Formation of Liquid Ordered Lamellar Phases in the Palmitic Acid

However, we report the formation of a lamellar liquid ordered. (lo) phase upon heating. In this phase, the acid and cholesterol molecules experience a...
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Langmuir 2001, 17, 5587-5594

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Formation of Liquid Ordered Lamellar Phases in the Palmitic Acid/Cholesterol System Chantal Pare´ and Michel Lafleur* De´ partement de Chimie and Groupe de Recherche en Transport Membranaire, Universite´ de Montre´ al, C.P. 6128, succursale Centre-ville, Montre´ al, Que´ bec, Canada H3C 3J7 Received February 15, 2001. In Final Form: June 25, 2001 The phase behavior of the palmitic acid (PA)-cholesterol system in aqueous buffer at pH 5.5 has been investigated using infrared spectroscopy and 2H NMR spectroscopy of both chain deuterated PA and partially deuterated cholesterol. The results indicate that at temperatures below 50 °C, both lipids exist in a crystalline phase and are not miscible. However, we report the formation of a lamellar liquid ordered (lo) phase upon heating. In this phase, the acid and cholesterol molecules experience an axial symmetric motion while the acid acyl chains are conformationally ordered. Despite this unusual composition, the lo phase formed by PA-cholesterol exhibits similar properties to the phospholipid-cholesterol lo phases. The acyl chain order, the thermal expansivity of the bilayer, and the orientation and the dynamics of cholesterol are similar to those previously measured on phospholipidic lo phases. A temperature-composition diagram is proposed for the PA-cholesterol system. This diagram includes the presence of a eutectic with a PA/ cholesterol molar composition of 30:70. It is proposed that melted palmitic acid provides a nonpolar environment for the solubilization of cholesterol and the latter induces a straightening of the fatty acid chains, promoting a molecular order compatible with bilayer formation. The implications of these findings in the understanding of the structure of the stratum corneum and in the development of non-phospholipidic liposomes are discussed.

Introduction It has been established that cholesterol affects considerably the physicochemical behavior of phospholipid matrixes and can induce the formation of a unique phase: the liquid ordered (lo) phase.1-5 This phase shares characteristics with both the gel and the liquid-crystalline lipid phases. The lateral and the rotational diffusions of lipids in the lo phase are similar to those of the fluid liquidcrystalline phase. However, the lipid acyl chains are highly ordered, similarly to the order observed in the gel phase.1,5 As a consequence, the presence of cholesterol in phospholipid membranes leads to the formation of membranes that are thicker and less permeable than those in the liquid-crystalline phase (which, in this context, is referred to as a liquid disordered phase). Most of the micromechanical properties of membranes are also influenced by cholesterol6,7 and are found to be intermediate between those of the liquid disordered and the gel phases. Therefore, cholesterol-containing phospholipid membranes are fluid, thick, and relatively rigid. These characteristics of the lo phase have been proposed to provide conditions that led to a turning point in the life evolution from procaryotes to eucaryotes.8 From a biotechnology point of view, these unique properties can be exploited to create drug-delivery liposomes that remain fluid but with an improved impermeability and an * Corresponding author. Telephone: (514) 343-5936. Fax: (514) 343-7586. E-mail: [email protected]. (1) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451-464. (2) Ipsen, J. H.; Karlstro¨m, G.; Mouritsen, O. G.; Wennerstro¨m, H.; Zuckermann, M. J. Biochim. Biophys. Acta 1987, 905, 162-172. (3) Thewalt, J. L.; Bloom, M. Biophys. J. 1992, 63, 1176-1181. (4) McMullen, T. P. W.; McElhaney, R. N. Biochim. Biophys. Acta 1995, 1234, 90-98. (5) Pare´, C.; Lafleur, M. Biophys. J. 1998, 74, 899-909. (6) Needham, D.; Nunn, R. S. Biophys. J. 1990, 58, 997-1009. (7) Needham, D.; McIntosh, T. J.; Evans, E. Biochemistry 1988, 27, 4668-4673. (8) Bloom, M.; Mouritsen, O. G. Can. J. Chem. 1988, 66, 706-712.

enhanced stability in vivo, with tunable molecular recognition performances.9,10 The formation of the lo phase has been first proposed in model membranes formed of dipalmitoylphosphatidylcholine (DPPC), a zwitterionic phospholipid with saturated acyl chains.1,2 The phenomenon has been extended to phosphatidylcholines with other chain compositions (including unsaturated chains)3,11 and to 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).5 2H NMR spectroscopy of lipids bearing deuterated chains is one of the most reliable methods to identify the presence of the lo phase as a spectrum typical of an axially symmetric system is associated with fluid phospholipids and large quadrupolar splittings reflect high orientational order and are characteristics of ordered chains. The formation of the lo phase in cholesterol-containing membranes has been associated to the ambivalent character of cholesterol.1,12,13 In a phospholipid membrane, cholesterol behaves as a foreign molecule as it is chemically significantly different from phospholipids. As an impurity, it prevents the formation of ordered phases, and this aspect has been reported in the early 1980s by the statement that cholesterol disorders the phospholipid gel phase.14,15 On the other hand, the sterol ring network of cholesterol has relatively flat surfaces that promote the ordering of the adjacent acyl chains that stretch to maximize the van der Waals interactions. In other words, cholesterol orders the phospholipid chains in the fluid phase. As a conse(9) Cullis, P. R.; Mayer, L. D.; Bally, M. B.; Madden, T. D.; Hope, M. J. Adv. Drug Delivery Rev. 1989, 3, 267-282. (10) Sideratou, Z.; Tsiourvas, D.; Paleos, C. M.; Tsortos, A.; Nounesis, G. Langmuir 2000, 16, 9186-9191. (11) Linseisen, F. M.; Thewalt, J. L.; Bloom, M.; Bayerl, T. M. Chem. Phys. Lipids 1993, 65, 141-149. (12) Bloom, M. Phys. Canada 1992, 48, 7-16. (13) Nielsen, M.; Miao, L.; Ipsen, J. H.; Zuckermann, M. J.; Mouritsen, O. G. Phys. Rev. E 1999, 59, 5790-5803. (14) Umemura, J.; Cameron, D. G.; Mantsch, H. H. Biochim. Biophys. Acta 1980, 602, 32-44. (15) Cortijq, M.; Chapman, D. FEBS Lett. 1981, 131, 245-248.

