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Hydrophobic Match between Cholesterol and Saturated Fatty Acid Is Required for the Formation of Lamellar Liquid Ordered Phases Jaclin Ouimet and Michel Lafleur* Department of Chemistry, Universite´ de Montre´ al, Montre´ al, Que´ bec, Canada, H3C 3J7 Received April 5, 2004. In Final Form: June 1, 2004 Palmitic acid and cholesterol have been shown to form, under certain conditions, bilayers in the liquid ordered (lo) phase. In the present work, the contribution of the hydrophobic match between cholesterol (chol), and the acyl chain of saturated fatty acids (FA) has been examined. The behavior of FA/chol mixtures where the FA acyl chain length was varied between 12 and 24 carbon atoms was investigated by infrared and 2H NMR spectroscopy, as well as by differential scanning calorimetry. It was found that only fatty acids with acyl chains of 14-18 carbon atoms lead to the formation of lo phase bilayers. The length of these chains corresponds, in fact, to the length of the long axis of the cholesterol molecule. Therefore, the hydrophobic match between the apolar parts of the molecular constituents appears to be a requisite for the formation of lamellar lo phases.

Introduction The contribution of hydrophobic matching in controlling the self-assembly of membranes has been demonstrated to be significant in various systems. Generally, the term “hydrophobic mismatch” is used to characterize the geometrical arrangement of the hydrophobic segments of molecules along the membrane normal. When molecules bear apolar segments that are considerably shorter or considerably longer than the apolar core thickness of the membrane in which they are embedded, there is an unfavorable free energy contribution associated with this mismatch, leading to some reorganization of the assembly. The hydrophobic mismatch has been shown to play a central role in the interactions between transmembrane peptides or protein segments and lipid membranes1,2 and has been proposed to play a significant role in phenomena like protein sorting3 and the formation of lipid rafts.4,5 Hydrophobic mismatch has also been shown to dictate the mixing properties of phospholipids in model mixtures. It has been observed, for example, that immiscibility in binary mixtures of saturated phospholipids is directly associated with hydrophobic mismatch of their acyl chains.6,7 Cholesterol is a fundamental component of mammal plasmic membranes. It is largely a stiff molecule, as the steroid ring network has very limited mobility. Once incorporated in fluid membranes, cholesterol aligns its long axis with the bilayer normal, with its hydroxyl, in position C3, being located at the interface; this arrangement is reported for lipid model membranes,8-11 biological * Author to whom correspondence should be addresed. Michel Lafleur, Department of Chemistry, C. P. 6128, Succ. Centre Ville, Universite´ de Montre´al, Montre´al, Que´bec, Canada, H3C 3J7. Fax: (514) 343-7586, E-mail: [email protected]. (1) Mouritsen, O. G.; Bloom, M. Biophys. J. 1984, 46, 141-153. (2) Killian, J. A. Biochim. Biophys. Acta 1998, 1376, 401-416. (3) Bretscher, M. S.; Munro, S. Science 1993, 261, 1280-1281. (4) London, E.; Brown, D. A. Biochim. Biophys. Acta 2000, 1508, 182-195. (5) Benting, J.; Rietveld, A.; Ansorge, I.; Simons, K. FEBS Lett. 1999, 462, 47-50. (6) Mendelsohn, R.; Liang, G. L.; Strauss, H. L.; Snyder, R. G. Biophys. J. 1995, 69, 1987-1998. (7) Cevc, G.; Marsh, D. Phospholipid Bilayers: Physical Principles and Models; Wiley: New York, 1987.

membranes such as human red blood cell membranes,12 and plasmic membranes of Acholeplasma laidlawii.13 Hydrophobic match between rigid cholesterol and lipids is a significant contribution in the membrane energetics, as proposed by several experimental studies. In monolayers, it was shown by AFM that a small amount of cholesterol was soluble in a palmitic acid monolayer, but no miscibility was observed for lignoceric acid.14 Similarly, the lateral heterogeneities in saturated phosphocholines (PC)scholesterol monolayers are influenced by the PC acyl chain length.15 In the study of the effect of cholesterol on a series of saturated PC bilayers,16 it was found that the shift of the gel-to-liquid-phase transition temperature caused by cholesterol is dependent on the hydrophobic mismatch between the sterol and the PC bilayers. It was also showed that the lateral miscibility of cholesterol was affected by the phosphoethanolamine (PE) chain length.17 In binary mixtures of PC of differing acyl chain length (specifically 12 and 16 carbon atom long), it was shown that cholesterol can promote or inhibit lateral phase separations, depending on its concentration.18 This behavior was rationalized partly by hydrophobic mismatch. Along the same line, nearest-neighboor recognition experiments have shown that sterol-phospholipid associations are, under certain conditions, influenced by lipid chain length.19-21 For example, it was recently reported (8) Pare´, C.; Lafleur, M. Langmuir 2001, 17, 5587-5594. (9) Fenske, D. B.; Thewalt, J. L.; Bloom, M.; Kitson, N. Biophys. J. 1994, 67, 1562-1573. (10) Dufourc, E. J.; Parish, E. J.; Chitrakorn, S.; Smith, I. C. P. Biochemistry 1984, 23, 6062-6071. (11) Marsan, M. P.; Muller, I.; Ramos, C.; Rodriguez, F.; Dufourc, E. J.; Czaplicki, J.; Milon, A. Biophys. J. 1999, 76, 351-359. (12) Kelusky, E. C.; Dufourc, E. J.; Smith, I. C. P. Biochim. Biophys. Acta 1983, 735, 302-304. (13) Monck, M. A.; Bloom, M.; Lafleur, M.; Lewis, R. N. A. H.; McElhaney, R. N.; Cullis, P. R. Biochemistry 1993, 32, 3081-3088. (14) Sparr, E.; Ekelund, K.; Engblom, J.; Engstro¨m, S.; Wennerstro¨m, H. Langmuir 1999, 15, 6950-6955. (15) Slotte, J. P. Biochim. Biophys. Acta 1995, 1238, 118-126. (16) McMullen, T. P. W.; Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1993, 32, 516-522. (17) McMullen, T. P. W.; Lewis, R. N. A. H.; McElhaney, R. N. Biochim. Biophys. Acta 1999, 1416, 119-134. (18) Silvius, J. R.; del Giudice, D.; Lafleur, M. Biochemistry 1996, 35, 15198-15208.

