; van der Spek, J.; Bras, W. J. Invest. Dermatol. 1991, 97, 1005. (4) Bouwstra, J. A.; Gooris, G. S.; Salomons-de Vries, M. A.; van der Spek, J. A.; Bras, W. Int. J. Pharm. 1992, 84, 205. (5) Hatta, I.; Ohta, N.; Inoue, K.; Yagi, N. Biochim. Biophys. Acta 2006, 1758, 1830. (6) Chen, Y.-L.; Wiedmann, T. S. J. Invest. Dermatol. 1996, 107, 15. (7) Ongpipattanakul, B.; Francoeur, M. L.; Potts, R. O. Biochim. Biophys. Acta 1994, 1190, 115. (8) Mendelsohn, R.; Flach, C. R.; Moore, D. J. Biochim. Biophys. Acta 2006, 1758, 923. (9) Schurer, N. Y.; Elias, P. M. Adv. Lipid Res. 1991, 24, 27.

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three main components of SC lipids.9-12 For each species, the models were more or less elaborate, depending on the goal of the study and the techniques that were used. The formation of the crystalline phase with orthorhombic chain packing is a property that can be associated to ceramides, as it has been shown that some pure ceramides could form this type of phase.13,14 However, it was shown that at least five ceramide species were required to reproduce the two lamellar spacings observed by X-ray diffraction.15-18 Several model mixtures included a single free fatty acid, while mixtures of fatty acids reproducing more closely the SC composition were used in others (e.g., refs 19 and 20). It was (10) Wertz, P. W.; Miethke, M. C.; Long, S. A.; Strauss, J. S.; Downing, D. T. J. Invest. Dermatol. 1985, 84, 410. (11) Downing, D. T.; Stewart, M. E.; Wertz, P. W.; Colton, S. W.; Abraham, W.; Strauss, J. S. J. Invest. Dermatol. 1987, 88, 2s. (12) Wertz, P. W. Acta Derm.-Venereol. 2000, Supplementum 208, 7. (13) Mendelsohn, R.; Moore, D. J. Methods Enzymol. 2000, 312, 228. (14) Moore, D. J.; Rerek, M. E.; Mendelsohn, R. J. Phys. Chem. B 1997, 101, 8933. (15) McIntosh, T. J.; Stewart, M. E.; Downing, D. T. Biochemistry 1996, 35, 3649. (16) Bouwstra, J. A.; Dubbelaar, F. E. R.; Ponec, M.; Gooris, G. S. J. Lipid Res. 2001, 42, 1759. (17) de Jager, M. W.; Gooris, G. S.; Dolbnya, I. P.; Bras, W.; Ponec, M.; Bouwstra, J. A. Chem. Phys. Lipids 2003, 124, 123. (18) de Jager, M.; Gooris, G. S.; Ponec, M.; Bouwstra, J. A. J. Lipid Res. 2005, 46, 2649. (19) de Jager, M. W.; Gooris, G. S.; Dolbnya, I. P.; Bras, W.; Ponec, M.; Bouwstra, J. A. J. Lipid Res. 2004, 45, 923. (20) Norlen, L.; Plascencia Gil, I.; Simonsen, A.; Descouts, P. J. Struct. Biol. 2007, 158, 386.

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recently shown21 that the chain length of the free fatty acid was a major factor dictating the lipid mixing and the phase behavior of ternary mixtures of nonhydroxylated ceramides, free fatty acids, and cholesterol. Finally, cholesterol has been widely used in mixtures mimicking SC lipids, as it is the most abundant sterol. Nevertheless, some mixtures also included cholesterol sulfate, with this sterol being a minor component in SC and for which an accumulation leads to severe skin pathologies.22,23 The presence of cholesterol sulfate was proposed to inhibit the formation of solid domains.24,25 Hydrophobic matching is a critical determinant of the mixing properties of lipids. The importance of this phenomenon is well established in phospholipid mixtures.26,27 The hydrophobic match has also been shown to be central in the phase behavior of mixtures of free fatty acids and cholesterol,28 two main components of SC membranes. In the present paper, our goal is to examine the phase properties of a simple model system that includes representatives of the three main SC lipid classes and for which hydrophobic matching is optimized. In order to hydrophobically match cholesterol (chol), ceramide bearing a nonhydroxylated palmitoyl alkyl chain (Cer16), and palmitic acid have been used. These species are present in the SC, even though they are not the most abundant of their kind. Palmitic acid represents up to 9% (w/w) of the SC free fatty acids, with the most abundant being docosanoic (C22), tetracosanoic (C24), and hexacosanoic (C26) acids.29,30 Similarly, only 3% (w/w) Cer16 is found in SC nonhydroxylated ceramide; Cer18 and Cer24 are the most abundant, corresponding to 30 and 26% (w/w), respectively.31 Nevertheless, the Cer16/PA/chol mixture constitutes an interesting model to assess the impact on membrane structure of hydrophobic matching in SC lipids, as the three lipid species display a “matched hydrophobic length”. It must be pointed out that, in model mixtures that reproduce the formation of crystalline phase with orthorhombic chain packing, a significant proportion (between 10 and 20%) of both ceramides and fatty acids has been found to form more fluid phases.21,32,33 These observations support the hypothesis that some noncrystalline phase lipids must exist to hold together the crystalline domains and to provide pathways across the SC intercellular membranes.34,35 Our conjecture upon embarking on this project was that hydrophobically matched lipids may be suited for forming such noncrystalline phases. We have thus investigated the thermal behavior of the equimolar Cer16/PA/ chol mixture using 2H NMR and vibrational spectroscopy to (21) Chen, X.; Kwak, S.; Lafleur, M.; Bloom, M.; Kitson, K.; Thewalt, J. Langmuir 2007, 23, 5548. (22) Williams, M. L. Adv. Lipid Res. 1991, 24, 211. (23) Zettersten, E.; Man, M.-Q.; Sato, J.; Denda, M.; Farrell, A.; Ghadially, R.; Williams, M. L.; Feingold, K. R.; Elias, P. M. J. Invest. Dermatol. 1998, 111, 784. (24) Arseneault, M.; Lafleur, M. Biophys. J. 2007, 92, 99. (25) Bouwstra, J. A.; Gooris, G. S.; Dubbelaar, F. E. R.; Ponec, M. J. Lipid Res. 1999, 40, 2303. (26) Mendelsohn, R.; Liang, G. L.; Strauss, H. L.; Snyder, R. G. Biophys. J. 1995, 69, 1987. (27) Cevc, G.; Marsh, D. Phospholipid Bilayers: Physical Principles and Models; Wiley: New York, 1987. (28) Ouimet, J.; Lafleur, M. Langmuir 2004, 20, 7474. :: (29) Norlen, L.; Nicander, I.; Lundsjo, A.; Cronholm, T.; Forslind, B. Arch. Dermatol. Res. 1998, 290, 508. (30) Nicollier, M.; Massengo, T.; Remy-Martin, J.-P.; Laurent, R.; Adessi G.-L. J. Invest. Dermatol. 1986, 87, 68. (31) Bouwstra, J. A.; Gooris, G. S.; Cheng, K.; Weerheim, A. M.; Bras, W.; Ponec, M. J. Lipid Res. 1996, 37, 999. (32) Fenske, D. B.; Thewalt, J. L.; Bloom, M.; Kitson, N. Biophys. J. 1994, 67, 1562. (33) Bouwstra, J. A.; Thewalt, J.; Gooris, G. S.; Kitson, N. Biochemistry 1997, 36, 7717. (34) Forslind, B. Acta Derm.-Venereol. 1994, 74, 1. (35) Kitson, N.; Thewalt, J. L. Acta Derm.-Venereol. Suppl. 2000, 208, 12.

