“Solid” Phase Formation in Models of Stratum Corneum Intercellular

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Fatty Acids Influence “Solid” Phase Formation in Models of Stratum Corneum Intercellular Membranes Xin Chen,£ Sungjong Kwak,‡ Michel Lafleur,*,‡ Myer Bloom,£ Neil Kitson,§ and Jenifer Thewalt*,# Department of Physics and Astronomy, UniVersity of British Columbia, VancouVer, British Columbia, Canada V6T 1Z1, Department of Dermatology and Skin Science, UniVersity of British Columbia, VancouVer, British Columbia, Canada V5Z 4E8, Departments of Molecular Biology and Biochemistry/ Physics, Simon Fraser UniVersity, Burnaby, British Columbia, Canada, V5A 1S6, and Department of Chemistry, UniVersite´ de Montre´ al, Montre´ al, Que´ bec, Canada, H3C 3J7 ReceiVed December 16, 2006. In Final Form: February 16, 2007 Stacked intercellular lipid membranes in the uppermost epidermal layer, the stratum corneum (SC), are responsible for skin’s barrier function. These membranes are unique in composition, the major lipids being ceramides (Cer), cholesterol, and free fatty acids (FFA) in approximately equimolar proportions. Notably, SC lipids include chains much longer than those of most biological membranes. Previously we showed that Cer’s small hydrophilic headgroup enabled SC model membranes composed of bovine brain ceramide (BBCer), cholesterol, and palmitic acid in equimolar proportion to solidify at pH 5.2. In order to determine the influence of FFA chain length on the phase behavior of such membranes, we used 2H NMR and FT-IR to study BBCer/cholesterol/FFA dispersions containing linear saturated FFA 14-22 carbons long. Independent of chain length, the solid phase dominated the FFA spectrum at physiological temperature. Upon heating, each dispersion underwent phase transitions to a liquid crystalline phase (only weakly evident for the membrane containing FFA-C22) and then to an isotropic phase. The phase behavior, the lipid mixing properties, and the transition temperatures are shown to depend strongly on FFA chain length. A distribution of FFA chain lengths is found in the SC and could be required for the coexistence of a proportion of solid lipids with some more fluid domains, which is known to be necessary for normal skin barrier function.

Introduction Lipid diversity has an influence on the function of biological membranes, particularly as mediated through membrane physical properties. An extreme biological example of the relationship among membrane lipid composition, membrane physical properties, and membrane functions is to be found in the intercellular membranes of the stratum corneum (SC). The SC is the outermost part of the mammalian skin’s epithelium, the epidermis, and provides an indispensable barrier to the environment. It is a composite material consisting of terminally differentiated epithelial cells (keratinocytes) containing proteins (particularly keratin), bounded by complex cellular envelopes, and linked to one another by mechanical junctions (desmosomes). The spaces between such cells are filled with stacks of unusual biological membranes that determine both the rate of water loss through the skin and absorption of exogenous molecules into the body. These stratum corneum intercellular membranes (SCIM) are unusual when compared to most mammalian cell membranes, with respect to lipid composition (mainly ceramide (Cer), cholesterol (Chol), and free fatty acid (FFA)) and physical organization (primarily “solid” rather than “fluid”, i.e., liquid crystalline, lamellae).1-3 Throughout this paper, * To whom correspondence should be addressed. Fax: (604) 291-3592 (J.T.); (514) 343-7586 (M.L.). E-mail: [email protected] (J.T.); [email protected] (M.L.). £ Department of Physics and Astronomy, University of British Columbia. § Department of Dermatology and Skin Science, University of British Columbia. # Simon Fraser University. ‡ Universite ´ de Montre´al. (1) Bouwstra, J. A.; Gooris, G. S.; Cheng, K.; Weerheim, A. M.; Bras, W.; Ponec, M. J. Lipid Res. 1996, 37, 999. (2) Bouwstra, J. A.; Gooris, G. S.; Bras, W.; Downing, D. T. J. Lipid Res. 1995, 36, 685. (3) White, S. H.; Mirejovsky, D.; King, G. I. J. Am. Chem. Soc. 1988, 27, 3725.

“solid” membranes are those with essentially motionless acyl chains from a NMR perspective, corresponding to crystalline chain packing as observed by wide-angle X-ray diffraction and IR spectroscopy. A proposed hypothesis is that the permeability of SCIM is determined by lipid organization, which is in turn determined to a great extent by lipid composition.4,5 In particular, the presence of a “solid” or crystalline phase within SCIM is an obvious factor restricting water permeability across the SC. Coordinated enzymatic modifications of membrane lipids during epidermal differentiation could produce such “solid” bilayers from the fluid lipid membranes that must necessarily be their precursors. It was previously shown6-8 that in model systems composed of the three classes of lipids that form the bulk of these membranes, formation of such a solid phase is strongly influenced by the nature of the sphingolipid headgroup and the pH of the hydrating medium. Both of these variables may plausibly influence phase behavior in vivo. The particular influence of FFAs on the solid phase is of interest for several reasons. First, FFA has the only ionizable head group, which under the influence of pH (or other properties of the hydrating medium) is susceptible to alteration in head group interfacial area and, therefore, phase behavior. Second, Bouwstra and colleagues9 found, using wide-angle X-ray diffraction, that model membranes composed only of SC ceramide (4) Forslind, B. Acta Dermato-Venereologica 1994, 74, 1. (5) Menon, G. K. AdV. Drug DeliVery ReV. 2002, 54, S3-S17. (6) Thewalt, J.; Kitson, N.; Araujo, C.; MacKay, A.; Bloom, M. Biochem. Biophys. Res. Commun. 1992, 188, 1247. (7) Kitson, N.; Thewalt, J.; Lafleur, M.; Bloom, M. Biochemistry 1994, 33, 6707. (8) Bouwstra, J. A.; Thewalt, J.; Gooris, G. S.; Kitson, N. Biochemistry 1997, 36, 7717. (9) Bouwstra, J. A.; Gooris, G. S.; Dubbelaar, F. E. R.; Weerheim, A. M.; Ponec, M. J. InVest. Dermatol. 1998, 3, 69.