10.1021/la0102410 CCC: $20.00 © 2001 American Chemical Society Published on Web 08/11/2001

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quence, the presence of the sterol in the membrane leads to a fluid and ordered phase. Despite reports of the formation of a lo phase with various phospholipids, it is unknown whether this phenomenon would be also observed for simpler lipids such as fatty acids. Actually, little is known about mixtures that include fatty acids and cholesterol as these molecules poorly hydrate individually, and in a first thought, their mixtures are not expected to lead to stable and hydrated macroassemblies. A recent paper reported that in monolayers with a subphase at pH 4, cholesterol has in fact a limited solubility in palmitic acid (less than 5%).16 At higher cholesterol content, domains were observed using atomic force microscopy. An X-ray diffraction study on cholesterol-fatty acid mixtures also reported phase separations, and crystalline phases formed by cholesterol and fatty acid were identified.17 Results obtained by vibrational spectroscopy showed that the addition of cholesterol to fatty acids (a mixture of palmitic and stearic acids in that case) induces only a small disordering effect on the acyl chains.18 The investigation of the fatty acid/ cholesterol system is interesting as it provides insights into the molecular requirements of the lipid matrixes for which cholesterol leads to the formation of a lo phase. Beyond this fundamental aspect, these mixtures are also of interest because fatty acids and cholesterol are two main components of the stratum corneum, the top layer of the epidermis.19,20 It is of interest to investigate the structures formed by fatty acids and cholesterol to examine whether these structures adopted by two major components of the stratum corneum are compatible with the proposed architecture. The present paper reports the phase behavior of the palmitic acid (PA)-cholesterol system and reveals the ability of cholesterol to induce formation of the lo phase with this saturated fatty acid. The polymorphism and the structure of the resulting macroassemblies have been characterized using infrared and 2H NMR spectroscopy. The behavior of the mixtures has been characterized at pH 5.5, an accepted value for the stratum corneum.21 In these conditions, the fatty acid was essentially protonated. Materials and Methods Palmitic acid, cholesterol (chol), and 2[N-morpholino]ethanesulfonic acid (MES) were obtained from Sigma Chemical Co. (St. Louis, MO). The fatty acid was deuterated according to the procedure described by Hsiao et al.22 2,2,4,4,6-d5 Cholesterol, purchased from Medical Isotopes (Pelham, NH), was recrystallized twice in ethanol prior to utilization. Deuterium-depleted water was obtained from Aldrich (Milwaukee, WI). Known amounts of palmitic acid and cholesterol were individually dissolved in a benzene/methanol (96:04, v/v) mixture, and appropriate volumes were mixed to provide the desired PA/ chol ratio. The sample was then freeze-dried. For IR spectroscopy, about 5 mg of the powder was hydrated with 20 µL of MES buffer (100 mM, containing 100 mM NaCl and 5 mM EDTA) with a pH 5.5. At this pH, the fatty acid molecules are all protonated as inferred from the IR results (data not shown). Several freeze-and-thaw cycles from above 60 °C to (16) Sparr, E.; Ekelund, K.; Engblom, J.; Engstro¨m, S.; Wennerstro¨m, H. Langmuir 1999, 15, 6950-6955. (17) Engblom, J.; Engstro¨m, S.; Jo¨nsson, B. J. Controlled Release 1998, 52, 271-280. (18) Ho¨ltje, M.; Fo¨rster, T.; Brandt, B.; Engels, T.; von Rybinski, W.; Ho¨ltje, H.-D. Biochim. Biophys. Acta 2001, 1511, 156-167. (19) Lampe, M. A.; Burlingame, A. L.; Whitney, J.; Williams, M. L.; Brown, B. E.; Roitman, E.; Elias, P. M. J. Lipid Res. 1983, 24, 120-130. (20) Schurer, N. Y.; Elias, P. M. Adv. Lipid Res. 1991, 24, 27-56. (21) O ¨ hman, H.; Vahlquist, A. Acta Derm.-Venereol. 1994, 74, 375379. (22) Hsiao, C. Y. Y.; Ottaway, C. A.; Wetlaufer, D. B. Lipids 1974, 9, 913-915.

