Monounsaturated Fatty Acids Reduce the Barrier of ... - ACS Publications

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Monounsaturated Fatty Acids Reduce the Barrier of Stratum Corneum Lipid Membranes by Enhancing the Formation of a Hexagonal Lateral Packing Enamul H. Mojumdar, Richard W. J. Helder, Gert S. Gooris, and Joke A. Bouwstra* Leiden Academic Center for Drug Research, Department of Drug Delivery Technology, Gorlaeus Laboratories, University of Leiden, 2333 CC Leiden, The Netherlands ABSTRACT: The effectiveness of the skin barrier underlies the outer layer of the skin: the stratum corneum (SC). However, in several skin diseases this barrier is impaired. In two inflammatory skin diseases, atopic eczema and Netherton syndrome, an increased level of monounsaturated fatty acids (MUFAs) has been observed as opposed to healthy skin. In the present study, we aimed to investigate the effect of MUFAs on the lipid organization and skin lipid barrier using an in vitro model membrane system, the stratum corneum substitute (SCS), mimicking the SC lipid composition and organization. To achieve our goal, the SCS has been prepared with increasing levels of MUFAs using various chain length. Permeation studies and trans-epidermal water loss measurements show that an increment of MUFAs reduces the lipid barrier in the SCS. The increased level of unsaturation exerts its effect by reducing the packing density in the lipid organization, while the lamellar phases are not affected. Our findings indicate that increased levels of MUFAs may contribute to the impaired skin barrier in diseased skin. hexagonal packing.14,16 Previous studies demonstrated that both the lamellar phases as well as the lateral packing are important in maintaining a proper skin barrier function.17−20 In this work our aim is to examine whether an increase in monounsaturated fatty acids (MUFAs) in the lipid mixture induces changes in the lipid organization and therefore impairs the skin lipid barrier. Focusing on the CER subclasses, the presence of an abundant level of CERs in a large variety of molecular structures is very characteristic for the SC lipid composition.16 The CERs are composed of an acyl chain linked to a sphingoid base through an amide linkage. Due to a variation in headgroup architecture, 12 subclasses of CERs have been identified in human SC so far.4,7,9,21 Each CER subclass shows a broad variation in acyl chain length, with some subclasses of CERs showing an exceptionally long ω-hydroxy fatty acid chain ester linked to linoleic acid, referred to as CER EO. As the LPP is important for a proper skin barrier function, the CER EOs are considered to play an important role to the skin barrier function.17,22 Diseased skin as well as dry skin are often characterized by an impaired barrier function. In several skin diseases (lamellar ichthyosis, psoriasis, Netherton syndrome, atopic dermatitis, etc.) it has been shown that the skin barrier function is significantly reduced.23−28 Besides other factors that play a role

1. INTRODUCTION The skin protects us from the environment and thus from allergens, pathogens, and microorganisms by providing an essential barrier. It also prevents the body from desiccation caused by an excessive trans-epidermal water loss. The outermost layer of the skin, the stratum corneum (SC), plays a crucial role in the skin barrier function.1 The SC consists of dead flattened cells, corneocytes, filled with keratin and water. In between the corneocytes in the intercellular space lipids adopt a highly organized matrix. As the corneocytes are almost impermeable to compounds due to the densely cross-linked protein envelope surrounding these cells, this lipid matrix serves as an important penetration pathway for compounds through the SC.2,3 The lipid matrix consists of mainly ceramides (CERs), cholesterol (CHOL), and free fatty acids (FFAs) in an approximately equimolar ratio.4−9 Traces of cholesterol sulfate but also cholesterol esters and other lipids are present as well. Together these lipids form the building blocks for a proper lipid organization in the SC: the lipids form two lamellar phases with repeat distances of approximately 13 and 6 nm, referred to as the long periodicity phase (LPP) and the short periodicity phase (SPP), respectively.10−13 Within the structure of the lamellae, the lipids are organized in the so-called lateral packing. Depending on the lipid composition, the lateral packing can be orthorhombic (highly dense packing), hexagonal (less dense packing), or liquid (loose packing).10,14,15 At skin temperature (∼32 °C), in human SC, the lipids adopt an orthorhombic packing with a small subpopulation of lipids forming a © 2014 American Chemical Society

