Preparation of a New Oligolamellar Stratum Corneum Lipid Model

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Preparation of a new oligolamellar stratum corneum lipid model Josefin Müller, Annett Schroeter, Roland Steitz, Marcus Trapp, and Reinhard Neubert Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00655 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 9, 2016

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Preparation of a new oligolamellar stratum corneum lipid model Josefin Mueller†, Annett Schroeter†, Roland Steitz‡, Marcus Trapp‡, Reinhard H. H. Neubert†* †

Institute of Pharmacy, Martin Luther University, Wolfgang-Langenbeck-Straße 4, 06120 Halle,

Germany ‡

Institute of Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin für Materialien

und Energie GmbH, Hahn-Meitner-Platz 1, 14109 Berlin, Germany

ABSTRACT In this study, we present a preparation method for a new stratum corneum (SC) model system, which is closer to natural SC than the commonly used multilayer models. The complex setup of the native SC lipid matrix was mimicked by a ternary lipid mixture of ceramide [AP], cholesterol and stearic acid. A spin coating procedure was applied to realize oligo-layered samples. The influence of lipid concentration, rotation speed, polyethyleneimine, methanol content, cholesterol fraction and annealing on the molecular arrangement of the new SC-model was investigated by X-ray reflectivity measurements.

The new oligo-SC-model is closer to native SC in the total number of lipid membranes found between corneocytes. The reduction in thickness provides the opportunity to study the effects of

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drugs and/or hydrophilic penetration enhancers on the structure of SC in full detail by X-ray or neutron reflectivity. In addition, the oligo-lamellar systems allows to infer not only the lamellar spacing, but also the total thickness of the oligo-SC-model and changes thereof can be monitored. This improvement is most helpful for the understanding of transdermal drug administration on the nanoscale.

The results are compared to the commonly used multilamellar lipid model systems and advantages and disadvantages of both models are discussed.


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INTRODUCTION The skin is the largest organ of the human body. It combines a variety of functions which are indispensable to the human body. Amongst those are the protection against external influences, thermoregulation and sensing for the transmission of different stimuli.1 Especially the outermost layer of the skin, the stratum corneum (SC), is responsible for the barrier function against external chemical, biological or physical influences. This includes also transdermally applied drugs, which permeate the skin into the systemic circulation. Consequently, their activity may be reduced or even inhibited by the strong barrier function of the SC. Nevertheless, for many drugs, the transdermal application route is advantageous, because of a reduction of side effects, the circumvention of the first-pass effect, which results in a higher bioavailability, and the possibility of controlled release.2 To improve the systemic availability of transdermally applied drugs, a reversible reduction in the tight structure of the SC is necessary. For this purpose, besides physical borne influences, and modern vehicle systems like microemulsions, penetration enhancers offer the possibility to reduce chemically the SC barrier function.1 Until now, several investigations were carried out to demonstrate the mode of action of penetration accelerators, reviewed by Williams and Barry.3 However, to improve detailed knowledge about the interactions on a molecular scale, the structure of the SC lipid matrix itself needs to be understood. The organization of the SC is comparable to a brick wall with dead corneocytes, representing the bricks and highly ordered multilamellar lipid structures, representing the mortar. Specifically this lipid matrix causes the extensive barrier function of the mammalian skin. It contains different subspecies of ceramides (CER) and free fatty acids (FFA) as well as cholesterol (CHOL) in a nearly equal molar ratio.4, 5, 6, 7, 8, 9 Up to now, there are several models, which try to explain the


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lipid arrangement and to evaluate the role of single lipid subspecies within the SC lipid layers.10, 11, 12, 13, 14

However, since these studies are contradictory in some aspects, and the detailed

structure of the SC on a molecular scale is not completely understood, further investigations, using simple SC lipid model systems, are necessary. So far, most studies on SC lipid model systems were carried out using multilamellar lipid assemblies.15,

