AFM Study of Lipid Monolayers: III. Phase Behavior of Ceramides

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AFM Study of Lipid Monolayers: III. Phase Behavior of Ceramides, Cholesterol and Fatty Acids E. Sparr,*,† L. Eriksson,†,‡ J. A. Bouwstra,§ and K. Ekelund‡ Department of Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden, and Department of Food Technology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden, and Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands Received February 25, 2000. In Final Form: July 11, 2000 The outer part of the skin, stratum corneum, is essential to the skin’s barrier function. Monolayer and bulk phase behavior of stratum corneum model lipids have thus been studied. Domain formation in Langmuir-Blodgett monolayers of synthetic ceramides (C16CerIII and C24CerIII), cholesterol, and free fatty acids (lignoceric acid, C24:0, and palmitic acid, C16:0) were investigated by atomic force microscopy. Binary, ternary, and more complex lipid mixtures were examined. It was shown that small amounts of ceramide are miscible in a cholesterol-rich phase with the miscibility dependent upon the ceramide chain length. Two phases are formed at low and intermediate cholesterol concentrations. In the ceramidecholesterol monolayers, very small rectangular-shaped ceramide domains thought to be two-dimensional single ceramide crystals are formed. Small domains were also found in more complex mixtures where the fatty acid is miscible in the ceramide phase, although these domains were not as regular in shape. Binary ceramide-cholesterol as well as ternary ceramide-cholesterol-lignoceric acid bulk mixtures were also studied by X-ray diffraction. The bulk lipid miscibility is consistent with the monolayer results.

Introduction Biological membranes have complex compositions of molecules with varying character. To study the membrane functions, simplified models such as Langmuir-Blodgett (LB) monolayers can be used. Two-dimensional phase separations and phase transitions have been intensively studied in LB films. Classically, the phase behavior of lipid mixtures at the gas-liquid interface is characterized by means of surface pressure-area isotherms.1,2 Fluorescence microscopy,3 Brewster angle microscopy,4 and X-ray diffraction5 have also been used to study the lateral arrangement of lipids at the interface. Atomic force microscopy (AFM) is a technique that can provide information on heterogeneous domain formation and nanometer-scale structures. There is a general acceptance that the structure of a monolayer at the air-water interface is correlated to that of the monolayer transferred to a solid substrate. For example, studies on the domain formation in phase-separated dipalmitoyl phosphatadylcholine (DPPC) monolayers show the same structures on the air-water interface as in the transferred LB films.3,6 Because of its high resolution, AFM has been widely exploited to investigate the detailed structure of phaseseparated LB films.7-9 * Corresponding author. † Department of Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University. ‡ Department of Food Technology, Center for Chemistry and Chemical Engineering, Lund University. § Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University. (1) Lo¨fgren, H.; Pascher, I. Chem. Phys. Lipids 1977, 20, 273-284. (2) Bibo, A. M.; Peterson, I. R. Adv. Mater. 1990, 2, 309-311. (3) McConnell, H. M.; Tamm, L. K.; Weis, R. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 3249-3253. (4) Wolthaus, L.; Schaper, A.; Mo¨bius, D. J. Phys. Chem. 1994, 98, 10809-10813. (5) Kenn, R. M.; Bo¨hm, C.; Bibo, A. M.; Peterson, I. R.; Mo¨hwald, H.; Als-Nielsen, J.; Kjaer, K. J. Phys. Chem. 1991, 95, 2095-2097. (6) Yang, X. M.; Xiao, D.; Xiao, S. J.; Wei, Y. Appl. Phys. A 1994, 59, 139-143.

Extensive work has been done on cell membrane lipid models.10 However, the focus of this work is the extracellular lipids in the stratum corneum, the outermost layer of the skin. The main function of human skin is to prevent unwanted water loss and influences from the environment. It has been known for about 40 years that the densely packed stratum corneum acts as the main barrier for diffusion of substances in the skin. The stratum corneum is a 1-20 µm thick layer of dead cells (corneocytes) embedded in a lipid lamellar matrix. The extracellular lipids are the only continuous regions of the stratum corneum: thus water and drugs must be transported through the lipid domains,11-13 where the lamellar organization of the lipids represents an almost ideal barrier toward both polar and nonpolar substances. The phase behavior of the stratum corneum lipids is of great importance for understanding the barrier function of skin, because the permeability of a bilayer is strongly determined by the structural state of the lipids. The main components of the stratum corneum lipids are ceramides, free fatty acids, and cholesterol. At least six different groups of ceramides (cer(1-6)), differing from each other by headgroup architecture and fatty acid chain lengths (16-30 C),14,15 are present in the stratum corneum, thus giving rather complex phase behavior. The stratum (7) Dufreˆne, Y. F.; Barger, W. R.; Green, J.-B. D.; Lee, G. U. Langmuir 1997, 13, 4779-4784. (8) Gliss, C.; Clausen-Schaumann, H.; Gu¨nther, R.; Odenbach, S.; Randl, O.; Bayerl, T. M. Biophys. J. 1998, 74, 2443-2450. (9) Kildemark Nielsen, L. Small Scale Lateral Organisation in Lipid Membranes - An Atomic Force Microscopy Investigation. Technical University of Denmark, Lyngby, Denmark, 2000. (10) Bloom, M.; Evans, E.; Mouritsen, O. G. Q. Rev. Biophys. 1991, 24, 293-397. (11) Potts, R. O.; Francouer, M. L. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3871-3873. (12) Bodde´, H. E.; van der Brink, I.; Koerten, H. K.; de Hann, F. H. N. J. Controlled Release 1991, 15, 227-236. (13) Simonetti, O.; Hoogstraate, A. J.; Bialik, W.; Kempenaar, J. A.; Schrijvers, A. H. G. J.; Bodde, H. E.; Ponec, M. Arch. Dermatol. Res. 1995, 287, 465-473. (14) Wertz, P. W.; Swartzendruber, D. C.; Madison, K. C.; Downing, D. T. J. Invest. Dermatol. 1987, 89, 419-425.