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provide a detailed description of the involvement of each lipid in the various existing phases. As a consequence, we have disproved our original conjecture and instead revealed the complexity of the mixing behavior of the lipids in these membranes.

Materials and Methods Cer16 and its palmitoyl-chain-perdeuterated analogue (Cer16d31) were purchased from Northern Lipids Inc. (Vancouver, BC). Palmitic Acid (PA) and cholesterol were obtained from Sigma Chemical Co. (St. Louis, MO). Perdeuterated palmitic acid (PAd31) was purchased from CDN Isotopes (Pointe-Claire, Quebec) or obtained from the catalytic deuteration of PA, according to an established procedure.36 2 H NMR. Equimolar quantities of Cer16 (hydrogenated or deuterated), PA (hydrogenated or deuterated), and cholesterol were weighed, mixed together codissolved in benzene/methanol, 95:5 (v/v), and freeze-dried. The dry lipid powders were then hydrated with a large excess of buffer prepared in deuteriumdepleted water containing 150 mM NaCl, 4 mM EDTA, and 100 mM citrate at pH 5.2, a pH typical of the SC.37,38 Due to the inherently low signal-to-noise ratio of 2H NMR acquisition, each sample contained 200 mg of lipid, in which either PA or Cer was deuterated. After hydration, the samples were transferred to NMR sample tubes (Li-Chan Mechanical Company Ltd., Taiwan), and, to prevent buffer evaporation, the threads at the top of the tubes were wrapped with Teflon tape and the tubes were sealed with Parafilm. Each sample was incubated in a 33 °C water bath for 2-3 weeks and then stored at 37 °C until the sample was fully equilibrated, typically an additional 6 weeks. The state of equilibration was determined by measuring the percentage of lipids in the solid phase using 2H NMR. The sample was considered fully equilibrated when the percentage of solid phase was above 80% (for PA-d31) and 90% (for Cer16-d31) and was stable over a period of days. 2 H NMR spectra were acquired using a quadrupolar echo acquisition sequence39 with a 90° pulse of 3.95 μs and interpulse delay of 40 μs. Spectra of membranes containing crystalline lipids were recorded using both long and short (300 ms) repetition times (reptime) to determine the proportion of nonsolid deuterated lipid (see the Supporting Information). To fully measure the presence of slow-relaxing solid components, the long reptime was set to five times the T1 of the spectral component with the slowest relaxation: 50 s for solid PA-d31 and 30 s for solid Cer16-d31. Spectra were acquired from 25 to 80 °C, usually with increments of 1 °C: typically two short reptime spectra of 10 000 averages that sandwiched the long reptime acquisition of 1000 averages. Above the melting temperature of the crystalline labeled lipid, only the short reptime was used. From the short- and long-reptime spectra, it was possible to determine how the deuterated species distribute among the different phases. We calculated the percentage of the deuterated lipid (PA-d31 or Cer16-d31) existing in a solid (%solid), a gel or fluid lamellar (%fluid), and an isotropic (%iso) phase. The method to extract these compositions is described in detail in the Supporting Information. The average spectral width (M1) was calculated from each of the spectra as a function of temperature.40 Vibrational Spectroscopy. The procedure for weighing, freeze-thawing, and hydrating the lipid mixtures used in the vibrational spectroscopy experiments was similar to the procedure for 2H NMR except that a MES buffer (100 mM MES, 100 mM NaCl, 5 mM EDTA) prepared in D2O was used to avoid (36) Hsiao, C. Y. Y.; Ottaway, C. A.; Wetlaufer, D. B. Lipids 1974, 9, 913. (37) Ohman, H.; Vahlquist, A. Acta Derm.-Venereol. 1994, 74, 375. (38) Wilhelm, D.; Elsner, P.; Maibach, H. I. Acta Derm.-Venereol. 1991, 71, 123. (39) Davis, J. H.; Jeffrey, K. R.; Bloom, M.; Valic, M. I.; Higgs, T. P. Chem. Phys. Lett. 1976, 42, 390. (40) Hsueh, Y. W.; Giles, R.; Kitson, N.; Thewalt, J. Biophys. J. 2002, 82 3089–95.