10.1021/la063640+ CCC: $37.00 © 2007 American Chemical Society Published on Web 04/03/2007

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and cholesterol resulted in hexagonal chain packing (which was insensitiVe to pH), typical of noncrystalline membranes, but that the inclusion of FFA produced an orthorhombic pattern (which was sensitiVe to pH). This is a strong evidence that FFA is an important determinant of SCIM structure, allowing the formation of a “solid” or crystalline bilayer organization (identified by orthorhombic chain packing). Furthermore, it has been shown that FFA and cholesterol, two major components of SC, can form fluid lamellar phases and the stability of these self-assemblies is dependent on the hydrophobic matching between the FFA chain length and the length of the long axis of the cholesterol molecule.10,11 For unknown reasons, SCIM contain FAs of significantly longer average chain length than the average length of the saturated acyl chains of their precursor membrane lipids. Typically, the chain length varies between C16 and C26, C24 and C22 being the most abundant.12-15 The present study aims at understanding the importance of the FFA chain length in the phase behavior of SCIM. The techniques of 2H NMR and FT-IR have been used to examine the phase behavior of SC model membranes: ternary equimolar mixtures of ceramides, cholesterol, and FFAs of chain length varying between C14 and C22. Bovine brain ceramide type III (BBCer) used in this study is identical in degree and position of hydroxylation and backbone structure to skin Cer type 2, the most prevalent of the Cer classes found in human SC.16 Mixtures formed of BBCer, cholesterol, and FFA-C16 (hereafter, these FFA will be referred to by their number of carbon atoms where, e.g., FFA-C16 refers to palmitic acid) have been well characterized by a combination of techniques, and the phase behavior is fairly well understood.6-8,17-24 This model mixture reproduces the dominant solid phase, which appears to be characteristic of SCIM, the orthorhombic chain packing, and the general acyl chain thermal disordering of SC lipids.7,20,25-29 However, it reproduces only the shortest of the two lamellar spacings observed by X-ray diffraction, the formation of both lamellar repeat distances requiring more complex mixtures that include at least five different ceramides.8,30-34 This should be kept in mind, and the findings reported here are likely more closely associated with the behavior (10) Ouimet, J.; Croft, S.; Pare´, C.; Katsaras, J.; Lafleur, M. Langmuir 2003, 19, 1089. (11) Ouimet, J.; Lafleur, M. Langmuir 2004, 20, 7474. (12) Lampe, M. A.; Burlingame, A. L.; Whitney, J.; Williams, M. L.; Brown, B. E.; Roitman, E.; Elias, P. M. J. Lipid Res. 1983, 24, 120. (13) Nicollier, M.; Massengo, T.; Re´my-Martin, J.-P.; Laurent, R.; Adessi, G.-L. J. InVest. Dermatol. 1986, 87, 68. (14) Wertz, P. W.; Swartzendruber, D. C.; Madison, K. C.; Downing, D. T. J. InVest. Dermatol. 1987, 89, 419. (15) Norle´n, L.; Nicander, I.; Lundsjo¨, A.; Cronholm, T.; Forslind, B. Arch. Dermatol. Res. 1998, 290, 508. (16) Downing, D. T.; Stewart, M. E.; Wertz, P. W.; Colton, S. W.; Abraham, W.; Strauss, J. S. J. InVest. Dermatol. 1987, 88, 2s. (17) Abraham, W.; Downing, D. T. Biochim. Biophys. Acta 1991, 1068, 189. (18) Abraham, W.; Downing, D. T. Pharm. Res. 1992, 9, 1415. (19) Fenske, D. B.; Thewalt, J. L.; Bloom, M.; Kitson, N. Biophys. J. 1994, 67, 1562. (20) Moore, D. J.; Rerek, M. E.; Mendelsohn, R. Biochem. Biophys. Res. Commun. 1997, 231, 797. (21) Velkova, V.; Lafleur, M. Chem. Phys. Lipids 2002, 117, 63. (22) Mendelsohn, R.; Rerek, M. E.; Moore, D. J. Phys. Chem. Chem. Phys. 2000, 2, 4651. (23) Chen, H.-C.; Mendelsohn, R.; Rerek, M. E.; Moore, D. J. Biochim. Biophys. Acta 2001, 1512, 345. (24) Percot, A.; Lafleur, M. Biophys. J. 2001, 81, 2144. (25) Parrott, D. T.; Turner, J. E. Biochim. Biophys. Acta 1993, 1147, 273. (26) Potts, R. O.; Francoeur, M. L. Drugs Pharm. Sci. 1993, 59, 269. (27) Ongpipattanakul, B.; Francoeur, M. L.; Potts, R. O. Biochim. Biophys. Acta 1994, 1190, 115. (28) Bouwstra, J. A.; Gooris, G. S.; van der Spek, J.; Lavvrijsen, S.; Bras, W. Biochim. Biophys. Acta 1994, 1212, 183. (29) Lafleur, M. Can. J. Chem. 1998, 76, 1501. (30) McIntosh, T. J.; Stewart, M. E.; Downing, D. T. Biochemistry 1996, 35, 3649. (31) de Jager, M. W.; Gooris, G. S.; Dolbnya, I. P.; Bras, W.; Ponec, M.; Bouwstra, J. A. Chem. Phys. Lipids 2003, 124, 123.