10.1021/la000271n CCC: $20.00 © 2001 American Chemical Society Published on Web 12/08/2000

Ceramides, Cholesterol and Fatty Acids

corneum lipid composition differs considerably from that of most other biological membranes, having longer-chain lipids and a lower content of phospholipids. This unique composition could be an important key in the understanding of the special properties of the stratum corneum. It has been established that the extracellular lipids in the stratum corneum mainly form a crystalline phase at ambient relative humidities and temperatures.16,17 Recently, electron diffraction results have shown that orthorhombic chain packing prevails over hexagonal lateral packing.18 X-ray diffraction studies on lipids extracted from porcine and human stratum corneum gave two repeated distances of the crystalline lamellar phase at approximately 6 and 13 nm.19,20 However, EPR and NMR experiments on extracted stratum corneum lipids and model systems of the stratum corneum lipids have indicated that a small fraction of the lipids are in the liquid crystalline state.21,22 The lipid phase can be envisioned as crystalline domains in a mosaic held together by lipids in a liquid crystalline state, according to the Domain Mosaic Model.23 The studies mentioned above all used techniques to investigate the bulk phase behavior of stratum corneum lipids, where a small fraction of liquid crystal might be hard to detect. Because even very small heterogeneities of the lipid matrix might be of great importance to the barrier function, it is important to use complementary techniques to directly resolve small-scale irregularities and structures of the lipid phase. By a combination of AFM with Langmuir-Blodgett technique, the twodimensional phase behavior of free fatty acids of different chain lengths (palmitic acid (C16:0) and lignoceric acid (C24:0)) and cholesterol were studied.24,25 To continue the study, mixtures including two synthetic ceramides related to group III of the natural ceramides of the skin26 have been investigated. The binary and ternary phase behavior of ceramide, cholesterol, and fatty acid mixtures are investigated, and finally more complex ceramidecholesterol-fatty acid stratum corneum lipid models are interpreted in consideration of our previous results. Materials and Methods The ceramides N-hexadecanoyl-phytosphingosine (C16CerIII), (Figure 1) and N-tetracosanoyl-phytosphingosine (C24CerIII) were prepared by Gist Brocades, Cosmoferm b.v. (Delft, The Netherlands) and made available by Beiersdorf AG (Hamburg, Germany). Palmitic acid (C15H31COOH) and lignoceric acid (C23H47COOH) (+99% purity) were purchased from Larodan Fine Chemicals (Malmo¨, Sweden), and cholesterol (+99% purity) was purchased from Sigma Chemicals (St Louis, MO). Monolayers (15) Norle´n, L.; Nicander, I.; Lundsjo, A.; Cronholm, T.; Forslind, B. Arch. Dermatol. Res. 1998, 290, 508-516. (16) White, S. H.; Mirejovsky, D.; King, G. I. Biochemistry 1988, 27, 3725-3732. (17) Bouwstra, J. A.; Gooris, G. S.; Salomons-de Vries, M. A.; van der Spek, J. A.; Bras, W. Int. J. Pharm. 1992, 84, 205-216. (18) Pilgram, G. S. K.; Engelsma-van Pelt, A. M.; Bouwstra, J. A.; Koerten, H. K. J. Invest. Dermatol. 1999, 113, 403-409. (19) Bouwstra, J. A.; Gooris, G. S.; Vanderspek, J. A.; Bras, W. J. Invest. Dermatol. 1991, 97, 1005-1012. (20) Bouwstra, J. A.; Gooris, G. S.; Weerheim, A.; Kempenaar, J.; Ponec, M. J. Lipid Res. 1995, 36, 496-504. (21) Hatcher, M. E.; Plachy, W. Z. Biochim. Biophys. Acta 1993, 1149, 73-78. (22) Kitson, N.; Thewalt, J.; Lafleur, M.; Bloom, M. Biochemistry 1994, 33, 6707-6715. (23) Forslind, B. Acta Derm. Venereol. 1994, 74, 1-6. (24) Ekelund, K.; Sparr, E.; Engblom, J.; Wennerstro¨m, H.; Engstro¨m, S. Langmuir 1999, 15, 6946-6949. (25) Sparr, E.; Ekelund, K.; Engblom, J.; Engstro¨m, S.; Wennerstro¨m, H. Langmuir 1999, 15, 6950-6955. (26) Bouwstra, J. A.; Gooris, G. S.; Dubbelaar, F. E. R.; Weerheim, A. M.; IJzerman, A. P.; Ponec, M. J. Lipid Res. 1998, 39, 186-196.

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Figure 1. The molecular structure of C16CerIII. were prepared on a Langmuir-Blodgett trough type 611 from Nima Technology (Coventry, U.K.). A 0.1 M acetate buffer, adjusted to pH 4.0, was used as a subphase. At this pH, the free fatty acids are assumed to be in an undissociated state, which facilitates the deposition of the monolayer.24 Water was deionized, distilled, and filtered through a Millipore Q purification system (Millipore Corporation, Bedford, MA). The lipids were dissolved in a chloroform/methanol mixture (molar ratio 5:1, 1 mg lipid/ml solvent) and spread at the air-water interface. Isotherms were reversible and reproducible. For deposition, sheets of freshly cleaved mica were immersed into the subphase. After the solvents were allowed to evaporate for 20 min, the monolayer film was compressed at a speed of 20 cm2/min to the pressure of deposition. The monolayer was left standing at the deposition pressure for 20 min before it was transferred to the substrate at a constant surface pressure of 22 mN/m and a dipping speed of 2 mm/min. The ceramides were mixed with cholesterol and fatty acids in different quantities to give a wide range of compositions. All samples were prepared in a clean room at a constant temperature of 19 °C. The transfer ratios of the monolayers were close to unity. Constant force AFM and lateral force AFM (LFM)27 measurements were performed on a commercial Nanoscope IIIa instrument (Digital Instruments, Santa Barbara, CA). Experiments were run under an air atmosphere at ambient temperature within 24 h from sample preparation. An E tube scanner with a 10 × 10 (x,y) × 2.5 (z) µm scan range was used for imaging. Microfabricated square pyramidal shaped tips of silicon nitride with a bending spring constant of 0.12 N/m (manufacture specified, Digital Instruments) were used as received. The scan rate was 2 Hz, and the applied force was in the order of 1-10 nN. To eliminate imaging artifacts, the scan direction was varied to ensure a true image. Images were obtained from at least five macroscopically separated areas on each sample. All images were processed using procedures for plane-fit and flatten in Nanoscope IIIa software version 4.22 (Digital Instruments) without any filtering. Dimensions of the domains were measured directly from the AFM height images. Area ratios and thickness variations were estimated from bearing and section analysis of the topographic images. For the X-ray diffraction studies, approximately 2 mg of lipid mixtures was solubilized in 80 µL of chloroform/methanol (2:1) at the desired composition and applied on mica. A detailed description is given elsewhere.20 The applied lipid mixtures were covered with 1-2 mL of acetate buffer at pH 5.0 (10 mM) and kept under nitrogen. The small-angle X-ray diffraction measurements were carried out at the Synchrotron Radiation Source at Daresbury Laboratory using station 8.2. This station has been built as part of a NWO/SERC agreement. The samples were put in a specially designed sample holder with two mica windows.19 The sample-detector distance was set to 1.7 m. Calibration of the detector was carried out using rat tail and cholesterol. From the scattering angle, the scattering vector (Q) was calculated as Q ) 4π(sin θ)/λ, where λ ) 0.154 nm is the wavelength at the sample position. No absolute intensities could be calculated. For this reason, the intensity of the tail of the diffraction curves was normalized. The diffraction curves were plotted as a function of Q. For comparison, the curves are staggered, and the intensity is given in arbitrary units. The periodicity of a lamellar phase was calculated from the positions of a series of peaks (Qn), using the equation Qn ) 2πn/d, in which d is the periodicity and n is the order of the diffraction peak. (27) Neubauer, G.; Cohen, S. R.; McClelland, G. M.; Horne, D.; Mate, C. M. Rev. Sci. Instrum. 1990, 61, 2296-2308.