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spectral interferences. The final overall lipid concentration was about 10 mg/mL. The state of equilibration of the samples was verified by the appearance of the splitting of the δCH2 and δCD2 band, and the amide I0 band shape (see the Results section for a detailed discussion on these bands). The 40 °C incubation was necessary to achieve stable ceramide crystalline domains, which showed the maximum splitting of the δCH2 band and the characteristic two components amide I0 band. During this period, PA-d31 crystalline domains were partially destabilized, as assessed by the appearance of a component at 1089 cm-1 in the δCD2 region. A subsequent incubation at 25 °C led to the recrystallization of PA-d31 with an orthorhombic chain packing, without affecting the ceramide bands in the spectra. At the end of the sample preparation, a waxy layer of the lipid particles was formed on the tube wall in a clear supernatant. For IR spectroscopy, some waxy particles were squeezed in between two CaF2 or BaF2 windows that were separated by a 5 μm thick Teflon spacer. The spectra were acquired on a BioRad FTS-25 spectrometer. Forty scans were coadded with a 2 cm-1 resolution for each spectrum, and the spectra were recorded from low to high temperatures, with a temperature-equilibration period of 6 min. The interferograms were Fourier transformed using a triangular apodization function with two levels of zero filling, leading to a digital resolution of 0.48 cm-1/point. The spectra were treated using GRAMS software (Galactic Industries Corporation). The positions reported here corresponded to the center of gravity of the top 5% of the band. For Raman spectroscopy, the waxy lipid particles were transferred on a round glass cover that was then mounted as the top of a container filled with buffer. This container included a Teflon cylinder that squeezed the lipid sample on the glass cover. This assembly was sealed with o-rings, and its temperature was regulated within (1 °C. This setup allowed the acquisition of Raman spectra on fully hydrated samples at well-defined temperatures. The Raman spectra were recorded using a Renishaw Raman imaging microscope WiRE (V 1.2, System 2000) (Renishaw, Gloucestershire, U.K.) containing a holographic grating (1800 grooves mm-1), a Leica microscope equipped with a long-working-distance 50 objective, and a Peltier-cooled CCD detector (600  400 pixels). The 514 nm line of an argon ion (Ar+) laser was used as the excitation source, with a power at the sample of ∼15 mW. With the optical setup used for the data collection, the size of the beam at the sample was ∼2.5 μm, as measured from the diameter of its reflection off a silicon surface. The configuration led to a spectral resolution of ∼2 cm-1. For the mapping, spectra were recorded between 1900 and 3200 cm-1. The acquisition of each spectrum required ∼30 s, and the digital resolution was one data point/1.6 cm-1. The sample was moved by a computercontrolled translational stage. Surfaces of 40  40 μm2 were scanned by moving the sample by 3 μm steps. Therefore, 196 spectra were recorded over the surface, for a total acquisition time of ∼2 h. The mapping was performed every 10 °C between 25 and 75 °C. The data analysis and the map generation were performed using GRAMS/32 (Galactic Industries Corp., Salem, NH) and the WiRE software (version 1.3) (Renishaw Spectroscopy Products Division, Gloucestershire, U.K.). The Raman intensities at a given wavenumber were measured from the baseline, directly on Table 1. Restrictions Applied for the Band Simulations of the C-D Stretching Region band no. 1 2 3 4 5 6 7 8

position (cm-1)

width (cm-1)

2051-2054 2070-2075 2097-2105 2123-2125 2145-2155 2167-2177 2192-2199 2212-2217

10-20 10-20 10-45 10-25 10-20 10-25 10-20 10-20

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the spectra. For the determination of the width of the CD stretching component at 2100 cm-1, the C-D stretching region, between 2019 and 2276 cm-1, was simulated with eight bands and the full width at half-height of the component at ∼2100 cm-1 corresponded to the reported ΔνCD. In order to obtain reasonable and reproducible numerical fits, the restrictions indicated in Table 1 were imposed on the least-squares fits. The height and proportion of the Gaussian/Lorentzian function for the band shape were left unrestricted. The full width at half-height of the CD symmetric stretching mode at 2100 cm-1 (ΔνCD) was estimated by the width at half-height of the simulated component at ∼2100 cm-1 (band 3).

Results H NMR. H NMR spectroscopy of hydrated Cer16/PA/chol equimolar mixtures was performed on membranes containing either deuterated PA or deuterated ceramide, in turn. This approach allowed us to characterize the phase behavior of these two lipid species in the mixture. Typical spectra are presented in Figure 1, and they were valuable for phase identification. The spectrum of the Cer16/PA-d31/chol mixture recorded at 25 °C displays a profile typical of a solid fatty acid.41 The signal is composed of two powder patterns with quadrupolar splittings of 114 and 35 kHz (measured between the 90° maxima); these are assigned to the methylene and terminal methyl deuterons, respectively. The fatty acid chain was essentially immobile, leading to practically identical (and therefore unresolved) powder patterns for the CD2 groups. Because of its rotation along the last C-C bond of the chain, the CD3 exhibited a reduced quadrupolar splitting. At 50 °C, the spectrum shows a signal characteristic of a liquid ordered (lo) phase observed in cholesterol-rich bilayers.28,42,43 It is composed of several overlapping powder patterns typical of the gradient of orientational order along the alkyl chains. The reduction of the quadrupolar splittings compared to those at 25 °C was in large part associated with the introduction of an axially symmetric rotation of PA-d31 along its long axis. The largest quadrupolar splitting was 46 kHz, a value characteristic of systems in lo phases. Upon further heating (see Figure 1, 74 °C), the spectrum is dominated by a narrow central peak, associated with a phase having isotropic motion on the NMR time scale. Therefore, in the Cer16/PA/chol equimolar mixtures, PA molecules experienced transitions from a solid to lo phase and then a lo to isotropic phase. The phase behavior of Cer16 was also determined by 2H NMR in the Cer16-d31/PA/chol equimolar mixture. At 25 °C, the spectrum, similar to that for PA-d31, is a very wide signal corresponding to practically immobile all-trans chains. The 90° maxima are not as sharp as those of the deuterated fatty acid, an observation that may be explained by some chain motions in the synthetic ceramide. Since the ceramide’s palmitoyl chain is attached to the sphingosine backbone, it is likely that its solid packing is intrinsically somewhat looser than that of PA. At 50 °C, the spectrum consists of an overlapping broad signal and a pattern typical of a lo phase, indicating the coexistence of solid and lo phase ceramides. In these specific conditions, we determined that about 60% of the ceramide molecules were in the solid phase while the remainder was in a fluid lipid matrix (see the Supporting Information). Upon heating to 74 °C, the 2H NMR spectrum of the Cer16-d31/PA/chol mixture includes a narrow central peak, indicating, as in the case of PA-d31, the formation of an isotropic phase; however, the fraction of Cer16-d31 contributing to the central peak is considerably smaller. Therefore, ceramide experienced a phase behavior generally similar to that of 2

2

(41) Davis, J. H. Biochim. Biophys. Acta 1983, 737, 117. (42) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451. (43) Lafleur, M.; Cullis, P. R.; Bloom, M. Eur. Biophys. J. 1990, 19, 55.

