Article pubs.acs.org/Langmuir
Probing the Role of Ceramide Headgroup Polarity in Short-Chain Model Skin Barrier Lipid Mixtures by 2H Solid-State NMR Spectroscopy Sören Stahlberg,† Stefan Lange,‡ Bodo Dobner,‡ and Daniel Huster*,† †
Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstrasse 16-18, 04107 Leipzig, Germany Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle, Germany
‡
S Supporting Information *
ABSTRACT: The thermoptropic phase behaviors of two stratum corneum model lipid mixtures composed of equimolar contributions of either Cer[NS18] or Cer[NP18] with stearic acid and cholesterol were compared. Each component of the mixture was specifically deuterated such that the temperature-dependent 2H NMR spectra allowed disentanglement of the complicated phase polymorphism of these lipid mixtures. While Cer[NS] is based on the sphingosine backbone, Cer[NP] features a phytosphingosine, which introduces an additional hydroxyl group into the headgroup of the ceramide and abolishes the double bond. From the NMR spectra, the individual contributions of all lipids to the respective phases could be determined. The comparison of the two lipid mixtures reveals that Cer[NP] containing mixtures have a tendency to form more fluid phases. It is concluded that the additional hydroxyl group of the phytosphingosine-containing ceramide Cer[NP18] in mixture with chain-matched stearic acid and cholesterol creates a packing defect that destabilizes the orthorhombic phase state of canonical SC mixtures. This steric clash favors the gel phase and promotes formation of fluid phases of Cer[NP] containing lipid mixtures at lower temperature compared to those containing Cer[NS18].
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INTRODUCTION The stratum corneum (SC) represents the outermost layer of the human skin, which maintains the most important barrier function.1 The SC is composed of flattened layers of dead corneocytes that are surrounded by intracellular lipids organized in a complicated and not fully understood multilamellar assembly.2,3 The most abundant lipids in these layers are ceramides, free fatty acids, and cholesterol (Chol) at approximately equimolar ratio.3,4 Although several models of the organization of the lipids in the SC exist, as of now, the exact organization of this skin layer is still not fully clear.3 Biophysical methods have contributed to our understanding of the complicated structure of the lipid phase of the SC. Traditionally, X-ray and neutron scattering experiments, the latter taking advantage of the contrast variation between protons and deuterons in the neutron scattering length density profiles, have been employed to study the mesoscopic structure of the lipid layers of the SC.5−8 Such measurements suggested that the lipids in complex SC mixtures are organized in two coexisting lamellar phases, one of which shows a long periodicity phase with a repeat spacing of ∼13 nm and a short periodicity phase where the repeat distance is about 6 nm.5−7 Furthermore, Fourier transformed infrared (FTIR) and Raman spectroscopy has helped in elucidating the conformational order and phase behavior in SC models.9−11 FTIR investigations revealed that SC lipid mixtures are organized in © XXXX American Chemical Society
crystalline bilayers with orthorhombic chain packing at skin temperature, while increase in temperature leads to hexagonal chain packing with gel-state bilayers or even liquid-crystalline phases.12−14 In particular, the chain length of the free fatty acids seems to influence the formation of orthorhombic chain packing and the solid phase in SC mixtures.13,15 Chain length differences of about three to four carbons already lead to a small degree of demixing.12,15 Furthermore, recent 2H NMR studies have demonstrated that different phases coexist in SC preparations.8,16,17 While older 2H NMR studies relied on conclusions derived from detecting 2H NMR spectra of deuterated free fatty acids,8,18 cholesterol,8,19 or even phospholipids,20,21 more recently, deuterated ceramide variants also became available,16,17,22 allowing direct study of the complex thermotropic phase behavior of all components of the SC mixtures in relatively simple models. These studies revealed that a SC mixture rarely exists in a single phase, but rather coexisting phases are often encountered. Typical phases that are detected include the crystalline phase, which prevails in SC mixtures at skin temperature, fluid phases (mostly liquid-crystalline) at intermediate temperature of 40−60 °C, and a surprising predominance for isotropic phases (such as cubic or micellar Received: November 12, 2015 Revised: February 1, 2016