Pare´ and Lafleur

Figure 1. Thermotropism of the palmitic acid probed using the position of the νC-D band for pure PA-d31 (9) and for the mixtures PA-d31/5 mol % chol (b), PA-d31/25 mol % chol (2), PA-d31/50 mol % chol (×), and PA-d31/75 mol % chol ((). liquid nitrogen temperature with periodical vortexing were performed on the samples. The sample was put between two CaF2 windows spaced out by a 5-µm-thick Teflon ring and fitted in a brass holder whose temperature was computer-controlled with thermopumps. For each spectrum, 100 scans were coadded with a nominal resolution of 2 cm-1 and Fourier transformed using a triangular apodization function. The spectra were recorded on a BioRad FTS-25 spectrometer equipped with a water-cooled globar source, a KBr beam splitter, and a MCT detector. For NMR spectroscopy, the samples were prepared similarly to those used for IR spectroscopy except that the lipid quantity was between 20 and 30 mg, the buffer quantity was adjusted accordingly, one component of the mixture was deuterated (PAd31 or chol-d5), and the buffer was prepared in deuterium-depleted water. The spectra were recorded on a Bruker DSX-300 spectrometer with a probe equipped with a 10-mm solenoid. Each spectrum was obtained with a quadrupolar echo pulse sequence. Between 1 000 and 20 000 scans were coadded, depending on the amount of deuterated material in the coil and the signal/ noise ratio required for the data processing. The 90° pulse was 4.2 µs, and the interpulse delay was 35 µs. The relaxation delay was 30 s for samples with solid chol-d5, 50 s for samples with solid PA-d31, and 0.5 s for the other samples. These delays correspond approximately to 5 T1. The temperature was controlled with a Bruker VT unit.

Results First, we have examined the thermotropism of several mixtures of PA-d31/chol, using the shift of the symmetric C-D stretching (νC-D) mode of the methylene groups observed as function of temperature in the IR spectra (Figure 1). As the samples were also used for 2H NMR spectroscopy, we used the deuterated form of the acid; this also presents the advantage of avoiding the spectral interference associated with cholesterol in the methyl and methylene stretching region. The melting point of PA-d31 was 59 °C as detected by the abrupt increase of the νC-D frequency by about 8 cm-1 because this vibrational mode is sensitive to the conformational chain disordering occurring during the transition.23 This melting point corresponds to the value provided by the manufacturer and that previously reported.24 This is a few °C lower than (23) Mantsch, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213-226.

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Figure 2. Variations of the frequency of the δCD2 band as a function of temperature for (A) pure PA-d31 and for the mixtures (B) PA-d31/25 mol % chol, (C) PA-d31/50 mol % chol, and (D) PA-d31/75 mol % chol.

the melting point of palmitic acid (64 °C25), but this shift is likely due to the isotopic labeling as already reported for phospholipids.26 The presence of cholesterol influences the thermotropism of the fatty acid. The presence of 5-25 mol % chol shifts the transition to 56-57 °C. The PAd31/50 mol % chol and PA-d31/75 mol % chol mixtures show a transition at 54 and 52 °C, respectively. This downshift is consistent with the decrease of the transition temperature by about 3 °C observed previously by differential scanning calorimetry for the PA/50 mol % chol system.24 We also report that cholesterol does not lead to any significant change of the νC-D band position below the transition. The observed value of about 2089 cm-1 is representative of highly ordered acyl chains.27 On the other hand, a decrease of the νC-D frequency is observed for temperatures above the transition temperature for the samples containing 25 mol % chol or more. At 60 °C, the νC-D band position shifts from 2097 cm-1 for pure PA-d31, a value typical of a very disordered phase,28 to 2092 cm-1 for the PA-d31/75 mol % chol samples, a value that has been observed for cholesterol-containing phospholipid matrixes in the lo phase.27,28 The CD2 scissoring (δCD2) mode of pure PA-d31 and PAd31/chol mixtures was also examined (Figure 2). Below the phase transition, this band is splitted into two components at about 1086 and 1091 cm-1. This splitting is associated to the interchain vibrational coupling when the chain packing is orthorhombic.29,30 Because the (24) Rehfeld, S. J.; Williams, M. L.; Elias, P. M. Arch. Dermatol. Res. 1986, 278, 259-263. (25) CRC Handbook of Chemistry and Physics, 61st ed.; CRC Press: Boca Raton, FL; 1980. (26) Davis, J. H. Biophys. J. 1979, 27, 339-358. (27) Lafleur, M. Can. J. Chem. 1998, 76, 1501-1511. (28) Silvius, J. R.; del Giudice, D.; Lafleur, M. Biochemistry 1996, 35, 15198-15208.