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Figure 2. Thermal evolution of the first moments extracted from the 2H NMR spectra of Cer16/PA-d31/Chol (b) and Cer16-d31/PA/ Chol (0) mixtures.

Figure 1. Thermal evolution of the 2H NMR spectra of Cer16/ PA-d31/chol (left) and Cer16-d31/PA/chol (right) mixtures.

PA, undergoing transitions between the solid and the lo phase and then the lo and the isotropic phase, except that its involvement in the lo phase was more limited at 50 °C and its isotropic component was less at 74 °C. In order to provide a more detailed picture of the thermal evolution of the phase composition of Cer16/PA/chol, the first moments (M1) of the 2H NMR spectra were plotted as a function of temperature (Figure 2). In the case of PA-d31, M1 was relatively constant between 25 and 42 °C. There was an abrupt drop at ∼45 °C; the M1 values decreased by about 50%. M1 was then roughly constant between 45 and 65 °C. Heating above 65 °C led to a progressive decrease of M1. In the Cer16-d31/PA/chol mixtures, M1 remained constant between 25 and 48 °C at a value similar to that of the corresponding deuterated PA-d31. At 48 °C, M1 dropped abruptly by about 20%. The M1 values were relatively stable between 50 and 60 °C, and then decreased progressively upon further heating. Quantitative determination of the distribution of the deuterated lipid species among the different phases (solid, lo/gel, and isotropic) was carried out (see the Supporting Information for details). Gel phase membranes lack the fast axially symmetric lipid rotation of the lo phases but have considerably more motional freedom than solid phase membranes. The temperature dependence of the proportions of PA-d31 and Cer16-d31 in the solid, lo/gel, and isotropic phases is shown in Figure 3 (for simplicity, we are not distinguishing between lo and gel phases). Below 40 °C, most of the lipids were in the solid phase. However, there was always a portion (between 10 and 20%) of Cer16 and PA that remained in a fluid or gel phase. The melting of the mixture at ∼45 °C involved both Cer16 and PA, but a higher proportion of PA compared to Cer16 became disordered over a narrow temperature range. For instance, at 55 °C, greater than 90% of PA was in a fluid phase, whereas this phase included only 7526 DOI: 10.1021/la9003643

35% Cer16, with the remainder being still in a solid phase. It is also shown that, even at 70 °C, there was a fraction of Cer16 that remained in the solid phase. Finally, these figures show that the isotropic phase giving rise to the narrow central peak involved, at 70 °C, about 40% PA and 15% Cer16. IR Spectroscopy. Figure 4 shows the thermal behavior of the Cer16/PA-d31/chol mixtures as probed by the position of the methylene symmetric stretching band, the νCH band for Cer16, and the νCD band for PA-d31. Between 25 and 40 °C, the νCH band was located below 2850 cm-1, indicating that acyl chains were highly ordered at this temperature range.44,45 Upon heating, the νCH band showed a small shift toward higher wavenumber between 42 and 52 °C. The νCH band was at ∼2850 cm-1 at 55 °C, which indicates that a large fraction of the ceramide molecules was still in a highly ordered phase. This was followed by a progressive increase that became significantly steeper above 60 °C. The νCH value reached, at 75 °C, ∼2853 cm-1, suggesting a melting of the solid ceramide to a disordered phase. The value did not reach a plateau though, and the position of the band was slightly lower than typical values for a disordered phase (∼2854 cm-1), indicating that the transition to a disordered phase was not yet complete. Below 35 °C, the frequency of the νCD band was lower than 2090 cm-1, indicating ordered acyl chains for PA-d31.45,46 Upon heating, the νCD band showed an initial shift at 55 °C to ∼2092 cm-1, corresponding to the value previously observed when the fatty acid formed a liquid ordered (lo) phase in similar ternary lipid mixtures with bovine brain ceramide45-47 or with the presence of only cholesterol.48,49 This initial increase was followed by a rather gradual shift to ∼2095 cm-1, an indication of further disordering of the lo phase. At 75 °C, the frequency of the band was lower than the value typically observed for a disordered phase (∼2096 cm-1), indicating that the order-disorder transition of PA-d31 was not complete. The splitting of the CD2 deformation (δCD2) band was observed for the Cer16/PA-d31/chol mixtures at low temperature (spectra not shown), giving rise to two components at ∼1086 and ∼1092 cm-1. Because the splitting is due to interchain coupling, (44) Moore, D. J.; Rerek, M. E.; Mendelsohn, R. Biochem. Biophys. Res. Commun. 1997, 231, 797. (45) Lafleur, M. Can. J. Chem. 1998, 76, 1501. (46) Chen, H.-C.; Mendelsohn, R.; Rerek, M. E.; Moore, D. J. Biochim. Biophys. Acta 2001, 1512, 345. (47) Velkova, V.; Lafleur, M. Chem. Phys. Lipids 2002, 117, 63. (48) Pare, C.; Lafleur, M. Langmuir 2001, 17, 5587. (49) Ouimet, J.; Croft, S.; Pare, C.; Katsaras, J.; Lafleur, M. Langmuir 2003, 19, 1089.

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Figure 3. Phase distribution of PA (a) and Cer16 (b) in the equimolar Cer16/PA/chol mixture as a function of temperature as inferred from the 2H NMR spectra.

its presence indicated that the PA-d31 in this mixture formed crystalline domains with an orthorhombic chain packing, composed almost exclusively of PA-d31.50-53 The domains must include at least 100 molecules, since the splitting was similar to that obtained with pure PA-d31. Upon heating, a peak at ∼1089 cm-1 grew while the intensity of the two peaks at ∼1086 and ∼1092 cm-1 decreased. Moreover, the splitting became smaller, indicating a decrease of the size and/or the PA-d31 content of the orthorhombic crystalline domains. For temperatures above 55 °C, the δCD2 deformation region was dominated by a broad band centered at ∼1089 cm-1. The CH2 deformation (δCH2) band also showed splitting (data not shown) at low temperature, indicating that ceramides were also in an orthorhombic crystalline phase. In this case, because of the overlap with the contribution from the δCH2 band of cholesterol and the amide II0 band of Cer16, the splitting components were not as well resolved as in the case of δCD2 (50) Snyder, R. G.; Goh, M. C.; Srivatsavoy, V. J. P.; Strauss, H. L.; Dorset, D. L. J. Phys. Chem. 1992, 96, 10008. (51) Snyder, R. G.; Srivatsavoy, V. J. P.; Cates, D. A.; Strauss, H. L.; White, J. W.; Dorset, D. L. J. Phys. Chem. 1994, 98, 674. (52) Snyder, R. G.; Strauss, H. L.; Cates, D. A. J. Phys. Chem. 1995, 99, 8432. (53) Moore, D. J.; Snyder, R. G.; Rerek, M. E.; Mendelsohn, R. J. Phys. Chem. B 2006, 110, 2378.