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that cholesterol favors phospholipids bearing 18 carbon chains over those with 14 carbon chains.21 Recently, we have shown that palmitic acid (PA) and cholesterol, despite the fact that these chemicals do not form fluid lamellar phases when hydrated individually, can form fluid bilayers in the liquid ordered (lo) phase when mixed together.8,22 These species are immiscible at low temperatures, but above 50 °C, cholesterol intercalates between palmitic acid molecules, stabilizing a lamellar architecture and rigidifying the PA acyl chains. The mixture behaves like a eutectic at a PA/chol molar composition of 30:70, as reported in the proposed temperature-composition diagram.8 In the present paper, we are examining the effect of acyl chain length of fatty acids (FA) on the formation of fluid lamellar phases with cholesterol to highlight the role of hydrophobic mismatch, and to explore the requirement at the FA acyl chain level to form these lamellar phases. We have examined, using differential scanning calorimetry (DSC), infrared (IR) and 2 H NMR spectroscopies, whether such lo bilayers could be formed with any fatty acid bearing saturated chains. The chain length was varied between 12 and 24 carbon atoms. We report here that hydrophobic matching is a relevant factor regarding the formation of this phase. Materials and Methods Deuterated lauric (dodecanoic) acid (LA-d23), deuterated myristic (tetradecanoic) acid (MA-d27), and deuterated lignoceric (tetracosanoic) acid (LignA-d47) were purchased from CDN Isotopes (Pointe Claire, Que´bec). Deuterated stearic (octadecanoic) acid (SA-d35) was obtained from Cambridge Isotope Laboratories (Andover, MA). Deuterium-depleted water and heavy water were purchased from Aldrich (Milwaukee, WI) while cholesterol and MES were obtained from Sigma Chemical Co. (St. Louis, MO). DSC. FA/chol mixtures were prepared by mixing appropriate volumes of individual lipid stock solutions prepared in a benzene/ methanol mixture (95/5 v/v). The organic solutions were freezedried for at least 15 h. The resulting lipid powder was hydrated with MES buffer (5 mM MES, 5 mM NaCl, and 0.5 mM EDTA), pH 5.5. To ensure proper hydration, the samples were submitted to at least three freeze-and-thaw cycles, from liquid nitrogen temperature to a temperature above the melting point of the pure acid. During this process, the samples were frequently vortexed. The pH was then measured and readjusted, if necessary. The final overall concentration of the suspensions was about 21 mg of total lipids/mL of buffer. The samples were formed by white macroscopic lipid particles and a clear supernatant. Controls showed that there was no lipid in solution in the supernatant. Upon heating above the transition temperature, the samples became turbid suspensions. The thermograms were recorded on a VP-DSC microcalorimeter (MicroCal, Northampton, MA), using a heating rate of 15 °C/h. The reference cell was filled with deionized water. Data acquisition and treatment were performed with the Origin software (Microcal software, Northampton, MA) IR Spectroscopy. For IR spectroscopy, the sample preparation was similar to that above, except that a Mes buffer (100 mM, 25 mM NaCl, 5 mM EDTA) pH 5.5 was used. The buffer was prepared in D2O to avoid the spectral interference from the H2Odeformation band, overlapping with the C-O stretching mode of the carboxylic group. 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 ring. The spectra were acquired on a BioRad FTS-25

Figure 1. DSC thermograms obtained for the different investigated fatty acids, with and without cholesterol. (A) Pure hydrated LA-d23, (B) LA-d23/chol mixture, (C) pure hydrated MA-d27, (D) MA-d27/chol mixture; (E) pure hydrated SA-d35, (F) SA-d35/chol mixture; (G) pure hydrated LignA-d47; and (H) LignA-d47/chol mixture. All the FA/chol mixtures were in equimolar proportion. The pure acids were examined over a limited temperature range because their signals served only as references for the binary mixtures. spectrometer using standard conditions:23 100 scans with a 2 cm-1 resolution for each spectrum, the spectra are recorded from low to high temperatures, with a temperature equilibration period of 12 min. 2H NMR Spectroscopy. For the 2H NMR spectroscopy, the sample preparation was similar to that described above, except that the buffer, prepared in deuterium-depleted water, contained 100 mM Mes, 100 mM NaCl, and 5 mM EDTA, (pH 5.5). The final lipid concentration was 225 mg of total lipids/mL of buffer. The spectra were recorded on a Bruker DSX-300 spectrometer operating at 44 MHz for 2H nuclei. A Bruker static probe equipped with a 5 mm coil was used. A quadrupolar echo sequence was used with a 90° pulse, and an interpulse delay of 3 and 40 µs, respectively. After the second pulse, 8192 points were recorded in quadrature mode, with a dwell time of 0.5 µs. The recycling time was 50 s for the spectra showing a solid component (its T1 was estimated to be around 10 s) or 500 ms in the absence of a slow-relaxation component. Typically 1350 FID were co-added for the spectra with a solid component, whereas 20 000 FID were performed in the absence of this component. The temperature was regulated using a Bruker VT-100 controller.