coupling occurs only when the neighboring oscillators have similar frequencies, the presence of the splitting of the δCD2 band indicates that the deuterated fatty acid exists in phase-separated crystalline domains where its neighbors have also deuterated chains. The splitting for pure PA-d31 is about 5 cm-1. Very similar splittings are observed for the examined cholesterol-containing PA-d31 samples. For all the samples, the two components merge into one located at 1088 cm-1, at the temperature for which a transition was detected from the νC-D band. 2H NMR spectra of pure PA-d 31 and cholesterolcontaining PA-d31 are presented in Figure 3. For pure PA-d31, the spectrum at 40 °C corresponds to two superimposed powder patterns with quadrupolar splittings between the maxima of 110 and 35 kHz. The broader pattern with sharp edges is associated to the CD2 groups of the all-trans chain conformation of the solid acid in absence of motion on the NMR time scale (which is in the order of few µs). The narrower powder pattern is assigned to the terminal CD3 that displays a quadrupolar splitting reduced by a factor of about 3 because of its rotation along the CD2-CD3 bond.26,31 At 65 °C, a narrow line representative of isotropic motions is observed, and this is consistent with the melting of the acid. For all the cholesterol-containing samples, the NMR spectra of PAd31 show, at 40 °C, the same profile characteristic of solid fatty acid, indicating the absence of motion. Above the transition temperature, however, the behavior of PA-d31 is influenced by cholesterol. For the PA-d31/75 mol % chol mixture, we observed, between 52 and 80 °C, spectra (29) Snyder, R. G.; Goh, M. C.; Srivatsavoy, V. J. P.; Strauss, H. L.; Dorset, D. L. J. Phys. Chem. 1992, 96, 10008-10019. (30) Snyder, R. G.; Strauss, H. L.; Cates, D. A. J. Phys. Chem. 1995, 99, 8432-8439. (31) Kitson, N.; Thewalt, J.; Lafleur, M.; Bloom, M. Biochemistry 1994, 33, 6707-6715.

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Figure 3. 2H NMR spectra of PA-d31 with various cholesterol contents (indicated at the top of each column), recorded at different temperatures (indicated on the left). The narrow line in the spectra containing 83 mol % chol has been truncated to provide a better display of the fluid-lamellar component.

formed by several overlapping powder patterns where several of them were partly resolved. This profile is typical of a fluid lamellar phase for which a gradient of orientational order is observed along the acyl chain.1,26,32 When the sample was heated, the spectrum remained characteristic of a fluid phase but the quadrupolar splittings were decreased. The fluid component was also observed for the sample containing 50 mol % chol for temperatures of g52 °C. In this case, it was always coexisting with other components. At 52 °C, the fluid-lamellar signal coexisted with a solid acid contribution (9% of the total area) and a narrow line (28% of the total area). At 55 °C, there was no longer a solid-phase component and the relative area of the narrow line was 37%. The contribution of the narrow line increased upon heating to reach 62% at 70 °C. The sample containing 83 mol % chol also displayed above the transition temperature the signal typical of the lamellar phase. In this case, a narrow signal was also observed but it represented only 15% of the total surface and was invariant upon heating. The order profiles associated with the fluid lamellar contribution in the spectra of the PA-d31/75 mol % chol samples were determined (Figure 4). The fluid component was dePaked,33 and the smoothed order profile was then (32) Lafleur, M.; Cullis, P. R.; Bloom, M. Eur. Biophys. J. 1990, 19, 55-62. (33) Sternin, E.; Bloom, M.; MacKay, A. L. J. Magn. Reson. 1983, 55, 274-282.

Figure 4. Orientational order profile for the PA-d31/75 mol % chol as a function of temperature: 52 °C (9), 60 °C (b), and 67 °C (2).

determined by dividing the region corresponding to the methylene groups of the dePaked spectra into 14 slices of equal areas and by defining as the quadrupolar splittings the midpoint of each slice.34 The quadrupolar splitting of the terminal CD3 was measured directly on the dePaked spectra. The quadrupolar splitting of the second carbon

Palmitic Acid/Cholesterol System

Figure 5. Orientational order profile for the PA-d31/75 mol % chol at 67 °C (b), POPC-d31/30 mol % chol at 10 °C (ref 32) (2), and POPE-d31/32 mol % chol at 32 °C (ref 5) (9); the spectra were recorded at a temperature 10 °C higher than the orderdisorder phase transition of the pure acid or phospholipid.

position (position n ) 1 being the carboxylic carbon), which has a peculiar value, was identified in the list of quadrupolar splittings, based on a previous assignment,35 whereas the rest of the smoothed order profile was determined by assuming a monotonic decrease of orientational order parameters from the positions near the headgroup to the end of the chain.34 The resulting orientational order profiles of the PA-d31 chain are indeed sensitive to temperature. The decrease of the order parameters induced by increased temperature is observed all along the palmitoyl chain, similarly to previous observations with phospholipid/cholesterol matrixes.5,32 However, for a given temperature, the order profile does not appear to be sensitive to the cholesterol content; the profiles obtained from the spectral component associated with the fluid lamellar phase were similar for the mixtures containing 25, 50, or 83 mol % chol (data not shown). This result suggests that the cholesterol content influences the proportion of the fluid phase in the mixtures but not the fatty acid chain order in this fluid phase. This later finding suggests that the phase composition does not vary. The order profile obtained from the PA-d31/75 mol % chol mixture at 67 °C is compared in Figure 5 with those obtained from POPE-d31/32 mol % chol at 32 °C5 and POPC-d31/30 mol % chol at 10 °C,32 the three systems being approximately 10 °C above the Tm of the pure lipid. The values of the order parameters obtained near the headgroup are about 0.4, suggesting highly ordered segments. The profiles obtained from these different cholesterol-containing systems are similar, indicating that cholesterol displays a similar ordering effect on the acyl chains of these various molecular species. The PA/75 mol % chol mixture, for which the fluid lamellar phase appears to be the most stable, has been also prepared using chol-d5, to get insights into the sterol behavior. The 2H NMR spectra obtained from this mixture are displayed in Figure 6. At 45 °C, we observed a single powder pattern with a quadrupolar splitting of 127 kHz (34) Lafleur, M.; Fine, B.; Sternin, E.; Cullis, P. R.; Bloom, M. Biophys. J. 1989, 56, 1037-1041. (35) Fenske, D. B.; Thewalt, J. L.; Bloom, M.; Kitson, N. Biophys. J. 1994, 67, 1562-1573.