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Figure 4. Thermal evolution of the acyl chain order of the equimolar Cer16/PA-d31/chol mixtures as probed, from IR spectroscopy, by (9) the νCH band position (to describe Cer16) and (b) the νCD band position (to describe the fatty acid). The two y-scales were selected to provide the same amplitude for a gel-to-liquid crystalline phase transition.

band. We could, however, estimate that the crystalline phase disappeared between 35 and 55 °C, as seen by the narrowing of the δCH2 band. This transition, based on the narrowing of δCH2 and DOI: 10.1021/la9003643

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Figure 5. Thermal evolution of the acyl chain order of the equimolar Cer16/PA-d31/chol mixtures as probed, from Raman spectroscopy, by (9) I2880/I2850 intensity ratios (to describe Cer16) and (b) ΔνCD band (to describe PA-d31). The two y-scales were selected to provide the same amplitude for a gel-to-liquid crystalline phase transition.

the position of the νCH band, was consistent with the previously reported solid-solid transition from an orthorhombic to a hexagonal chain packing.46,54 Raman Spectroscopy. Figure 5 presents the evolution of the hydrocarbon chain order of the fatty acid and ceramide in the Cer16/PA-d31/chol mixture as a function of temperature. For PA-d31, the hydrocarbon chain order was characterized by the full width at half-height of the CD symmetric stretching mode at 2100 cm-1 (ΔνCD), whereas that of ceramide was probed by the intensity ratio of the Raman signal at 2880 cm-1 to that at 2845 cm-1 (I2880/I2845); these spectral features are known to be sensitive to the degree of the lipid alkyl chain ordering and have been commonly used to study the thermal phase behavior of phospholipids.55-57 For each temperature, these parameters were measured on the spectrum resulting from the averaging of the 196 spectra recorded over an area of 40  40 μm2. Consequently, they should be representative of the overall behavior of the mixture. At 25 °C, the I2880/I2845 value was ∼1.4, which is typical for gel phase phospholipid vesicles, indicating ordered ceramide acyl chains. Above 40 °C, the I2880/I2845 value decreased progressively as a function of increasing temperature, an indication of the disordering of the Cer16 chain. At 75 °C, the I2880/I2845 value reached ∼1.1; this value was slightly higher than those typically reported for phospholipid liquid disordered phases (I2880/I2845 = 0.9).56,58 This observation agreed well with the conclusion inferred from the IR and 2H NMR results, stating that not all the ceramide hydrocarbon chains were completely disordered at 75 °C. In particular, 2H NMR indicates that, at 75 °C, 70% of the ceramide chains are in the lo phase and the remainder highly disordered, that is, undergoing isotropic motion (Figure 3b). The ΔνCD value was ∼15 cm-1 at 25 °C, a value consistent with the high chain order expected for solid phase PA-d31. The ΔνCD value increased sharply between 45 and 55 °C, probing the disordering of the PA-d31 hydrocarbon chains. The width reached ∼30 cm-1 at 75 °C, indicating that PA-d31 was primarily in a disordered phase. Again, this agrees with the 2H NMR result: at 75 °C, 62% (54) Chen, H.-C.; Mendelsohn, R.; Rerek, M. E.; Moore, D. J. Biochim. Biophys. Acta 2000, 1468, 293. (55) Bunow, M. R.; Levin, I. W. Biochim. Biophys. Acta 1977, 487, 388. (56) Huang, C.; Mason, J. T.; Levin, I. W. Biochemistry 1983, 22, 2775. (57) Mendelsohn, R.; Koch, C. C. Biochim. Biophys. Acta 1980, 598, 260. (58) Kouaouci, R.; Silvius, J. R.; Graham, I.; Pezolet, M. Biochemistry 1985, 24, 7132.

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of the PA-d31 is undergoing isotropic motion, with the remainder being lo phase (Figure 3a). To investigate the spatial distribution of each component in the equimolar Cer16/PA-d31/chol mixture, a similar but simplified analysis of the spectra as previously described24 was performed. Areas of 40  40 μm2 were investigated by recording 196 (14  14) spatially resolved Raman spectra. For each sampling element, we performed two integrations. The first, between 2780 and 3100 cm-1, corresponds to the CH stretching region (ACH), associated with both ceramide and cholesterol. The second, between 2015 and 2250 cm-1, is the CD stretching region (ACD), associated with PA-d31. The areas of the two regions were not equal due to the different Raman activities of these vibration modes. In addition, the area of a given region varied as a function of temperature, a consequence of the change in chain order. Therefore, we normalized the area values by dividing them by their average value, calculated from the 196 spectra over the investigated region. It led to normalized areas of the CH (AnCH) and CD (AnCD) stretching regions, where n stands for normalized. To describe the proportion of PA-d31 relative to the total lipid content in each sampling element (FPA-d31), the normalized area of the CD stretching regions was divided by the total of the normalized areas of the CH and CD stretching regions, assuming that the relative Raman activity coefficients of the integrated C-H and C-D stretching regions were not considerably affected by the acyl chain order variations. FPAd31 ¼

AnCD þ AnCH

AnCD

ð1Þ

FPA-d31 larger than 0.5 indicated a sampling element relatively enriched in PA-d31, while a value lower than 0.5 indicated a PA-d31-depleted sampling element. Second, we estimated the proportion of Cer16 in the sampling elements. The peak at 2845 cm-1 corresponds to the symmetric CH2 stretching vibration mode of ceramide and cholesterol. However, cholesterol’s Raman spectrum was dominated in the CH stretching region by a band at 2930 cm-1 while the contribution at this wavenumber was much less intense in the spectrum of Cer16. From the intensities of these two spectral features (I2930 and I2845), it was possible to calculate FCer: F Cer ¼