Received: March 12, 2014 Revised: May 5, 2014 Published: May 12, 2014 6534

dx.doi.org/10.1021/la500972w | Langmuir 2014, 30, 6534−6543

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in these diseases, the lipid composition in the SC often deviates from that in SC of healthy skin. This change in lipid composition may be one of the underlying factors responsible for a reduced skin barrier function. One of the important changes in lipid composition is a reduction in the level of CER EO subclasses.23,29−32 As reported in literature in (pre)clinical studies, the level of CER EO subclasses may contribute to the formation of the LPP and consequently be a factor for a proper skin barrier.17,30 Previously, studies performed using a synthetic model lipid membrane, referred to as the stratum corneum substitute (SCS),33 showed that a drop in the level of CER EO reduced the formation of LPP and subsequently impaired the SC lipid barrier.22 Apart from the CER subclasses, the FFA subclasses also play an important role in the skin barrier function. Recently, it has been shown that in atopic dermatitis and Netherton syndrome there is a significant increase in the level of MUFAs.34,35 In Netherton syndrome patients, the level of MUFAs can approach around 80−90% of the total level of FFAs.34 In previous studies unsaturated fatty acids, such as oleic and linoleic acid, have been studied, as they were considered to be potent penetration enhancers.36,37 In our present study, we aimed to investigate the effect of MUFAs on the lipid barrier of SCS model membrane. The SCS is prepared from CERs, CHOL, and FFAs and mimics the lipid organization in human SC very closely. In the present study, saturated FFAs were replaced by MUFAs. The lamellar organization was examined by small-angle X-ray diffraction (SAXD), while the lateral packing was investigated by Fourier transform infrared spectroscopy (FTIR). Finally, the barrier functionality was tested by measuring the trans-epidermal water loss across the SCS and by performing permeation studies. Our studies show that the presence of MUFAs enhances the permeability of the SCS by inducing the formation of the hexagonal lateral packing and that a substantial level of MUFAs may be one of the underlying factors of a reduced skin barrier.

Figure 1. Molecular structure of the synthetic CER subclasses used in our current study. The CERs consists of an acyl chain that ranges from 16 to 30 carbon attached to a sphingoid base through an amide linkage. CER NP subclass is used with two different acyl chain lengths (C24 and C16).

2. EXPERIMENTAL SECTION 2.1. Materials. Five subclasses of synthetic CERs were used in our studies. These are (1) the ester-linked ω-hydroxyl acyl chain [abbreviation EO, 30 carbons in the acyl chain (C30)] with a sphingosine chain (abbreviation S, C18), referred to as CER EOS (C30); (2) a nonhydroxy acyl chain (abbreviation N, C24) linked to a sphingosine base (C18), referred to as CER NS (C24); (3) a nonhydroxy acyl chain (C24 or C16) linked to a phytosphingosine base (abbreviation P), referred to as CER NP (C24) and CER NP (C16), respectively; (4) an α-hydroxy chain (abbreviation A) linked to a sphingosine base, referred to as CER AS (C24); and (5) an α-hydroxy acyl chain (C24) linked to a phytosphingosine base referred to as CER AP (C24). The number in parentheses indicates the number of carbon atoms in the acyl chain of the CER. The molecular structure of the synthetic CERs are provided in Figure 1. All the CERs were generously provided by Evonik (Essen, Germany). Palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), eicosanoic acid (C20:1), behenic acid (C22:0), erucic acid (C22:1), tricosanoic acid (C23:0), lignoceric acid (C24:0), nervonic acid (C24:1), cerotic acid (C26:0), and CHOL were obtained from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). The drug molecule hydrocortisone was purchased from Larodan (Malmo, Sweden). The molecular structures of the unsaturated FFAs and the drug compound hydrocortisone are given in Figure 2. Nucleopore polycarbonate filter disks (pore size 50 nm) were obtained from Whatman (Kent, UK). All solvents were of analytical grade and supplied by Labscan (Dublin, Ireland). The water was of Millipore quality.

Figure 2. (A) The structure of the unsaturated FFAs (ω-9 FFAs) used in our study and their corresponding generic names are given. (B) The structure of the steroid drug molecule hydrocortisone used for permeation studies. 2.2. Preparation of the Model Lipid Mixtures. For the preparation of the SCS, synthetic CERs, CHOL, and FFAs were used in an equimolar ratio. The CER composition was CER EOS (C30), CER NS (C24), CER NP (C24), CER AS (C24), CER NP (C16), and CER AP (C24) in a 15:51:16:4:9:5 molar ratio. This specific mixture is referred to as the sCER mixture. Its composition is 6535


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based on a CER subclass composition of pig SC reported earlier.38 The FFA mixture was composed of C16:0, C18:0, C20:0, C22:0, C23:0, C24:0, and C26:0 at a molar ratio of 1.8:4.0:7.7:42.6:5.2:34.7:4.1. This FFA composition is based on that reported to be present in human SC.39 Besides this FFA mixture, four additional FFA mixtures were also used. In three of these FFA mixtures, one of the saturated FFAs was replaced by its unsaturated counterpart. That is, FFA with a chain length of C20 was replaced by C20:1 (FFA C20:1) or the FFA C22 by C22:1 (FFA C22:1) or the FFA C24 by C24:1 (FFA C24:1). The fourth FFA mixture was prepared by replacing the above-mentioned three saturated FFAs by their unsaturated counterparts (FFA3FA:1). In total, five different model lipid mixtures were prepared. The composition of these model mixtures with their corresponding unsaturated FFA molar ratios are provided in Table 1.

