Influence of a Novel Dimeric Ceramide Molecule on the Nanostructure

Article pubs.acs.org/Langmuir

Influence of a Novel Dimeric Ceramide Molecule on the Nanostructure and Thermotropic Phase Behavior of a Stratum Corneum Model Mixture Sören Stahlberg,†,∇ Adina Eichner,‡,∇ Stefan Sonnenberger,†,‡,∇ Andrej Kovácǐ k,†,§ Stefan Lange,‡ Thomas Schmitt,‡,⊥ Bruno Demé,∥ Thomas Hauß,⊥ Bodo Dobner,‡ Reinhard H. H. Neubert,‡,# and Daniel Huster*,† †

Institute for Medical Physics and Biophysics, Leipzig University, Leipzig, Germany Institute of Pharmacy and #Institute of Applied Dermatopharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany § Skin Barrier Research Group, Faculty of Pharmacy in Hradec Králové, Charles University, Akademika Heyrovského 1203, 50005 Hradec Králové, Czech Republic ∥ Institute Laue-Langevin (ILL), Grenoble, France ⊥ Institute of Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin für Materialien und Energie, Berlin, Germany ‡

S Supporting Information *

ABSTRACT: The stratum corneum (SC) is the outermost layer of the skin and is composed of a multilayered assembly of mostly ceramids (Cer), free fatty acids, cholesterol (Chol), and cholesterol sulfate (Chol-S). Because of the tight packing of these lipids, the SC features unique barrier properties defending the skin from environmental influences. Under pathological conditions, where the skin barrier function is compromised, topical application of molecules that rigidify the SC may lead to a restored barrier function. To this end, molecules are required that incorporate into the SC and bring back the original rigidity of the skin barrier. Here, we investigated the influence of a novel dimeric ceramide (dim-Cer) molecule designed to feature a long, rigid hydrocarbon chain ideally suited to forming an orthorhombic lipid phase. The influence of this molecules on the thermotropic phase behavior of a SC mixture consisting of Cer[AP18] (55 wt %), cholesterol (Chol, 25 wt %), steric acid (SA, 15 wt %), and cholesterol sulfate (Chol-S, 5 wt %) was studied using a combination of neutron diffraction and 2H NMR spectroscopy. These methods provide detailed insights into the packing properties of the lipids in the SC model mixture. Dim-Cer remains in an all-trans state of the membrane-spanning lipid chain at all investigated temperatures, but the influence on the phase behavior of the other lipids in the mixture is marginal. Biophysical experiments are complemented by permeability measurements in model membranes and human skin. The latter, however, indicates that dim-Cer only partially provides the desired effect on membrane permeability, necessitating further optimization of its structure for medical applications.

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Several conditions and diseases such as psoriasis,26 atopic dermatitis,27−29 and irritant/allergic contact dermatitis27 are known to be associated with a depletion of the ceramide concentration within the SC, which leads to a disturbance of its structure and a compromised barrier function. On a molecular level, modifications of the thermotropic phase behavior, altered lipid packing, and compromised permeability of SC model systems have been revealed.6,7,30 In the literature, quite a lot of substances have been described, which enhance skin penetration to relevant drugs and cosmetic actives, the so-called penetration enhancer.31,32 In contrast, there is limited knowl-

INTRODUCTION The outermost layer of human skin, the stratum corneum (SC), represents nature’s most important barrier against all environmental influences.1 The SC consists of flattened layers of dead corneocytes, which are surrounded by intracellular lipids organized in a relatively complicated multilamellar lipid assembly.2−4 In these layers, the most important lipid species are ceramides (Cer), free fatty acids (FFA), and cholesterol (Chol) as well as cholesterol sulfate (Chol-S).3,5 Numerous studies involving quantitative biophysical tools have described mixtures of various SC lipids in great detail, highlighting that most lipids in the SC at physiological skin temperature are organized in a rigid orthorhombic phase state, which is responsible for the robustness of the outermost skin layer and the well-defined barrier properties.3,6−25 © 2017 American Chemical Society

Received: April 24, 2017 Revised: August 16, 2017 Published: August 18, 2017 9211

DOI: 10.1021/acs.langmuir.7b01227 Langmuir 2017, 33, 9211−9221

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Figure 1. Structures of the dimeric ceramide (dim-Cer) and deuterated variant dim-Cer-d12.

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edge about substances that are able to reduce skin penetration by stabilizing the SC lipid structure.33 One approach to stabilizing the integrity of the lipid layer of the SC is to incorporate molecules that induce the orthorhombic lipid phase possibly at low concentrations. To this end, the synthesis of a novel dimeric ceramide molecule (dim-Cer) has been described recently.34 The structure of dimCer is shown in Figure 1 and consists of two phytosphingosine moieties linked by a long artificial nonhydroxyl fatty acid, rendering this molecule a derivative of Cer[NP], which is one of the most abundant ceramides in human SC.35,36 The long lipid chain was designed to promote the orthorhombic lipid phase in order to restore the barrier function of the outermost layer in the skin. Applying such putatively rigid molecules is promising because it has been shown that even small quantities of ceramides with altered structure have a profound impact on the microstructure and basic properties such as the permeability of skin lipid membranes.37,38 Here, we have investigated the influence of the dim-Cer molecule on the thermotropic phase behavior of an SC mixture consisting of Cer[AP18] (55 wt %), cholesterol (Chol, 25 wt %), steric acid (SA, 15 wt %), and cholesterol sulfate (Chol-S, 5 wt %). 2H NMR spectroscopy was applied to investigate the phase state of the major components of the mixture as well as lipid chain order and packing properties. Neutron diffraction experiments were carried out to localize the deuterated central segment of dim-Cer in the SC multilamellar lipid layers by exploiting the difference in the coherent scattering lengths bcoh of neutrons for hydrogen (−0.374 × 10−12 cm) and deuterium (0.667 × 10−12 cm). Thus, the influence of this new ceramide analog on the crucial properties of the lipid organization in SC models could be evaluated. Although 2 H NMR can quantitatively reveal the phase composition of complicated lipid mixtures, neutron diffraction provides detailed insights into the structure of the highly ordered orthorhombic phase, thus the combination of the two methods is highly useful for a comprehensive description of the dynamic structural arrangement of stratum corneum model membranes. We complement our structural results by permeability data on membrane models and human skin specimens to provide some physiological data on the effect of the dim-Cer molecule.