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of the lipid structure with the short lamellar spacing. The use of simplified model mixtures in our study is advantageous to focus on the influence of FFA chain length on the phase behavior of SC model membranes. The interactions between the lipid components that affect the observed phase behavior are discussed. Materials and Methods Sample Preparation. Deuterated FFAs were either obtained from Cambridge Isotope Labs (Andover, MA) or prepared according to a published method,35 while cholesterol and BBCer were obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. BBCer was prepared by the action of phospholipase C on bovine brain sphingomyelin, and the amide-linked chains were mainly stearic (18:0), nervonic (24:1), and lignoceric (24:0). Equimolar quantities of lipids were mixed together by weight, dissolved in benzene/methanol, 7:3 (v/v), and freeze-dried. The resulting white powders were hydrated using a pH 5.2 buffer prepared in deuterium-depleted water containing 150 mM NaCl, 4 mM EDTA, and 100 mM citrate. Hydration was performed at about 90 °C, and the resulting dispersions (about 7:1 w/w buffer/lipid) were alternately frozen in liquid nitrogen and then heated to at least 90 °C for five cycles. The samples were annealed at a temperature below the onset of the solid-phase melting transition until a maximum proportion of solid was obtained, at least 24 h. The formation of solid phase was probed by the splitting of the methylene deformation, in IR spectroscopy, and by the intensity of the solid spectral component in 2H NMR spectroscopy. NMR Methods. 2H NMR spectra were obtained using the quadrupolar echo technique36 with a locally built spectrometer operating at 46.2 MHz. The typical spectrum resulted from 1000 to 10000 repetitions of the two-pulse sequence with a 90o pulse length of 4 µs, an interpulse spacing of 40 µs, and a dwell time of 2 or 5 µs. The delay between acquisitions was typically 300 ms (for spectra with only fluid components) or 50 s (for spectra including a solid component). The data were collected in quadrature with 8-cyclops phase cycling. Spectral half-moments37 were calculated from Fourier-transformed spectra. The proportion of FFA in the solid phase was calculated by the following formula: [ND/(ND - 3)][(EHL - EHS)/EHL], where ND is the number of deuterons per molecule, EHL is the echo height of the long repeat time (50 s) experiment, and EHS is the echo height of the short repeat time (300 ms) experiment.7 The carbondeuteron order parameter, SCD, was calculated using ∆υ/252 kHz, where ∆υ is the 0° splitting of a given doublet, calculated from dePaked spectra.38 FT-IR Methods. For FT-IR spectroscopy, the sample preparation was similar to that above except that a Mes buffer (100 mM, 25 mM NaCl, 5 mM EDTA) pH 5.2 was used. In addition, the buffer was D2O-based to avoid the spectral interference from the H2O deformation band, overlapping with the CO stretching mode of the carboxylic acid group, and the Amide I band of the ceramide. The final overall lipid concentration was 150 mg of total lipids/mL of buffer. An aliquot of the sample was placed between two CaF2 windows separated by a 5-µm-thick Teflon spacer. The sample was then introduced into a brass sample holder whose temperature was computer controlled. The spectra were acquired on a BioRad FTS25 spectrometer. Forty scans were co-added with a 2 cm-1 resolution for each spectrum. The spectra were recorded from low to high temperatures, with a temperature-equilibration period of 6 min. They were analyzed using GRAMS software (Galactic Industries Cor(32) de Jager, M. W.; Gooris, G. S.; Dolbnya, I. P.; Ponec, M.; Bouwstra, J. A. Biochim. Biophys. Acta 2004, 1684, 132. (33) de Jager, M. W.; Gooris, G. S.; Dolbnya, I. P.; Bras, W.; Ponec, M.; Bouwstra, J. A. J. Lipid Res. 2004, 45, 923. (34) de Jager, M.; Gooris, G. S.; Ponec, M.; Bouwstra, J. A. J. Lipid Res. 2005, 46, 2649. (35) Hsiao, C. Y. Y.; Ottaway, C. A.; Wetlaufer, D. B. Lipids 1974, 9, 913. (36) Davis, J. H.; Jeffrey, K. R.; Bloom, M.; Valic, M. I.; Higgs, T. P. Chem. Phys. Lett. 1976, 42, 390. (37) Davis, J. H. Biochim. Biophys. Acta 1983, 737, 117. (38) Sternin, E.; Bloom, M.; MacKay, A. L. J. Magn. Reson. 1983, 55, 274.

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Figure 1. Temperature dependence of 2H NMR spectra of equimolar dispersions of (A) BBCer/chol/FFA-C14-d27, (B) BBCer/chol/FFAC18-d35, (C) BBCer/chol/FFA-C20-d39, and (D) BBCer/chol/FFA-C22-d43, all in excess buffer, pH 5.2. poration). The peak positions of the symmetric methylene stretching modes correspond to the center of gravity of the top 5% of the band.39

Results 2H

NMR. As a first step, the behavior of the fatty acid in ternary BBCer/chol/FFA dispersions was investigated using 2H NMR spectroscopy. Figure 1 illustrates the temperature dependence of the 2H NMR spectrum for these mixtures containing perdeuterated FFA of different lengths. As shown in Figure 1, there are three classes of spectrum, corresponding to three different phases of the SC model system. At low temperatures, the observed spectrum is dominated by a superposition of two Pake doublets with quadrupolar splittings of 126 and 38 kHz. The former doublet (39) Cameron, D. G.; Kauppinen, K;, J., Moffatt, D. J.; Mantsch, H. H. Appl. Spectrosc. 1982, 36, 245.

is the maximum observable powder splitting for C-2H bonds in a hydrocarbon, indicating that conformational averaging is not occurring, and results from the methylene deuterons on the “solid” FFA chains. The narrower doublet is assigned to the terminal methyl deuterons that still reorient rapidly even at low temperature. As temperature is increased, the intensity of the “solid” doublet in the spectrum gradually decreases and another spectral component, whose width is less than half that of the “solid” Pake doublet, appears. This narrower spectrum is consistent with motional averaging about the long axis of the fatty acid, characteristic of a lamellar liquid crystalline phase (“fluid”). The “fluid” spectrum features many resolved doublets since signals from different methylenes no longer overlap, reflecting the increasing conformational freedom of chain segments at increasing distances from the aqueous interface. We also observe a sharp central peak in some spectra due to FFA

Fatty Acids in Model SC Membranes

Figure 2. Temperature dependencies of the average spectral width, M1, of the 2H NMR spectra. (O) BBCer/chol/FFA-C14-d27; (b) BBCer/chol/FFA-C16-d31 (from ref 7); (∆) BBCer/chol/FFA-C18d35; (2) BBCer/chol/FFA-C20-d39; (0) BBCer/chol/FFA-C22-d43.