throughout the experiment. The acceptor phase was perfused at a flow rate of approximately 2 mL/h with a flow pump (Ismatec IPC pump; IDEX Health & Science GmbH, Germany) and the acceptor compartment was stirred with the magnetic stirrer at 160 rpm. A fraction collector (Isco Retriever IV; Teledyne Isco) was used to collect the acceptor fluid in 10 mL glass vials. The collection time interval was 1 h. The diffusion was measured over a period of 20 h. During the permeation studies, the temperature of the diffusion cells was controlled at a physiological skin temperature of approximately 32 °C by a thermostated water bath (thermostat from Lauda Dr. Wobser GmbH; Lauda-Koenigshofen, Germany). After the diffusion studies, the volume per collected fractions was determined by weighing. During the steady-state conditions, the permeation studies of hydrocortisone across the SCSs can be described by Fick’s first law of diffusion:

Jss =

Table 1. Various Model Lipid Mixtures with Their Composition and Molar Ratios Used in the Present Studya lipid model type SCSsat SCSC20:1 SCSC22:1 SCSC24:1 SCS3FA:1 a

composition and molar ratio (1.1.1) sCER/CHOL/FFA sCER/CHOL/FFA with FFA C20:0 replaced by FFA C20:1 sCER/CHOL/FFA with FFA C22:0 replaced by FFA C22:1 sCER/CHOL/FFA with FFA C24:0 replaced by FFA C24:1 sCER/CHOL/FFA with FFA C20:0, C22:0, and C24:0 replaced by C20:1, C22:1, and C24:1, respectively

KDCd h

Jss represents the steady-state flux of hydrocortisone across the SCS (μg/cm2/h), while D is the hydrocortisone diffusion coefficient in the SCS (cm2/h). K is the partition coefficient between donor compartment and the lipid membrane. Cd is the drug concentration in the donor compartment (μg/cm3), and h is the pathway length in the SCS (cm). The Jss values were calculated at a time interval between 5 and 15 h from the cumulative plot of the flux values of hydrocortisone. The slope of the linear part of the cumulative plot represents Jss. 2.5. HPLC Analysis. The analysis of hydrocortisone was carried out by high-performance liquid chromatography (HPLC). The HPLC system consisted of a Spectra System UV2000 detector (Thermo Separation Products Inc.), a high-precision pump model 300 (Thermo Separation Products Inc.), and a solvent recycler, complemented with an autoinjector (Gilson 234 Autoinjector; Gilson Inc., Middleton, WI). The C18 reversed phase column (Grace, 5 μm i.d., 125 × 4.6 mm) was used during the experiment. The mobile phase used for the HPLC analysis contained helium-degassed mixtures of acetonitrile:Millipore water at 50:50 (v/v) ratio. A UV-detection wavelength of 243 nm was used for the detection of hydrocortisone. The flow rate of the mobile phase was set to 1 mL/min. A series of calibration samples were run in parallel with each series of the permeation samples to quantify hydrocortisone. The software ADchrom was used to analyze and integrate the HPLC data. 2.6. TEWL Measurements. Trans-epidermal water loss (TEWL) measurements were performed using an AquaFlux (model no. AF200, Biox systems Ltd.). The setup for the TEWL measurements mimics the experimental procedure for permeation, except that the donor compartment was empty. The TEWL device was coupled vertically with the donor compartment using a measurement cap (Biox Systems Ltd.) in order to seal the compartment without touching the membrane and for recording the TEWL values. Therefore, this sealing ensured vapor tight connectivity and did not affect the readings. As controls, the supporting membrane and isolated human SC were also measured. For each data point the readings were recorded for about 7 min. The TEWL values of the SCS were recorded over a period of 5 h with 1 h intervals. After which an average value was calculated for each SCS (n = 3) as a mean value of the 5 h TEWL readings. 2.7. SAXD Studies. SAXD was used to determine the lipid lamellar organization in the SCS. The scattering intensity I (in arbitrary units) was measured as a function of the scattering vector q (in reciprocal nm). The latter is defined as q = (4π sin θ)/λ, in which θ is the scattering angle and λ is the wavelength. From the positions of a series of equidistant peaks (qn), the periodicity or d-spacing of a lamellar phase was calculated using the equation d = 2nπ/qn, with n being the order number of the diffraction peak. One-dimensional intensity profiles were obtained by transformation of the 2D SAXD detector pattern from Cartesian (x,y) to polar (ρ,θ) coordinates and subsequently integrating over θ. All measurements were performed at the European Synchrotron Radiation Facility (ESRF, Grenoble) using station BM26B. The X-ray wavelength and the sample-todetector distance were 0.1033 nm and 2.1 m, respectively. Diffraction

FFA unsaturation (molar % of FFA) 7.7 42.6 34.7 85

The unsaturated FFA molar ratio in each mixture is also presented.