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Results & Discussion The present studies may be considered as a first step toward linking the organization of well-defined ceramide mixtures with that of the complicated ceramide mixtures present in the stratum corneum. In the experiments described in this paper, a ceramide present in the stratum corneum, CerIII, has been studied. CerIII was chosen because it is readily available and represents up to 20% w/w of the total ceramide content in human stratum corneum. We have focused on CerIII and free fatty acids containing either C24 or C16 acyl chains because in the stratum corneum, lipids of C24 acyl chain length are dominantly present but a small fraction of the ceramides have C16 acyl chains.14 Surface Pressure-Area Isotherms. The surface pressure-area isotherms of the systems were carefully studied before depositing the monolayers on the mica support. Surface pressure-area isotherms were recorded for monolayers of the ceramides and their mixtures with cholesterol, lignoceric acid (C24:0), and palmitic acid (C16:0). From the isotherms, the surface area per molecule in the condensed monolayer, A0, can be calculated as 49 Å2 for C16CerIII and 51 Å2 for C24CerIII. The isotherms for the pure ceramides show no clear transition from the expanded to the condensed states of the monolayer. However, at low surface pressures the low slope and the rounded shape of the isotherms indicate a rearrangement of the molecules at the air-water surface (Figure 2). Lo¨fgren and Pascher have reported surface pressurearea isotherms for a range of different synthetic ceramides,1 showing analogous monolayer phase behavior. To quantify the monolayer transition, the authors defined the area at the “onset of condensation” (Aonset) as the area per molecule at a surface pressure of 1.5 mN/m. Adopting this terminology, ∆Aonset ) Aonset - A0 can be estimated as 10 and 1 Å2 for C16CerIII and C24CerIII, respectively. The isotherms show that the ceramides spontaneously condense at the air-water surface and form a dense monolayer even at low surface pressures. The C24CerIII isotherm has a higher slope and a lower ∆Aonset than the C16CerIII isotherm, indicating condensation at a lower surface pressure. The explanation for this is that the longer-chain lipid has a higher tendency to form a condensed monolayer. The surface pressure-area isotherms for both C16CerIII and C24CerIII change significantly upon addition of a small amount of cholesterol. As the cholesterol content is increased, the slope gradually increases and finally exhibits surface pressure-area behavior analogous to that of pure cholesterol. Figure 2a shows the isotherms for C24CerIII, cholesterol, and their mixtures with molar ratios of 1:0.01 and 1:1. For cholesterol, A0 is 38 Å2/ molecule. We have previously reported surface pressure-area isotherms of lignoceric acid, palmitic acid, and cholesterol.24,25 Surface pressure-area isotherms of binary mixtures of ceramide-fatty acid exhibit similar trends for both ceramides. The extended phase transition of lignoceric acid24 is not visible in the ceramide-lignoceric acid isotherms. Figure 2b shows that the early condensation of the ceramides is visible for the lignoceric acid mixture whereas the palmitic acid mixture resembles the isotherm of the fatty acid. This could be a sign of higher miscibility in palmitic acid-ceramide mixtures than in lignoceric acid-ceramide mixtures. For the three- and four-component monolayers, the early condensation of the ceramides diminishes and the surface pressure-area

Figure 2. Surface pressure-area isotherms at 19 °C for monolayers of (a) C24CerIII, cholesterol,25 and their mixtures, with molar ratios for C24CerIII-cholesterol of 1:0.01 and 1:1, and (b) C24CerIII, palmitic acid, lignoceric acid,24 and their binary equimolar mixtures.

isotherms more closely resemble the cholesterol-palmitic acid isotherm.25 A0 for the fatty acids is 22 Å2/molecule. Ceramide-Cholesterol Monolayers. Table 1 summarizes the number of phases, the differences in thickness between the phases, and the characteristics and estimated area fractions of the domains in monolayers of different compositions. The deposited monolayer of C16CerIII shows a rough surface with some defects and holes in the film. Upon addition of a small amount of cholesterol (C16CerIIIcholesterol, molar ratio of 1:0.05), no domain formation is seen in the transferred monolayer (Figure 3a), although a vague structure is visible. Two phases are observed upon addition of more cholesterol. The deposited monolayer of C16CerIII and cholesterol (molar ratio of 1:0.4) at first view looks homogeneous. However, when studied at higher resolution (2 × 2 µm), very small rectangular domains are seen as a fine texture of the film (Figure 3b). These domains are thicker than the surrounding continuous phase, although they are too small and closely packed to be accurately measured in height and size. As the cholesterol concentration is increased to a molar ratio of 1:1, the small domains become more prominent as they are less densely packed. An AFM image of the deposited monolayer of C16CerIII-cholesterol (molar ratio of 1:2) is shown in Figure 3c. The domains retain their rectangular shape but increase in size with increasing cholesterol content. The sizes of the domains are 10 × 100 to 20 × 120 nm2 for C16CerIII-cholesterol (molar ratio of 1:1) and 20 × 150 nm2 for C16CerIII-cholesterol (molar ratio of 1:2). The height difference between the two phases is 0.4 ( 0.1 nm, which is in good agreement with the predicted difference between the C16CerIII [16(0.127) + 0.015 )