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Figure 6. 2H NMR spectra of the PA/75 mol % chol mixture as a function of temperature (indicated on the left). The spectra on the left were recorded with a PA-d31/75 mol % chol sample, whereas those on the right were obtained from a PA/75 mol % chol-d5 sample. Table 1. 2H NMR Parameters Associated with Cholesterol in the PA/75 mol % Chol-d5 Mixtures

position 2,4-2H2 axa 2,4-2H2 eqa 6-2Ha 2,4-2H2 axb 2,4-2H2 eqb 6-2Hb

βo′ derived calcd expt ∆νQc expt ∆νQd from calcd ∆νQ PA/chol-d5 DMPC/chol-d6 ref 40 Smol (kHz) (kHz) (kHz) 45.8 34.6 4.4 45.3 32.3 3.6

44.6 31.3 2.4 43.4 30.3 2.2

74.1 66.2 55.8 74.1 66.2 55.8

0.95 0.94

a 55 °C. b 65 °C. c Our results with PA/75 mol % chol-d . 5 30 mol % chol-d6 (ref 36).

d

48 32 3 47 31 3 DMPC/

between the 90° peaks; this spectrum is typical of solid cholesterol.35 This single component is expected for immobile cholesterol since the static quadrupolar coupling is similar for alkyl and alkenyl groups36,37 and therefore, all the five deuterated positions should give rise to similar splittings for the immobile molecule. At 55 and 65 °C, three reasonably well resolved powder patterns were observed. These powder patterns coexisted with a remaining solid cholesterol contribution that corresponded to 40 and 15% of the total area at 55 and 65 °C, respectively. The solid fraction was estimated by subtracting a spectrum of the same sample acquired with a short recycling delay (300 ms) in order to isolate the signal from the “solubilized” cholesterol (the relaxation time of solid cholesterol-d5 is about 4 s,38 whereas that of the “bilayer-solubilized” form is less than 14 ms for all the labeled positions39). The values of the quadrupolar splitting obtained for the solubilized chol-d5 at 55 °C are reported in Table 1 (they were actually measured on a spectrum recorded with the relaxation delay of 300 ms since the saturation of the solid signal simplified the analysis and the shorter acquisition (36) Dufourc, E. J.; Parish, E. J.; Chitrakorn, S.; Smith, I. C. P. Biochemistry 1984, 23, 6062-6071. (37) Seelig, J. Q. Rev. Biophys. 1977, 10, 353-418. (38) Monck, M. A.; Bloom, M.; Lafleur, M.; Lewis, R. N. A. H.; McElhaney, R. N.; Cullis, P. R. Biochemistry 1993, 32, 3081-3088. (39) Dufourc, E. J.; Smith, I. C. P. Chem. Phys. Lipids 1986, 41, 123-135.

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time allowed us to perform more scans and get a better signal/noise ratio). The assignments are based on previous studies of DMPC/chol-d6 systems,36,40 as the splittings measured on the PA/chol-d5 samples were comparable. Discussion At low temperatures, the 2H NMR spectra of PA-d31 and chol-d5 indicate that both components are in the solid phase for all the investigated sample compositions. This is supported by the low values of the νC-D frequency that are observed for those samples at low temperatures in the IR spectra. Moreover, the splitting of the δCD2 band before the phase transition is indicative that the palmitic acid is in the crystalline form in which the chains are packed in an orthorhombic symmetry.29,30 It is also inferred from this result that the fatty acid and the cholesterol are phase separated as the coupling giving rise to the splitting is only possible if the neighboring functional groups vibrate at the same frequency as that of the δCD2 mode. The splitting has a value similar to that observed for the pure PA-d31, and as a consequence, this implies that the PA-d31 domains are almost pure and that they include at least 100 molecules.29,30,41 Therefore, in these conditions, we have coexisting crystalline domains of cholesterol and fatty acids. Upon heating, we observed the melting of PA-d31 from the drastic change of the 2H NMR spectrum profile, going from a solid-type spectrum to a narrow line typical of isotropic motions, an abrupt increase of the νC-D frequency up to 2097 cm-1, and from the merge of the two δCD2 components into one located at 1088 cm-1. The presence of cholesterol modifies the behavior of the acid. The NMR spectra obtained from the PA-d31/chol mixtures include a component which is typical of a lo phase: the spectra are composed of overlapping powder patterns typical of systems with axial symmetry and the measured quadrupolar splittings are in the order of those previously reported for cholesterol-containing phospholipid matrixes in the lo phase.1,3,5 The formation of this phase is also supported by the progressive decrease of the νC-D frequency observed at temperatures higher than the transition temperature upon increasing proportions of cholesterol, suggesting that the acyl chains are not as disordered as in the liquid disordered phase. The order profile that was obtained for palmitic acid in these mixtures is actually similar to those obtained for other cholesterol-containing matrixes forming lo phases, as inferred from Figure 5. First, the order profile of palmitic acid in the PA/chol mixtures displays a region near the headgroup where the order parameter value does not vary considerably, the plateau region. This is followed by a rapid decrease of the order toward the end of the chain. Such a profile is characteristic of fluid phospholipid bilayers.26,32 Second, the order parameter values in the plateau region are relatively high (S ≈ 0.4 whereas an all-trans chain has a S of 0.5). These values indicate rather stiff fatty acid acyl chains. On the basis of a proposed relation between orientational order parameters and the thickness of the hydrophobic part of the bilayer,42 it is calculated that the hydrophobic thickness associated with PA-d31 is about 32-33 Å. This represents an average thickness of about 16 Å for each fatty acid layer. This value corresponds to the length of cholesterol molecules.16 The hydrophobic length match (40) Marsan, M. P.; Muller, I.; Ramos, C.; Rodriguez, F.; Dufourc, E. J.; Czaplicki, J.; Milon, A. Biophys. J. 1999, 76, 351-359. (41) Mendelsohn, R.; Moore, D. J. Chem. Phys. Lipids 1998, 96, 141157. (42) Douliez, J.-P.; Le´onard, A.; Dufourc, E. J. J. Phys. Chem. 1996, 100, 18450-18457.