For each of the lipid mixtures the appropriate amount of individual lipids was combined, after which the organic solvent chloroform:methanol (2:1) was evaporated under a flow of nitrogen. Then the lipids were redissolved in a hexane:ethanol (2:1) solution to a final concentration of 3 mg/mL. This mixture was used to prepare the SCSs as described below. 2.3. Preparation of the SCSs. In order to prepare the SCSs, the lipid mixture was sprayed on top of a Whatman nucleopore polycarbonate filter disk using a Camag Linomat IV (Muttenz, Switzerland) device with an extended y-axis arm. A Hamilton syringe (100 μL) was inserted in the Linomat and used to spray a selected volume of sample solution from a distance of approximately 1 mm onto the porous filter substrate under a gentle stream of nitrogen. With the y-axis arm, the Linomat is capable of spraying lipids in a square shape (8 mm by 8 mm), by a continuous zigzag movement. The spraying flow rate was set to 5.0 μL/min and 0.6 mg of lipids per membrane was sprayed. After spraying, the SCS was equilibrated for 12 min between 75 and 85 °C. The equilibration temperature depended on the selected lipid mixture but was close to the temperature range at which melting occurs. After equilibration, the membrane was gradually cooled to room temperature. 2.4. Permeability Studies. The permeation studies were performed using Permegear inline diffusion cells (Bethlehem, PA). The diffusing area between donor and acceptor compartment is 0.282 cm2. The SCS was mounted in the diffusion cell. Phosphate-buffered saline (PBS) was used in the acceptor phase. The PBS solution (0.1 M) was prepared by adding NaCl, Na2HPO4, KH2PO4, and KCl in water at a concentration of 8.13, 2.87, 0.20, and 0.19 g/L respectively. The pH of the buffer was adjusted to 7.4 and subsequently filtrated and degassed for about 15 min with helium to remove the dissolved air. The composition of the donor phase was 0.34 mg/mL hydrocortisone (saturated solution, MW = 364) in acetate buffer (pH 5.0), and 1 mL of hydrocortisone solution was applied in the donor compartment. A saturated concentration was used to obtain a maximum thermodynamic activity. The donor compartment was closed with adhesive tape for maintaining occlusive conditions 6536


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data were collected on a PILATUS 1M detector with 1043 × 981 pixels of 172 μm spatial resolution. The spatial calibration of this detector was performed using silver behenate (d = 5.838 nm for AgBeh). The SCS (prepared on the polycarbonate filter disk) was mounted parallel to the primary beam in a sample holder with mica windows. The diffraction data were collected for 5 min at 25 and 32 °C, respectively. 2.8. FTIR Sample Preparation. The preparation method for samples used for FTIR studies was the same as described above except that 1.5 mg of lipids at a concentration of 7.5 mg/mL was dissolved in chloroform:methanol (2:1) solution. This solution was sprayed on an area of 1 × 1 cm2 on a ZnSe window. The samples were equilibrated for about 12 min between 80 and 85 °C, depending on the sample mixture, and cooled gradually to room temperature. Subsequently, the lipid layers were covered with 25 μL of deuterated acetate buffer (pH 5.0) and stored at 37 °C for 15 h to fully hydrate the samples. Although the FTIR sample preparation is slightly different compared to the SCS membrane preparation, the SCS term will be used throughout the paper for clarity to explain the differences in different lipid mixtures. 2.9. FTIR Studies. The FTIR setup consisted of a Bio-Rad FTS4000 FTIR spectrometer (Cambridge, MA) equipped with a broad-band mercury cadmium telluride detector, cooled by liquid nitrogen. The sample cell was closed by two ZnSe windows. The sample was under continuous dry air purge starting 30 min before the data acquisition. The spectra were collected in transmission mode with a coaddition of 256 scans at 1 cm−1 resolution during 4 min. In order to detect phase transitions, the sample temperature was increased at a heating rate of 0.25 °C/min, resulting in a 1 °C temperature rise per recorded spectrum. The spectra were collected between 0 and 90 °C and deconvoluted using a half-width of 4 cm−1 and an enhancement factor of 1.7. The software used for data reduction was Win-IR pro 3.0 from Bio-Rad. The FTIR stretching vibration frequencies were selected to examine the orthorhombic−hexagonal phase transition in a temperature range between 20 and 40 °C and the hexagonal−liquid transition in the range between 50 and 80 °C. The linear regression curve fitting method was used to determine the transition temperature of the lipid mixtures. 2.10. Statistical Analysis. Statistical analysis was performed using an Excel spreadsheet (version 2013). Unpaired two-tailed t test and one-way ANOVA were performed, both with a significance level set at P < 0.05.

Figure 3. Mean permeation flux of hydrocortisone drug molecule across the SCSs prepared from various lipid mixtures. The figure legend describes the different model membranes used for the study (n = 5). For the detailed composition and abbreviations of the SCSs, see Table 1

decided to examine the TEWL values of the various SCSs. The TEWL readings are shown in Figure 4.

Figure 4. TEWL readings of the corresponding model lipid membranes used for the permeation studies. Figure legend shows various SCS-associated TEWL curves (n = 3).