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Table 1. Number of Phases, Characteristics, and Estimated Area Fractions of the Domains and the Differences in Thickness between the Phases in Monolayers of Different Compositions

sample

no. of phases

C16CerIII-chol 1:1 C24CerIII-chol 1:1 C16CerIII-C(16:0) 1:1 C24CerIII-C(16:0) 1:1 C16CerIII-C(24:0) 1:1 C24CerIII-C(24:0) 1:1 C16CerIII-chol-C(16:0) 1:1:1 C24CerIII-chol-C(16:0) 1:1:1 C16CerIII-chol-C(24:0) 1:1:1

2 2 1 1 2 2 2 2c 3

C24CerIII-chol-C(24:0) 1:1:1 C16CerIII-C24CerIII 1:1 C16CerIII-C24CerIII-chol 1:1:2

2 1d 3

C16CerIII-C24CerIII-chol-C(16:0) 1:1:2:2 C16CerIII-C24CerIII-chol-C(24:0) 1:1:2:2

2c 3

domain characteristicsa

area fraction covered by domains/%

height difference relative to the thinner phase/nm

I I

25 50

0.4 ( 0.1 0.8 ( 0.1

III III I II/III III I II

(40) (85) b 50 45 15 (65)

1.0 ( 0.2 0.9 ( 0.1 b 0.8 ( 0.1 1.3 ( 0.2 0.5 ( 0.2 1.2 ( 0.2

I I I/II I II

10 40 60 25 25

1.0 ( 0.1 0.5 ( 0.1 0.4 ( 0.1 1.6 ( 0.1 0.4 ( 0.1

a The domain characteristics at each composition are classified as follows: (I) oblong domains of similar shape and size, (II) irregularly shaped domains of varying size with a “diameter” smaller than 300 nm, and (III) domains larger than 300 nm. b Domains are too closely packed to accurately measure height differences in the images. c The thicker phase forms domains of two different shapes and sizes. d Reference 29.

Figure 3. Topographic AFM image (2 × 2 µm) of a transferred monolayer of C16CerIII-cholesterol. (a) Molar ratio of 1:0.05. No domains are observed, but a vague structure can be seen in the film. (b) Molar ratio of 1:1. Two phases can be observed; one thick ceramide-rich phase is embedded in a thinner cholesterol phase. (c) Molar ratio of 1:2. The ceramide domains have increased in size, and approximately 75% of the area is occupied by the thin, flat cholesterol-rich phase. The difference in thickness between the phases is 0.4 ( 0.1 nm. (d) Molar ratio of 1:5. One cholesterol-rich phase is observed. Z range, 4 nm.

2.0 nm] and cholesterol (1.6 nm) monolayers. Further, the area ratio between the thicker domains and the surrounding thinner phase decreases with increased cholesterol content. The area ratios and the monolayer height differences indicate that the thick phase consists of C16CerIII and the thin phase is rich in cholesterol. The ceramide phase in Figure 3c covers approximately 25% of the surface. One can conclude from the headgroup areas of the different lipids that for a total phase separation approximately 40% of the surface area should be covered by the ceramide phase. Consequently, the thin phase must include a non-negligible amount of ceramide. At a higher cholesterol concentration (molar ratio of 1:5), only one

cholesterol-rich phase is observed (Figure 3d), and the monolayer appears smoother than that of C16CerIII. The exact composition at the phase boundary to the one-phase cholesterol-rich monolayer was not thoroughly investigated in this work, although it can be estimated using the area ratios to be approximately a molar ratio of 1:4 (C16CerIII-cholesterol). The qualitative phase behavior of C24CerIII-cholesterol monolayers is similar to that of C16CerIIIcholesterol. At a very low cholesterol content (molar ratio of 1:0.01), the monolayer is smooth and homogeneous. The friction images of the films show a smooth surface and no indications of differences in surface elasticity.

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Figure 4. Topographic AFM image (2 × 2 µm) of a transferred monolayer of C24CerIII-cholesterol: (a) molar ratio of 1:1 and (b) molar ratio of 1:2. Small sticklike ceramide-rich domains are embedded in a thinner cholesterol phase. The domains become bigger and the area covered by the domains decreases at increased cholesterol content. The difference in thickness between the phases is 0.8 ( 0.1 nm. Z range, 4 nm.

Similar results were obtained for the monolayer of C24CerIII-cholesterol (molar ratio of 1:0.05), although the surface was not as smooth as in the sample with a lower cholesterol content. At higher cholesterol contents, two phases are clearly observed. Figure 4a shows the AFM image of a transferred monolayer of C24CerIII-cholesterol (molar ratio of 1:1). The domains are rectangular, like those observed for C16CerIII-cholesterol, although they are significantly larger than the C16CerIII domains at the corresponding cholesterol concentration. Upon further addition of cholesterol, the domains become even larger (Figure 4b). The thicker phase is assumed to consist of C24CerIII, and the thinner phase is rich in cholesterol. The domains have a size of 20 × 150 to 30 × 200 nm2 at a molar ratio of 1:0.4, 40 × 250 nm2 at a molar ratio of 1:1, and 70 × 500 to 80 × 600 nm2 at a molar ratio of 1:2. The ratio between the long and the short edges remains almost constant as the domains grow bigger. The thick phase covers approximately 50% of the surface at a molar ratio of 1:1 and approximately 33% at a molar ratio of 1:2. Assuming total phase separation, one would expect the ceramide phase to cover 57% and 40% of the surface for these compositions, respectively. Using the area ratios from these different samples, the phase boundary to a two-component cholesterol-rich phase can be estimated to be a molar ratio of 1:12 (C24CerIII-cholesterol). The height difference between the two phases in the C24CerIII-cholesterol monolayers is 0.8 ( 0.1 nm, which is significantly lower than the predicted difference of 1.5