MATERIALS AND METHODS

Materials. dim-Cer was synthesized with a perdeuterated hexamethylene-d12 block in the middle of the artificial fatty acid as described in the literature.34 Cer[AP18] (N-(α-hydroxyoctadecanoyl)-phytosphingosine) (98% purity) was a gift from Evonik Industries AG (Essen, Germany). Cholesterol (Chol), cholesterol sulfate (Chol-S), and stearic acid (SA) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. For the neutron diffraction experiments, quartz slides (Spectrosil 2000, 25 × 65 × 0.3 mm3) were purchased from Saint-Gobain (Wiesbaden, Germany). An airbrush instrument (Harder & Steenbeck, Norderstedt, Germany) was used to spray the lipids onto the quartz plate. Chloroform and methanol (GC grade) were purchased from Merck (Darmstadt, Germany). Sample Preparation for 2H NMR Measurements. Two SC model systems were prepared that contained Cer[AP18] (55 wt %), cholesterol (Chol, 25 wt %), steric acid (SA, 15 wt %), and cholesterol sulfate (Chol-S, 5 wt %). One system contained 20 wt % of the dimeric ceramide (dim-Cer). In each sample, one of the components was deuterated, i.e., either Cer[AP18]-d35, SA-d35, Chol-(2,2,3,4,4,6)-d6, or dim-Cer-d12. Thus, a total of three samples in the absence and four samples in the presence of dim-Cer were prepared. For sample preparation, aliquots of each component were dissolved in chloroform/methanol (2:1) and mixed. The solvent was removed using a rotary evaporator. The remaining lipid film was dissolved in cyclohexane and lyophilized overnight, leaving a fluffy powder. For hydration, a 50 wt % aqueous buffer (100 mM NaCl, 100 mM MES, 5 mM EDTA, prepared in deuterium-depleted H2O at pH 5.4) was added. Samples were homogenized by rigorous vortex mixing and stirring.6,7 To avoid dehydration of the samples during the NMR measurements, they were filled into 4 mm magic-angle spinning rotors and sealed with an airtight Kel-F cap for the 2H NMR measurements. Subsequently, all samples underwent 10 freeze−thaw cycles; i.e., they were frozen in liquid nitrogen and subsequently placed in a water bath at 80 °C before incubating them for at least 24 h at 22 °C. The incubation assures the formation of orthorhombic domains of fatty acid in the sample, and it avoids different kinetics in phase formation.6 Sample Preparation for Neutron Diffraction Measurements. To determine the effect of dim-Cer, a well-characterized SC lipid model membrane was used as a reference.39,40 dim-Cer and dim-Cerd12 were added in ratios of 5, 10, and 20 wt % to observe the increasing impact of dim-Cer on the lipid membranes. The fully protonated samples were required as a reference for the localization of the deuterated segments in the samples containing dim-Cer-d12. The samples for the neutron diffraction measurements were prepared as described in the literature.41 All lipid components were dissolved 9212


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Langmuir Table 1. Overview of the Composition of the SC Lipid Model Systems Studied in Neutron Diffraction Experiments SC lipid model system Cer[AP18]/Chol/SA/Chol-S Cer[AP18]/Chol/SA/Chol-S Cer[AP18]/Chol/SA/Chol-S Cer[AP18]/Chol/SA/Chol-S Cer[AP18]/Chol/SA/Chol-S Cer[AP18]/Chol/SA/Chol-S

+ + + + + +

ratio (wt %)

5% dim-Cer 10% dim-Cer 20% dim-Cer 5% dim-Cer-d12 10% dim-Cer-d12 20% dim-Cer-d12

55/25/15/5 55/25/15/5 55/25/15/5 55/25/15/5 55/25/15/5 55/25/15/5

N

⎛⎛

∑ ⎜⎜∫ i=1

⎝⎝

90 °

θ=0

AP_dim-Cer5 AP_dim-Cer10 AP_dim-Cer20 AP_ dim-Cer5-d12 AP_ dim-Cer10-d12 AP_ dim-Cer20-d12

d = (2hπ )/Q h

(2)

Here, h is the Miller index of the h00 lamellar reflections or the Bragg peak order. Scattering vector Q correlates with scattering angle 2θ of the incoming neutrons by Q = 4π sin θ /λ . The absolute values of structure factor Fh were determined by

Fh =

hAhIh

(3)

with h as the Lorentz factor, Ah as the absorption correction, and Ih as the integrated intensity of the hth-order Bragg peak. The thickness of the sample (7 μm) and a linear absorption coefficient of 6 cm−1 were used for the absorption correction. The performed contrast variation enabled the determination of the Fh phase signs. With a linear correlation between Fh and the D2O/H2O ratio, the received phase signs were proven by the slope of the linear fit (Figure S1). The phase signs have a positive (+) or negative (−) slope, which is correct for centrosymmetric bilayers.42 The calculation of the neutron scattering length diffraction (NSLD) profiles ρs(x) was done by the Fourier transformation of Fh45