As can be observed from Figure 4, after 1 h a steady-state situation was already reached in all SCSs. The SCSsat and SCSC20:1 resulted in very low TEWL values (see Table 2) that were not significantly different. In the case of SCSC22:1 and SCSC24:1 with an increased level of MUFA, a significant increase in TEWL values was achieved compared to that of SCSsat, while the highest TEWL value was obtained with SCS3FA:1, being about 5 times higher compared to the TEWL value obtained for SCSsat. Apart from the SCSs, as a control, the TEWL values were also examined across the supporting membrane (data not shown in the figure). The mean TEWL value across the supporting polycarbonate membrane was 105.3 ± 1.8 g−2 h−1. Therefore, the supporting membrane did not contribute to diffusion resistance of the SCS. In addition, TEWL values were also measured across ex vivo human SC. The mean value of 5.28 ± 0.2 g−2 h−1 was obtained, which is not significantly different from the values obtained for SCSsat and SCSFA20:1. As differences were observed in the Jss flux of hydrocortisone and the TEWL values when using the various SCSs, it was decided to study the lamellar and lateral organization of these lipid mixtures in detail. 3.3. The Effect of Unsaturated Fatty Acids on the Lamellar Phase Behavior. SAXD provides information about the presence of the lamellar phases and their corresponding repeat distances. The diffraction profiles of the various SCSs are provided in Figure 5.

3. RESULTS 3.1. The Presence of MUFAs Affects the Permeability across SCS. To investigate the effect of MUFAs in the SCS on the lipid barrier function, permeation studies were performed using hydrocortisone as a model drug. Figure 3 displays the mean permeation flux profiles of all the lipid mixtures used in the current study. The abbreviations of the various SCSs are provided in Table 1. The SCSsat displayed a low Jss of hydrocortisone and a steady state situation was already achieved after 1 h. When replacing the low level of FFA C20 by its unsaturated counterpart resulting in SCSC20:1, there was no significant difference in the values obtained for Jss of hydrocortisone compared to SCSsat. However, the hydrocortisone Jss values across the SCSC22:1 and SCS C24:1 (replacing FFA C22:0 and C24:0 by their monounsaturated counterparts) were significantly higher than those of the SCSsat and SCSC20:1, respectively. Finally, the SCS with the combined MUFAs, SCS3FA:1, resulted in a significant increase in Jss compared to both SCSC22:1 and SCSC24:1. The flux across SCS3FA:1 was almost 14 times higher compared to the hydrocortisone flux across SCSsat. 3.2. The Degree of Unsaturation Increases the TEWL Values of the SCS. As the TEWL is often used in (pre)clinical studies to measure the inside−outside skin barrier function, we 6537


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Table 2. Lipid Lamellar and Lateral Organization, the Midpoint Transition Temperature, and the Conformational Ordering Values of the Various Lipid Models and the Permeability Flux of Drug Compound Hydrocortisone and the TEWL Values of Different Lipid Models with Their Standard Deviation lipid model type SCSsat SCSC20:1 SCSCx22:1 SCSC24:1 SCS3FA:1 a

lamellar organization (32 °C) LPP, LPP, LPP, LPP, LPP,

SPP SPP SPP SPP SPP

lateral packinga (32 °C) OR+HEX OR+HEX HEX+low level OR HEX HEX

mid-transition temp (°C) 72.1 70.7 67.4 66.2 60.9

conformational ordering (32 °C)

permeation flux (μg/cm2/h) (32 °C)

2850.1 2850.2 2850.4 2850.5 2850.8

0.05 0.03 0.29 0.23 0.71

± ± ± ± ±

0.1 0.1 0.1 0.1 0.2

TEWL (g (32 °C) 3.1 3.9 9.3 7.3 15.9

−2 −1

± ± ± ± ±

h )

0.4 0.6 0.7 0.9 0.7

The abbreviations OR and HEX stand for orthorhombic and hexagonal lateral packing, respectively.

Figure 5. SAXD pattern of the various SCSs detected at 25 °C. The Arabic numbers (1, 2, 3, etc.) in the SAXD profile indicate the different diffraction orders of the LPP. The Roman numbers (I, II, etc.) indicate the SPP diffraction orders. The peaks originating from crystalline CHOL domains are indicated by an asterisk (*). The additional phase noticed in the SCS3FA:1 is indicted by the hash (#) sign (panel E). Table 1 explains the abbreviations used.

Stretching Frequencies. Besides the lamellar organization, the lateral packing and conformational disordering also play important roles in maintaining the barrier. Therefore, the lipid mixtures were subsequently studied to determine the lateral packing and conformational disordering by using FTIR. The CH2 symmetric stretching frequency of the hydrocarbon chains provides information about the conformational ordering of the lipid tails. A low (∼2848 cm−1) wavenumber of the CH2 symmetric stretching vibrations indicates the presence of a highly ordered lipid organization (either hexagonal or orthorhombic), while a high (∼2853 cm−1) wavenumber indicates a liquid disordered phase.40 The thermotropic response of the CH2 symmetric stretching frequencies of the various lipid mixtures are provided in Figure 6. At 10 °C, in the spectrum of the SCSsat and the SCSC20:1, the CH2 symmetric stretching frequencies are 2849.5 and 2849.6 cm−1 respectively, indicating a conformational ordering of the lipid tails. The