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nm between an extended lignoceric acid chain (C24:0) (3.1 nm) and cholesterol (1.6 nm). Therefore, the packing of C24CerIII cannot be a vertical all-trans state of the lignoceric acid part of the ceramide. One alternative is that the ceramides adopt a tilted arrangement in the monolayer, resulting in decreased thickness. However, a tilting of the molecules would lead to a significantly larger interfacial area per molecule for C24CerIII than for C16CerIII. The surface pressure-area isotherm results show no large difference in headgroup areas, and we can therefore conclude that tilting of the chains cannot be the only explanation for the discrepancy between the observed and estimated height differences. Ceramides of type III are composed of a fatty acid amide linked to a phytosphingosine base. The phytosphingosine chain consists of 16 carbon atoms (Figure 1). Because of the different chain lengths of the double-chained ceramide, a close packing of the full lignoceric acid chain in C24CerIII is not possible and a liquid zone is expected above the condensed hydrocarbon part of the monolayer, where the end part of the longer hydrocarbon chain is tilted above the side chain.28 Consequently, the lignoceric acid chain of C24CerIII should not be in an all-trans state in the condensed monolayer. The miscibility of C24CerIII in the cholesterol monolayer can be explained by assuming such a packing of C24CerIII in the monolayer. To analyze the height differences, the shorter chain, the phytosphingosine, is considered the longest extended hydrocarbon chain of C24CerIII. The thickness of the C24CerIII domains is then estimated as approximately 2.3 nm. The corresponding thickness of the C16CerIII domains is in the same way estimated as 2.0 nm. Thus, the thicknesses of the ceramide monolayers are in a similar range, and the ceramides are therefore assumed to be miscible. Miscibility in monolayers of an equimolar mixture of these two ceramides has been demonstrated by ten Grotenhuis et al.29 Although the monolayers of cholesterol and the synthetic ceramides C24CerIII and C16CerIII show qualitatively very similar phase behaviors, quantitative differences exist. The domains at corresponding compositions are larger in the C24CerIII-cholesterol monolayers than in the C16CerIII-cholesterol monolayers. Furthermore, C24CerIII shows a lower miscibility in the cholesterolrich monolayer than C16CerIII. Previous studies have revealed that the acyl chain length mainly determines the association of cholesterol with lipids in a monolayer. Studies using phospholipids of different chain lengths have shown the best “match” for cholesterol and saturated acyl chains having 14-17 carbon atoms.30,31 The interaction can be explained by the steric packing constraints of the hydrocarbon chains because of the character of the cholesterol molecule. The same mechanism, if applied to the interaction between cholesterol and the ceramide acyl chains, explains the quantitative differences in the ceramide-cholesterol monolayers. Recently, we have studied the monolayer phase behavior of cholesterol and free fatty acids of different chain lengths. A small amount of cholesterol is miscible with palmitic acid (C16:0), but excess cholesterol forms a rougher cholesterol-rich phase. No miscibility is observed in monolayers of lignoceric acid (28) Abrahamsson, S.; Dahle´n, B.; Lo¨fgren, H.; Pascher, I.; Sundell, S. Molecular arrangement and conformation of lipids or relevance to membrane structure. In Structure of Biological Membranes; Abrahamsson, S., Pascher, I., Eds.; Plenum Press: New York, 1977; pp 1-23. (29) ten Grotenhuis, E.; Demel, R. A.; Ponec, M.; Boer, D. R.; van Miltenburg, J. C.; Bouwstra, J. A. Biophys. J. 1996, 71, 1389-1399. (30) Slotte, J. P. Biochim. Biophys. Acta 1995, 1238, 118-126. (31) Hagen, J. P.; McConnell, H. M. Biochim. Biophys. Acta 1997, 1329, 7-11.


(C24:0) and cholesterol.25 On the basis of these observations one would expect low miscibility of C24CerIII and cholesterol. We have shown that the cholesterol-rich monolayer must incorporate some C24CerIII. However, the miscibility of C24CerIII in cholesterol is significantly lower than that of the better matching C16CerIII. Ternary CerIII-Cholesterol Mixtures. When adding cholesterol to an equimolar mixture of ceramides (C16CerIII-C24CerIII-cholesterol, molar ratio of 1:1: 2), three phases are observed (Figure 7b). The thickest phase forms large rectangular domains that have a size of 30 × 200 to 50 × 300 nm2 and cover approximately 10% of the surface, whereas the smaller rectangular domains of the intermediately thick phase are 20 × 100 nm2 and cover approximately 40% of the surface. The height differences relative to the thinner, continuous phase are 1.0 ( 0.1 and 0.5 ( 0.1 nm, respectively, which is slightly larger than the corresponding height differences in the binary monolayers. It can be concluded from the area ratios of the different phases that the intermediate C16CerIIIrich domains must include some C24CerIII, which slightly increases the thickness of the phase. In the presence of C16CerIII, it is unlikely that the thin cholesterol-rich phase incorporates as much C24CerIII as in the binary C24CerIII-cholesterol monolayer, explaining the increased difference of thickness between the C24CerIII and the cholesterol-rich phases. In an earlier monolayer study, it was shown that C16CerIII and C24CerIII are miscible in an equimolar mixture.29 Interestingly, cholesterol is able to segregate the ceramides, resulting in a dispersion of both C24CerIII-rich and C16CerIII-rich domains in a cholesterol-rich phase. Binary Ceramide-Fatty Acid Monolayers. Miscibility in monolayers of free fatty acids and ceramides was investigated. The fatty acids used were palmitic acid (C16:0) and lignoceric acid (C24:0), which have the same chain lengths as the amide-linked fatty acids of the ceramides. Palmitic acid is miscible in monolayers of both ceramides at equimolar compositions. The transferred monolayer of C16CerIII-palmitic acid appears smoother than the C16CerIII monolayer, and the C24CerIIIpalmitic acid monolayer is slightly rougher with a few holes and ripples. In the C16CerIII-palmitic acid mixtures, the similarity in hydrocarbon chain lengths may allow denser packing in the monolayer, explaining the smoother surface. Miscibility of palmitic acid and C24CerIII is explained by the suggested packing of the hydrocarbon chains in the C24CerIII monolayer. In contrast, lignoceric acid is not entirely miscible with any of the ceramides. The AFM image of a transferred monolayer of the equimolar mixture of C16CerIIIlignoceric acid shows two phases with a height difference of 1.0 ( 0.2 nm. The height difference between the phases is close to the expected difference between the extended molecules of C16CerIII (2.0 nm) and lignoceric acid (3.1 nm). The domains are irregular in shape and size, and some of them are much larger than those in the previously described samples. Because of the heterogeneity in the sample, the area ratio of the thicker phase is roughly estimated to be approximately 40%. Thus, the lipids must have very low miscibility, and the thin phase is rich in ceramide. The monolayers of C24CerIII-lignoceric acid (molar ratio of 1:1) also form two separate phases (Figure 5). The domains are large and irregularly shaped, and the height difference between the two phases is 0.9 ( 0.1 nm. The thick phase covers approximately 85-90% of the surface, and we assume it to consist of lignoceric acid and a considerable amount of ceramide. Partial miscibility of C24CerIII and lignoceric acid is probably due to the