⎞ ⎛3 1 ⎞ dθ cos⎜ χ (3 cos2 θ − 1)SCDit ⎟ sin θ ⎟ ⎝4 2 ⎠ ⎠

⎞ exp(t LBi π )⎟ ⎠

designation

5% dim-Cer 10% dim-Cer 20% dim-Cer 5% dim-Cer-d12 10% dim-Cer-d12 20% dim-Cer-d12

were rotated in angular increments of 0.05° from Ω = 0 (membrane stack parallel to the incoming beam) to 12° (detector position at γ = 12.5°) for the first to the third diffraction order and from Ω = 8 to 20° (γ = 27°) for the fourth to the fifth diffraction order. To simulate in vivo conditions, a skin temperature of 32 °C was chosen for the experiments. A relative humidity (RH) of 98% was achieved through the vapor phase of a saturated solution of K2SO4 (Sigma-Aldrich, Steinheim, Germany) in several D2O/H2O mixing ratios. Each sample was measured at 8/92, 50/50, and 100/0% (mol/mol) D2O/H2O, referred to as contrast variation. The sample environment was constituted by lockable aluminum chambers, realizing constant measurement conditions. After each contrast change, the samples were equilibrated for at least 8 h in order to achieve the optimized level of hydration.15 The detector efficiency was corrected using a water calibration, and an empty aluminum chamber measurement was used as background. ILL software LAMP was used for the data reduction. Software package IGOR Pro, version 6.34A (WaveMetrics Inc., Portland, OR, USA) was used to determine the Bragg peak positions and Gaussian fitting of their intensities. From the position of equidistant Bragg peaks Qh, the distance d between the scattering planes was calculated, which is identical to the repeat distance of the stacked bilayers:

separately in chloroform/methanol (2:1 v/v), reaching a concentration of 10 mg/mL and mixed according to the final sample composition listed in Table 1. The airbrush technique was used to deposit 1.2 mL of the final lipid solution onto heated (80 °C) quartz wafers. The lipid membranes covered an area of 4.5 × 2.5 cm2. Storage under vacuum for at least 12 h guaranteed the total removal of residual solvent. Subsequently, annealing cycles were carried out involving three heating periods (85 °C for 90 min and two times for 30 min), each followed by a cooldown to room temperature (30 min, two times for 15 min). This procedure is known to result in a higher state of lamellar lipid order.42,43 A higher signal-to-noise ratio was gained by a subsequent carbonate buffer treatment.39 2 H NMR Spectroscopy. Static solid-state NMR measurements were carried out using a Bruker Avance 750 WB NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) operating at a deuterium resonance frequency of 115.1 MHz. A solids probe equipped with a 5 mm solenoid coil was used. In all experiments, we applied a spectral width of ±250 kHz, quadrature detection, and a phase-cycled quadrupolar echo sequence with two π/2 pulses of 2 to 2.7 μs length. The interpulse delay was 30 μs, and a repetition time of 50 s between successive scans was chosen to allow for full relaxation of the excited coherences. Such conditions are required for the quantification of the 2 H NMR spectra.10 All samples were measured at three temperatures: skin temperature of 32 °C and elevated temperatures of 50 and 70 °C. Acquired spectra were processed and analyzed using a self-written Mathcad (MathSoft, Cambridge, MA) script, the details of which are published elsewhere.44 2 H NMR spectra were simulated using a superposition of N 2H NMR Pake doublets, scaled by the appropriate order parameter (SCD) and whenever necessary an isotropic line. The rigid limit quadrupolar coupling was 125.25 kHz, assuming a quadrupolar coupling constant of χ = 167 kHz for the C−2H bond. Time domain data were simulated as free induction decays (FID) with an increment of the powder angle θ of 0.0625° according to FID(t ) =

+ + + + + +

(1)

In eq 1, LB is the line-broadening factor and θ is the angle for powder averaging. All NMR spectra were simulated using programs written in MathCad. The relative proportions of the individual phases observed in the 2H NMR spectra were directly determined from these fits. As 2 H NMR spectra were acquired at a relaxation delay of 50 s to account for the long T1 relaxation times (up to 10 s) of the crystalline phase,10 the relative contributions of each phase were determined from the relative proportion of each individual line shape for each phase that gave rise to the final NMR spectrum of the respective sample. Neutron Diffraction Experiments. For the collection of the neutron diffraction data, the D16 diffractometer at Institut LaueLangevin (ILL, Grenoble, France) was used. The highly ordered pyrolytic graphite monochromator selected a neutron wavelength of λ = 4.5 Å. The distance between the samples and the position-sensitive two-dimensional 3He detector (area = 320 × 320 mm2, spatial resolution = 1 × 1 mm2) was 955 mm. The reflection mode was used for the measurements. Sample alignment was performed by goniometers. During the sample rocking scans (Ω scans), the samples

ρs (x) = a + b

2 d

hmax

⎛ 2πhx ⎞ ⎟ d ⎠

∑ Fh cos⎜⎝ h=1

(4)

with a and b as constants for the relative normalization of ρs(x). Using the structure factors of the deuterated samples Fh_deut and those from the protonated control sample Fh_prot, NSLD profiles ρdeut(x) and ρprot(x) were calculated. The deuterium difference ρdiff(x) was calculated by

ρdiff (x) = ρdeut (x) − ρprot (x)

(5)

The maxima in ρdiff(x) indicate the position of the deuterated segments in the lipid bilayer. Preparation of Model SC Membranes for Permeability Experiments. The lipid mixtures (Cer[AP18]/SA/Chol/Chol-S and Cer[AP18]/SA/Chol/Chol-S + 20 wt % dim-Cer) were dissolved in hexane/96% ethanol 2:1 (v/v) to a concentration of 4.5 mg/mL. 9213