In the SCSsat the lipids form both the LPP and SPP (Figure 5A). In the diffraction profile, the peak positions at q = 0.52, 1.06, 1.57, and 2.1 nm−1 are the first, second, third, and fourth diffraction orders attributed to the LPP with a repeat distance of 12.1 nm. The diffraction peaks at q = 1.18 and 2.36 nm−1 are the first and second orders of the SPP with a repeat distance of 5.3 nm. When focusing on the SCS prepared with MUFAs, no differences have been observed in the diffraction pattern (Figure 5B−E) compared to SCSsat except for minor changes in the diffraction pattern of SCS3FA:1. Besides the formation of the LPP and SPP, the lipids in the SCS3FA:1 display an additional phase (Figure 5E) indicated by the presence of a peak at q = 1.41 nm−1 corresponding to a spacing of 4.4 nm. In all SCSs no differences in the presence of the lamellar phases were noticed between 25 °C and at 32 °C. 3.4. The Level of Unsaturation Affects the Conformational Disordering and Lateral Packing. CH2 Symmetric 6538


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increase in temperature from 55 to 85 °C results in large frequency shifts from 2849.6 to 2853.7 cm−1 for SCS and 2851.2 to 2853.8 cm−1 for SCSC20:1. This demonstrates the formation of a liquid phase in this temperature range with a midpoint transition temperature of 72.1 °C for SCS and 70.7 °C for SCSC20:1, respectively. For the SCSC22:1 and SCSC24:1, the thermotropic response of the CH2 symmetric frequencies is very similar. At 10 °C the CH2 symmetric stretching frequencies in the FTIR spectra is located at 2849.9 cm−1 and continued steadily until 24 °C. Between 24 and 40 °C, the frequency shifts gradually from 2850.2 to 2850.9 cm−1, suggesting a phase transition from an orthorhombic to a hexagonal packing. Upon increasing the temperature to 60 °C, there is only a slight frequency shift in the spectrum. When increasing the temperature beyond 60 °C, a large shift in frequencies for both SCSC22:1 and SCSC24:1 is observed. These large shifts in frequencies are characterized by a phase transition from hexagonal to the liquid phase. The midpoint transition temperature of the SCSC22:1 and SCSC24:1 is 67.4 and 66.2 °C, respectively. Finally, in the spectrum of the SCS3FA:1 at 10 °C, the CH2 symmetric frequency has a value of 2850.4 cm−1, indicating conformational disordering. The frequency increases gradually until 40 °C to a value of 2850.9 cm−1. Upon increasing the temperature beyond 40 °C, a larger shift in frequency is

Figure 6. Thermotropic CH2 symmetric stretching frequencies of the various SCSs used in our studies. The figure legend describes the different SCSs. A detailed composition is given in Table 1. The conformational disordering values of the different SCSs at skin temperature (32 °C) at which the permeation studies were performed are given in Table 2

frequencies gradually shift to higher values upon increasing the temperature between 24 and 40 °C from 2849.4 to 2850.6 cm−1 for SCS and from 2849.6 to 2850.7 cm−1 for SCSC20:1. These shifts in frequency are based on a phase transition from an orthorhombic to a hexagonal lateral packing. Between 40 and 55 °C, the frequency does not shift for the SCSsat, while in the spectrum of SCSC20:1 a slight increment in frequency is observed with a value of 2851.2 cm−1 at 56 °C. A further

Figure 7. FTIR spectra of the rocking vibration frequencies as a function of temperature. The spectra are shown in the temperature region from 20 to 60 °C. At 32 °C, the curves are shown as a magenta line as the diffusion studies were performed at that particular temperature. For clarity, this is also the case at 20, 40, 50, and 60 °C. 6539


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observed over a very large temperature range until 85 °C. At this temperature the frequency is 2854.2 cm−1, indicating the formation of a liquid phase. The midpoint transition temperature for this SCS was 60.9 °C. CH2 Rocking Frequencies. The FTIR rocking vibrations provide information about the lateral packing present in the lipid lamellae. The CH2 rocking frequencies of all the model lipid mixtures are shown in Figure 7. The FTIR spectrum of the SCSsat reveals a strong doublet in the rocking vibration frequencies at approximately 720 and 731 cm−1 at 20 °C (Figure 7A). This splitting is caused by the short-range coupling of the adjacent lipid tails, indicating the presence of an orthorhombic lateral packing.41 A gradual increase in temperature results in a disappearance of the splitting contour between temperature 36 and 40 °C. At 40 °C, only one peak is observed, demonstrating the absence of an orthorhombic lateral packing. The SCSC20:1 and SCSC22:1 also reveal a doublet in the FTIR spectrum at 20 °C with rocking vibration frequencies at 731 and 720 cm−1, respectively, suggesting the presence of an orthorhombic packing at this temperature (Figure 7B,C). The 731 cm−1 shoulder present at 20 °C in the spectrum of the SCSC22:1 is much weaker compared to the shoulder present in the spectra of the SCSsat and SCSC20:1, suggesting that a smaller fraction of lipids forms the orthorhombic lateral packing. Upon increasing the temperature, the doublets start to disappear, and at 38 °C only a singlet is observed in the spectrum of both SCSs, indicating the absence of an orthorhombic packing. A shift in temperature up to 60 °C does not change the rocking frequency. When focusing on the SCSC24:1, at 20 °C, the rocking spectrum is characterized by a weak doublet with frequencies at approximately 721 and 731 cm−1 (Figure 7D). This indicates that a very small fraction of lipids is forming an orthorhombic phase at this temperature range but that a large population forms a hexagonal packing. The 731 cm−1 contour in the spectrum vanishes when increasing the temperature to around 30 °C. At this temperature the rocking vibrations are represented by a singlet, demonstrating the absence of the orthorhombic packing. A further increase in temperature until 60 °C does not change the rocking contours in the spectrum. The FTIR rocking contour of the sample prepared with SCS3FA:1 exhibits a broad intense contour at around 721 cm−1 with weak shoulder at 20 °C, indicating the lipids mostly adopting hexagonal packing while a small subpopulation of the lipids is also present in an orthorhombic lattice (Figure 7E). The high conformational disordering (see Figure 6) also suggests that a small proportion of lipids might also be present in a liquid phase at this temperature. A gradual increase in temperature causes the weak shoulder to disappear and the contour becomes a singlet at around 32 °C, demonstrating the absence of the orthorhombic packing at this temperature. A further increase to 60 °C does not affect the shape of the contour.