A
DOI: 10.1021/acs.langmuir.5b04173 Langmuir XXXX, XXX, XXX−XXX
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Langmuir phases) at high temperature above ∼70 °C has been found.16,17 This roughly summarizes the typical thermotropic phase behavior of mixtures containing the most abundant species of the SC such as Cer[NS24], lignoceric acid (C24:0), and cholesterol. It seems clear that a complex network of molecular interactions between the individual lipids of SC mixtures with small amounts of water determine the free energy of the complex lipid assembly. This energy landscape further determines the respective phase state of a SC lipid mixture at a given temperature. As the majority of lipids in the SC are uncharged, largely hydrogen bonding in the headgroup region and van der Waals interactions between the chain segments and the tetracyclic ring structure of Chol appear relevant but configurational entropy also has to be considered in the free energy function.23,24 As the packing density of the lipids in the SC is very high, it has been shown that small structural alterations such as the exchange of the sphingosine backbone for dihydrosphingosine led to rather drastic consequences in the thermotropic phase behavior of SC mixtures.17 Thus, the presence of an additional hydroxyl group as presented in phytosphingosine-containing ceramides should also alter the interaction networks between neighboring lipids and water.10,11,14 In atopic dermatitis patients, the level of shorter chain ceramides carrying a palmitoyl or stearoyl fatty acid at the expense of the longer C24:0 containing species has been observed. As the barrier properties of the SC are influenced by the acyl chain length of these molecules, such patients experience compromised skin barrier function.25,26 It was previously shown that the replacement of Cer[NS24] by the short chain analogue Cer[NS16] led to the formation of a (more permeable) gel phase even at skin temperature.17,17,27 However, the Cer[NS16] was studied in mixture with lignoceric acid (C24:0), which represents a significant chain length mismatch that could have been responsible for the unexpected formation of the gel phase. In this work we address two questions: (i) What is the phase organization of chain length-matched short Cer[NS18] in equimolar mixture with stearic acid (C18:0) and Chol? (ii) How is the phase behavior of such mixtures influenced when the polarity of the ceramide headgroup is altered by the introduction of an additional hydroxyl group as present in Cer[NP18]? Previous papers have also studied the influence of the number of hydroxyl groups in pure ceramide preparations using molecules carrying a sphingosine backbone11 as well as in ceramides with sphingosine (e.g., Cer[NS]) and phytosphingosine backbones (e.g., Cer[NP])10,14,28 using FTIR and Raman spectroscopy. These studies already highlighted the variations in the thermotropic phase behavior and complex hydrogen bond pattern. In the current paper, we further elaborate these issues in ternary SC model mixtures of ceramide, free fatty acid, and cholesterol locking at all three lipids individually.
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Synthesis of Cer with Perdeuterated Acyl Residue. Phytosphingosine and sphingosine were purified by column chromatography with freshly distilled CHCl 3 and MeOH (SiO2:substance 10:1, CHCl3:MeOH:0.5% ammoniaaqueous, discontinuous increase of polarity, initial concentration: 100:0, final concentration: [phytosphingosine] 89:11 [sphingosine] 94:6) before use. SA, SA-d35, and PYBOP were directly used without purification. In a 250 mL round-bottom flask (oven-dried, cooled under an atmosphere of argon) 0.1 mmol SA-d35 and 0.11 mmol PYBOP were dissolved in 150 mL dried CH2Cl2. Then, 0.2 mmol Hunig’s base was added, and the mixture was stirred at room temperature for 20 min. Afterward, 0.1 mmol phytosphingosine or sphingosine was added, and the mixture was stirred at room temperature for 24 h. The reaction was monitored by thin layer chromatography (TLC) using precoated aluminum sheets (normal SiO2; Merck 60 F254; CHCl3:MeOH:NH3 95:5:0.5 Cer-d35[NP18] Rf = 0.3, Cer-d35[NS18] Rf = 0.4). Following this, the organic layer was washed 2 times with 100 mL distilled water, once with 100 mL brine, dried over Na2SO4, filtered, and concentrated under vacuum. After column chromatography with freshly distilled CHCl 3 and MeOH (SiO 2 :Cer 