16, 17, 18

As the SC lipids are naturally arranged in a stack of up to 20 bilayers

only,19 oligolamellar lipid models serve as more realistic models. Furthermore, an oligolamellar model will give additional information on the lipid structure, especially with respect to the application of penetration enhancers, as it is much more sensitive to small structural changes than a multilayer model. In this study, we developed and optimized a new, oligolamellar SC lipid model (oligo-SCmodel), designed to address specifically the possibility of applying hydrophilic penetration enhancers in prospective studies. To keep the models as simple as possible, we used lipid mixtures, composed of three different lipid species, each representing one of the main components of the native SC, i.e. CER[AP], CHOL and stearic acid (SA) (see Figure 1). CER[AP]-C18/18 was chosen with respect to the 4 OH groups in the head group region, focusing on the potential to strongly interact with hydrophilic penetration enhancers. With an amount of ~ 9 % in a total quantity of meanwhile 19 subclasses of CERs, it is the sixth common CER species.20 To match the chain length of the CER, SA was then determined, as the most abundant short chain FFA.21 All lipid species used were synthetically derived to reduce their diversity. The molar ratio of 1:0.7:1 (CER:CHOL:SA) was based on findings for native SC with a slightly lower CHOL content to avoid CHOL separation.


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Applying different physical methods, these simple mixtures allow for direct conclusions about the structure-function relationship of special lipid species. Techniques like Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, Nuclear Magnetic Resonance spectroscopy or Differential Scanning Calorimetry are commonly used to describe the physicochemical properties of SC lipid models.22,

23, 24

Insights into the inner structure and organization of the

lamellar lipid models on the nanoscale can be obtained by scattering methods. Consequently, as characterization methods we used X-ray reflectivity and Infrared spectroscopy. While the FTIRtechnique offers additional information on the lipid phase state, X-ray reflectivity measurements yield information about the structure, interface roughness and thickness of the lipid layer. An additional advantage of the oligolamellar model used in this study is a sample thickness in the order of several tens of nanometers. In case of reflectivity measurements, this results in pronounced Kiessig fringes and Bragg peaks and thus gives detailed information about the sample thickness and lamellar distances. Precondition is that the sample is homogeneously layered and the roughness of the individual layers is small compared to its thickness. For lipids and especially phospholipids, like DMPC, oligolamellar lipid arrangements have already been prepared and characterized.25, 26 In the present study a mixture of three lipid species, which are structurally different from phospholipids, is used. Therefore, the given sample preparation process had to be adapted.


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Figure 1: Structural formula of the investigated SC lipid mixture containing ceramide [AP], cholesterol and stearic acid.

EXPERIMENTAL Materials Ceramide [AP]-C18 (N-(α-hydroxyoctadecanoyl)-phytosphingosine, 92.3 %) was provided by Evonik Industries AG (Essen, Germany) and purified using column chromatography. Cholesterol (99+ %), stearic acid (≥ 99.5 %), tetracosanoic acid (≥ 99.0 %), branched polyethylenimine (PEI) (50 % (w/v), Mw ≈ 750 kDa, Mn ≈ 60kDa), deuterium dioxide (D2O, purity ≥ 99.9 atom % D), ammonium hydroxide, hydrogen peroxide and ethanol absolute were purchased from Sigma Aldrich GmbH (Taufkirchen, Germany). Chloroform (≥ 99.9 %) was from Carl Roth GmbH (Karlsruhe, Germany) and methanol (≥ 99.9 %) from VWR Chemicals (Fontenay-sous-Bois, France). Ultrapure Water was received by a Milli-Q purification system (http://www.millipore.com/; resistivity > 18.2 MΩ cm). Disc-shaped silicon wafers with a diameter of 60 mm and a thickness of 10 mm were provided by Sil’Tronix (Archamps, France). On two opposing ends these wafers are inclined by an angle of 45°. Small rectangular wafers of about 20 mm x 25 mm and 0.625 mm in thickness were cut from silicon wafers, purchased from CrysTec GmbH (Berlin, Germany). The silicon wafers had a roughness of less than 5 Å and had an orientation of (100). All rectangular wafers were one-side polished, whereas the disc-shaped wafers were polished on both sides and on the inclined surfaces as well.