Results (19) Davidson, S. M. K.; Liu, Y.; Regen, S. L. J. Am. Chem. Soc. 1993, 115, 10104-10110. (20) Sugahara, M.; Uragami, M.; Regen, S. L. J. Am. Chem. Soc. 2003, 125, 13040-13041. (21) Sugahara, M.; Uragami, M.; Regen, S. L. J. Am. Chem. Soc. 2002, 124, 4253-4256. (22) Ouimet, J.; Croft, S.; Pare´, C.; Katsaras, J.; Lafleur, M. Langmuir 2003, 19, 1089-1097.

DSC. Every fatty acid studied in aqueous milieu gives rise to a single and narrow peak in their thermograms (the width at half-height was between 0.3 and 1.0 °C) (Figure 1). The measured melting temperatures (maxima (23) Pare´, C.; Lafleur, M. Biophys. J. 1998, 74, 899-909.

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of the peaks) are estimated to be 38.8, 49.1, 64.2, and 78.9 °C for LA-d23, MA-d27, SA-d35, and LignA-d47, respectively. These temperatures are 5-6 °C below the reported melting point of the acids,24 the literature data being obtained on anhydrous and perhydrogenated fatty acids. These shifts are a combined effect of the deuteration of the chains and the hydration of the acids. The decrease of the gel-tofluid-phase transition temperature by a few degrees, associated to acyl chain deuteration, is well established in the case of phospholipids.25 The thermal behavior of the fatty acids is influenced by the presence of cholesterol. In the equimolar mixture of LA-d23 and cholesterol (Figure 1B), two transitions are observed. The main transition at 36.2 °C is 2.6 °C lower than that observed for the hydrated pure fatty acid. Its width remains small (a width at half-height of 0.6 °C). A second transition with a considerably smaller enthalpy is observed at 73 °C. This temperature corresponds to that observed for the dehydration of crystalline cholesterol.26,27 In the case of the MA-d27/chol mixture, two very close peaks are obtained at 45.7 and 46.2 °C. They appear about 3 °C lower than the transition observed for hydrated myristic acid. No transition is observed between 70 and 75 °C. The thermal behavior of stearic acid is drastically affected by the presence of cholesterol. Two maxima at about 57.6 and 61.1 °C are observed. The first peak is almost 3 times wider than the second one. Again, no transition between 70 and 75 °C could be detected. In the case of LignA-d47/chol equimolar mixtures, two transitions are observed at 73.3 and 75.0 °C. The first peak corresponds to the temperature where crystalline cholesterol dehydration is observed. The second peak is about 4 °C lower than the transition observed for the pure hydrated fatty acid. It has to be noted that the changes in heat capacity observed as inflections were often detected but were not reproducible. They are thought to be associated with artifacts due to the sample macroscopic heterogeneity. IR Spectroscopy. The thermal behavior of the fatty acids was examined using the band associated with the symmetric C-D stretching (νCD) mode (Figure 2). This mode is mainly sensitive to trans-gauche isomerization along the acyl chains and to interchain coupling, providing a sensitive probe for transitions involving the introduction of chain conformational disorder.28,29 In our study, the use of the deuterated analogues has also the advantage of preventing spectral interferences from cholesterol. The melting of the pure hydrated fatty acids (filled symbols) is easily detected by the abrupt shift of the νCD band, from ≈2086 to 2096-2097 cm-1, except in the case of lauric acid, for which the νCD band is observed at 2090 cm-1 at low temperatures. Values below 2090 cm-1 are generally indicative of highly ordered chains, while values around 2096 cm-1 are characteristic of disordered chains. This well-established interpretation is fully consistent with the fatty acids undergoing a transition from a crystalline to a liquid state. The transition temperatures determined from the νCD mode correspond to those obtained by DSC. (24) Gunstone, F. D.; Harwood, J. L.; Padley, F. B. The Lipid Handbook, 2nd ed.; Chapman & Hall Chemical Database: New York, 1994. (25) Davis, J. H. Biophys. J. 1979, 27, 339-358. (26) Epand, R. M.; Bach, D.; Borochov, N.; Wachtel, E. Biophys. J. 2000, 78, 866-873. (27) Loomis, C. R.; Shipley, G. G.; Small, D. M. J. Lipid Res. 1979, 20, 525-535. (28) Mantsch, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213-226. (29) Kodati, V. R.; El-Jastimi, R.; Lafleur, M. J. Phys. Chem. 1994, 98, 12191-12197.

Ouimet and Lafleur

Figure 2. Thermal behavior of the fatty acid as probed by the position of the νCD band. (A) Pure hydrated LA-d23 (9) and LAd23/chol mixture (4); (B) pure hydrated MA-d27 (9) and MAd27/chol mixture (4); (C) pure hydrated SA-d35 (9) and SA-d35/ chol mixture (4); and (D) pure hydrated LignA-d47 (9) and LignA-d47/chol mixture (4). All the FA/chol mixtures were in equimolar proportion.