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Figure 5. Topographic AFM image (2 × 2 µm) of a transferred monolayer of C24CerIII-lignoceric acid with a molar ratio of 1:1. Two phases are observed. The difference in thickness relative to the thinner phase is 0.9 ( 0.1 nm. Z range, 4 nm.

matching hydrocarbon chains. Similarly, the poor miscibility of the long chain lignoceric acid and C16CerIII is due to the difference in hydrocarbon chain lengths. Ternary Equimolar Ceramide-Cholesterol-Fatty Acid Monolayers. To understand the phase behavior of complex multicomponent mixtures, the phase behavior of more simple monolayers of ceramides, cholesterol, and fatty acids is used.24,25 By a comparison of differences in height, area ratios, and domain shapes in the simple models, features of the complex mixtures can be identified. AFM images of deposited equimolar monolayers of C16CerIII-cholesterol-palmitic acid resemble those of the equimolar monolayer of C16CerIII-cholesterol (Figure 3b). Rectangular domains are formed, although the edges are more rounded than those in the binary cholesterolceramide mixture. The domains are too small and closely packed to be accurately measured in height and size. Palmitic acid is typically miscible in C16CerIII monolayers and is miscible with cholesterol to some extent. However, the shape and size of palmitic acid-rich domains in binary palmitic acid-cholesterol monolayers are very different from what is observed in the ternary system.25 Thus, the thick phase is expected to be rich in ceramide and the thin phase is rich in cholesterol. Palmitic acid is considered to be present in both phases with a preference for the ceramide phase. The phase behavior of monolayers of C24CerIII-cholesterol-palmitic acid (molar ratio of 1:1: 1) is similar to that of the previous sample. Two phases are observed, although the shape and size of the domains are different. The thick phase forms irregularly shaped domains of various sizes, and it covers approximately 50% of the surface. The coverage is larger than the total area of the ceramide phase. On the basis of the binary monolayer phase behavior of these lipids together with the area ratio, one can assume the thick phase to be rich in ceramide and the thin phase to be rich in cholesterol. The fatty acid must be present in both phases. The height difference between the two phases is 0.8 ( 0.1 nm. This is consistent with the height difference in the C24CerIIIcholesterol monolayer. Figure 6a shows an AFM image of the deposited monolayer of C16CerIII-cholesterol-lignoceric acid (molar ratio of 1:1:1). Three phases are seen. Thick, large domains of irregular shape (ca. 45% of the area) and thinner, small, rectangular domains (ca. 15% of the area) are embedded in a thin, flat monolayer. The height differences relative to the thin phase are 1.3 ( 0.2 and 0.5 ( 0.2 nm, respectively. The thick, large domains have a smooth perimeter and resemble the dense lignoceric acid

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Figure 6. Topographic AFM images (2 × 2 µm) of a transferred monolayer. (a) C16CerIII-cholesterol-lignoceric acid with a molar ratio of 1:1:1. Small sticklike domains and big domains of irregular shape are observed. The differences in thickness relative to the thinner phase are 0.5 ( 0.2 and 1.2 ( 0.2 nm, respectively. Z range, 4 nm. (b) C24CerIII-cholesterollignoceric acid with a molar ratio of 1:1:1. Chainlike domains are trailing over the surface. The difference in thickness relative to the thinner phase is 1.2 ( 0.2 nm. Z range, 4 nm.

domains observed in monolayers of lignoceric acidpalmitic acid-cholesterol.24 The rectangular domains resemble those in binary ceramide-cholesterol monolayers, although the difference in thickness relative to the thinner phase is somewhat larger. Using the domain shapes and differences in thickness, the different phases can be assigned as rich in lignoceric acid, rich in ceramide, and rich in cholesterol, respectively. Monolayers of C24CerIII, cholesterol, and lignoceric acid (molar ratio of 1:1:1) form two separate phases (Figure 6b). The thick phase resembles a fine chain trailing across the surface. The chainlike structure resembles the interconnection of the rectangular domains in the binary C24CerIII-cholesterol mixture (compare Figure 6b and Figure 4a), although the width of the chain domains is slightly larger than the width of the rectangular domains in the binary C24CerIII-cholesterol monolayers. The thick phase covers 60-70% of the surface, and the difference in thickness is 1.2 ( 0.2 nm, which is somewhat larger than that in the binary ceramide-cholesterol monolayer. Height differences, area ratios, and the miscibility of lignoceric acid and C24CerIII, as well as the shape of the domains, imply that the thicker phase contains both lignoceric acid and ceramide.