I 2845 I 2845 þ I 2930

ð2Þ

This Raman intensity ratio was found to be mainly sensitive to the Cer16 content relative to the (Cer16 + cholesterol) content. For example, at 25 °C, its value varied from 0.67 for pure Cer16 to 0.46 for pure cholesterol. The average value of FCer for the Cer16/PAd31/chol mixture was 0.60 and did not vary considerably ((0.01 over the investigated temperature range). Note that, in contrast to FPA-d31 that was relative to the total lipid content (ceramide + cholesterol + PA-d31), FCer was relative to the sum of the ceramide and cholesterol contents only. This allowed us to estimate the regional composition of the remaining lipids when the PA-d31 content had been determined. Figure 6 shows the thermal evolution of the distribution of the individual values of FPA-d31 (left panel) and FCer (right panel) for the equimolar Cer16/PA-d31/chol mixture, collected over a 40  40 μm2 area. Both the FPA-d31 and the FCer values showed a broad distribution below 50 °C, and a considerably narrower distribution range over 50 °C, indicating a greater inhomogeneity in composition below the solid-fluid transition temperature. The associated maps are shown in Figure 7. PA-d31-enriched domains Langmuir 2009, 25(13), 7523–7532

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Figure 7. Evolution of the PA-d31 distribution in the sample as a function of temperature. The relative PA-d31 content (FPA-d31), as calculated using eq 1, is reproduced in false colors according to the scale presented below. Each map was obtained, at different temperature, from the same area (40  40 μm2).

Figure 6. Thermal evolution of the distribution of the spatial composition of the equimolar Cer16/PA-d31/chol mixtures. FPA-d31 represents the PA-d31 relative content, as calculated using eq 1, while FCer represents the Cer16 relative content, as calculated using eq 2. As a reminder, FPA-d31 > 0.5 is indicative of a sampling element enriched in PA-d31 and FPA-d31 < 0.5 is indicative of one which is depleted. The average value of FCer for the mixture was 0.60; FCer > 0.6 is indicative of a sampling element enriched in Cer16 relative to cholesterol while FCer < 0.6 is indicative of a Cer16-depleted one. Each histogram was obtained from 196 spectra sampled over an area of 40  40 μm2.

(red pixels) and the regions where PA-d31 was depleted compared to the average content (blue pixels) are clearly shown for the maps obtained at 25 °C. The shape of the domains was rather undefined, and their length along one dimension varied from a few μm up to ∼20 μm. Upon heating up to 45 °C, the composition and morphology of the domains in the equimolar Cer16/PA-d31/ chol mixture remained fairly unchanged. When the temperature was increased to 55 °C and above, the chemical maps clearly indicated a homogenization of the sample, as the domains became Langmuir 2009, 25(13), 7523–7532

smaller and less contrasted. Moreover, the shape and position of the enriched regions varied considerably between the maps obtained at 55, 65, and 75 °C, suggesting a fluid environment. The acquisition of a large number of spatially resolved spectra of a sample allowed us to extract correlations between spectral features to provide a more detailed description of the lipid behavior. Graphs presenting the correlation between the FPA-d31 and the FCer values for the equimolar Cer16/PA-d31/chol mixture are shown in Figure 8 (left panels). For the data acquired between 25 and 45 °C, no correlation was found, indicating that cholesterol, PA-d31, and Cer16 were mainly phase-separated, leading to a heterogeneous mixture where the domain size was on the order of or larger than the sampling laser beam (diameter of 2.5 μm). For the sampling elements depleted in PA-d31, the lipids present may be mainly cholesterol, mainly Cer16, or a mixture of these: there was a broad range of Cer16/cholesterol composition, uncorrelated with the PA-d31 content. Upon heating, a correlation between the FPAd31 and the FCer values was observed between 55 and 75 °C. In these cases, the sampling elements depleted in PA-d31 were enriched in ceramide relative to cholesterol. Conversely, the sampling elements enriched in PA-d31 were depleted in ceramide relative to cholesterol or, in other words, contained a cholesterol content higher than the average. This positive correlation between cholesterol and the PA-d31 indicated that, above the melting temperature of PA-d31, cholesterol was preferentially solubilized in fluid regions with PA-d31. Ceramide Is More Disordered When Mixed with Fluid PA/Cholesterol. We have also examined the chain conformational order of ceramide, using the I2880/I2845 ratio, as a function of the lipid spatial distribution. The ratio values are represented as a function of FPA-d31 for a series of spectra measured over a 40  40 μm2 area of the Cer16/PA-d31/chol mixture (Figure 8, middle panels). Between 25 and 45 °C, the averaged I2880/I2845 values were 1.3-1.4, consistent with the values measured on the averaged spectra. Between 55 and 75 °C, these values decreased to about 1.1 and a correlation between the I2880/I2845 and the FPA-d31 DOI: 10.1021/la9003643

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Figure 8. Correlations graphs derived from the chemical maps obtained using Raman spectroscopy. Left column: Correlation between the relative PA-d31 content, FPA-d31, calculated using eq 1, and the relative Cer16 content, FCer, calculated using eq 2. Middle column: Correlation between the relative PA-d31 content, FPA-d31, calculated using eq 1, and the ceramide chain order, as expressed by the I2880/I2850 intensity ratio. Right column: Correlation between the relative PA-d31 content, FPA-d31, calculated using eq 1, and PA-d31 chain order, as expressed by ΔνCD. The results from the spectra with FPA-d31 < 0.2 are not included because their weak signal/noise ratios led to inaccurate ΔνCD values.