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Figure 2. 2H NMR spectra of Cer[AP18]-d35 (left column), SA-d35 (middle column), and Chol-d6 (right column) of the Cer[AP18]/SA/Chol/ Chol-S mixture (50 wt % buffer at pH 5.4) at temperatures of 32 °C (bottom line), 50 °C (middle line), and 70 °C (top line). Red lines indicate the best-fit numerical simulations of the 2H NMR spectra. All 2H NMR spectra were acquired at a relaxation delay of 50 s. Then, the lipid solutions/suspensions (3 × 100 μL, 1.35 mg/cm2) were sprayed onto Nuclepore polycarbonate filters with 15 nm porosity (Whatman, Kent, U.K.) under nitrogen using a Linomat V (Camag, Muttenz, Switzerland) equipped with additional y-axis movement.46 The prepared lipid membranes were dried in vacuum over P4O10 and solid paraffin. The day before the permeation experiments, the lipid membranes were annealed at 90 °C for 10 min and then slowly (3 to 4 h) cooled to 32 °C. During this process, a lamellar structure was created. Afterward, the membranes were equilibrated at 32 °C and 45 ± 5% relative humidity for at least 24 h. The procedure of model SC membranes preparation is carefully described in the Supporting Information. Membrane Permeability Measurements. The membranes were sandwiched between Teflon holders with an available diffusion area of 0.5 cm2 and mounted in the Franz diffusion cells (7.5 ± 0.2 mL) with the lipid film facing the upper (donor) compartment. The bottom (acceptor) compartment was filled with PBS at pH 7.4 containing 50 mg/L gentamicin. After a 12 h equilibration at 32 °C, the water loss through the membrane (TEWL) and electrical impedance were measured (Supporting Information). Then, membranes received 100 μL of either 5% theophylline (TH) or 2% indomethacin (IND) in 60% propylene glycol, and the cells were stirred at 32 °C. Samples of the acceptor phase (300 μL) were withdrawn every 2 h over 8 h and were replaced by the same volume of PBS. During this period, a steady-state situation was reached (Supporting Information).38 Skin-Permeability Measurements. The skin-permeation experiments were performed using Franz diffusion cells with an available diffusion area of 1 cm2. The skin fragments were slowly thawed immediately before use, cut into squares of approximately 2 × 2 cm2,

and mounted into the diffusion cells. The acceptor compartments of the cells (16.5 ± 1.5 mL) were filled with PBS. The diffusion cells were stirred at 32 °C. After a 12 h equilibration, TEWL and electrical impedance were measured (see below).47 Afterward, 100 μL of a 1% suspension of dim-Cer in propylene glycol/ethanol 7:3 (v/v) or 100 μL of the vehicle solvent without lipids was applied to the skin. The diffusion cells were incubated at 32 °C. After 14 h, the donor samples were carefully removed using cotton swabs; the skin surface was rinsed with PBS and dried. After 2 h, TEWL and electrical impedance were measured (data are presented as a fold change after/before treatment).

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RESULTS AND DISCUSSION Thermotropic Phase Behavior of the SC Model System in the Absence of Dimeric Ceramide. As a reference, we first looked at the 2H NMR spectra of the mixture containing Cer[AP18]/SA/Chol/Chol-S with one lipid species deuterated, respectively, which are shown in Figure 2. Each column displays the 2H NMR spectra of the mixture with one deuterated component in the sample, and each row shows the NMR spectra at one of three temperatures. At a physiological skin temperature of 32 °C, the 2H NMR spectra of Cer[AP18] and SA are dominated by a broad Pake spectrum with a quadrupolar splitting of about 120 kHz that arises from the all-trans methylene groups and a smaller spectral component with a quadrupolar splitting of 40 kHz, which is ascribed to the terminal methyl groups of the chains. The NMR spectrum of the deuterated Chol shows a major 9214


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Table 2. Quantitative Analysis of the Phase Composition of the Cer[AP18]/SA/Chol/Chol-S Mixture As Derived from Numerical Simulation of the 2H NMR Spectra Shown in Figure 2 Cer[AP18]

stearic acid

cholesterol

temperature

cryst

fluid

isotr

cryst

fluid

isotr

cryst

fluid

isotr

32 °C 50 °C 70 °C

70% 58% 13%

30% 42% 86%

− − 1%

67% 62% 32%

33% 38% 68%

− − −

49% 27% −

51% 73% 100%

− − −

contribution from the rigid Pake spectrum with 120 kHz quadrupolar splitting. Such 2H NMR spectra are indicative of a solid-phase state that has similarly been found for several other SC lipid mixtures.6,7,10−14 In addition, the fractions of all components also form a more fluid phase as indicated by smaller spectral features superimposed on the broad 2H NMR spectra stemming from the solid phase. As described in the Materials and Methods section, we were able to analyze all 2H NMR spectra in a quantitative manner; i.e., we could calculate the relative contribution of one of the deuterated molecules in the samples to any of the observed phases from the line shape simulation indicated by the red lines in all NMR spectra in Figure 2. This analysis reveals that 70% of the Cer[AP18], 67% of the SA, and 49% of the Chol form a rigid orthorhombic phase at skin temperature (Table 2). The 2H NMR spectra also demonstrate that some of the lipids are found in a fluid phase indicated by narrow spectral components. For the Cer[AP18] and the SA, about 30 and 33% of the molecules form this fluid phase, respectively. The 2H NMR spectrum of Chol-d 6 shows very well resolved quadrupolar splittings that agree with the bilayer-like behavior of the molecule and rotational diffusion about the long axis as well described in the literature.46 According to our line-shape simulations, 51% of the cholesterol forms the fluid phase. All proportions of the solid and fluid phases for each component of the mixture at the corresponding temperatures are listed in Table 2. Raising the temperature to 50 °C increases the fluid and decreases the solid phase proportions for all investigated lipid components. A total of 42% of Cer[AP18] and 38% of the SA are fluid under these conditions. The 2H NMR spectra of Cer[AP18] and SA also display the typical superposition of Pake spectra with varying quadrupolar splittings that are indicative of a lamellar liquid-crystalline phase state as known from phospholipid membranes. Such spectra can be used to calculate order parameter profiles along the chain as presented in Figure 3. Order parameter profiles could be derived from the 2 H NMR spectra of Cer[AP18] and SA, respectively. In agreement with previous work, they feature an order gradient along the lipid chain. Similar order parameter profiles indicate that Cer[AP18] and SA mix relatively well at 50 °C.44 Order parameters of the upper chain segments are relatively high (>0.4), which demonstrates that a significant amount of cholesterol must be present in that phase as well to condense the chains. Indeed, the 2H NMR spectrum of the Chol component is dominated by spectral features that agree with mobile Chol,48 which accounts for 73% of the spectral intensity. At 70 °C, all components of the mixture show a preference for the liquid-crystalline phase. Only about 13% of Cer[AP18] and 30% of SA remain to be crystalline, while virtually all cholesterol is found in the fluid phase. Although higher temperature would normally decrease the chain-order parameters, the larger relative Chol content induces more condensation of the lipid chains and the order parameters at