Figure 2 shows the influence of cholesterol on the thermal behavior of the fatty acids. Below the transition temperature, cholesterol slightly increases the wavenumber of the νCD mode by about 1-2 cm-1. Upon heating, the disordering of the acyl chain of the fatty acids, giving rise to the increase in νCD frequency, is observed about 3 °C lower for the FA/chol equimolar mixtures than for the pure hydrated fatty acids. This finding is in very good agreement with the DSC results. The band shifts occur abruptly (over 1 or 2 °C), except in the case of SA-d35/chol mixtures where the shift is spread over a 5 °C interval, with an inflection in the middle of the transition. The derivative of this curve shows, in fact, a striking resemblance with the DSC trace obtained for this mixture (data not shown). For temperatures higher than the transition temperature, cholesterol has no significant effects for the mixtures with lauric and lignoceric acids, whereas it causes a decrease in the νCD frequency for MA-d27/chol and SAd35/chol mixtures. The effect of cholesterol on the position of the νCD band in the fluid phase is summarized in Figure 3. At low temperatures, the IR spectra of all the investigated hydrated fatty acids, as well as all the FA/chol equimolar mixtures, show a splitting of the CD2 deformation band at 1090 cm-1 (data not shown). Even though the splitting is slightly larger for the long-chain fatty acids than for the short-chain ones (4.5 cm-1 for lauric acid up to 7.0 cm-1 for lignoceric acid), they have the same magnitude with and without cholesterol. These splittings are indicative of the formation of a crystalline phase where the acyl chain packing adopts an orthorhombic symmetry.30,31 The fact that the splitting is not affected by the presence of cholesterol indicates that, at low temperatures, not only the fatty acid remains in the crystalline phase with an orthorhombic chain packing but the two components are also extensively phase separated.31,32 It was (30) Snyder, R. G.; Goh, M. C.; Srivatsavoy, V. J. P.; Strauss, H. L.; Dorset, D. L. J. Phys. Chem. 1992, 96, 10008-10019. (31) Snyder, R. G.; Strauss, H. L.; Cates, D. A. J. Phys. Chem. 1995, 99, 8432-8439.

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Figure 3. Effect of the fatty acid chain length on the shift of the νCD band induced by the addition of cholesterol in an equimolar proportion to the fatty acid. The shift is measured at about 10 °C above the melting temperature. The data for palmitic acid (C16) were obtained from ref 8.

estimated that, to get the maximum splittings such as in our systems, the domains should include at least 100 molecules of fatty acid.30,31 In all the investigated mixtures, the components of the methylene deformation band merge to give rise to a single component at 1088 cm-1 at the melting temperature. Similar to the results obtained from DSC and from the shift of the νCD band in the IR spectra, the merge is observed 3-6 °C lower for the cholesterolcontaining mixtures compared to that of the pure hydrated fatty acids (data not shown). The region associated to the CdO stretching (νCO) mode of the carboxylic group, between 1650 and 1750 cm-1, has also been examined (Figure 4). For all the samples, no band could be observed around 1550 cm-1, the position where the C-O stretching mode of the carboxylate is expected.33 Therefore, it is concluded that, at pH 5.5, the acid is essentially all protonated. This is consistent with the pKaapp value of 8.4 previously found for palmitic acid mixed with cholesterol in equimolar proportions.22 In the case of the pure hydrated fatty acids, below the transition temperature, the νCO band shows similar profiles composed of at least two components located at 1685-1686 cm-1 and 1704-1707 cm-1, as determined by spectral simulation. Upon melting, the band is shifted toward the high frequencies, a change which is likely associated to the weakening of the hydrogen-bond network between the carboxylic groups upon melting. This shift is also associated to a narrowing of the band, this effect being more pronounced for the acids with longer acyl chains. In the presence of cholesterol, the νCO band, at low temperatures, is generally narrower than that observed for pure hydrated FA, irrespective of the acyl chain length. Two components at around 1697 and 1684 cm-1 are observed. The observed weak component at 1669 cm-1 was observed in the spectrum of monohydrate crystalline cholesterol and is likely associated to a CdC stretching mode of the sterol.34 Upon heating the mixtures above their transition temperature, the νCO band is shifted toward high-frequency values, suggesting a decrease in (32) Mendelsohn, R.; Moore, D. J. Chem. Phys. Lipids 1998, 96, 141157. (33) Go´mez-Ferna´ndez, J. C.; Villalaı´n, J. Chem. Phys. Lipids 1998, 96, 41-52. (34) Silverstein, R. M.; Webster, F. X. Spectrometric Identification of Organic Compounds; John Wiley & Sons: Toronto, 1998.

Figure 4. Region of the IR spectra associated with the νCO mode of the carboxylic group. (A) Pure hydrated LA-d23 (25, 35, 40, 47 °C); (B) LA-d23/chol mixture (25, 30, 40, 54 °C); (C) pure hydrated MA-d27 (30, 40, 51, 60 °C); (D) MA-d27/chol mixture (30, 40, 51, 65 °C); (E) pure hydrated SA-d35 (20, 55, 64, 70 °C); (F) SA-d35/chol mixture (25, 50, 60, 70 °C); (G) pure hydrated LignA-d47 (20, 60, 76, 82 °C); and (H) LignA-d47/chol mixture (25, 70, 76, 80 °C). All the FA/chol mixtures were in equimolar proportion. The spectra have been slightly offset to facilitate the identification of the associated temperature: the temperature at which the spectra were recorded is increasing from the top spectrum to the bottom spectrum.