100:1, CHCl3:MeOH:0.5% ammoniaaqueous) and a discontinuous increase of polarity (initial concentration: 100:0:0, final concentration: Cer[NP18]-d35 96:4:0.5%, Cer[NS18]-d35 98:2:0.5%), the ceramides were obtained as a white solid (yield: Cer-d35[NP18] 85%, Cer[NS18]/ Cer[NS18]-d3581%). The purification was checked by TLC. Moreover, deuterated Cer[NP18]-d35 and Cer[NS18]-d35 were checked by HRMS (Cer[NP18]-d35 calcd: 619.7809 [M+H]+, found: 619.7788 [M+H]+; Cer[NS18]-d35: calcd: 599.7558 [M−H]− 635.7325 [M +HCl]−, found: 599.7598 [M−H]− 635.7360 [M+HCl]−) and HPLC (see also Supporting Information). Sample Preparation. For each model system, three samples were prepared, each of them containing one deuterated component: Cer-d35 (either Cer[NS18]-d35 or Cer[NP18]-d35 with a perdeuterated C18:0 acyl chain)/SA/Chol (1:1:1, mol/mol/mol); Cer/SA-d35 (deuterated)/Chol (1:1:1, mol/mol/mol); Cer/SA/Chol-d6 (deuterated) (1:1:1, mol/mol/mol). Aliquots of Cer, free fatty acid, and Chol were dissolved in chloroform/methanol (2:1) and the solvent was evaporated using a rotary evaporator. Subsequently, the sample was dissolved in cyclohexane and lyophilized at ∼0.1 mbar overnight yielding a fluffy powder, which was then hydrated with 50 wt % deuterium-depleted water. At such very low water content, buffer solutions are not capable of buffering the large amount of lipid molecules. In order to prepare homogeneous samples, we used our previously developed protocol.17 The lipid−water dispersions were first filled into 4 mm magic-angle spinning (MAS) rotors, which were used as sample containers, and sealed for static 2H NMR measurements to avoid sample dehydration. Subsequently, the samples were frozen in liquid nitrogen and heated to 80 °C. These freeze−thaw cycles were repeated 10 times. As shown in a recent study, 4−10 h are required for the formation of orthorhombic domains in samples containing Cer[NS18],29 all samples were incubated for 24−30 h at 22 °C to allow sufficient time for sample equilibration and avoid different kinetics in phase formation. Although other studies suggested that the formation of crystalline phases in Cer/palmitic acid/cholesterol mixtures containing either bovine brain Cer[NP] (acyl chain lengths mainly C18, C24 and C24:1) or Cer[NS16] required longer periods,16,30 a previous study on Cer[NS]-based systems showed that the IR spectra did not change with incubation longer than 24 h.31 In addition, no changes in the permeability of two model drugs of such SC models were found between 1 and 14 days.31 Our 2H NMR spectra of identical samples remained very similar over time periods of several weeks. 2 H NMR Spectroscopy. A Bruker Avance 750 WB NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) was used to acquire all NMR spectra at a deuterium resonance frequency of 115.1 MHz and a spectral width of ±250 kHz. The probe was equipped with a 5 mm solenoid coil. For the NMR measurements, quadrature detection and a phase-cycled quadrupolar echo sequence with two 2− 2.5 μs π/2 pulses separated by 30 μs delay were applied. In order to
MATERIALS AND METHODS
Materials. Perdeuterated stearic acid (SA-d35) was purchased from SIGMA Aldrich (Germany). PyBOP was acquired from carbolution CHEMICALS, GmbH (Germany). Cer[NP18], Cer[NS18], phytosphingosine (2S, 3S, 4R) and sphingosine (2S, 3R, 4E) were donated by Evonik Industries, AG (Germany). Cholesterol was purchased from Avanti Polar Lipids (Alabaster, AL), and cholesterol-(2,2,3,4,4,6)-d6 was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). B
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Figure 1. 2H NMR spectra of the equimolar mixture of Cer[NS18]/SA/Chol at varying temperatures and 50 wt % hydration. The left column shows the 2H NMR spectra of the Cer[NS18]-d35, the middle column depicts the 2H NMR spectra of the SA-d35, and the right column displays the 2H NMR spectra of the Chol-d6 in the mixture. NMR spectra were acquired with a relaxation delay of 50 s. The red lines illustrate the best fit numerical simulations. monotonic variation of quadrupolar splittings along the chain was assumed. The 2H NMR spectrum gel phase was simulated using a superposition of two C−2H bond vectors undergoing 2-site exchange with a hop angle of 120° for each methylene group in the chain and one methyl group undergoing fast rotation. The 2-site exchange line shape was simulated using NMR-WEBLAB.34 From the simulated line shapes of each sample, the percentage of each phase contributing to the resulting 2H NMR spectra could be determined.