Methods Sample preparation


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Before use, all silicon wafers were cleaned with chloroform in a sonicator to remove remaining organic material. Thereafter, the commonly used RCA wafer cleaning procedure,27,

28, 29

and

more precisely the first step “RCA standard clean 1” was used to make sure that all organic material was removed from the wafer surface. Additionally, the surface becomes more hydrophilic. In this procedure, the silicon wafer was immersed in a solution of ultrapure water, NH4OH 29 % (w/w) and H2O2 30 % (w/w) (5:1:1) which was heated to 70-75 °C for 10 minutes. Subsequently, the wafers were rinsed 21 times with ultrapure water. During the optimization process a polyethyleneimine (PEI) coating of the wafer surface became necessary to improve the lipid binding at a solid/liquid interface. Therefore, the wafers were stored in a PEI solution (0.01 monomol/l, pH 10) for 20 minutes, then removed and stored 3 times for 2 minutes in ultrapure water at room temperature. Thereafter, 10 mg/ml stock solutions of CER[AP], CHOL and the FFA in pure chloroform or a mixture of chloroform and methanol with a ratio by volume of 2:1 (v/v) were prepared. As FFA in most cases SA, but also tetracosanoic acid (TA) was used. In a second step, respective amounts of these stock solutions were mixed together to obtain a molar lipid ratio of 1:0.7:1 (CER[AP]: CHOL: FFA). Using either pure chloroform or a mixture of chloroform and methanol (2:1 (v/v)) the lipid mixtures were adjusted to distinct concentration. The lipids were deposited on a silicon wafer surface either by spin coating or air-brushing, immediately after wafer pretreatment. The spin coating was performed with a spin coater (P-6708D Cookson Electronics (Langenfeld, Germany)) according to Mennicke and Salditt.26 For rectangular wafers (surface ∼ 5 cm2) an amount of the lipid solution (in chloroform/methanol) of 0.7 ml and for disc-shaped wafers (surface ∼ 28 cm2) an equivalent amount of 3.5 ml was deposited on the wafer surface. The spin


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coating procedure was started typically with a rotation speed of 500 rounds per minute (rpm) for 1 second followed by a rotation speed of 4000 rpm for 60 seconds. In the presented experiments the final rotation speed, as well as the concentration of the lipid solution were varied. For the preparation of multilamellar samples by air-brushing the disc-shaped silicon wafers were placed on a heating block at a temperature of 50 °C. 2 ml of a lipid solution with a concentration of 5 mg/ml (in chloroform/methanol) was sprayed homogeneously onto the wafer surface at a constant flow. For the annealing procedure the sample was stored at 100 % humidity in a horizontal position and alternately heated to 75 °C and cooled to room temperature according to Schroeter and coworkers.15 The first cycle comprises 1 hour heating and 30 minutes cooling. During the two subsequent cycles the sample is heated for 30 minutes and cooled down for 15 minutes in each cycle.

X-ray reflectivity measurements All samples were either measured on a Bruker D5000 Diffractometer (Bruker Optik GmbH, Germany) with a reflectivity add-on or on a home-built instrument. In both cases the copper Kα Line with a wavelength of 1.541 Å was used for the measurements. In the case of the D5000, the incident beam was defined by the line focus of the x-ray tube and a slit of 0.1 mm, in the home built setup, the diaphragm also had a width of 0.1 mm. The resolution of the instrument was set to δQz = 0.003 Å−1. The reflected beam was in both cases monochromatized and detected by scintillation detectors. For low angles where the high intensities would saturate the detector a 0.1 mm copper absorber in the case of the D5000 and a nickel absorber for the home-built instrument were inserted into the reflected beam.