the H-bond strength in which the carbonyl of the carboxylic acid is involved. This shift is concomitant with the melting of the chain reported by the νCD mode. The peak observed at high temperatures seems to include one main component located at around 1705-1707 cm-1. The νCO band is narrower for the LA-d23/chol and LignA-d47/chol mixtures, with a width at half-height of about 27-29 cm-1, relative to those measured for MA-d27/chol and SA-d35/chol mixtures, for which the width at half-height of the νCO band varies between 39 and 46 cm-1. 2 H NMR Spectroscopy. At 22-23 °C, the 2H NMR spectra of the FA/chol systems are formed from two powder patterns (Figure 5). The quadrupolar splittings associated with these, measured between the maxima, correspond to 34 and ≈115 kHz. This signal is characteristic of all trans chains that do not experience any motion (on the 2H NMR time scale). All the methylene groups contribute to the larger-powder pattern, whereas the terminal methyl is associated to the narrower one. This reduced quadru-

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Figure 6. Effect of the acyl chain length on the lamellar phase proportion present in the FA/chol systems, as estimated from the 2H NMR spectra recorded 6 °C above the melting temperature of the fatty acid. The data for PA-d31/chol system is obtained from ref 8.

Figure 5. 2H NMR spectra of the FA/chol systems. LA-d23/ chol mixture at (A) 23, and (B) 37 °C; MA-d27/chol mixture at (C) 22, and (D) 52 °C; SA-d35/chol mixture at (E) 22, and (F) 66 °C; and LignA-d47/chol mixture at (G) 22, and (H) 73 °C. All the FA/chol mixtures were in equimolar proportion. In the spectra in (D) and (F), the narrow line, referenced to 0 kHz, has been truncated to highlight the fluid component.

polar splitting is attributed to the rotation of the methyl group along the CD2-CD3 bond.25,35,36 These spectra are very similar to those recorded for the pure hydrated fatty acids (data not shown). It is interesting to note that the maxima of the wider powder pattern of the spectrum of the LA-d23/chol system are considerably broader than for those of the fatty acids with longer chains (the feature is also observed on hydrated LA-d23). For this system, the spectra were recorded relatively close to the melting point of the FA, and it is likely that some motions near the end of the acyl chains become detectable by 2H NMR spectroscopy. Above the transition temperature, the spectra of the FA/chol systems are very different. For the lauric and lignoceric acids, the spectra are dominated by a narrow line centered at the isotropic chemical shift (referenced to 0 kHz). It was verified that this narrow signal is the only one observed up to 66 and 79 °C for LA-d23/chol and Lignd47/chol systems, respectively. Such a signal is characteristic of a phase presenting isotropic motions. This is a property shared by several lipid systems, including liquid, solution, micelles, cubic phases, etc.36 It is, therefore, impossible to assess the geometry of this phase on the (35) Lafleur, M.; Cullis, P. R.; Bloom, M. Eur. Biophys. J. 1990, 19, 55-62. (36) Seelig, J. Q. Rev. Biophys. 1977, 10, 353-418.

basis of only this result. In the case of the MA-d27/chol and SA-d35/chol systems, the spectra are considerably different, as they show the coexistence of the narrow line with a broader signal that is typical of fluid bilayers. This broad component is, in fact, the superposition of partly resolved powder patterns associated to the different CD2 groups along the fatty acid acyl chains, as previously observed for phospholipids in the fluid lamellar phase25,35 and also for the PA/chol system above the melting point of the acid.8 Therefore, it is inferred that, under these conditions, MA-d27 and SA-d35 are partly incorporated in a fluid lamellar structure, leading to a 2H NMR signal typical of axially symmetric systems. The proportion of the lipid existing in this fluid lamellar phase can be estimated by the relative area of the broad spectral component over the total spectrum surface. This proportion, when measured at about 6 °C higher than the respective melting temperature of the different fatty acids, shows a remarkable dependence on chain length (Figure 6). For short (C12) and long (C24) chain length, no lamellar phase is actually formed. In the C14-C18 interval, the fluid lamellar phase is actually the one adopted by the majority of the fatty acid molecules. The systems forming a lo lamellar phase share a similar behavior as a function of temperature (Figure 7). The proportion of the FA participating in the lamellar phase decreased as a function of increasing temperatures. About 60% of the fatty acids were in a fluid lamellar phase 6 °C above the melting point, whereas they represented 35% at around 70 °C. There is almost a linear decrease of the lamellar phase proportion as a function of temperature, and the slope is 1.7 ( 0.3 and 2.2 ( 0.2%/° C for MA-d27, and SA-d35, respectively. This is consistent with the value of 1.7 ( 0.2%/° C that was found for the PA/chol system.8 Another way to quantify the thermal stability of the lamellar phase is to identify the temperature for which half the fatty acid molecules are in the lamellar phase. This temperature was estimated to be 60 °C for the MAd27/chol system, which represents about 15 °C above the melting point of the fatty acid. This proportion of lamellar

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relatively constant order parameters near the headgroup, followed by a rapid decrease in orientational order toward the end of the chain.35,36 The values of orientational order parameters, SC-D, near the interface are relatively high, being in the order of 0.4 (the maximum value, measured for an all-trans chain would be 0.5). These values are similar to those recently reported for palmitic acidcholesterol mixtures8 above the melting temperature. They are slightly higher than those measured for lamellar systems in the lo phase in cholesterol-containing phospholipid bilayers (with at least 30 mol% cholesterol),23,35,38 which are typically around 0.38. Once normalized for the chain length difference, the order profiles obtained for the MA-d27/chol and SA-d35/chol systems are very similar, compared at the same absolute or relative (to the melting temperature) temperature. The agreement is slightly better when the comparison is made for the same absolute temperature scale, but it is a rather marginal difference. Figure 7. Variation of the proportion of fatty acid in the lamellar phase (9) and in the phase associated with the narrow line (0) as a function of temperature. (A) Equimolar MA-d27/ chol mixture and (B) equimolar SA-d35/chol mixture.