obtain NMR spectra for quantitative analysis, the delay between successive scans was 50 s.18 Samples were measured at temperatures of 32 °C, 50 °C, and 75 °C. For further processing of the acquired spectra, a self-written Mathcad (MathSoft, Cambridge, MA) script was used; more details are reported in the literature.32 2 H NMR Line Shape Simulation. The measured 2H NMR spectra were simulated by using a superposition of N 2H NMR Pake doublets each scaled by the appropriate order parameter (SCDi) and typically one isotropic line.17 Assuming a quadrupolar coupling constant of χ = 167 kHz for the C−2H bond, the rigid quadrupolar coupling was 125.25 kHz. Using an increment of the angle θ of 0.0625° for powder averaging, NMR time domain data were simulated as free induction decays (FID) according to N
FID(t ) =
⎛⎛
∑ ⎜⎜∫ i=1
⎝⎝
90 °
θ=0
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RESULTS H NMR of the Cer[NS18]/SA/Chol Mixture. The 2H NMR spectra of each molecular component of the Cer[NS18]/ SA/Chol mixture are shown in Figure 1. The spectra are organized in columns according to the deuterated component in the sample, i.e., the 2H NMR spectrum of chain deuterated Cer[NS18]-d35 in the mixture is shown in the left column, the 2 H NMR spectrum of perdeuterated SA-d35 component is displayed in the middle column, and the 2H NMR spectrum of the deuterated Chol-d6 in the mixture is shown in the right column. Each row represents the temperature at which the samples were measured, starting at 32 °C on the bottom, 50 °C in the middle, and 75 °C in the top row. Hence, Figure 1 summarizes the thermotropic phase behavior of this specific ceramide mixture. At the physiological skin temperature of 32 °C, the 2H NMR spectrum of the Cer[NS18] component of the mixture is dominated by a spectral line shape that is indicative of a solid phase as characteristic for other ceramide subspecies.16,17,35
⎞ ⎛3 1 ⎞ dθ cos⎜ χ · (3 cos2 θ − 1) · SCDi· t ⎟·sin θ ⎟ ⎝4 2 ⎠ ⎠
⎞ · exp(t ·LBi · π )⎟ ⎠ where LBi represents the line broadening factor for each Pake doublet, and θ is the angle for powder averaging. As some spectra showed slight magnetic field induced orientation, the spherical distribution function sin θ was replaced by an ellipsoidal distribution function:33
2πc 2 sin θ
(sin θ + 2
c2 a2
cos2 θ
2
)
with the parameters c and a as eccentricities. From these line shape simulations of the experimental 2H NMR spectra, the order parameters as a function of carbon position SCDi could be determined. A C
2
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Table 1. Quantitative Analysis of the Phase Composition of Equimolar Ternary Cer[NS18]/SA/Chol Mixtures Determined from Numerical Simulation of the 2H NMR Spectra Shown in Figure 1a Cer[NS18]
a
stearic acid
cholesterol
temperature
cryst./gel
fluid
isotr.
cryst.
fluid
isotr.
cryst.
fluid
isotr.
32 °C 50 °C 75 °C
83% 53% 9%
17% 46% 37%
1% 54%
96% 48% -
2% 48% 28%
2% 4% 72%
81% -
14% 93% 81%
5% 7% 19%
The experimental error in the phase composition is judged to be ≤10%.