values could be observed. On the basis of the I2880/I2845 values, ceramides in the PA-d31-enriched (i.e., ceramide-depleted) sampling elements were less ordered than those in the PA-d31-depleted (i.e., ceramide-enriched) elements. This implied that the small amount of ceramides mixed in the PA-d31 and cholesterol regions had rather disordered hydrocarbon chains while in the regions that were enriched in ceramides the hydrocarbon chains of ceramide were more ordered. These correlations established that, in addition to regions with different chemical compositions, the lipid mixtures displayed regions with different chain order. In addition to acyl chain order, the I2880/I2845 values were found to be slightly affected by the cholesterol content of a sampling element. The ratio measured on the pure cholesterol spectrum recorded at 75 °C was 1.09. The cholesterol contribution could be subtracted from the average spectrum of the ternary mixture at 75 °C to isolate the contribution of Cer16 in the CH stretching region. The I2880/I2845 value measured for this difference spectrum was 1.11. Indeed, simulated spectra of mixtures of cholesterol and Cer16, 7530 DOI: 10.1021/la9003643

obtained by adding these two contributions in different proportions, led to values between 1.09 and 1.11, a variation of 2%. The limited effect of extreme variations of cholesterol fraction on I2880/I2845 values at high temperature means that the correlations observed between 55 and 75 °C are essentially representative of a correlation between PA-d31 content and Cer16 chain disorder. At temperatures below the transition, the I2880/I2845 values varied from 1.1 for the pure cholesterol spectrum to 1.5 for the Cer16 contribution, isolated by spectral difference. Under these conditions, the cholesterol might have contributed to the broadening of the intensity ratio distribution but it is likely minor as no correlation could be observed between FCer and I2880/I2845 (data not shown). Fatty Acid Chain Order Does Not Depend on Local Membrane Composition. The acyl chain order was characterized by the width of the symmetric CD2 stretching, and there was no correlation between this parameter and the PA-d31 content in the sampling elements (Figure 8, right panels). At Langmuir 2009, 25(13), 7523–7532

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low temperatures, ΔνCD was ∼20 cm-1, irrespective of the PA-d31 content. Upon heating, the values increased, as a consequence of the disordering of the PA-d31 hydrocarbon chains. The ΔνCD values did not show any correlation with FPA-d31, indicating that most of the PA-d31 molecules were mainly uniformly disordered and randomly distributed, at least at the microscopic level. Remember that, at temperatures of 55 °C and above, 2H NMR showed that essentially all of the fatty acid was in a fluid or isotropic phase (see Figure 3), and therefore, this homogeneous disordering of PA-d31 was expected.

Discussion Combining results from complementary spectroscopic techniques provides detailed knowledge of the phase behavior of the ternary Cer16/PA/chol mixture. The approach presented here includes some original aspects. The characterization of ternary mixtures containing deuterated ceramide or palmitic acid by 2H NMR provides a quantitative description of the involvement of both lipids in each phase. Moreover, the analysis of 196 spatially resolved Raman spectra, with a resolution of about 3 μm, allowed us to extract novel correlations between chemical composition and alkyl chain order. Below 40 °C, the lipids are mainly phaseseparated and exist in the crystalline phase with orthorhombic chain packing. The solid phase formed by both Cer16 and PA is clearly identified by the shape of their 2H NMR spectra. The symmetry of the chain packing for Cer16 and PA-d31 is assessed by the splitting of the methylene deformation modes. Under our conditions, small crystalline domains (on the order of a few μm) with very heterogeneous composition are observed. According to the quantitative analysis of the 2H NMR spectra, a small proportion of Cer16 and PA, between 10 and 20%, does not form a crystalline phase. It is hypothesized that regions of noncrystalline lipid would be located between the solid domains. Between 40 and 55 °C, the lipids undergo a transition from the solid phase to a lo phase, as identified by the 2H NMR spectra. The drop in M1, calculated from the 2H NMR spectra of the Cer16-d31/PA/chol and Cer16/PA-d31/chol mixtures, and the relative shifts of the νsCH and νsCD bands both indicate that the lo phase includes a larger proportion of PA than that of Cer16. At 50 °C, almost 85% of PA is in the lo phase while only 35% of Cer16 contributes to this phase. Most likely, this phase includes most of the cholesterol if not all. It was shown that lo phases could be formed exclusively with cholesterol and PA.48,49 In fact, the PA/chol mixtures display an eutectic behavior at a molar PA/chol composition of 3/7. At pH 5.5, a PA/chol mixture with the eutectic composition was shown to exist in a crystalline phase up to 52 °C where a solid-to-lo sharp phase transition was observed.48 In the Cer16/PA-d31/chol mixture, the involvement of Cer16 in the solid-to-lo phase transition slightly reduces the transition temperature. The analysis of the spectral data obtained by Raman vibrational microspectroscopy reveals that, upon heating, the onset of the mixture’s melting leads to the formation of domains that include predominantly cholesterol and fatty acids but also contain some ceramide. The acyl chain of these ceramides is relatively disordered compared to that of the ceramide molecules in the ceramide-enriched domains, which maintain solid characteristics. At about 65 °C, it appears that the increased proportion of Cer16 and/or the conformational chain disorder of the lipids leads to the destabilization of the lamellar phase and to the formation of a highly disordered phase (as inferred from vibrational spectroscopy) where both ceramide and PA experience isotropic motions (on the NMR time scale, 10-5 s). Despite its simplicity, the investigated model mixture reproduces some features of the SC lipid phase, defining the minimal Langmuir 2009, 25(13), 7523–7532