reorienting isotropically on the NMR time scale. The presence of this central peak is reversible with temperature and is very likely due to micelle or ‘oily droplet’ formation. It is not, however, due to cubic phase formation, since small-angle X-ray diffraction patterns of similar model membranes containing FFA-C16 show no evidence of the cubic phase.8 The spectra of all the BBCer/chol/FFA membranes (Figure 1) except those with FFA-C22 (Figure 1D) clearly show that, upon heating from room temperature to 80 °C, the labeled fatty acid transforms from a predominantly solid phase to a mainly fluid phase and then to an isotropic phase. In the lipid mixture with the shortest FFA, FFA-C14 (Figure 1A), the spectra are “solid” dominant below 37 °C. Above 40 °C, they are “fluid” dominant, until at 53 °C the central peak begins to grow and develops upon further heating to above 60 °C. In BBCer/chol/FFA-C16 (spectra shown in Figure 3b of ref 7), BBCer/chol/FFA-C18, and BBCer/ chol/FFA-C20 (Figure 1B, C) similar transformations occur, although at higher temperatures for increasing chain lengths. FFA-C16 begins to change from a “solid” dominant spectrum to a “fluid” dominant spectrum at about 41 °C, FFA-C18 at about 49 °C, and FFA-C20 at about 52 °C. The central peak begins to develop at about 53 °C for FFA-C16, about 57 °C for FFA-C18, and about 62 °C for FFA-C20. The lipid dispersion having the longest FFA, FFA-C22 (Figure 1D), behaves differently from the others in two ways: at no temperature does the “fluid” spectrum predominate and the central peak grows through the whole temperature range. The central peak is so strong that it is necessary to truncate it at high temperatures in order to observe the weak underlying “fluid” and residual “solid” spectra (insets, Figure 1D). Below 61 °C, the spectra are essentially a combination of “solid” and “isotropic” signals, with a very small fluid signal that is observed from 56 °C. Above 78 °C, only the isotropic signal remains. The FFA disordering within each model SC lipid mixture is shown clearly by the temperature dependence of the average width of its 2H NMR spectrum, represented by the first moment, M1 (Figure 2). Plots of the temperature dependence of M1 for the BBCer/chol/FFA dispersions share similar characteristics, except, to some extent, those containing FFA-C14 and C22, as discussed below. At low temperature, the solid spectrum is dominant and

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Figure 3. Order parameter profiles of the FFA in the SC model mixtures. (O) BBCer/chol/FFA-C14-d27, at 44 °C; (b) BBCer/chol/ FFA-C16-d31 (from ref 7), at 50 °C; (∆) BBCer/chol/FFA-C18-d35, at 58 °C; (2) BBCer/chol/FFA-C20-d39, at 61 °C.

the high M1 values show a plateau. It is interesting to observe how insensitive the proportion of “solid” spectrum is to chain length at room temperature. For all studied dispersions, the spectrum is ca. 80 ( 3% solid between 25 and 35 °C. Upon heating, the transition to the fluid-dominant spectrum begins and, as a result, the M1 drops sharply. The percent solid value begins to decrease at the same temperature where the first drop of the M1 is observed, and the solid phase disappears (i.e., % solid e 10) at 41 (C14), 49 (C16), 58 (C18), 63 (C20), and 65 °C (C22), respectively (the temperature interval over which the solid percentage decreases from 75% to 10% is shown in Figure 7). During this transition, the solid and the fluid phases coexist. For all the investigated mixtures, except that including FFAC22, the FFA spectrum in the transition region can be obtained by summing appropriate fractions of representative spectra from the solid (drawn from the plateau region of M1 at low temperature) and fluid phases (from the region after the first sharp drop of the M1), confirming that the process is a real two-phase transition. Following the first sharp drop, the M1 shows a rather moderate reduction and then decreases sharply again as the remaining fluid phase transforms into the isotropic phase. In the case of FFA-C14, the M1 reaches a well-defined plateau after the first drop, corresponding to a value associated with a fluid-phase spectrum. No fluid-dominant spectrum is observed with FFAC22, and consequently, the drop of the M1 is mostly attributed to the growth of the isotropic component. Each BBCer/chol/FFA dispersion’s powder pattern has a spectral component characteristic of a lamellar liquid crystalline phase over a certain temperature range.37,40 To compare these “fluid” phases in detail, the variation of conformational order as a function of position along the FFA chain was calculated; since BBCer/chol/FFA-C22 has little fluid signal, this sample could not be examined. The sample temperature was selected to be 9 °C above the temperature at which the fluid phase spectrum first appears in order to ensure that the model SC membranes are in the fluid-dominant phase. Smoothed order parameter profiles41 were determined (Figure 3). Assignments from carbon number 3 (C(3)) to the ends of the chains were made on the basis of monotonically decreasing order parameters. For FFA-C16, the (40) Lafleur, M.; Cullis, P. R.; Bloom, M. Eur. Biophys. J. 1990, 19, 55. (41) Lafleur, M.; Fine, B.; Sternin, E.; Cullis, P. R.; Bloom, M. Biophys. J. 1989, 56, 1037.