Figure 3. 2H NMR order parameters of Cer[AP18]-d35 (squares) and SA-d35 (triangles) in the Cer[AP18]/SA/Chol/Chol-S SC model system in the absence of dim-Cer at 50 °C (black) and 70 °C (red) calculated from best-fit simulations of the 2H NMR spectra shown in Figure 2.

70 °C show only marginal differences. As opposed to other ceramide mixtures,6,7 the Cer[AP18]/SA/Chol/Chol-S system shows no preference of an isotropic phase. We observe an interesting phenomenon when comparing the order parameters of Cer[AP18] and SA at 50 and 70 °C. Although the upper part of the lipid chains has lower order parameters at higher temperature as expected, the lower half of the chain is more ordered at higher temperature. This effect is likely related to the redistribution of the cholesterol component of the mixture. Although all cholesterol partitions into the fluid phase at 70 °C, the cholesterol content in the fluid phase is lower at 50 °C, which leads to alterations of the chain order parameter profiles. Recent work highlighted the influence of the cholesterol side chain on the lipid chain order,49 which may modify the order parameters of the ceramide-containing mixtures in the lower half of the chains. Order parameter profiles determined for fluid membranes contain information about the length of the lipid chains in the respective preparation.50−52 From the order parameter profiles shown in Figure 3, chain lengths of the respective lipids can be determined, which are 16.1 and 15.8 Å for Cer[AP18] and SA at 50 °C, respectively. At 70 °C, these values increase a bit to 16.5 and 15.9 Å, respectively. This indicates that the fluid portions of Cer[AP18] and SA are well mixed. Thermotropic Phase Behavior of the SC Model System in the Presence of Dimeric Ceramide. Figure 4 presents the 2H NMR spectra of the Cer[AP]/SA/Chol/CholS mixture in the presence of 20 wt % dim-Cer. In the figure, an additional column is added showing the 2H NMR spectra of the partially deuterated dim-Cer ceramide at the three different temperatures. Two conclusions can be derived from the 2H 9215


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Figure 4. 2H NMR spectra of lipid mixture Cer[AP18]/SA/Chol/Chol-S in the presence of a 20 wt % dim-Cer mixture at a hydration level of 50 wt % buffer at pH 5.4. 2H NMR measurements were carried out at temperatures of 32, 50, and 70 °C. Each column depicts the acquired NMR spectra for a single deuterated component in the mixture (black). Red lines show the best numerically calculated fit of the spectral line shape. For all 2H NMR spectra, a relaxation delay of 50 s was applied.

Table 3. Quantitative Analysis of the Phase Composition of the Cer[AP18]/SA/Chol/Chol-S Mixture in the Presence of 20 wt % dim-Cer as Derived from Numerical Simulation of the 2H NMR Spectra Shown in Figure 4 dim-Cer

Cer[AP18]

stearic acid

cholesterol

temperature

cryst

fluid

isotr

cryst

fluid

isotr

cryst

fluid

isotr

cryst

fluid

isotr

32 °C 50 °C 70 °C

96% 94% 92%

4% 5% 6%

− 1% 2%

72% 63% 16%

28% 37% 81%

− − 3%

57% 65% 37%

43% 35% 63%

− − −

24% − −

76% 100% 100%

− − −

packing in the orthorhombic rigid phase formed by dim-Cer and the majority of Cer[AP18] and SA. However, this redistribution has only a small effect on the order parameters of the Cer[AP18] and SA forming a fluid phase as reported in Figure 5. Because virtually all dim-Cer is found in the rigid phase, the order parameters of the lipids in the fluid phase are not influenced, as seen by comparison between Figures 3 and 5. At 70 °C, the lipid mixture is very comparable to the mixture prepared in the absence of dim-Cer with very similar proportions of Cer[AP18], SA, and Chol in the rigid and fluid phases, respectively. All of the dim-Cer is found in the rigid phase along with 16% of the Cer[AP18] and 37% of SA. The rigid phase is again totally depleted of cholesterol. The fluid phase of the mixture shows very similar order parameters as observed in the absence of dim-Cer as reported in Figure 5. With respect to the numbers for the chain length, at 50 °C, Cer[AP18] and SA both feature a chain length of 15.7 Å, and at 70 °C, these values are a slightly different, 16.5 Å for Cer[AP18] and 15.7 Å for SA, indicating some nonideal mixing of the two lipids at elevated temperature. Lipid Organization in the SC Lipid Model System in the Presence of Dimeric Ceramide. Neutron diffraction experiments are particularly well suited to characterizing the