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All x-ray reflectivity experiments were performed at ambient humidity at room temperature. Details on the home-built instrument and its operational mode can be found elsewhere.30

Analysis of X-ray reflectivity data During the optimization of the sample preparation, the influence of different parameters was investigated. The aim of this study was a reproducible protocol for the preparation of an oligolamellar stack of membranes. Specular X-ray reflectivity data were normalized by time and initial beam intensity. All data are displayed as a function of wavevector transfer ସగ

ܳ = ܳ௭ = ቀ ቁ ‫ߠ݊݅ݏ‬ ఒ

1

perpendicular to the sample surface, with the incident wavelength λ, θ being the reflection angle and analyzed as described by Kreuzer et al.31 The incoming X-ray beam is reflected at interfaces with different electron densities, which are at least the air/lipid and the lipid/solid interfaces. Constructive and destructive interferences occur, which appear as the typical Kiessig oscillations and Bragg peaks, respectively. Applying Bragg‘s law ݊ ߣ = 2݀ ‫ߠ ݊݅ݏ‬

2

the spacing between adjacent Kiessig maxima (minima) provides information about the overall sample thickness via the combination of equations 1 and 2 ‫ = ݐ‬2ߨ/߂ܳ௄௜௘௦௦௜௚

3,

whereas the spacing between adjacent Bragg peak maxima can be used to calculate the thickness ݀ = 2ߨ/߂ܳ஻௥௔௚௚

4

of an individual bilayer membrane. In case higher orders are visible in the reflectivity curve, the peak positions can be plotted as a function of order n. Equations 3 and 4 can then be rewritten as


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∆ܳ௄௜௘௦௦௜௚ =

ଶగ ௧

݊ and ∆ܳ஻௥௔௚௚ =

ଶగ ௗ

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݊, respectively. The corresponding layer thickness t or d

are then calculated from the slope of a linear fit to the data. Errors given correspond to the error obtained by the fitting procedure. A non-zero y-intercept indicates a not repeated contribution like in our case the PEI layer. The number of lipid bilayers ܰ in the sample can then be determined as the ratio of the overall sample thickness and the thickness of one single bilayer ܰ = ‫ݐ‬/݀

5

ATR-FTIR measurements Infrared measurements were carried out at an FTIR spectrometer Vertex 70 (Bruker Optik GmbH, Germany) at Helmholtz-Zentrum Berlin. All spectra were recorded by the Opus 6.5 software package (Bruker Optik GmbH, Germany) in the range of 1000 to 6000 cm-1. The silicon wafer was placed onto a Teflon cell which was filled with D2O. The background was determined once, using the cleaned silicon wafer against D2O. As detector a nitrogen cooled mercury cadmium telluride (MCT) detector was used.

RESULTS AND DISCUSSION X-Ray reflectivity Lipid concentration According to investigations on phospholipids25 different concentrations, i.e. 10 mg/ml, 5 mg/ml, 3,5 mg/ml and 2.5 mg/ml, were spin coated at 4000 rpm directly on the wafer surface in a first set of experiments. As can be seen in Figure 2, the reflectivity curve of films from the lipid concentrations of 10 mg/ml (black line) and 5 mg/ml (blue line) show pronounced Bragg peaks