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splitting of C(2) was independently measured in a similar lipid mixture having the fatty acid specifically labeled at position 2.19 The assignment of the C(2) peaks in spectra of dispersions containing other FFAs was made assuming that the splitting of C(2) vs FFA chain length varies in a similar manner as that of other carbon positions, i.e., the quadrupolar splitting from deuterons bound to a specific carbon decreases gradually as the FFA is elongated. All the resulting order profiles are indicative of the high order of the acyl chain, combined with the axial symmetric motion of the molecule. The SCD values at the plateau, i.e., near the headgroup, are ∼0.4, a value close to what would be observed for an all trans chain (0.5) indicating that these lamellar liquid crystalline membranes are in the liquid-ordered (lo) phase. This phase is a fluid lamellar phase (for which axially symmetric powder patterns are observed) where the conformational freedom of the acyl chains is restricted, leading to high orientational order parameters. In the data presented here, the values observed in the plateau region and those of the terminal methyl group increase somewhat as the FFA chain length is reduced from 20 to 14 carbons, implying that the orientational disorder of FA chain segments increases with the chain length. FT-IR. The methylene stretching modes provide sensitive probes for the phase transition of skin lipids.20,26,29 In addition, by using deuterated FFAs, the information on thermal behavior of ceramide, as well as that of FFAs, can be obtained simultaneously, providing comparative and complementary data to those of the model membranes obtained by 2H NMR method. The CH (∼2850 cm-1) and CD (∼2090 cm-1) symmetric stretching bands have been used as a probe to investigate the thermotropism of ceramide and FFAs, respectively. Figure 4 shows the thermal behavior of the CH symmetric stretching (νsCH) band. For all the systems, the νsCH band is located below 2850 cm-1 between 25 and 35 °C, which indicates that acyl chains are highly ordered in this temperature range.20,21 Upon heating, the νsCH band shifts toward higher wavenumber, an indication of the disordering of the acyl chains. Overall, this shift can be characterized by two steps, on the basis of the rate of change upon heating; the first shift from below 2850 to ∼28512852 cm-1 is followed by a further increase to over 2853 cm-1. The first shift appears over the temperature range between ∼35 and 57 (C14), ∼40 and 57 (C16), ∼41 and 57 (C18), ∼45 and 55 (C20), and ∼45 and 55 °C (C22). The νsCH band position after this shift is similar to those of the lo phase observed previously,21,42 suggesting that this first shift is the result of the conversion of BBCer from a solid to lo-dominant phase. This first shift begins at the lowest temperature for BBCer/chol/FFAC14 and progressively at higher temperatures as the FFA chain lengthens, except for BBCer/chol/FFA-C22, which shows similar behavior with BBCer/chol/FFA-C20. Contrary to the fact that the beginning of the first νsCH band shift temperature increases as a function of the FFA chain length, it ends almost at the same temperature (∼55-57 °C). Therefore, the first shift of BBCer/ chol/FFA-C14 appears over a much broader temperature range than that of BBCer/chol/FFA-C22. These BBCer melting temperatures are much lower than that of the pure form. Upon hydration, BBCer melts at 80 °C.43 All the investigated model membranes show a second part of the chain disordering process that occurs between 60 and 70 °C. The band finally reaches ∼2853.5 cm-1 at 75 °C, a typical value for a disordered phase, suggesting that this second shift is related to the participation of BBCer from a lo-dominant to a disordered phase. This transition (42) Pare´, C.; Lafleur, M. Langmuir 2001, 17, 5587. (43) Moore, D. J.; Rerek, M. E.; Mendelsohn, R. J. Phys. Chem. B 1997, 101, 8933.

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Figure 4. Thermotropism of equimolar mixtures of BBCer/chol/ FFA (prepared with deuterated FFA) as probed with the νsCH and νsCD band. (9) νsCH band position, (O) νsCD band position. (A) BBCer/chol/FFA-C14-d27, (B) BBCer/chol/FFA-C16-d31, (C) BBCer/ chol/FFA-C18-d35, (D) BBCer/chol/FFA-C20-d39, and (E) BBCer/ chol/FFA-C22-d43 mixtures.

occurs over a slightly lower temperature range for BBCer/chol/ FFA-C18 to C22 (∼55-70 °C) than BBCer/chol/FFA-C14 and BBCer/chol/FFA-C16 (∼57-75 °C). Figure 4 also shows the CD symmetric stretching band (νsCD) position as a function of temperature. Below 35 °C, the frequency of the band is lower than 2090 cm-1 for all the model membranes, indicating ordered acyl chains.29,42 Upon heating, the νsCD band shifts toward higher wavenumber. For BBCer/chol/FFA-C14 and BBCer/chol/FFA-C16, this shift occurs in a two-step fashion; first there is a rather abrupt increase of the band wavenumber followed by a gradual increase over a wider temperature range. The first shift appears between ∼33 and 43 (C14), and ∼40 and 51 °C (C16). The νsCD band is located at ∼2092 cm-1 after this first shift, which corresponds to the lo phase that is previously observed for similar ternary lipid mixtures.21,42 Then, the νsCD band gradually undergoes the second shift to ∼2096 cm-1, a value that was previously associated with a disordered phase.21,42 The results suggest that the thermotropism of the FFAs in BBCer/

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Figure 5. (A) δCD2 band of BBCer/chol/FFA-C14-d27 (left panel), and BBCer/chol/FFA-C22-d43 (right panel) showing the merger of the band. (b) The intensity ratio, IM/IT, of the δCD2 band. (O) BBCer/chol/FFA-C14-d27; (b) BBCer/chol/FFA-C16-d31; (∆) BBCer/chol/FFAC18-d35; (2) BBCer/chol/FFA-C20-d39; (0) BBCer/chol/FFA-C22-d43.