Figure 8. Smoothed order profiles of the lamellar fraction of the MA-d27/chol equimolar mixture (at 52 (b) and 66 °C (1)) and the SA-d35/chol equimolar mixture (at 66 °C (X)).

phase was obtained at 65 °C for the SA-d35/chol system, about 5 °C above the melting point of the fatty acid. This parameter was found to be 61 °C for the PA/chol system on the basis of the data previously presented.8 Therefore, it seems that the stability of the lamellar phase is mainly dictated by the temperature on the absolute scale and shows little dependence on the acyl chain length of the fatty acid. By using a longer intersequence delay, it was verified that no solid acid was present in the mixtures in these conditions. No solid-like signal was observed above 49 °C and above 60 °C for the MA-d27/chol and SA-d35/chol systems, respectively (data not shown). It should be noted that a significant proportion of solid-like SA-d35, coexisting with some fluid lamellar phase, was detected at 56 °C, a temperature intermediate between the two transitions detected by DSC for this system. The smoothed-order profiles along the fatty acid acyl chain (Figure 8) have been determined for the fluid lamellar phase components of the spectra using a standard procedure.9,37 The profiles show the main characteristic of the fluid lamellar phase, with a plateau region with

Discussion It was recently reported that the PA/chol system has the ability to form lamellar lo phases under certain conditions.8,22 The results presented here indicate that the hydrophobic matching between the fatty acid acyl chain and cholesterol is required to form this phase. The complementary techniques used in the present work allowed us to accumulate evidences of the formation of the lamellar lo phase with the MA-d27/chol and SA-d35/ chol systems. This phase is observed for temperatures higher than the melting temperature of the fatty acids. The 2H NMR spectra obtained for these systems constitute the most reliable indication of the formation of lo phase, with spectra characteristic of acyl chains that are both fluid (illustrated by the spectra typical of axially symmetric systems) and ordered (illustrated by the large quadrupolar splittings). In addition, the frequency of the νCD mode in the IR spectra display values that were observed for cholesterol-containing phospholipid and PA/chol systems in the lo phase.8,18,39 The shorter LA and the longer LignA do not form lamellar lo phase in equimolar mixtures with cholesterol. The 2H NMR signal of these cases is a narrow line, indicating fast isotropic motions that can be associated to several types of lipid structures. The νCD mode of the IR spectra indicates that the fatty acid chains are highly disordered, similar to that observed for the melted fatty acids. In addition, the thermograms of the LA-d23/ chol and LignA-d47/chol systems show a small endothermic peak at 73 °C, corresponding to the dehydration of crystalline cholesterol.26,27 It is, therefore, proposed that, for these two systems, the fatty acid and cholesterol are immiscible over the whole temperature range. Consequently, the fatty acid would undergo a transition from a crystalline to liquid phase, while cholesterol would be in the hydrated crystalline cholesterol form up to 73 °C, where dehydration would occur. For the MA-d27/chol and SA-d35/chol systems, the order profiles derived from the lamellar part of the 2H NMR spectra are characteristic of the lo phase.8,35,38 It is possible from the orientational order parameter values to estimate the acyl chain length of these FA in the lo phase.40-42 It (37) Lafleur, M.; Fine, B.; Sternin, E.; Cullis, P. R.; Bloom, M. Biophys. J. 1989, 56, 1037-1041. (38) Thewalt, J. L.; Bloom, M. Biophys. J. 1992, 63, 1176-1181. (39) Lafleur, M. Can. J. Chem. 1998, 76, 1501-1511. (40) Ipsen, J. H.; Mouritsen, O. G.; Bloom, M. Biophys. J. 1990, 57, 405-412. (41) Seelig, A.; Seelig, J. Biochemistry 1974, 13, 4839-4845. (42) Douliez, J.-P.; Le´onard, A.; Dufourc, E. J. Biophys. J. 1995, 68, 1727-1739.

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is calculated that the fatty acid chain length was 14-15 Å for the MA-d27/chol system (evaluated between 45 and 66 °C) and 18-19 Å for the SA-d35/chol system (evaluated between 59 and 74 °C). These values are consistent with the previous dimension reported for the PA/chol system (16-17 Å).8 The chain lengths of these systems are relatively close to the length of cholesterol, which is estimated at about 16 Å. It has been shown previously that cholesterol is oriented with its long axis parallel with the normal of the bilayer in PA/chol bilayers and in other similar systems.8,10,11,13 Therefore, these three saturated acids are excellent candidates for hydrophobic matching with cholesterol in the lamellar phase. From the variations of order profiles as a function of temperatures, we have also extracted the thermal expansion coefficients of the bilayers.43 They were calculated according to