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between the PA layer thickness and the cholesterol is therefore relatively good. The role of this parameter in the formation of the lo phase described in this paper requires further investigation; however, considering the fundamental role of this parameter in lipid-transmembrane peptide interactions43 and considering that cholesterol is a rather stiff molecule, the good match that we observed between the chain length of the flexible PA molecules and the cholesterol length is most likely partly responsible for the stability of the bilayers. The fatty acid layer thickness that we measured is also typical of cholesterol-containing phospholipid matrixes in the lo phase,44,45 another feature shared with our non-phospholipid lo phase. The temperature dependence of the orientational order of the PA-d31/75 mol % chol mixture has been examined in more detail. We observed smaller order parameters when the temperature is increased as a consequence of the introduction of increased thermal disorder in the chains. Because bilayers usually behave as incompressible fluids, this membrane thinning should be accompanied with an expansion of molecular area. The thermal expansion coefficient of PA/chol bilayers has been estimated from the variation of the bilayer thickness, as described previously.42,46 The thermal expansion coefficient has been evaluated to 2.4 × 10-3/°C (the average value over the 52-65 °C interval). This value is similar to that of cholesterol-containing phosphatidylcholine7,11 and cholesterol-containing dielaidoylphosphatidylethanolamine (DEPE)47 bilayers. It should be pointed out that the dependence of the bilayer thermal expansivity on cholesterol content has been shown to be nonmonotonic and it seems that there is no typical value for the lo phase.7,11,47 The results presented here however indicate that despite the peculiar chemical composition, the fluid bilayers that are formed with the PA-d31/chol system display thermal expansivity properties expected for fluid bilayers. Upon the melting of the palmitic acid, we observe the fluidification of cholesterol. This conclusion is inferred from the apparition upon heating of three well-resolved powder patterns with reduced quadrupolar splittings of the 2H NMR spectra of chol-d5 mixed with palmitic acid. The orientation of the cholesterol molecules solubilized in the fluid bilayers seems to be similar to that determined for the DMPC/30 mol % chol-d6 mixtures36,40 as the quadrupolar splittings that we obtained are similar to those measured for that system (Table 1). Since there is very little intramolecular mobility at the labeled positions,40 the quadrupolar splittings measured with chol-d5 are essentially representative of the orientation of the C-D bonds relative to the axis of fast rotation of cholesterol and of the orientation and fluctuations of this symmetry axis relative to the bilayer normal. This latter contribution is quantitatively expressed by the molecular order parameter, Smol, describing the orientation and fluctuations of the whole cholesterol molecule relative to the bilayer normal.40 If we assume that the angles between the C-D bonds and the axis of fast rotation of cholesterol determined on the DMPC/chol systems are transferable to the PA/chol system, we can determine Smol that provides the best reproduction of the experimental quadrupolar split(43) Mouritsen, O. G.; Bloom, M. Biophys. J. 1984, 46, 141-153. (44) Ipsen, J. H.; Mouritsen, O. G.; Bloom, M. Biophys. J. 1990, 57, 405-412. (45) Nezil, F. A.; Bloom, M. Biophys. J. 1992, 61, 1176-1183. (46) Bloom, M.; Evans, E.; Mouritsen, O. G. Q. Rev. Biophys. 1991, 24, 293-397. (47) Takahashi, H.; Sinoda, K.; Hatta, I. Biochim. Biophys. Acta 1996, 1289, 209-216.