NMR spectra of all components of the mixture. (i) dim-Cer remains in a rigid conformation at all temperatures investigated, indicative of an all-trans conformation of the bilayer-spanning chain even at 70 °C where most of the other components of the mixture are fluid. (ii) The presence of bilayer-spanning dim-Cer imposes only very small alterations on the thermotropic phase behavior of the mixture. In more detail, at a physiological skin temperature of 32 °C, similar proportions of Cer[AP18] and SA are found in the orthorhombic phase (72 and 57%, respectively; see Table 3) as in the mixture prepared without dim-Cer. However, the amount of crystalline cholesterol is reduced from 49% in the absence to 24% in the presence of dim-Cer. Obviously, the long bilayer-spanning chain of dim-Cer induces a restructuring of the rigid phase, which expels all molecules that perturb this rigid packing, which interestingly is the cholesterol. In spite of the chain-length mismatch,11 Cer[AP18] and SA appear to fit the rigid phase packing of dim-Cer very well. At 50 °C, the relative proportions of Cer[AP18] and SA in the fluid phase are again very similar compared to the mixture in the absence of dim-Cer. Again, a notable exception is the cholesterol, which is found only in the fluid phase at 50 °C. Obviously, cholesterol is very incompatible with the rigid 9216


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values according to eq 3, the neutron scattering length diffraction (NSLD) profiles ρs(x) for all D2O/H2O ratios across the unit cell were obtained (reproduced in Figure S3). The profiles are nearly congruent in the middle of the unit cell, where the lipid alkyl chains are localized. Only at the edges, where the lipid headgroups are localized, are significant differences observed. Because of the water distribution function, we can conclude that there is no indication of the presence of water in the aliphatic alkyl chain regions; water is exclusively found in the polar headgroup regions. Boundary xHH between the hydrophobic and hydrophilic unit cell areas is localized at x = ±16.3 Å (Figure S3). Thus, the hydrophobic thickness of the bilayer is about 32.5 Å, and each polar region covers a thickness of 6.9 Å in the presence of 5 wt % dim-Cer. In the SC lipid model system in the absence of dim-Cer, we determined a hydrophobic−hydrophilic boundary xHH of 16.3 Å,39 which is identical to the results described here. Furthermore, the hydrophobic part of the bilayer is comparable as well and has been determined to be 32.6 Å in the absence of dim-Cer. The higher d spacing detected for the dim-Cer-containing mixture translates to a larger polar region increased by about 1.2 Å on either side. This is quite interesting because the dimeric ceramide increases the unit cell scale only by stretching the polar membrane areas. Localization of the Central Segments of the Dimeric Ceramide in the SC Lipid Model System. The specific deuteration of the dim-Cer-d12 variant enables the localization of the deuterated segments within the SC lipid model system. From the H/D contrast differences, the NSLD difference profiles ρdiff(x) was calculated for the dim-Cer containing SC mixtures and is displayed in Figure 6. Alterations in the values of ρdeut(x) and ρprot(x) indicate the putative location of the six deuterated methylene segments of dim-Cer-d12. The most prominent alterations in the two NSLD profiles are found in the center of the bilayer between ±7.0 Å. Because of truncation artifacts caused by the relatively small number of Bragg peaks, an oscillation on the NSLD profiles is observed, and the interpretation of these local maxima/minima should be done with caution especially because the differences in the NSLD profiles are small. This particularly concerns the regions

Figure 5. 2H NMR order parameters of Cer[AP18]-d35 (squares) and SA-d35 (triangles) in the Cer[AP18]/SA/Chol/Chol-S SC model system in the presence of 20 wt % dim-Cer at 50 °C (black) and 70 °C (red) calculated from best-fit simulations of the 2H NMR spectra shown in Figure 4.

atomistic details of rigid lipid phases. Our measurements, showing five lamellar diffraction orders, revealed a single ordered phase in the system. Figure S2 displays the typical Bragg peak pattern of sample AP_dim-Cer5-d12. Using eq 2, the d spacing for all samples was calculated from the diffraction data. Compared to a sample without dim-Cer,39 membranes containing dim-Cer show a distinctly increased d spacing. In the absence of dim-Cer, a d spacing of 43.9 Å was reported,39 which increases to 46.3, 46.8, and 47.7 Å in the presence of 5, 10, and 20 wt % dim-Cer, respectively, indicating that dim-Cer is part of the SC lipid model membranes and likely increases the membrane thickness of the rigid orthorhombic domains of the lipid membranes. The phase signs of all samples were − + − + − for the first to the fifth Bragg peak orders. They were determined by a linear fit of the correlation between Fh and the several D2O/H2O ratios (Figure S1). By Fourier transformation of the absolute Fh

Figure 6. NSLD profiles ρs(x) of sample AP_dim-Cer5 (thin dotted line) and sample AP_dim-Cer5-d12 (thin dashed line) at 50% D2O, 98% RH, and 32 °C. The solid black line represents the deuterium difference ρdiff(x), indicating the position of deuterium labels. The red curve represents a hypothetical distribution function of the central deuterated segments of dim-Cer. 9217


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Figure 7. Structural model of the lipid mixture based on Cer[AP18], Chol, SA, Chol-S, and dim-Cer. The gray areas represent a rough interface region, where few water molecules are bound to the lipid headgroups only, likely avoiding contact with the aliphatic chain segments that may also be localized in this region.