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In summary, the addition of palmitic acid to C16CerIIIcholesterol monolayers does not have a profound effect on the size and shape of the domains. The size and the arrangement of domains in monolayers of C24CerIIIcholesterol-lignocericacidresemblethoseintheC24CerIIIcholesterol monolayers. This is explained by the good matching between the hydrocarbon chains of the ceramide and the free fatty acid. One could expect the fatty acid to be incorporated into the ceramide phase without considerably disrupting the preferred packing of the ceramide molecules. On the other hand, in monolayers where the matching of the hydrocarbon chains is not as good (C16CerIII-cholesterol-lignoceric acid and C24CerIIIcholesterol-palmitic acid), large and irregularly shaped domains are formed. This is a sign of disturbance of the preferred packing of the ceramides or segregation within the ceramide-fatty acid phase. One explanation for the large domains in the C24CerIII-cholesterol-palmitic acid monolayers could be that palmitic acid mixes with cholesterol instead of the long chain ceramide. In the presence of palmitic acid, cholesterol forms a denser phase, which may prevent the ceramide from dispersing into small domains. Monolayers of C16CerIII, C24CerIII, Cholesterol, and Free Fatty Acids. Several features of the binary and ternary mixtures previously described can be recognized in the monolayers of C16CerIII-C24CerIIIcholesterol-palmitic acid (molar ratio of 1:1:2:2). Rectangular-shaped domains such as those in C16CerIIIcholesterol-palmitic acid monolayers as well as large, irregularly shaped domains resembling those in C24CerIII-cholesterol-palmitic acid monolayers are observed. The height difference relative to the thin, continuous phase is 0.4 ( 0.1 nm for both types of domains. It is therefore difficult to determine whether these domains have the same composition or not. Ceramides tend to form rectangular domains in the presence of cholesterol, whereas fatty acids form large domains of irregular shape.24,25 Together, the thicker phases cover more than 60% of the surface. Consequently, the thicker phases are assigned as rich in ceramides and fatty acids and the thin phase is rich in cholesterol. The height difference is consistent with height differences measured in C16CerIIIcholesterol, palmitic acid-cholesterol,25 and C16CerIIIcholesterol-palmitic acid monolayers. It is, however, significantly lower than the height differences measured in ternary C24CerIII-cholesterol-palmitic acid monolayers. One explanation for this discrepancy is that the presence of C16CerIII in the ceramide-rich phase reduces the thickness compared to the binary and ternary mixtures of C24CerIII. Monolayers of C16CerIII-C24CerIII-cholesterol-lignoceric acid (molar ratio of 1:1:2:2) (Figure 7a) also show many recognizable features. Both the characteristic chainlike domains (compare with Figure 6b) and the thinner, small, rectangular domains (compare with Figures 3b and 7b) are observed. The thickest phase is 1.6 ( 0.1 nm thicker than the thinnest phase, and it covers approximately 25% of the surface. This suggests that it consists mainly of lignoceric acid. However, the area ratio implies that some ceramide must also be incorporated in this phase. The height difference between the rectangular domains of intermediate thickness and the thinnest phase is 0.4 ( 0.1 nm, and the rectangular-shaped domains cover approximately 25% of the surface. It is therefore proposed that the rectangular domains consist mainly of ceramide and the thin phase is rich is cholesterol. It is likely that the longer-chain ceramide is preferentially located in the thicker phases.


Figure 7. Topographic AFM image (5 × 5 µm) of a transferred monolayer. (a) C16CerIII-C24CerIII-cholesterol-lignoceric acid with a molar ratio of 1:1:2:2. Small, oblong domains and thicker, chainlike domains are observed. The differences in thickness relative to the thinner phase are 0.4 ( 0.1 and 1.6 ( 0.1 nm, respectively. (b) C16CerIII-C24CerIII-cholesterol with a molar ratio of 1:1:2. Two types of sticklike domains of disperse size and thickness are observed. The differences in thickness relative to the thinner phase are 0.5 ( 0.1 and 1.0 ( 0.1 nm, respectively. The thickest phase covers approximately 10% and the phase of intermediate thickness covers around 40% of the surface. Z range, 4 nm.

Shape and Size of Domains. Monolayer studies on ceramides, cholesterol, and fatty acids24,25 have shown that the character of the lipids has a very big influence on the shape of the domains. We find the very small size of the domains we have observed particularly remarkable. The domain size is mainly determined in the formation moment, and the kinetics for domain growth is thereafter relatively slow.32 Small domains have been observed in monolayers including ceramides and cholesterol as well as in the monolayer of ceramides, cholesterol, and fatty acids. In monolayers of phase-separated fatty acids and cholesterol, the areas of the domains are hundreds of times larger compared to the domains in binary ceramidecholesterol monolayers. Thus, one concludes that both ceramides and cholesterol are required for the formation of the very small domains. Domains of the same size but not as regular in shape are formed in monolayers of extracted pig skin ceramides and cholesterol.29 This, together with our results from studying ceramidecholesterol-fatty acid monolayers, indicates that the (32) Ekelund, K.; Eriksson, L.; Sparr, E. Biochim. Biophys. Acta 2000, 1464, 1-6.

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tendency to create small ceramide domains is very high. Lipid domains of magnitude 10-100 nm have also been observed in monolayers of DPPC6 and in bilayers of binary mixtures of synthetic lecithins.8 It is of utmost importance to study these small heterogeneities to better understand lipid membrane function. Small lipid domains may have important functions in membrane transport and intracellular signaling.33 The unusual and regular shapes of the small domains are also interesting from a physical point of view. In a parallel study, it has been discussed how cholesterol acts as an impurity, initiating nucleation in the formation of two-dimensional single ceramide crystals.32 The number of nucleation sites increases with decreasing cholesterol content, which results in smaller crystal domains. If the crystal size is below the resolution of the experimental technique, then the monolayer appears homogeneous. This is observed at very low cholesterol concentrations (typically below a molar ratio for CerIII-cholesterol of 1:0.05). Finally, it is also noted that the rectangular domains in the C16CerIII-cholesterol monolayers intersect with a preferred angle of 54°.32 This lateral arrangement is observed in the binary monolayer of C16CerIII-cholesterol (Figure 3c) and in the ternary monolayer of C16CerIII-cholesterol-lignoceric acid (Figure 6a). Currently, we have no explanation for this. X-ray Diffraction of C16CerIII, C24CerIII, Cholesterol, and Lignoceric Acid. In Figure 8a, the diffraction curves of the equimolar mixtures of C16CerIIIcholesterol and C24CerIII-cholesterol are plotted. At least two phases coexist in these mixtures. In the diffraction pattern of the C16CerIII-cholesterol mixture, the peaks at a spacing of 3.62 and 1.82 are attributed to a phase with a periodicity of 3.62 nm. The two peaks located at 3.46 and 1.74 nm are attributed to a phase with a periodicity of around 3.46 nm. Because these 3.46 and 1.74 nm spacings are located close to those of the reflections of crystalline cholesterol (3.34 and 1.69 nm), this phase may be formed by mainly cholesterol, with a low content of C16CerIII, as suggested for the binary C16CerIIIcholesterol monolayer. However, a small fraction of crystalline cholesterol might also be present in addition to these phases, as indicated by the presence of the 1.69 nm reflection. The 3.34 nm reflection has probably been obscured by the 3.46 nm peak. Similar phase behavior is observed for the equimolar C24CerIII-cholesterol mixture. One phase is present with a periodicity of 4.22 nm, of which the first-, second-, and third-order diffraction peaks are located at 4.22, 2.13, and 1.40 nm spacings, and the appearance of the 3.46 and 1.74 nm peaks indicates the presence of a similar phase as in the C16CerIIIcholesterol mixtures. Finally, crystalline cholesterol is also present, which is indicated by the small shoulder at the right-hand side of the 1.74 nm peak. Addition of lignoceric acid to the CerIII-cholesterol mixtures results in more complicated phase behavior; see Figure 8b. In the C16CerIII-cholesterol-lignoceric acid mixtures (molar ratio of 1:1:1), two additional phases are formed next to the 3.60 nm phase and the 3.40 nm phase, with periodicities of 4.20 nm (second order at 2.08 nm spacing) and 5.65 nm (second order at 2.87 nm spacing), respectively. Addition of lignoceric acid to the C24CerIIIcholesterol mixture (C24CerIII-cholesterol-lignoceric acid, molar ratio of 1:1:1) results in an additional phase with a periodicity of 5.45 nm. In both fatty acid containing mixtures, the 3.40 nm phase probably consists mainly of cholesterol, with a low content of either fatty acid or CerIII. (33) Simons, K.; Ikonen, E. Nature 1997, 387, 569-572.