Palmitic Acid/Cholesterol System

Figure 7. Proposed temperature-composition diagram for PA/ chol system. × represents the limits of the transitions observed by IR spectroscopy, * represents the boundaries obtained by spectral subtraction previously proposed (as in ref 1), and 3 represents the boundaries obtained from eq 1. Conditions for which the 2H NMR spectra of PA-d31 were typical of a solid phase (9), a lamellar phase (]), a solid-lamellar phase coexistence (b), a lamellar-isotropic phase coexistence (0), and a solid-lamellar-isotropic phase coexistence (2). The lines are not strict boundaries but limit regions for which the same phase(s) is (are) observed.

tings. When this calculation is performed, it is found that a Smol of 0.94 reproduces the three measured quadrupolar splittings within 2 kHz, strongly suggesting that the cholesterol molecules in the PA/chol matrix experience similar orientation and dynamics compared to those in the lo phase formed with DMPC/chol matrixes, for which Smol was 0.96.40 The high Smol value indicates that the axis of rotation of cholesterol is roughly parallel to the bilayer normal and that there is very little fluctuation of the axis. Interestingly, the quadrupolar splittings of cholesterol-d5 measured in this non-phospholipidic bilayer are also similar to those measured in model mixtures of stratum corneum lipids35 as well as real biological membranes such as human red blood cell membranes48 and membranes of the mycoplasma Acholeplasma laidlawii (strain B),38 suggesting that the molecular design of cholesterol strongly dictates its orientation in lipid bilayers with no considerable dependence on the composition of the matrix. Our data are put together in the temperaturecomposition diagram of the PA/chol system presented in Figure 7. The phases present at a certain temperaturecomposition were determined by the visual inspection of the 2H NMR spectra of the PA-d31/chol mixtures (note that only PA-d31 is probed), and they are represented using different symbols on the diagram. In this analysis, we have neglected the presence of an isotropic line for the PA-d31/83 mol % chol samples as its contribution was always less than 15% of the total area. The limits of the transition detected by the variations of νC-D position observed by infrared spectroscopy are also included. In addition, the borders of the phase coexistence regions (lo(48) Kelusky, E. C.; Dufourc, E. J.; Smith, I. C. P. Biochim. Biophys. Acta 1983, 735, 302-304.

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isotropic) and (lo-solidchol) were determined using two approaches. First, the spectral subtraction method previously proposed1 was applied to determine the frontiers of the region in which the lo phase and a phase associated to a narrow line coexist. In this approach, pairs of normalized spectra that included the two different phases, recorded at the same temperature but with different cholesterol proportions, were subtracted one from the other to isolate the lo phase component or the narrow line. From the subtraction factors, it is possible to determine the position of the borders of the coexistence region as indicated elsewhere.1 The applicability of this approach in the lo-isotropic coexistence region implies the similarity of the spectral components associated to the two phases. As stated in the results section, the spectral component associated to the lo phase was practically identical in the coexisting region at a given temperature; this finding indicates that for a given temperature and pressure, the proportions but not the compositions of the two phases vary as a function of cholesterol content, in agreement with the Gibbs phase rules, and that the lever approach is valid. Second, the 2H NMR spectra of both cholesterol (-d5) and palmitic acid (-d31) were recorded independently for the PA/75 mol % chol mixture. The relative proportion of solid and lo forming lipids were determined for both PA and cholesterol from their relative area in the spectra. Knowing these proportions, it was simple to calculate the composition of the lo phase boundary, using

Xfluid chol

)

Xchol%fluid chol fluid Xchol%fluid chol + (1 - Xchol)%PA

(1)

where Xfluid chol is the molar fraction of cholesterol corresponding to the boundary of the solid cholesterol/lo coexisting phase region, Xchol is the molar fraction of cholesterol in the investigated mixture (here 0.75), and fluid %fluid are the percentages of chol and PA, chol and %PA respectively, in the fluid phase in the investigated mixture. This approach was performed at three different temperatures. At low temperatures, both constituents are in the solid phase and immiscible. An atomic force microscopy study has also reported the formation of domains in monolayers made of PA and cholesterol,16 and the authors suggested the formation of cholesterol-rich and PA-rich domains. By examining the phase behavior of PA/chol monolayers with different proportions, they have established that the solubility of cholesterol in the PA phase appears to be very limited because some domains were already observed when the proportion of cholesterol was 5%. In our study, the strongest evidence for extensive phase separation is provided by the splitting of the δCD2 band which has the maximal value (i.e., that obtained for pure PA-d31). Nevertheless, the presence of a small proportion of foreign molecules may not affect the splitting in a measurable manner. We have then decided to omit the limits of the (solid PA)-(solid cholesterol) coexistence region from the phase-composition diagram. Between 0 and about 15 mol % chol, the melting of the acid occurs over a slightly broader temperature range and is shifted toward lower temperatures. This is likely associated with the presence of cholesterol acting as an impurity. Above 15 mol % chol, the PA/chol system forms a lo phase above 50 °C. The presence of the fatty acid showing 2H NMR spectra typical of an axially symmetric system and displaying large quadrupolar splittings is clearly indicative of a lo phase. Moreover, in the IR spectra, the