Some minor fraction of the molecule displays a NMR spectrum characteristic of a more fluid phase. The SA of the mixture almost entirely forms a crystalline phase and so does the cholesterol. As the 2H NMR experiments were acquired with a sufficiently long relaxation delay of 50 s, the relative contributions to the respective line shapes can be quantified and are reported in Table 1.18 This analysis reveals that >80% of each molecular component of the mixture is found in a crystalline phase at skin temperature, while smaller fractions are fluid and trace amounts of the molecules are found in an isotropic phase. In detail, the majority of Cer[NS18] (83%), SA (96%) and Chol (81%) forms a crystalline phase. In this rigid phase, only the methyl groups of the ceramide and the fatty acid are undergoing fast axially symmetric rotation and possibly some small angle librations. The fraction of fluid molecules amounts to 17%, 2%, and 14% for Cer[NS18], SA, and Chol, respectively, while 2% of the SA and 5% of the Chol are isotropic. These results suggest the superposition of several phases in the mixture. This is in good agreement with chain matched mixtures of Cer[NS24]/lignoceric acid (C24:0)/Chol reported earlier.17 Raising the temperature to 50 °C leads to quite drastic alterations of the phase composition of the mixture. Very evident in all NMR spectra is the occurrence of a fluid liquidcrystalline phase highly indicative of lamellar structures as known from phospholipid membranes.36,37 This phase is detected for 46% of the Cer[NS18], 48% of the SA, and 93% of cholesterol. In addition, 53% of the Cer[NS18] form a more rigid phase, most likely a gel phase, and 48% of the SA are crystalline. Minor fractions of each lipid are found to be isotropic. The well-resolved quadrupolar splittings observed for the fluid components of the mixture are reminiscent of liquidcrystalline membranes as known from phospholipids. Therefore, order parameter profiles36,37 can be determined for the fluid acyl chains and are displayed in Figure 2. Typical order parameter profiles with a plateau value for the upper chain segments and decreasing lipid chain order toward the end of the chain are calculated. However, compared to liquid crystalline phospholipid membranes, higher order parameters are found, which may suggest that the phase state is liquid ordered. Such liquid ordered phases have also been identified in other ceramide containing lipid mixtures.16,18,38 The 2H NMR spectrum of the fluid cholesterol also shows the characteristic features of cholesterol in fluid membranes undergoing axially symmetric reorientations.39 Heating the samples to 75 °C abolishes most crystalline or gel phase features from the 2H NMR spectra, and the fluid and isotropic phases dominate the 2H NMR spectra. Most of the Cer[NS18] (54%) and SA (72%) is found in an isotropic phase, while only 19% of Chol shows this isotropic phase behavior. The predominant phase for Chol is still the liquidcrystalline phase accounting for 81% of the molecules. Lipid chain order parameters of the fluid phase proportions of the
Figure 2. 2H NMR order parameters of Cer[NS18]-d35 and SA-d35 of an equimolar mixture of Cer[NS18]/SA/Chol at 50 °C (filled symbols) and 75 °C (open symbols) and a hydration level of 50 wt %.
Cer[NS18] and SA are shown in Figure 2. It is worth noting that at higher temperature the upper chain segments are more disordered as expected, but the lower chain segments show similar (SA) or even higher order parameters (Cer[NS18]). Ceramide chains sometimes display a kink at carbon position #2 that reduces the quadrupolar splitting and the apparent order parameter. Overall, the phase behavior of the Cer[NS18]/SA/Chol mixture is very similar to that of Cer[NS16]/ palmitic acid/Chol, reported in the literature.16 Using the diamond lattice model,40,41 the average chain length of each fluid lipid chain and the average number of gauche defects can be calculated; these numbers are reported in Table 2. Table 2. Average Order Parameter ⟨S⟩, Chain Length L, and Number of Gauche Defects in the Chains of the Individual Ceramide-Containing Lipid Mixtures Calculated Using the Diamond Lattice Model40 Cer[NS]-d35/ SA/Chol ⟨S⟩ L/Å # gauche defects
Cer[NS]/SAd35/Chol
Cer[NP]/SAd35/Chol
50 °C
75 °C
50 °C
75 °C
50 °C
75 °C
0.287 16.0 3.9
0.296 16.2 3.8
0.292 16.1 3.8
0.268 15.6 4.3
0.281 15.9 4.0
0.281 15.9 4.0
2
H NMR of the Cer[NP18]/SA/Chol Mixture. An overview of the measured 2H NMR spectra as well as the respective line shape simulations and calculated phase proportions of Cer[NP18]/SA/Chol are shown in Figure 3 and Table 3, respectively. At physiological skin temperature, the 2H NMR spectra of SA and Chol in the mixture are dominated by a crystalline D
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Figure 3. 2H NMR spectra of the equimolar mixture of Cer[NP18]/SA/Chol at varying temperatures and 50 wt % hydration. The left column shows the 2H NMR spectra of the Cer[NP18]-d35, the middle column depicts the 2H NMR spectra of the SA-d35, and the right column displays the 2H NMR spectra of the Chol-d6 in the mixture. NMR spectra were acquired with a relaxation delay of 50 s. The red lines illustrate the best fit numerical simulations.
Table 3. Quantitative Analysis of the Phase Composition of Equimolar Ternary Cer[NP18]/SA/Chol Mixtures Determined from Numerical Simulation of the 2H NMR Spectra Shown in Figure 3a Cer[NP18]
a
Stearic Acid
Cholesterol
temperature
gel/cryst.
fluid
isotr.
cryst.
fluid
isotr.
cryst.
fluid
isotr.