chol/FFA-C14 and BBCer/chol/FFA-C16 includes two transitions, from a solid to a lo-dominant, and from a lo-dominant to a disordered phase. For the FFA in BBCer/chol/FFA-C18, the νsCD band begins to shift to higher wavenumber at 45 °C, shows an inflection at ∼57 °C, and then increases further to 2096 cm-1. At ∼57 °C, the band is at ∼2091 cm-1, a value compatible with the existence of a lo phase under these conditions. Model membranes with long-chain FFAs, BBCer/chol/FFA-C20 and BBCer/chol/FFA-C22, show only one very abrupt shift of the νsCD band centered at 63 and 65 °C, respectively, suggesting a direct transition from a solid to a disordered phase. The above observation of the two-step transition and the formation of the lo phase for BBCer/chol/FFA-C14 to C18 agree well with the 2H NMR data. For BBCer/chol/FFA-C20 and BBCer/chol/FFAC22, the presence of the lo phase is detected by 2H NMR, but its proportion is small compared to that observed with BBCer/ chol/FFA-C14 to C18. The maximum proportion of the lo phase reaches only ∼50% and ∼30% for BBCer/chol/FFA-C20 and -C22, respectively, coexisting with a solid phase. For all the FFAs investigated, the melting temperatures are lower than their pure forms. Hydrated deuterated FFAs melt at 49, 59, 64, 70, and 73 °C for C14-C22, respectively.11 The splitting of the CD2 deformation (δCD2) band was observed for all the model membranes at low temperature, giving rise to two components at ∼1086 and ∼1092 cm-1. In Figure 5A, the δCD2 bands at different temperatures are shown for BBCer/ chol/FFA-C14 and -C22. This splitting indicates that the FFAs in these mixtures form nearly pure crystalline domains, at least 100 molecules in size, having orthorhombic chain packing.20,44 Upon heating, a middle band at ∼1089 cm-1 appears along with the two original components, indicating a loss of the orthorhombic chain packing. Subsequently, there is a decrease of the split component intensity, and eventually the three bands merge into one. To analyze the δCD2 band behavior more in detail, a curvefitting procedure was established. The spectra in this region were simulated using four bands. The middle band position was restricted between 1088 and 1089 cm-1 and the width below 5 cm-1, which are the values obtained from the spectra at 75 °C. The position of the two outer bands was allowed to vary between 1085 and 1093 cm-1, which correspond to the maximum splitting observed at 25 °C. Their width was kept below 3.5 cm-1. An additional broad band at 1083 ( 2 cm-1 was required to simulate (44) Snyder, R. G.; Strauss, H. L.; Cates, D. A. J. Phys. Chem. 1995, 99, 8432.

a low-wavenumber shoulder. The intensity of the middle band (IM) was then divided by the sum of the intensities of all three major bands (IT), and the variation of the IM/IT ratio is plotted as a function of temperature in Figure 5B. Note that the middle band intensity is not zero at low temperatures, suggesting that a fraction of the FFA exists in a non-orthorhombic phase. This fraction could correspond to the 20% non-solid phase detected by 2H NMR spectroscopy at those temperatures. The IM/IT value does not change much until a characteristic temperature is reached. Then, as the middle band grows, the IM/IT value increases, eventually reaching unity where there is a complete merger of the split components. The increase of the IM/IT value begins at about 31 (C14), 39 (C16), 43 (C18), and 49 °C (C20). For BBCer/ chol/FFA-C22, the middle band first grows very gradually whereas an abrupt increase is observed at ∼61 °C. The complete merger occurs at 39 (C14), 49 (C16), 57 (C18), 63 (C20), and 67 °C (C22). This merger is correlated with the drop of the 2H NMR % solid data, indicating that the merger is the result of the melting of the FFAs into a non-solid phase. The CH2 deformation (δCH2) band also showed a splitting (data not shown) at low temperature, indicating that BBCer is in an orthorhombic crystalline phase. The δCH2 band splitting is not as clear as that of the δCD2 band, probably because of the heterogeneous alkyl chain composition. For the δCH2 band, the width was calculated at 90% of its height. A decrease of the bandwidth associated with the collapse of the splitting was observed upon heating. The δCH2 band merger occurred over the temperature range ∼31-43 (C14), ∼37-47 (C16), ∼39-51 (C18), ∼43-55 (C20), and ∼45-57 °C (C22) (see Figure 7). This merger appears over the same temperature range with the first shift of the νsCH band. The region between 1540 and 1800 cm-1 includes the Amide I′ vibrational mode of the ceramides at ∼1620 cm-1 and the carbonyl stretching (νCOOH) associated with the carboxylic group of the FFAs at ∼1700 cm-1. Figure 6 shows the FT-IR spectra of this region for BBCer/chol/FFA-C14 (left panel) and BBCer/chol/FFA-C22 (right panel). Below 50 °C, the Amide I′ band shows a common feature for all the mixtures. This vibrational mode includes three components at 1630, 1615, and 1597 cm-1. Upon heating, the component at 1597 cm-1 first disappears, and subsequently, the remaining two components become a broad band centered at ∼1630 cm-1. The low-wavenumber component disappears over the same temperature range with the completion

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Figure 6. Region between 1570 and 1790 cm-1 of the IR spectra of BBCer/chol/FFA-C14-d27 (left panel) and BBCer/chol/FFA-C22d43 (right panel).

of the first shift of the νsCH band. The remaining two components merge during the second shift of the νsCH band (see Figure 7). Below 35 °C, the νCOOH band is found at 1695 cm-1 with a shoulder at 1683 cm-1 for all the model membranes. There was no signal associated with the carboxylate group, at 1560 cm-1, indicating that the free fatty acids were completely protonated, in agreement with previous results.21 Upon heating, the band shifts to 1706 cm-1, and in parallel, the low-frequency shoulder disappears. The shift of the νCOOH band occurs in a concomitant manner with the merger of the δCD2. This shift is also correlated with the first shift of the νsCD band for BBCer/chol/FFA-C14 to -C18 and the abrupt shift of the νsCD band for BBCer/chol/ FFA-C20 and -C22 (see Figure 7).

Discussion Our results show that variation of free fatty acid chain length produces significant effects on the ensemble phase behavior of biomimetic SC model membranes. The effect is strongly dependent on temperature. There were also similarities in phase behavior among the ensembles, despite the variation in FFA chain length. The most striking was that at low temperatures all the investigated mixtures formed coexisting crystalline domains rich in either FFA or BBCer. A spectral component due to solid FFA is observed by 2H NMR, and the formation of the orthorhombic crystal has been detected by FT-IR spectroscopy for both BBCer and FFAs. The formation of phase-separated crystalline domains and limited miscibility in BBCer/chol/FFA-C16 mixtures have been observed previously for a wide range of lipid compositions.7,17,20,29 The present data extend this conclusion showing that ternary mixtures with FFAs having longer and shorter chains than PA also form separated solid domains at low temperatures. It is estimated by the 2H NMR data that 80% of the FFAs exist in this solid phase irrespective of their chain length. We propose this solid-phase separation to be a general and unique feature of these SC lipid model membranes and strongly suspect such separation may have biological significance in vivo. Upon heating the SC model membranes containing crystalline domains, the chain length of FFA plays an essential role in determining the composition and the stability of the disordered phases obtained. When the FFA chain length is between C14 and C18, the acid undergoes a transition involving cholesterol and ceramides to lead to the formation of a lo phase. For these FFAs, this first transition is characterized by the 2H NMR M1 drop, a