outside the center (±7.0 Å). To guide the eye, we have drawn a hypothetical distribution function for the deuterated segments. Clearly, the data suggests that the long lipid alkyl chain is stretched over the complete unit cell. Assuming 1.27 Å per an all-trans C−C bond,53 a total length of 40.6 Å for the C32 chain can be estimated. The data suggests that the central region between ±7.0 Å indicates the most probable localization of the deuterated segments, allowing us to conclude that one polar headgroup of the dim-Cer molecule may protrude into the polar region of the opposing bilayer. This could possibly explain the local minima in the NSLD difference profile at ±18 Å, which could be caused by the depletion of the aliphatic region by the ceramide headgroups. Such a scenario would impose packing differences, which could be responsible for the segregation of cholesterol from the orthorhombic phase observed in the 2H NMR data. Nevertheless, such an arrangement is surprising because it suggests that dim-Cer is not able to incorporate completely and symmetrically into a single bilayer. Although dim-Cer is increasing the unit cell dimension compared to a lipid model membrane in the absence of dim-Cer, the increase in bilayer thickness does not lead to a perfect length match of all molecules in the mixture. This could be caused by the presence of Cer[AP18], which seems to impose a stronger influence on the membrane-assembling process than does dim-Cer. The confidence levels of the NSLD

profile of the deuterated sample are not overlapping with the NSLD profile of the protonated sample in the areas we have assigned to the positions of the deuterated methylene segments of dim-Cer. The impact of dim-Cer on the nanostructure of the SC lipid model system has been observed in penetration studies, which revealed an enrichment of dim-Cer within the native SC.54 A model illustrating the arrangement of the lipids in the mixture is shown in Figure 7. The novel dim-Cer stretches over one unit cell and protrudes into the opposing bilayer. This position would explain the stabilizing effect of dim-Cer on the SC barrier function. Because of the comprehensive bilayer interlocking, the penetration of exogenous substances through the SC would be more difficult. Thus, the application of dimCer in cutaneous formulations would enable proper barrier properties of diseased skin. Effect of dim-Cer on the Permeability of Cer[AP18] Model Membranes. We tested the effect of dim-Cer on membrane permeability using several assays and systems. First, we prepared model membranes composed of Cer[AP18]/SA/ Chol/Chol-S in the absence (controls) and in the presence of 20 wt % dim-Cer. The membrane permeability was evaluated using four parameters: transepidermal water loss (TEWL); electrical impedance; the flux of theophylline (TH), a small 9218


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Figure 8. Effects of dim-Cer on the membrane and skin permeability. Permeabilities of the studied model membranes composed of Cer[AP18]/SA/ Chol/Chol-S (control, white) or Cer[AP18]/SA/Chol/Chol-S/dim-Cer (black). (A) Water loss through the membrane (TEWL). (B) Electrical impedance of the membrane. (C) Permeation profile for TH. (D) Flux value for TH. (E) Permeation profile for IND. (F) Flux value for IND. Permeability of human skin: the fold change in the TEWL (G) and electrical impedance (H), induced by topical dim-Cer (red bar) or the control (white bar). Data are presented as the mean ± SEM, n = 5. The asterisks show statistically significant differences compared to the control (p < 0.05).

molecule with balanced lipophilicity; and the flux of indomethacin (IND), a large lipophilic molecule. A summary of the results is presented in Figure 8. The value of the water loss of the control membranes reached 10.6 ± 0.9 g/h/m2, compared to those based on Cer[AP18] with the addition of dim-Cer, i.e., 11.9 ± 1.1 g/h/m2. The addition of dim-Cer to model membranes slighly increased the electrical impedance to 20 ± 3 kΩ × cm2, and the electrical impedance of control membranes was 14 ± 1 kΩ × cm2. The flux of TH through the control membrane was 0.7 ± 0.1 μg/cm2/h. The addition of dim-Cer significantly decreased the membrane permeability (0.2 ± 0.1 μg/cm2/h). The permeability to IND of the membranes with dim-Cer decreased over that of the control. The permeation profiles for both TH and IND are shown in Figure 8C,E. Permeability of Human Skin after Topical Application of dim-Cer. The permeability results from model membranes were verified using human skin (Figure 8G,H). The topical application of dim-Cer in propylene glycol/ethanol 7:3 (v/v) increased the TEWL almost 2.5-fold (from 15.1 ± 2.8 g/h/m2 before treatment to 38.6 ± 2.0 g/h/m2), a similar effect to that of the control (Figure 8G). Thus, topical application of dimCer on intact human skin partially disturbs the permeability barrier to water. The electrical impedance after the dim-Cer application (Figure 8H) confirmed a partial barrier impairment.

artificial ceramide-like molecules that are able to stabilize the skin barrier may represent interesting therapeutic approaches. We have investigated the influence of an artificial dimeric ceramide (dim-Cer) on the structure and thermotropic phase behavior of a typical SC lipid model system. Our results indeed show that the dim-Cer molecule can form a very rigid orthorhombic phase along with Cer[AP18] and SA as desired. Even at elevated temperature, this rigid phase persists. Only cholesterol does not seem to be compatible with this rigid orthorhombic phase state. The neutron diffraction studies reveal an incorporation of dim-Cer into the applied SC lipid model system. It increases the lamellar repeat distance because of its immense chain length. The stabilizing effect of the lipid variant in the SC barrier is visible by its interlocking arrangement beyond one unit cell, extending into the next one. This indeed suggests that dim-Cer carries the potential to stabilize the SC, and whether it is also used to treat skin diseases and the question of how to apply it in medical formulations remains subjects for further studies. First permeability measurements demonstrate that TEWL is slightly increased in the presence of dim-Cer, but the membrane permeability of TH and IND is decreased in the presence of dim-Cer. This indicates that the molecular structure of dim-Cer needs to be further optimized to induce the desired effect of restoring the barrier function of human skin.