32 °C 50 °C 75 °C
57% 44% 16%
43% 56% 80%
4%
60% 27% -
39% 67% 35%
1% 6% 65%
69% -
28% 96% 100%
3% 4% -
The experimental error in the phase composition is judged to be ≤10%.
phase (>60% for each component; see Table 3). However, in contrast to the Cer[NS18] containing mixture, about 57% of the Cer[NP18] clearly forms a gel phase. This has also been found by Raman spectroscopy on isolated Cer[NP] preparations.14 Residual spectral intensity with a quadrupolar splitting >125 kHz may also indicate the presence of a fraction of the ceramide in a very small crystalline phase.16 Furthermore, in contrast to the Cer[NS18] containing mixture, the proportions of fluid Cer[NP18], SA, and Chol are significantly larger, while the contributions from the isotropic phase are comparable. This suggests a significant restructuring of the mixture induced by the additional hydroxyl group of Cer[NP18]. The number of molecules that prefer the fluid phase at the expense of the crystalline phase is at least double (almost 20 times in the case of SA). While the gel phase of Cer[NP18] remains relevant at an elevated temperature of 50 °C, the crystalline SA becomes a less significant phase in the mixture and Chol is almost entirely fluid. In particular, the fluid phase is more dominating in the
Cer[NP18]-containing mixture compared to the Cer[NS18]containing mixture. The 2H NMR spectrum of the Chol component shows almost identical features as the Cer[NS]containing mixture. Order parameters of SA-d35 in the mixture can be determined and are plotted in Figure 4. The final heating step to 75 °C again reveals further interesting differences between the Cer[NP18]- and Cer[NS18]-containing lipid mixtures. For SA and Chol, the dominating phase is the isotropic phase at this temperature, while the gel phase persists for the Cer[NP18] with 16% contribution, but 80% of the Cer are fluid. Only a very small portion (4%) of the Cer[NP18] forms an isotropic phase as oppose to the 54% of Cer[NS18] found in this phase in the other mixture. The fluid phase found for Cer[NP18] at 75 °C shows some spectral characteristics that largely differs from all other fluid phases observed so far. The 2H NMR spectrum of Cer[NP18] in the mixture is enlarged in Figure 5 and in fact shows two fluid phases in addition to the gel phase and the isotropic phase. The first fluid phase is very similar to the one E
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the acyl chains of both ceramides6,7,12,17,27,31 and the free fatty acid,13 the degree of unsaturation of the free fatty acid,15,19 or the ceramide backbone.17,42 Here, we focus our attention on the influence of the polarity of the ceramide headgroup, in particular the influence of the number of hydroxyl groups on the thermotropic phase behavior of equimolar mixtures of short chain-matched ceramide and stearic acid with cholesterol. We compare the thermotropic phase behaviors of Cer[NS18] and Cer[NP18] that comprise two or three hydroxyl groups in the headgroup, respectively. From the stereochemical point of view, the hydroxyl group in position 3 is not allylic in Cer[NP], and there is also a double bond missing. We started the investigation of the thermotropic phase behavior at the physiological skin temperature of 32 °C. However, it should be mentioned that the chain lengths of the lipid mixtures we investigate are somewhat unphysiological as the mixture lacks the most relevant long chain ceramide and free fatty acid species. A first obvious difference between these preparations at 32 °C is that the majority of the molecules in the Cer[NS18] containing mixture form a classical orthorhombic phase, while the Cer[NP18] in the other mixture is organized in a gel phase (57%) and a very likely separated fluid phase (43%) that carries the signature of the liquid ordered phase.43−45 The majority of the stearic acid and Chol in the Cer[NP18] containing mixture is organized in a crystalline phase, but significantly larger proportions of these lipids are fluid as oppose to the Cer[NS18] containing mixture, where these proportions are smaller. Apparently, the additional hydroxyl group in the phytosphingosine of Cer[NP18] creates a packing defect that prevents formation of the densest lipid packing in an orthorhombic phase. This is visualized in Figure 6, which shows the structures
Figure 4. 2H NMR order parameters of SA-d35 of an equimolar mixture of Cer[NP18]/SA/Chol at 50 °C (filled symbols) and 75 °C (open symbols) and a hydration level of 50 wt %.