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The solubility of the fatty acids shows similar trends for the monolayer and bulk mixtures. Lignoceric acid is not completely miscible in either of the CerIII monolayers. In the three-dimensional lattice, a separate phase with a periodicity of 5.65/5.45 nm is formed after addition of free fatty acid. This phase must include lignoceric acid because it is not present for the ceramide-cholesterol mixtures. Lignoceric acid shows higher miscibility in C24CerIIIcholesterol mixtures than in C16CerIII-cholesterol mixtures. Most probably, the limited fatty acid solubility in these mixtures is due to the highly ordered lattice. This limited miscibility is in agreement with recently published data regarding the presence of eutectic mixtures between palmitic acid and bovine brain ceramides.34 Conclusions

Figure 8. Small-angle X-ray diffraction curves. (a) Equimolar mixtures of CerIII and cholesterol: C16CerIII (top) and C24CerIII (bottom). (b) Equimolar mixtures of CerIII, cholesterol, and lignoceric acid: C16CerIII (top) and C24CerIII (bottom).

Comparison with the Formation of Lamellar Phases in Bulk Mixtures. Extrapolating the findings of the monolayer approach to the bulk mixtures, the following remarks can be made. When adding cholesterol to either C16CerIII or C24CerIII, even at a very low concentration a CerIII phase will coexist with a cholesterolrich phase in the monolayer. Two phases in the binary CerIII-cholesterol mixtures are also observed by X-ray diffraction, using the same lipid composition. The situation is different when adding cholesterol to a mixture of isolated stratum corneum ceramides. Then, cholesterol is dissolved into the ceramide mixture up to a ceramide-cholesterol molar ratio of 1:0.4,26 most likely because the fatty acid chain length distribution is greater in the isolated stratum corneum ceramide mixture than in the mixtures we have used. The greater chain length distribution in the isolated ceramide mixture probably increases the solubility of cholesterol. Interestingly, in the monolayer approach cholesterol induces a phase separation of C16CerIII and C24CerIII. Such a phase separation has also been observed in monolayers prepared from isolated stratum corneum ceramides29and corresponds to a recently proposed molecular model for the 13 nm lamellar phase.26

Biological membranes often incorporate many different components, resulting in complex phase behavior that is relevant for the membrane’s function. To predict the phase behavior of such systems is no trivial task. In this study, we have, together with previous results,24,25 identified the phases in monolayers and in bulk of four-component model systems of stratum corneum lipids. The surface pressurearea isotherms, the direct AFM observations, and the X-ray diffraction results give a consistent picture of the phase behavior. The ceramides show a low-to-moderate miscibility in a cholesterol-rich phase, with the miscibility dependent upon the ceramide chain length. The AFM results show large variations in size and shape of the domains, which are determined by molecular packing and interactions in the monolayer. In light of this, the character of the domains can be used as a tool for identifying different phases. Cholesterol shows a tendency to phase-separate ceramides that are miscible in the absence of cholesterol. In ceramide-cholesterol monolayers, small rectangular domains, referred to as two-dimensional single ceramide crystals,32 are formed. We have shown that several lipid phases coexist in model systems of stratum corneum lipids. The lipids are divided into small domains of different compositions. The regularly shaped, spontaneously condensed ceramide monolayer forms a crystalline phase, whereas the cholesterol-rich phase is considered to be less dense. The findings for small domains of different composition and packing do not oppose the Domain Mosaic Model of stratum corneum lipids but instead indicate a high heterogeneity at submicrometer resolution. With AFM at this resolution, one cannot distinguish between liquid and solid phases, and therefore a correlation of AFM results with results from techniques such as X-ray diffraction and NMR is important. In the future, it will be of great interest to study the effect of cholesterol sulfate on phase behavior in monolayers. It has recently been found that cholesterol sulfate induces the formation of a liquid phase and facilitates the solubility of cholesterol in stratum corneum lipid mixtures.35 The results of these studies may provide insight into the localization of this liquid phase. Acknowledgment. E.S., L.E., and K.E. gratefully acknowledge Håkan Wennersto¨m and Sven Engstro¨m for fruitful discussions. Crafoordska Stiftelsen funded the acquisition of the Langmuir-Blodgett trough. LA000271N (34) Neubert, R.; Rettig, W.; Wartewig, S.; Wegener, M.; Wienhold, A. Chem. Phys. Lipids 1997, 89, 3-14. (35) Kitson, N.; Monck, M.; Wong, K.; Thewalt, T. J.; Cullis, P. Biochim. Biophys. Acta 1992, 1111, 127-133.

AFM Study of Lipid Monolayers: III. Phase Behavior of Ceramides

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