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CONCLUSIONS The synthesis of new ceramide molecules potentially stabilizing SC lipid layers, particularly in skin diseases that are characterized by the loss of ceramide concentration in the SC, involves the topical application of these molecules to the skin in order to strengthen and recover the barrier function of the diseased skin. The ceramides are very important constituents that reduce the permeability of the SC by inducing a very dense and rigid packing of all lipids. In clinical situations, where a loss in this lipid component of the SC is encountered,

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b01227. Normalized Fh values of the samples. Correlation between absolute Fh and the D2O/H2O ratio. Neutron diffraction pattern of sample AP_ dim-Cer5-d12. NSLD profiles of D2O. Linear fit of the water distribution 9219


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(10) Kitson, N.; Thewalt, J.; Lafleur, M.; Bloom, M. A model membrane approach to the epidermal permeability barrier. Biochemistry 1994, 33, 6707−6715. (11) Paz Ramos, A.; Lafleur, M. Chain length of free fatty acids influences the phase behavior of stratum corneum model membranes. Langmuir 2015, 31, 11621−11629. (12) Brief, E.; Kwak, S.; Cheng, J. T.; Kitson, N.; Thewalt, J.; Lafleur, M. Phase behavior of an equimolar mixture of N-palmitoyl-D-erythrosphingosine, cholesterol, and palmitic acid, a mixture with optimized hydrophobic matching. Langmuir 2009, 25, 7523−7532. (13) Ouimet, J.; Lafleur, M. Hydrophobic match between cholesterol and saturated fatty acid is required for the formation of lamellar liquid ordered phases. Langmuir 2004, 20, 7474−7481. (14) Chen, X.; Kwak, S.; Lafleur, M.; Bloom, M.; Kitson, N.; Thewalt, J. Fatty acids influence ″solid″ phase formation in models of stratum corneum intercellular membranes. Langmuir 2007, 23, 5548− 5556. (15) Engelbrecht, T. N.; Schröter, A.; Hauss, T.; Deme, B.; Scheidt, H. A.; Huster, D.; Neubert, R. H. The impact of ceramides NP and AP on the nanostructure of stratum corneum lipid bilayer. Part I: neutron diffraction and 2H NMR studies on multilamellar models based on ceramides with symmetric alkyl chain length distribution. Soft Matter 2012, 8, 6599−6607. (16) Mendelsohn, R.; Selevany, I.; Moore, D. J.; Mack Correa, M. C.; Mao, G.; Walters, R. M.; Flach, C. R. Kinetic evidence suggests spinodal phase separation in stratum corneum models by IR spectroscopy. J. Phys. Chem. B 2014, 118, 4378−4387. (17) Mendelsohn, R.; Rabie, E.; Walters, R. M.; Flach, C. R. Fatty acid chain length dependence of phase separation kinetics in stratum corneum models by IR spectroscopy. J. Phys. Chem. B 2015, 119, 9740−9750. (18) Oguri, M.; Gooris, G. S.; Bito, K.; Bouwstra, J. A. The effect of the chain length distribution of free fatty acids on the mixing properties of stratum corneum model membranes. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 1851−1861. (19) Groen, D.; Poole, D. S.; Gooris, G. S.; Bouwstra, J. A. Is an orthorhombic lateral packing and a proper lamellar organization important for the skin barrier function? Biochim. Biophys. Acta, Biomembr. 2011, 1808, 1529−1537. (20) Mojumdar, E. H.; Gooris, G. S.; Barlow, D. J.; Lawrence, M. J.; Deme, B.; Bouwstra, J. A. Skin lipids: localization of ceramide and fatty acid in the unit cell of the long periodicity phase. Biophys. J. 2015, 108, 2670−2679. (21) Rowat, A. C.; Kitson, N.; Thewalt, J. L. Interactions of oleic acid and model stratum corneum membranes as seen by 2H NMR. Int. J. Pharm. 2006, 307, 225−231. (22) Rerek, M. E.; Chen, H. C.; Markovic, B.; van Wyck, D.; Garidel, P.; Mendelsohn, R.; Moore, D. J. Phytosphingosine and sphingosine ceramide headgroup hydrogen bonding: Structural insights through thermotropic hydrogen/deuterium exchange. J. Phys. Chem. B 2001, 105, 9355−9362. (23) Moore, D. J.; Rerek, M. E.; Mendelsohn, R. FTIR spectroscopy studies of the conformational order and phase behavior of ceramides. J. Phys. Chem. B 1997, 101, 8933−8940. (24) Pham, Q. D.; Topgaard, D.; Sparr, E. Tracking solvents in the skin through atomically resolved measurements of molecular mobility in intact stratum corneum. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E112−E121. (25) Mieremet, A.; Rietveld, M.; Absalah, S.; van, S. J.; Bouwstra, J. A.; El, G. A. Improved epidermal barrier formation in human skin models by chitosan modulated dermal matrices. PLoS One 2017, 12, e0174478. (26) Cho, Y.; Lew, B. L.; Seong, K.; Kim, N. I. An inverse relationship between ceramide synthesis and clinical severity in patients with psoriasis. J. Korean Med. Sci. 2004, 19, 859−863. (27) Berardesca, E.; Barbareschi, M.; Veraldi, S.; Pimpinelli, N. Evaluation of efficacy of a skin lipid mixture in patients with irritant contact dermatitis, allergic contact dermatitis or atopic dermatitis: a multicenter study. Contact Dermatitis 2001, 45, 280−285.

function’s gain of sample AP_dim-Cer5-d12. Preparation of model SC membranes. Human skin. Membrane permeability. Transepidermal/transmembrane water loss. Electrical impedance. High-performance liquid chromatography. Data treatment. (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49 (0) 341 97-15701. Fax: +49 (0)341 97-15709. ORCID

Daniel Huster: 0000-0002-3273-0943 Author Contributions ∇

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The study was supported by the Deutsche Forschungsgemeinschaft (DFG HU 720/13-1, NE 427/30-1, and DO 463/6-1). A.K. is grateful for a stipend from the Czech Science Foundation (16-25687J) and Charles University (SVV 260 401). We thank the scientists supporting the operation of instrument D16 at the ILL (Grenoble, France) for their assistance and the allocation of beam time for the neutron diffraction measurements.

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