as reduction in the level of CER EO, and a simultaneous increase in the level of short chain CERs and short chain FFAs have been reported.17,34,51,52 Recently, it has been shown that the level of MUFAs also increased in the SC of these patients.34,35 In previous studies, we noticed that a reduction or complete abduction in the CER EO leads to the absence of the LPP in the SCS, resulting in a reduced lipid barrier function.22,53 In subsequent studies, we noticed that an increased level of short chain FFAs also revealed a higher permeability of the SCS. This was due to both a change in the lipid lateral packing and lamellar organization.20 However, we had not yet studied the effect of MUFAs on the lipid barrier. Therefore, in this study we aimed to elucidate the role of increased level of MUFAs on the lipid barrier function in the SC using the SCS. In order to accomplish our endeavor, we prepared the SCS by varying the level of MUFAs while keeping the CER composition and chain length constant. Although our CER composition is not entirely mimicking the CER composition in human SC, the rationale behind the selected CER composition is that (i) mixtures prepared with this CER composition, CHOL, and FFAsat mimic the lipid organization in human and pig SC as well as in mixtures prepared from isolated pig and human CERs and (ii) the synthetic counterparts of the CER subclasses in pig SC are available, while a large number of CER subclasses present in human SC are not.38,54,55 Our studies demonstrate that a drastic increase in the level of MUFAs may have an important contribution to the impaired SC lipid barrier, as discussed below. 4.1. The Choice of the FFA Composition. The saturated FFAs that we used in our current study are based on the FFA distribution reported in SC.39 The MUFAs show considerable variation in abundancy in patients varying from 5% (healthy human skin) up to 85% of the total FFA content (for Netherton syndrome patients). In our approach, we decided to replace the most abundant saturated FFAs by the monounsaturated counterparts, while keeping the FFA chain length distribution the same. In this way we were able to only study the effect of the level of unsaturation on the lipid phase behavior and lipid barrier function. 4.2. Correlation between TEWL and Hydrocortisone Jss values. In the present study we decided to monitor the diffusion profiles and TEWL values. When comparing the results of these measurements, an approximately linear correlation is observed between Jss values and TEWL values, with a correlation coefficient of 0.96. As in clinical studies, TEWL values are often recorded as a measure for the skin barrier function; our results indicate that as far as the lipid organization is concerned, the transport of low MW drugs may be correlated with the TEWL. However, of course, in the in vivo situation, the SC structure is more complex with the presence of corneocytes. The penetration pathway of more lipophilic low MW compounds is expected to take preferentially the intercellular route, while water is able to partition into the corneocytes.56−58 These differences in penetration pathway may be one of the factors that makes a comparison between TEWL and drug permeation in SC much more complex. 4.3. The Lipid Barrier Function Decreases with Increasing the Degree of Unsaturation. The results reported in the present paper show that the TEWL values and the Jss values both increase when the level of MUFA in the SCS increases. The SCS3FA:1 shows significantly higher TEWL and Jss values for hydrocortisone than when using SCS prepared with a single MUFA. Furthermore, the higher MUFA levels of

4. DISCUSSION Although over the last 20 years many studies were performed to examine the lipid organization in SC,10−12,33,42−48 there are not many studies related to lipid composition that are specifically mimicking the deviations in lipid composition encountered in diseased skin.49,50 In diseases of the skin, such as atopic dermatitis and Netherton syndrome, changes in the CER:CHOL/FFA ratio, the composition of the CER subclasses such 6540