Figure 5. 2H NMR spectrum of Cer[NP18]-d35/SA/Chol at 75 °C and 50 wt % hydration showing coexisting gel and fluid phases. Fluid phases I and II can be considered as liquid ordered and liquid disordered, respectively. Panel A shows fluid phase II enlarged. Panel B shows the 2H NMR spectrum of the Chol-d6 in the same mixture, where again, two fluid phases can be detected. Figure 6. Structural models of Cer[NS18] and Cer[NP18] illustrating the effect of the putative packing defect created by the hydroxyl group of the phytosphingosine of Cer[NP18]. Red arrows point toward the additional hydroxyl group of Cer[NP18] that appears to perturb lipid packing.
observed at 50 °C featuring order parameters that are in agreement with a liquid ordered phase behavior. In addition, there are Cer[NP18] molecules in a fluid phase that is characterized by much smaller quadrupolar splittings, which reach a maximum of ∼16 kHz, which would translate into a maximal order parameter of 0.13, which is characteristic for the liquid disordered phase at such high temperatures.
of both ceramides. It can clearly be seen how the additional hydroxyl group of Cer[NP18] “sticks out”, which likely causes a defect in the packing rendering the gel phase energetically more favorable than the orthorhombic phase. It is interesting to consider that the additional possibility of a hydroxyl group to form a hydrogen bond, for instance with the carboxyl group of a neighboring ceramide or stearic acid, does not improve the packing properties in the mixture. Due to a possible steric clash, this hydroxyl group may in fact even prevent the most optimal contacts between Cer[NP18] and cholesterol. Due to these packing differences, the gel phase is preferred for the Cer[NP]containing mixtures and the phase transitions to liquid crystalline are observed at lower temperature.
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DISCUSSION The stratum corneum represents the major skin barrier and is characterized by a densely packed layer of stacked ceramide rich lipid layers. Subtle structural differences between the individual ceramide classes exist and even minor structural alterations represent critical determinants of the thermotropic phase behavior of artificial lipid mixtures that mimic the composition of the human outermost skin layer, typically consisting of ceramide, free fatty acid, and cholesterol. Such critical parameters for the structure of the SC include the length of F
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order parameter, chain length, and average number of gauche defects) are relatively low (Table 2). Another interesting feature is observed for the Cer[NP18]containing mixture at 75 °C. The 2H NMR spectra of Cer[NP18] and Chol-d6 exhibit two major fluid contributions, that can be attributed with the molecules in a more ordered fluid phase such as the liquid-ordered phase38 as well as a very disordered fluid phase that shows the characteristics of the liquid-disordered phase state (Figure 5).43,44 In both 2H NMR spectra, the magnitude of the quadrupolar splittings of both lipids in the disordered fluid phase are about 1/3 of those of the ordered fluid phase. This could suggest the coexistence of a lamellar and an inverse hexagonal phase (HII), for which the ratio between the two sets of quadrupolar splittings would have to be 1/2 or smaller.47 Often, HII lipid chains are more disordered than bilayer chains due to the cone shaped space they can now explore, which reduces splitting more than the factor of 2 that stems solely from the extra degree of orthogonal averaging (diffusion of lipids around the cylinder axis in addition to rotational diffusion of the lipids about their long axes). Therefore, the observation of a factor of 3 reduction is very comparable to the observations reported by Pare and Lafleur.48 Other nonlamellar phases are also possible, involving, for instance, small radii of curvature, which give rise to additional averaging of the electric field gradient tensor. Given the very large difference in the quadrupolar splittings, it is rather unlikely that the narrow component of the 2H NMR spectrum comes from lipids in a lamellar phase. While the 2H NMR spectrum of the Cer[NP18] is too convoluted to allow for a reliable quantification, such a quantitative analysis was possible for the NMR spectrum of the Chol in the mixture (Figure 5B). It is interesting to note that the 2H NMR spectrum measured for the more rigid fluid phase is very similar to data on Chol-d6 ion mixture with DMPC at a 30:70 molar ratio, a mixture that would typically be referred to as liquid-ordered.39 However, for the much narrower 2H NMR spectrum of Chol in the more disordered fluid phase, no reference data are known. Nevertheless, the assignment of a coexisting liquid-ordered and liquid-disordered phase is contradictory. Our quantitative analysis revealed that about 46% of the cholesterol is associated with the disordered fluid phase and 54% with the more ordered fluid phase. Although it is believed that cholesterol is associated with both the liquid-ordered and the liquid-disordered phases, the majority of the cholesterol should be associated with the liquid ordered phase.43−45 This indicates the limits of the comparison of ceramide containing lipid mixtures with lipid raft mixtures. Taken together, the additional hydroxyl group of the phytosphingosine containing ceramide Cer[NP18] in mixture with chain-matched SA and cholesterol creates a packing defect that destabilizes the orthorhombic phase state of canonical SC mixtures. Although the hydrogen bonding pattern was found to be less strong in sphingosine-based ceramides than in phytosphingosine-containing ceramides,10 this steric clash favors the gel phase and promotes formation of fluid phases at lower temperature compared to Cer[NS18]-containing mixtures. At the same time, the Cer[NP18]-containing mixture stabilizes the fluid lamellar phase at high temperature. These observations underline the highly specific structure-forming properties of ceramides in the human skin.