requirements to obtain these specific features. The Cer16/PA-d31/ chol mixture exhibits a solid phase with orthorhombic chain packing, a geometry observed for SC lipids.31,59-63 It experiences a transition where acyl chain disorder is introduced, similar to the SC lipids for which an analogous transition is also observed between 60 and 80 °C.64-66 However, the lipid mixing in this mixture optimized for hydrophobic matching seems to be limited compared with analogous mixtures including a larger number of chemical species. Analogous mixtures prepared from nonhydroxylated ceramides prepared from sphingomyelin extracted from bovine brain (CerIII) have been previously characterized.32,33,44,47,67-72 This sphingolipid is isolated on the basis of the polar headgroup, but its alkyl chains are heterogeneous. The amide-linked chains were mainly stearic (18:0), nervonic (24:1), and lignoceric (24:0). In the present case, ceramide bears a well-defined hexadecanoyl chain, providing, to a certain extent, simpler mixtures. In the ternary CerIII/PA-d31/chol mixture,44,45 the solid-to-lo phase is accompanied by a larger shift of the νsCH band (by at least 1 cm-1 (refs 44 and 45)) and a large reduction of M1 of the 1H NMR spectra,68 two parameters that show a larger change than those observed for the Cer16/PA-d31/chol mixture. In addition, the transition is initiated at about 40 °C for the CerIII/PA-d31/chol mixture, a temperature slightly lower than that observed for the Cer16/PA-d31/chol mixture investigated here and about 15 °C lower than that of the PA/chol system. Because CerIII has a greater influence on this transition than does Cer16, it can be inferred that the miscibility of Cer16 within PA/ chol lo matrices is more limited than that of CerIII. This finding reveals that the acyl chain heterogeneity of ceramides is an important factor for enhancing lipid miscibility. This parameter appears to be more important than hydrophobic chain matching, as Cer16 bears chains that should have optimal matching with cholesterol and PA. Even though lipid miscibility is shown to be a critical factor determining the structure of the self-assemblies prepared from cholesterol and fatty acids,28 this parameter seems to be subjugated by ceramide chain heterogeneity. This finding was rather unexpected because a considerable proportion of the CerIII alkyl chains are too long (C24) for optimized hydrophobic matching with PA and cholesterol.28 A likely explanation for this finding is the fact that CerIII contains a significant amount of nervonylceramide, which has a monounsaturated C24 chain. Nervonylceramide has a broad melting transition near 52 °C,73 which is much lower than the Cer16 melting point, 93 °C.74 Thus, it is probable that the nervonylceramide destabilizes the CerIII/ (59) Parrott, D. T.; Turner, J. E. Biochim. Biophys. Acta 1993, 1147, 273. (60) Chen, Y.-L.; Wiedmann, T. S. J. Invest. Dermatol. 1996, 107, 15. (61) Bouwstra, J. A.; Gooris, G. S.; Bras, W.; Downing, D. T. J. Lipid Res. 1995, 36, 685. (62) Bouwstra, J. A.; Gooris, G. S.; Salomons-de Vries, M. A.; van der Spek, J. A.; Bras, W. Int. J. Pharm. 1992, 84, 205. (63) Bouwstra, J. A.; Gooris, G. S.; van der Spek, J. A.; Bras, W. J. Invest. Dermatol. 1991, 97, 1005. (64) Golden, G. M.; Guzek, D. B.; Harris, R. R.; Mckie, J. E.; Potts, R. O. J. Invest. Dermatol. 1986, 86, 255. (65) Potts, R. O.; Francoeur, M. L. Drugs Pharm. Sci. 1993, 59, 269. (66) Ongpipattanakul, B.; Francoeur, M. L.; Potts, R. O. Biochim. Biophys. Acta 1994, 1190, 115. (67) Kitson, N.; Thewalt, J.; Lafleur, M.; Bloom, M. Biochemistry 1994, 33, 6707. (68) Thewalt, J.; Kitson, N.; Araujo, C.; MacKay, A.; Bloom, M. Biochem. Biophys. Res. Commun. 1992, 188, 1247. (69) Abraham, W.; Downing, D. T. Pharm. Res. 1992, 9, 1415. (70) Abraham, W.; Downing, D. T. Biochim. Biophys. Acta 1991, 1068, 189. (71) Moore, D. J.; Rerek, M. E. Acta Derm.-Venereol. Suppl. 2000, 208, 16. (72) Percot, A.; Lafleur, M. Biophys. J. 2001, 81, 2144. (73) Pinto, S. N.; Silva, L. C.; de Almeida, R. F. M.; Prieto, M. Biophys. J. 2008, 95, 2867. (74) Sot, J.; Aranda, F. J.; Collado, M.-I.; Goni, F. M.; Alonso, A. Biophys. J. 2005, 88, 3368.

DOI: 10.1021/la9003643

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PA/chol solid phase and, upon melting, incorporates into the PA/ chol liquid crystalline phase. Similar melting temperatures of the components may be a better predictor of miscibility than hydrophobic matching. Interestingly, it was shown that fatty acid heterogeneity leads to an enhanced miscibility with the ceramide, in mixtures including CerIII, cholesterol, and fatty acid.17 This conclusion was obtained by comparing the phase behavior, as described by X-ray diffraction, of CerIII/PA/chol and CerIII/FFA/chol mixtures, where FFA stands for a free fatty acid mixture that includes different linear chain lengths: C16:0, C18:0, C20:0, C22:0, C22:3, C24:0, and C26:0 at a respective molar proportion of 1.3, 3.3, 6.7, 41.7, 5.4, 36.8, and 4.7%. Again, most of these acids are not hydrophobically matched with cholesterol. Phase-separated PA was observed in the CerIII/PA/chol, while only a weak signal that could be associated with phase-separated FFA could be observed in the CerIII/FFA/chol mixture. One conclusion of our present work is that ceramides with heterogeneous chains (but homogeneous head groups) play a similar homogenizing role. Recently, it was shown that SC model mixtures including five types of ceramides and seven types of fatty acids form a crystalline phase with an orthorhombic chain packing that includes both ceramides and fatty acids,75 suggesting that the heterogeneity of the ceramides and the fatty acids may promote lipid miscibility even in the crystalline phase. In natural SC, the distribution of the various lipid components is not known. Because the lipids of SC intercellular domains are known to be hydrophobically mismatched (ranging from cholesterol to long chain fatty acids, e.g.) and yet are also known to form lipid crystals, we imagine SC intercellular lipids in vivo to be made (75) Gooris, G. S.; Bouwstra, J. A. Biophys. J. 2007, 92, 2785.

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up of complex and heterogeneous crystals that for the most part are not compositionally demixed. SC crystalline intercellular lamellae may well include compositionally distinct, crystalline domains however. The composition of the lamellae that oppose the keratinocytes is likely specialized in order to tightly pack with the Cer-1-rich cell envelope, for example. The composition of the lamellae near desmosomes may also be specialized. A variety of such domains could form spontaneously at the interface between the stratum granulosum and the stratum corneum. These distinct domains might be essential for SC function, providing barrier function for water and toxins, and yet allowing for elasticity and bending required of the tissue. Further work is needed to understand the particular physical qualities of fatty acids and ceramides that govern lipid miscibility in model SC membranes. Such information will shed light on lipid interactions in the SC itself. In the present work, we have shown that, for a mixture optimized for hydrophobic matching, ceramide, cholesterol, and fatty acid spontaneously separate within crystalline membranes. Acknowledgment. The authors thank the Natural Sciences and Engineering Research Council of Canada for the financial support of this Collaborative Health Research Project. We also thank Prof. Felix Go~ ni for helpful discussions. M.L. is also grateful to the Fonds Quebecois de la Recherche sur la Nature et les Technologies (FQRNT). Supporting Information Available: Method providing the calculated percentage of the deuterated lipid (PA-d31 or Cer16-d31) existing in a solid (%solid), a gel or fluid lamellar (%lo/gel), and an isotropic (%iso) phase. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2009, 25(13), 7523–7532