(∂d/∂T)σ (∆〈SCD〉/∆T) ≈ d (〈SCD〉 + 0.5) where (δd/δT)σ/d is the thermal expansion coefficient at constant tension, T is the temperature, d is the hydrophobic thickness of the bilayer, and 〈SCD〉 is the averaged order parameter, calculated over the whole acyl chain. In this approach, bilayers are considered as incompressible fluid, and the reduction in bilayer thickness is concerted with an expansion of the bilayer surface. From these calculations, taking strictly into account the contribution of the fatty acid (i.e., strictly the change in FA area), it was found that the thermal expansion coefficient was 3.2 ( 0.4 × 10-3 and 2.0 ( 0.3 × 10-3 °C-1 for MA-d27 (average value between 45 and 66 °C) and SA-d35 (average value between 59 and 74 °C), respectively. These values are close to the coefficient reported for the PA/chol system, which was 2.4 × 10-3 °C-1.8 It seems that the coefficient shows a small dependence on chain length, being slightly higher for shorter chains. These values are comparable to phospholipid systems in the fluid phase.44-46 However, the thermal expansion coefficients reported above for our systems are strictly associated to FA and do not reflect the overall expansion of the bilayer since it does not take into account the presence of cholesterol. Since cholesterol is actually the major component of these bilayers and since the thermal expansion of cholesterol is very limited, the overall thermal expansion coefficients of these bilayers (taking into account both the fatty acid and cholesterol) are expected to be unusually small. This would be consistent with the fact that cholesterol cannot increase considerably its interfacial area without leading to the exposure of the hydrophobic bilayer interior to the aqueous environment. More direct measurements of this property should be performed to assess the validity of this inference. Upon melting, the bilayer-forming FA/chol systems experience a significant reduction of the strength of the H-bonding as revealed by the shift of the νCO band toward higher frequency (Figure 5). These frequencies, however, indicate that there is still considerable H-bonding above the transition temperature in all the investigated systems; the position of the νCO band of palmitic acid in solution in CHCl3, a non-hydrogen-bonding milieu, is 1745 cm-1.39 (43) Bloom, M.; Evans, E.; Mouritsen, O. G. Q. Rev. Biophys. 1991, 24, 293-397. (44) Needham, D.; McIntosh, T. J.; Evans, E. Biochemistry 1988, 27, 4668-4673. (45) Linseisen, F. M.; Thewalt, J. L.; Bloom, M.; Bayerl, T. M. Chem. Phys. Lipids 1993, 65, 141-149. (46) Takahashi, H.; Sinoda, K.; Hatta, I. Biochim. Biophys. Acta 1996, 1289, 209-216.

Ouimet and Lafleur

This observation is in agreement with the conclusion obtained from the investigation of mixtures of long-chain alcohols and fatty acids, stating that the presence of a H-bond network between the polar headgroups is required for the formation of stable bilayers with single-chain surfactants.47 In addition, the νCO band of MA-d27/chol, and SA-d35/chol systems is significantly broader than that of the non-bilayer-forming systems. This broadening could originate from an enhanced hydration of the carboxyl group once the fluid lamellar phase is formed. Such a broadening upon hydration was also observed for the νCO band of the ester links in phospholipids bilayers48,49 and in monooleoylglycerol/water systems.50 A temperature increase leads to the destabilization of the bilayers and an increased proportion of the phase with isotropic motions, giving rise to the narrow peak in the NMR spectra. A similar behavior was observed for the PA/chol system,8 indicating little dependence on chain length. Similarly our results show that the orientational order profile of the lamellar phase is comparable for the lamellar systems formed with different fatty acids in the presence of cholesterol. It is established that bilayers are formed when there is a correspondence between the effective interfacial area defined by the polar headgroups and the cross-sectional area defined by the acyl chains.51,52 The area occupied by fatty acids at the collapse in monolayers is relatively independent from the chain length.53,54 Therefore, their headgroups should provide similar cross-sectional areas for the acyl chains in a bilayer structure. This phenomenon is likely the origin of the high orientational order observed for the fatty acids in lo phase, irrespective of the chain length. In addition, it is likely that, because of the very limited flexibility of the interfacial polar groups (-COOH for the acid and -OH for the cholesterol), the bilayers cannot accommodate a considerable chain disorder, which would lead to an increase of the area subtended by the chain in the hydrophobic region. As a consequence, the fatty acid molecules involved in the bilayer structure could, upon heating, progressively leave this lamellar phase and be incorporated into the phase with isotropic motions. At this point, we do not know the fate of cholesterol during this transformation. It is possible that cholesterol has a more pronounced tendency to remain in the lamellar phase, leading to an increased cholesterol proportion in the lamellar phase and, as a consequence, buffering the chain disorder induced by thermal agitation. A similar phenomenon has been found for bicelles formed by melittin, a peptide extracted from bee venom.55 It was shown that when bicelles formed by melittin, phosphocholine, and cholesterol were heated, they would get richer in cholesterol (i.e., phospholipids were selectively excluded from the bicelles). It was proposed that this phenomenon was limiting the chain disorder induced by the temperature increase and, therefore, preserved the hydrophobic (47) Hargreaves, W. R.; Deamer, D. W. Biochemistry 1978, 17, 37593768. (48) Wong, P. T. T.; Mantsch, H. H. Chem. Phys. Lipids 1988, 46, 213-224. (49) Salgado, J.; Villalaı´n, J.; Go´mez-Ferna´ndez, J. C. Biochim. Biophys. Acta 1995, 1239, 213-225. (50) Nilsson, A.; Holmgren, A.; Lindblom, G. Chem. Phys. Lipids 1994, 71, 119-131. (51) Israelachvili, J. Intermolecular and Surface Forces, 2nd ed.; Academic Press, Inc.: London, 1992. (52) Tilcock, C. P. S.; Cullis, P. R. Ann. N. Y. Acad. Sci. 1987, 492, 88-101. (53) Gaines,G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley Interscience: New York, 1966. (54) Mingotaud, A.-F.; Mingotaud, C.; Patterson, L. K. Handbook of Monolayer; Academic Press, Inc.: San Diego, 1993. (55) Pott, T.; Dufourc, E. J. Biophys. J. 1995, 68, 965-977.