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νC-D frequency of the fatty acid is considerably decreased and reaches values characteristic of a lo phase in other phospholipids.27,28 Despite its unusual composition, this lo phase shares several features with those previously reported for some cholesterol-containing phospholipid matrixes. The orientational order profile measured for PA-d31 in this phase and the orientation and wobbling of the cholesterol molecules described by the result obtained with chol-d5 are all very similar to those obtained for the phospholipidic lo phase. Moreover, the bilayer thermal expansion coefficient, inferred from the variation of quadrupolar splittings as a function of temperature, is in the same order of magnitude as that measured for the phospholipidic lo phase, suggesting that the PA/chol lo phase has also probably similar micromechanical properties. The temperature-composition phase diagram displays a eutectic behavior. The composition of the eutectic corresponds to 65-70 mol % chol, a consistent composition obtained from two independent NMR approaches. Between 15 and 70 mol % chol, there is coexistence of the lo phase with a phase whose isotropic motions lead to a narrow line in the 2H NMR spectra. This phase most likely corresponds to melted PA in which about 15-25 mol % chol is solubilized. Similarly, when the cholesterol content is higher than 75 mol %, the lo phase coexists with some solid-state cholesterol as clearly indicated by the NMR spectra of the PA/chol-d5 samples. At the eutectic composition, the mixture exhibits a sharp transition from the solid to the lo phase, a transition that involves the fluidification of both PA and cholesterol. A previous differential scanning calorimetry study24 has proposed that PA and cholesterol form a eutectic mixture at an equimolar ratio. This conclusion was based on the observation that the transition of this mixture was shifted toward a lower temperature but remained sharp. However, the authors investigated only this lipid composition and it is not clear that it corresponds exactly to the eutectic composition. The packing parameter49 has a value of 1.10 for the lo phase formed by a PA/chol mixture with the eutectic composition (assuming Vchol ) 605 Å3,50 Achol ) 37 Å2,50 VPA ) 496 Å3,25 and APA ) 20 Å2 51 and based on the additivity of these geometrical parameters as previously assumed50,52). This value predicts that the macroassembly should adopt a lamellar form in agreement with our experimental findings. The formation of lamellar phases by related systems has already been reported. Cholesteryl hemisuccinate, a charged derivative of cholesterol, has been shown to form a lamellar phase.53,54 Similarly, palmitic acid in water can form a lamellar phase when a significant fraction of (49) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 1976, 72, 1525-1568. (50) Chen, Z.; Rand, R. P. Biophys. J. 1997, 73, 267-276. (51) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley Interscience: New York, 1966. (52) Lafleur, M.; Bloom, M.; Eikenberry, E. F.; Gruner, S. M.; Han, Y.; Cullis, P. R. Biophys. J. 1996, 70, 2747-2757.

Pare´ and Lafleur

the acid is in the deprotonated state.55 In these two cases, some interfacial ionized groups had to be present to form bilayers whereas their neutralization led to the destabilization of the lamellar phase.54,55 In the PA-cholesterol system, no charges are involved in the formation of the lamellar phase. It appears that bilayers can be formed by mixing these species so the melted palmitic acid can provide a nonpolar environment for the solubilization of cholesterol and the latter can induce a straightening of the fatty acid chains, promoting a molecular order compatible with bilayer formation. The present findings have some impact in at least two different areas. First, we have clearly shown that cholesterol and palmitic acid, two major components of the stratum corneum lipids, can form, when present together, a lo phase. This finding suggests that this structure can exist in the top layer of the skin. It has been proposed that the lipid fraction of the stratum corneum would be responsible for the impermeability of the skin barrier by adopting a structure that could be described as a mosaic of small crystalline domains surrounded by a more fluid lipid matrix.56 It has been shown, using deuterium-labeled molecules, that PA and cholesterol participate in the formation of a fluid phase in the ternary ceramide/PA/ cholesterol mixture.31,35 On the basis of the present results, it is not compulsory that the fluid phase includes ceramide as cholesterol and fatty acid can form a fluid lamellar phase by themselves. It follows from this that additional experiments are needed to clearly define the participants in the skin lipid fluid phase. Second, liposomes made of molecules other than phospholipids (non-phospholipid liposomes or NPL) are developed as they can show some interesting physicochemical and biochemical properties.57 The formation of a fluid and ordered lamellar phase by the PA/chol system, as reported here, indicates that fatty acid-cholesterol systems are potentially a novel class of NPL that would be made of naturally occurring and cheap molecules, resisting several enzymatic degradations, providing a neutral surface, and being potentially pH sensitive. The investigation of this aspect is currently under investigation in our group. Acknowledgment. This work was supported by the Natural Sciences and Engineering Research Council of Canada and by the Que´bec Fonds FCAR. LA0102410 (53) Janoff, A. S.; Kurtz, C. L.; Jablonski, R. L.; Minchey, S. R.; Boni, L. T.; Gruner, S. M.; Cullis, P. R.; Mayer, L. D.; Hope, M. J. Biochim. Biophys. Acta 1988, 941, 165-175. (54) Hafez, I. M.; Cullis, P. R. Biochim. Biophys. Acta 2000, 1463, 107-114. (55) Cistola, D. P.; Hamilton, J. A.; Jackson, D.; Small, D. M. Biochemistry 1988, 27, 1881-1888. (56) Forslind, B. Acta Derm.-Venereol. 1994, 74, 1-6. (57) Philippot, J. R.; Milhaud, P. G.; Puyal, C. O.; Wallach, D. F. H Liposomes As Tools in Basic Research and Industry; Phillippot J. R., Schuber, F., Eds.; CRC Press: Boca Raton, FL, 1995; pp 41-57.