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Figure 7. Summary of the thermal behavior of the model membranes observed from a combination of 2H NMR and FT-IR spectroscopic parameters. Represented thermal changes: for BBCer, the disappearance of the low wavenumber shoulder of the Amide I′ band (Amide I′ band 3f2), the merger of the Amide I′ band (Amide I′ band 2f1), the δCH2 band merger; for FFA, the CO band shift, the δCD2 band merger, the NMR % solid drop, NMR isotropic signal growth.

shift of the νsCD band, the merger of the δCD2 splitting, and the CO band shift (Figures 2, 4, and 7). The 2H NMR spectra clearly indicate that the FFA forms a lo phase. For BBCer, this first transition is characterized by the shift of the νsCH band, the merger of the δCH2 splitting, and the disappearance of the lowwavenumber component of the Amide I′ band (Figures 4 and 7). The νsCH values are compatible with those obtained for lipids in the lo phase.29,42,45 The formation of the lo phase has been reported previously for ternary mixtures of BBCer/chol/FFAC16.7,21,29 From these studies, it was concluded that cholesterol acts as a promoter of a lo phase in the SC models, a feature shared with phospholipid membranes.46-48 Moreover, a high cholesterol content (BBCer/chol/FFA-C16; 1:1:3) was shown to induce a sharp solid-lo transition at 55 °C in which both BBCer and PA were involved.21 That lo phase was stable over the investigated temperature range, up to 75 °C. Therefore, a relatively high cholesterol concentration is needed for inducing exclusively a lo phase involving all BBCer and FFA molecules. When the cholesterol concentration is lower, the proportion of FFA and BBCer molecules participating in the lo phase progressively increases as a function of increasing cholesterol concentration.21 Recently, it has also been found that cholesterol can form stable lo phase in the presence of FFAs.10,11,42 For example, an equimolar binary mixture of FFA-C16 and cholesterol forms a stable lo phase. These results and our observations suggest that for the model membranes with short-chain FFAs (C14-C18), FFAs and cholesterol are mainly responsible for the formation of the lo phase. This phase appears to include some BBCer molecules, and their proportion increases upon heating. This behavior is most pronounced with BBCer/chol/FFA-C14, where the solid-lo transition is characterized by a lo phase initially very rich in FFA-C14 whose BBCer concentration increases gradually as the temperature is raised (Figures 2 and 4). A related behavior was previously reported for the BBCer/chol/FFA-C16 equimolar mixture.20 Note that the transition of BBCer is (45) Silvius, J. R.; del Giudice, D.; Lafleur, M. Biochemistry 1996, 35, 15198. (46) Bloom, M.; Mouritsen, O. G. Can. J. Chem. 1988, 66, 706. (47) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451. (48) Ipsen, J. H.; Mouritsen, O. G.; Bloom, M. Biophys. J. 1990, 57, 405.

Fatty Acids in Model SC Membranes

progressively shifted toward high temperature as the FFA chain becomes longer, whereas the melting of the FFAs and their incorporation in the lo phase happens over a larger temperature range. This suggests that the interactions between FFAs and cholesterol that result in the formation of the lo phase become less favorable as the FFA chain becomes longer in these mixtures. When the model membranes include FFAs with long alkyl chains (C20 and C22), the thermal phase behavior is different. In this case, the significant hydrophobic mismatch between FFA and cholesterol appears to prevent the full participation of FFAs in the formation of a lo phase. As a consequence, the phase behavior of these model membranes has features distinct from those observed in membranes having shorter chain FFAs. First, the ceramide component’s behavior for models containing FFAC20 and -C22 differs from that observed in the presence of the shorter chains FFA-C14 to -C18 (Figures 4, and 7) in being insensitive to the FFA chain length. We therefore conclude that FFA chain length has little effect on the ceramide thermotropism in these model membranes. Second, the number of FFA molecules in the lo phase is very limited for FFA-C20 and -C22 and the FFA thermotropism is dominated by a direct transition from a solid to a disordered isotropic phase. We interpret this to be due to poor hydrophobic matching between the long-chain FFAs and cholesterol. In fact, a recent report demonstrates the necessity of hydrophobic matching between FFA and cholesterol for the formation of a lo phase.11 In that study, among the binary mixtures of Chol/FFAs with FFA chain length varying between C12 and C24 (C20 and C22 were not included), only those with good hydrophobic matching (∼C14-C18) were found to form a lo phase. FFAs with too short (C12) or too long (C24) alkyl chain were not able to form a lo phase with cholesterol. Similar results were reported by the previous studies on the association of phospholipids with cholesterol in monolayers, and it has been shown that a good match in acyl chain length is found with phospholipids with carbon units of 14-17.49-51 Our present findings suggest that the hydrophobic mismatch between FFAs with carbon units C20 and C22 and cholesterol is unfavorable to the formation of a lo phase. Consequently, the transitions of these model membranes with FFA-C20 and -C22 observed at 50-60 °C is likely a solid-lo phase change involving mostly BBCer and cholesterol. A similar transition obtained for BBCer/ chol binary mixtures21 supports this conclusion. Being excluded from the lo phase formed by BBCer and cholesterol, FFA-C20 and -C22 are mostly in a solid crystalline phase up to 55 °C (% solid ≈ 70). Further heating the model membranes destabilizes the lo phase, and lipids undergo a transition toward a disordered phase. For BBCer, this second transition is characterized by the shift of the νsCH band to ∼2854 cm-1 (Figure 4) and the formation of a broad Amide I′ band centered at ∼1630 cm-1 (Figure 7). These IR features are characteristic of a disordered phase such as, for example, micelles.29,43 The appearance of the 2H NMR narrow signal (Figure 7), the resulting drop of M1 (Figure 2), and the shift of the νsCD band to ∼2096 cm-1 (Figure 4) indicate the disordering of FFAs. For all the investigated model membranes, this transition occurs over a similar temperature range (60-70 °C). However, the origin of these transitions is likely dependent on the FFA chain length. For ∼C14-C18, the progressive solubilization of BBCer in the FFA/Chol-based lo phase destabilizes the lamellar structure. On the other hand, for C20 and C22, the abrupt melting of the FFA crystalline phase suddenly (49) Mattjus, P.; Hedstro¨m, G.; Slotte, J. P. Chem. Phys. Lipids 1994, 74, 195. (50) Slotte, J. P. Biochim. Biophys. Acta 1995, 1238, 118. (51) Hagen, J. P.; McConnell, H. M. Biochim. Biophys. Acta 1997, 1329, 7.