Formation of Lamellar Liquid Ordered Phases

matching between melittin and the bicelle thickness. FA/ chol fluid bilayers seem to exhibit a similar behavior. Below the melting temperature of the fatty acids, the behavior of the FA/chol systems appears independent of the chain length. The present results indicate that, as in the case of PA/chol system,8 the fatty acids and cholesterol are immiscible. This conclusion is inferred from the 2H NMR spectra that are, at low temperatures, characteristic of solid fatty acids from the CD2 deformation band showing a splitting indicative of the phase-separated fatty acids in the crystalline form with orthorhombic chain packing and from the low frequency of the νCD mode also characteristic of highly ordered chains. This behavior is reinforced by the fact that, in the case of PA/chol systems, the 2H NMR spectrum of cholesterol-d5 (labeled at positions 2, 2, 4, 4, and 6) was also found to be indicative of solid cholesterol.8 The fact that the CD2 deformation band of the FA/chol systems displayed a splitting identical to that measured for the pure fatty acids indicates the formation of domains of almost pure fatty acid that include at least 100 molecules. It should be noted, however, that the νCO mode of the carboxylic group in the IR spectra are considerably different for the pure hydrated fatty acids and the FA/chol systems. For temperatures below the melting temperature of the fatty acids, the νCO band is systematically narrower in the presence of cholesterol, even though its components do not appear to be shifted by the presence of the sterol. Therefore, it seems that the H bonds are probably still as strong as those observed in solid fatty acids, but cholesterol interacts at the interface with the fatty acid molecules. At this point, it is not defined whether these interactions occur in the plane of the bilayer interface or are associated with inter-bilayer contacts. Cholesterol behaves like an impurity in these mixtures, as it decreases the melting point of the fatty acids, as seen by DSC and IR spectroscopy. These effects of cholesterol suggest that the cholesterol and fatty acid molecules are relatively close to one another, and a mosaic of crystallites seems to be an organization that reconciles all the experimental observations. Conclusion It is possible to prepare fluid ordered bilayers when fatty acids and cholesterol are mixed together. Such structure was reported for the PA/chol system,8,22 and this study extends this phenomenon to two other saturated linear fatty acids. The hydrophobic matching between cholesterol and the acyl chain of the saturated fatty acid is a significant energy contribution and, in fact, is decisive in the resulting assemblies for FA/chol systems above the melting temperature of the acids. The contribution of the cholesterol-acyl chain hydrophobic matching reported here is likely playing a role in defining the phase behavior of other systems. Interestingly, it has been shown that

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the oxidation of cholesterol in PC/chol monolayers by cholesterol oxidase is dependent on the PC chain length.56 The oxidation is minimal when the PC chain length varies between 14 and 17 carbon atoms, whereas it becomes significantly higher when the lipid chains include 10-12 carbon atoms, or 18-20. This illustration of the effect of hydrophobic matching between cholesterol and lipid acyl chains is in agreement with our findings. Cholesterol and fatty acids are two major components of the lipid fraction of the stratum corneum, the top layer of the epidermis.57-59 In this layer, the fatty acids are heterogeneous from a chain-length point of view, varying mainly between 14 and 24 carbon atoms.57,58 It is established that stratum corneum lipids display a rich polymorphism that includes phase separations (for example, refs 60-63). The results presented here suggest that hydrophobic mismatch may be a significant contribution in the formation of these phase domains, and this hypothesis is currently under investigation. The formation of condensed complexes of cholesterol and phospholipids has been proposed in monolayers.64,65 This concept was recently extended to monolayers made of myristic acid and dihydrocholesterol,66 where it was found that two fatty acids can replace one phospholipid in the condensed complexes. The results presented here are certainly consistent with a large condensation of the fatty acids by the presence of cholesterol. The article reporting the formation of fatty acid-cholesterol complexes in monolayers concluded by the comment “It is not evident, however, that bilayers based on cholesterol and fatty acids will be stable”. The findings presented here show the existence of such bilayers under certain conditions and identify one structural prerequisite in order to form stable bilayers. Acknowledgment. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Fonds FCAR. J.O. is grateful to NSERC for his M.Sc. scholarship. LA0491293 (56) Mattjus, P.; Hedstro¨m, G.; Slotte, J. P. Chem. Phys. Lipids 1994, 74, 195-203. (57) Ponec, M.; Boelsma, E.; Weerheim, A.; Mulder, A.; Bouwstra, J. A.; Mommaas, M. Int. J. Pharm. 2000, 203, 211-225. (58) Schurer, N. Y.; Elias, P. M. Adv. Lipid Res. 1991, 24, 27-56. (59) Elias, P. M. J. Invest. Dermatol. 1983, 80, 44s-49s. (60) Bouwstra, J. A. Colloids Surf., A 1997, 123-124, 403-413. (61) Percot, A.; Lafleur, M. Biophys. J. 2001, 81, 2144-2153. (62) Kitson, N.; Thewalt, J.; Lafleur, M.; Bloom, M. Biochemistry 1994, 33, 6707-6715. (63) Rehfeld, S. J.; Plachy, W. Z.; Williams, M. L.; Elias, P. M. J. Invest. Dermatol. 1988, 91, 499-505. (64) Radhakrishnan, A.; McConnell, H. M. Biophys. J. 1999, 77, 1507-1517. (65) Radhakrishnan, A.; McConnell, H. M. J. Am. Chem. Soc. 1999, 121, 486-487. (66) Okonogi, T. M.; Radhakrishnan, A.; McConnell, H. M. Biochim. Biophys. Acta 2002, 1564, 1-4.