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SCSC22:1 and SCSC24:1 compared to the levels of MUFAs in SCSC20:1 resulted in significantly higher TEWL and Jss values. However, as we varied both the level of unsaturation and the chain length simultaneously, we performed an additional study, in which the level of unsaturation in one particular chain length, namely, SCSC22:1, was gradually increased. In that experiment, the elevation in MUFA levels also gradually increased TEWL values (data not shown here), similarly as for the different chain length FFA. 4.4. The MUFAs Enhance the Conformational Disordering While Reducing the Level of Orthorhombic Packing. To unravel the mechanisms underlying the impaired lipid barrier, we examined the lipid organization by FTIR. Our results indicate a higher conformational disordering and a reduced level of orthorhombic packing, suggesting less chain compactness and subsequently reduced lipid barrier when increasing the level of MUFA. The SCS3FA:1 exhibit a higher conformational disordering compared to the other SCS throughout the complete temperature range (10−90 °C). This may indicate that at skin temperature (32 °C) a subpopulation of the lipids already forms a liquid phase, while the rocking vibrations indicate mostly the absence of an orthorhombic lateral packing. The high conformational disordering and the increased presence of the hexagonal packing may account for the high Jss and TEWL readings in our model membranes. When focusing on the SCSC22:1 and SCSC24:1, the degree of conformational disordering and the presence of the orthorhombic lateral packing are in between those of the lipids in SCS3FA:1 and SCSsat. As the Jss and TEWL values are also intermediate of the values of the SCS3FA:1, SCSsat, and SCSC20:1, this again suggests that the lateral packing, as well as the conformational disordering, plays a role in the impaired lipid barrier when the level of MUFA is increased. The interaction between other MUFAs such as oleic acid and the SC lipids has been investigated extensively, as these were potential enhancers aiming to increase the penetration of drugs across the SC. Oleic acid was shown to phase separate, while in the present study the MUFAs induce an orthorhombic to hexagonal phase transition. Whether phase separation plays also a role in the present studies is not entirely clear: an increase in the conformational ordering is observed at high levels of MUFAs. This may indicate some phase separation.36,47,59,60 Another important parameter providing a measure for the conformational disordering is the midpoint temperature of the order−disorder transition. Although the midpoint temperature of the SCS does not carry much extra information as far as the lipid barrier is concerned, we observed that a higher degree of unsaturation reduces the midpoint temperature. This indicates that a high level of MUFAs in the SCS results in the formation of a disordered phase at a reduced temperature, suggesting a greater susceptibility to be in a disordered state even at lower temperature. 4.5. Unsaturated FFAs Do Not Affect the Lipid Lamellar Organization. In all SCSs, the lipids form both the LPP and SPP at 25 °C and at physiological skin temperature (32 °C). This demonstrates that the lipid lamellar organization does not change when varying the MUFA levels in SCSs up to around 40%. Only at higher MUFA levels (85% for SCS3FA:1), an additional phase was observed along with LPP and SPP. Therefore, the impaired lipid barrier function when increasing the MUFA levels is only attributed to the changes in lateral packing and the increase in conformational disordering.

4.6. In Vitro Studies Are Related to Findings in (Pre)Clinical Studies. Clinical studies with patients and control subjects revealed that, in the control subjects, the level of MUFA is significantly increased in the lesional skin of the atopic dermatitis patients.35 In Netherton syndrome patients, the level of MUFA is even further increased to around 25%, but in individual patients the level of MUFA reaches 80−90% of the total FFA.34 The enzyme stearoyl-coA desaturase 1, which is responsible for the conversion of FFAs into MUFAs, is overexpressed while the elongases (ELOVL 1 and 6) responsible for the elongation of the FFAs are less abundantly expressed in patients with Netherton syndrome.34 The combined effect of these two enzymes leads to the reduction of long chain FFAs and an increment of MUFAs in these patients. It has been shown that a reduction in chain length of CERs and FFAs and a reduced lipid/protein level correlates with the impaired skin barrier function monitored by TEWL.17,51 However, the results shown in the present study indicate that a high levels of MUFAs may also contribute to the impaired skin barrier by increasing the level of hexagonal packing. A reduced skin barrier has been demonstrated in a mouse model study mimicking features of Netherton syndrome.23 As shown in this work, the formation of the lamellar phases is not very sensitive to the presence of MUFA. Therefore, changes in the lipid lamellar phases observed in atopic dermatitis and Netherton syndrome patients cannot be contributed by increased levels of MUFA in these patients. However, when additional changes occur, such as (i) reduced chain length of FFAs or CERs, (ii) reduced levels of CER EO, or (iii) unsaturated acyl chains in CERs, a higher level of MUFA may amplify changes in the lipid organization in the SC of these patients. This will be the subject of future research.

5. CONCLUSION In conclusion, the present study demonstrates that high levels of MUFAs reduces the lipid barrier function: a high level of MUFAs shifts the lateral packing from an orthorhombic to a hexagonal packing and increases the conformational disordering of the lipid tails, thereby inducing a reduced lipid barrier. Therefore, our in vitro results show that besides a reduced chain length of the lipids, a substantial increase in the level of MUFAs may also contribute to an impaired skin barrier function. However, the in vivo the situation is much more complex, as various changes in lipid composition occur simultaneously. Therefore, in the future, the impact of changes in CER composition (including reduced chain length) combined with fatty acid unsaturation on the lipid organization and SCS permeability will be examined.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 00 31 71 527 4208. Fax: 00 31 71 527 4565. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We would like to thank the company Evonik for their generous provision of CERs and the personnel at the DUBBLE Beamline 26b at the ESRF located at Grenoble, France, for their assistance during the X-ray diffraction measurements. We also 6541


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like to thank Uchiyama Masayuki for his assistance in TEWL measurements.

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