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enriches the lo phase, which previously consisted mainly of BBCer and cholesterol, in FFA, leading to a transition to a disordered phase. Therefore, it is concluded that both ceramide and FFA must be present to obtain the formation of this isotropic phase. When these species are both involved in the disordered phase, the cholesterol content in this phase may not be sufficient to maintain the system in a lamellar structure. This conclusion is supported by the previous studies on the binary (1:3) BBCer/ chol,21 and (3:7) PA/chol42 systems, as well as the ternary 1:3:1 BBCer/chol/FFA-C16d31 system, showing that, with a higher cholesterol molar fraction, these mixtures remain mainly in a lo phase up to 70 °C. The structure of this isotropic phase has not been determined yet. On the basis of the increased intensity of the narrow line in the 2H NMR spectra of the BBCer/chol/PAC16 equimolar mixture in the presence of oleic acid at 25 °C, the formation of oily droplets in which SC lipid components would be dissolved has been proposed.52 Such a proposition is also consistent with the systems investigated here at high temperature. The present study shows that hydrophobic matching is a very important factor defining lipid mixing properties and phase stability in SC model membranes. At low temperatures, two crystalline phases, enriched in FFA and BBCer, respectively, coexist. We have demonstrated that the solid-lo transition of the domains begins close to skin temperature when the FFA chain is sufficiently short (C14). As the FFA chain lengthens, the hydrophobic mismatch with cholesterol becomes more pronounced and, as a consequence, the mixing between FFA and cholesterol in the lo phase becomes less favorable. Therefore, the intermediate cholesterol-rich lo phase is initially composed mainly of either FFA (for mixtures including FFA-C14 to -C18) or BBCer (for mixtures including FFA-C20 and -C22). Upon heating, the lo phase is enriched by the other lipid; this leads to a reduction of the cholesterol mole fraction in this phase and its eventual destabilization. At high temperatures, the formation of a disordered phase such as oily droplets is inferred from all the spectroscopic parameters. In human SC, FFAs with chain length ranging between C16 and C26 have been extracted.12-15 Among those FFAs, molecules with shorter chain length display a preferential association with cholesterol while those with longer ones can be involved preferentially in the crystalline domains. The “domain mosaic model” of SCIM predicted that crystalline domains are formed by long-chain lipids and are surrounded by liquid crystalline border of relatively short chain lipids and free fatty acids in order that SC can achieve its barrier function and proper mechanical properties at the same time.4 Our present study provides molecular details that could explain the origin of this proposed phase coexistence. It should be noted that model mixtures formed with equimolar mixtures of BBCer, cholesterol, and FFA, where the FFA chain length distribution is similar to that of the SC, display at room temperature phase-separated cholesterol and FFA.31 Comparing these results with those obtained from analogous model mixtures prepared from a single FFA (namely FFA-C16),8 it was concluded that the presence of the mixture of FFA promote significantly the mixing of ceramides and FFA, at the X-ray diffraction length scale. The phase behavior of more complex systems, including mixtures of FFA with different chain length and ceramide mixtures reproducing the short and long lamellar spacings, should definitely be examined to determine how the phase mixing propensities determined here might be extrapolated to models containing SC ceramides. In the meantime, we point out some possible biological implications. First, the solid lipid domains (that we assume are (52) Rowat, A. C.; Kitson, N.; Thewalt, J. L. Int. J. Pharm. 2006, 307, 225.

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critical to SC barrier function) must form through lipid crystallization and phase separation at physiological temperatures. We cannot conceive how such crystallization could occur within a biological membrane capable of normal functions such as fusion and exocytosis. Second, although crystallization could perhaps begin within the membrane contents, but not the outer membrane, of the lamellar body (lamellar granule), larger domains could logically only occur following extrusion from the keratinocyte, and therefore within the intercellular spaces of cell layers entering the SC compartment. The rate and extent of lipid crystallization during epidermal differentiation must be determined by local physical conditions of course, but we presume the crystallization will begin to occur at some reasonably predictable point in epidermal differentiation, when lipid modifications result in inevitable phase separation. We presume also that the entire crystallization process, and resultant barrier formation, would be altered by ambient physical and pathological conditions, inflammation being an obvious example. Epidermal differentiation is accelerated in inflammatory

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conditions such as psoriasis, and one might expect that the extent of lipid crystallization would be reduced as a result. The physiological consequence of reduced SC lipid crystallization might be impaired barrier function. Acknowledgment. The authors thank the Natural Sciences and Engineering Research Council of Canada for the financial support of this Collaborative Health Research Project. M.L. is also grateful to the Fond Que´be´cois de la Recherche sur la Nature et les Technologies (FQRNT) through its financial support to the Center for Self-Assembled Chemical Systems (CSACS). J.T. thanks Dr. Denis Langlais for his expert help. Supporting Information Available: The amount of fatty acid in the solid phase as a function of temperature, as determined by 2H NMR, in the model membranes FFA-C14, -C18, -C20, or -C22. This material is available free of charge via the Internet at http:// pubs.acs.org. LA063640+