Ceramide gel phases in mixtures with free fatty acid and cholesterol are exclusively observed for shorter chain ceramides or dihydroceramides.16,17 However, the lignoceric acid in mixtures of Cer 24/LA/Chol also formed a gel phase at 37 °C, but not the ceramide.38 Although the amount of date on the phase state of the ceramide molecules in SC mixtures is still rather limited as deuterated ceramides have only become available recently, at the current point of time, this may support the idea that an additional hydroxyl group of the phytosphingosine of Cer[NP18] creates a packing defect. In a longer chain, more methylene segments can contribute van der Waals interactions (∼6 kJ/mol per CH2 group46), which amount to a sizable free energy that can overcome the entropic losses that are introduced in the headgroup due to the packing defect. In agreement with our results, it has previously been found that optimal H-bonding of the hydrated phytosphingosine headgroup is not compatible with orthorhombic chain packing.10 The perturbation of the orthorhombic lipid packing by Cer[NP18] is likely not connected to the missing trans double bond in the phytosphingosine backbone of Cer[NP18]. In a previous study, we studied Cer[NS24]- and Cer[NDS24]containing mixtures where sphingosine and dihydrosphingosine backbones were directly compared.17 The Cer[NDS24]containing mixture with the dihydrosphingosine backbone showed a preference for the crystalline phase along with a more pronounced isotropic phase, but the proportion of the fluid phase was drastically reduced in comparison to the Cer[NS] containing mixture.17 The preference of the Cer[NP18]-containing mixture for less ordered and more fluid phases represents a trend that was also observed at elevated temperature. At 50 °C, this particular applies to the SA. The SA lipids that are in a fluid phase at this intermediate temperature show 2H NMR spectra that are very characteristic of the liquid ordered phase featuring very high order parameters. Using the simple diamond lattice model,40 the average chain length of the Cer[NS18] and the SA in both mixtures can be calculated. Overall, very similar chain lengths are observed for both lipid chains at both temperatures as reported in Table 2. As all chains are very similar in length, the liquid-crystalline chains of SA and Cer[NS18] are relatively highly ordered with comprising on average about 4 gauche defects per chain. The preference of SA for the fluid phase in a Cer[NP18] containing mixture was also found in a combined neutron scattering/2H NMR study, but at lower Chol concentration of 20 mol %.8 However, the current analysis for the first time shows this phenomenon for all three lipid components of this mixture. At 75 °C, large portions of all lipids are found in an isotropic phase state that is characterized by liquid phases or highly curved lipid structures. However, the proportion of lipids in the isotropic phase are significantly lower compared to longer chain Cer[NS24] and Cer[NDS24] mixtures with length-matched lignoceric acid and cholesterol as well as not length matched Cer[NS16] in mixture with lignoceric acid and cholesterol.17 In the shorter C18:0 ceramide mixtures, a fluid phase is preferred, which is apparently not stable in longer chain ceramides or significantly mismatched mixtures of ceramide and free fatty acids. In the Cer[NS18] containing mixture, 37% of the ceramide, 28% of the SA, and 81% of the Chol form a fluid phase that features the characteristics of the liquid ordered phase. Differences in the chain packing properties (average G
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b04173. HPLC chromatograms of purified Cer[NS18]-d35 and Cer[NP18]-d35 along with high resolution mass spectra of both lipids (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Address: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstrasse 16-18. Tel: 49 (0) 341 97 15701. Fax: 49 (0) 341 97 15709. E-mail: daniel.huster@ medizin.uni-leipzig.de. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to thank Dr. Alexander Vogel for preparing Figure 6, Dr. Christian Ihling for the HRMS spectra, and Manuela Woigk for the HPLC chromatograms. The study was supported by the Deutsche Forschungsgemeinschaft (DFG HU 720/13-1 and DO 463/6-1) and Evonik Industries, AG.
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REFERENCES
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