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and some rather indistinct Kiessig fringes. By lowering the lipid concentration to 3.5 mg/ml, well defined Kiessig fringes up to high Q values occur (green line). The lowest tested concentration of 2.5 mg/ml also induced many well-shaped Kiessig fringes (red line), although they are distinctly broader. The corresponding bilayer (d) and total thickness (t) are summarized in Table 1. As expected, the total sample thickness decreased drastically from (472.4 ± 8.4) Å to (172.1 ± 1.8) Å by reducing the concentration of the lipid mixture for spin coating from 10 mg/ml to 2.5 mg/ml. Furthermore, a comparable lipid arrangement can be estimated for all samples, since the respective bilayer thickness does not differ significantly. The calculated repeat distances for this phase, named phase A, between 43.46 – 45.43 Å are slightly smaller than those commonly described in the literature for similar mixtures.14, 32 This is most likely due to different humidity levels. As already presented in the literature,33 with increasing humidity the lamellar repeat distance expands. Since in our system the humidity is very low (ambient conditions against air), a smaller repeat distance than for the reference systems at 60 - 100 % humidity is likely. The reflectivity curves of films from lipid mixtures with concentrations of 3.5 mg/ml and 2.5 mg/ml show Bragg peaks and, in addition, well-defined Kiessig fringes. The appearance of the latter are the signature of well-aligned films with little roughness and -from the Bragg peaks- well pronounced interlamellar ordering.


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Figure 2: Reflectivity curves of films from lipid mixtures (CER[AP]: CHOL: SA; 1:0.7:1 molar ratio; in CHCl3/MeOH (minimum methanol content)) spin coated on rectangular wafers at a rotation speed of 4000 rpm using concentrations of 10 (black line), 5 (blue line), 3.5 (green line) and 2.5 mg/ml (red line). Curves are shifted vertically for clarity of presentation.

Table 1: Extracted bilayer, d, and total thickness, t, of the SC model systems displayed in Figure 2, prepared by using different lipid concentrations. 10 mg/ml

5 mg/ml

3.5 mg/ml

2.5 mg/ml

d (Å)

45.43 ± 0.99

44.08 ± 0.89

43.46 ± 1.51

43.92 ± 0.58

t (Å)

472.4 ± 8.4

223.0 ± 5.0

192.3 ± 3.2

172.1 ± 1.8

Rotation speed Varying the rotation speed of the spin coating process from 1500 rpm up to 4000 rpm is in agreement with results by Kreuzer et al.25 Highest speed (4000 rpm) produced the most distinct Kiessig fringes. In contrast, with decreasing rotation speed, the Kiessig fringes flattened considerably (data not shown).

Polyethyleneimine All experiments were performed against ambient humidity. With regard to the planned investigations of hydrophilic penetration enhancers, an aqueous sample environment is needed at a later stage. Consequently, the SC lipids have to be stable also at a solid/liquid interface in this latter case. Therefore, samples were incubated in water for several hours. X-ray reflectivity experiments conducted on the withdrawn samples revealed removal of the lipids from the silicon support due to insufficient attractive forces (Figure 3, blue curves). From the loss in intensity and


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the disappearance of the Kiessig fringes an almost complete loss of the lipid layers and a fragmented remaining coating is evident. According to previous findings, where similar detachment problems occurred in case of polyelectrolyte multilayer films,34 we decided to use PEI as an anchor for the SC lipid mixture. Our investigations indicate, that the PEI surface layer pre-prepared on the silicon support needs to be covered immediately by the SC lipids to avoid intermediate drying of the PEI film, which is known to alter the polymer structure35, 36 and in our case worsens the molecular ordering of the lipid coating. Strictly following the outlined protocol ensures that the PEI interlayer sandwiched between silicon backing and lipid fronting, strongly enforces the adhesion of the lipid film to its solid support. Figure 3 (black and grey curve) illustrates the observed effect on lipid film stability. The film withstands extended incubation in excess water. The positively charged PEI molecule seems to have the potential to bind the negatively charged stearic acid with its neighboring lipids. The total thickness of the sample before water treatment of (116.9 ± 4.8) Å did not significantly change after water treatment (111.9 ± 9.5) Å. This finding approves the application of a PEI linker layer as a reliable tool to stabilize the complex SC lipid mixture at the silicon wafer surface for measurements at a solid/liquid interface.


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Figure 3: Reflectivity of a lipid coating from 2.5 mg/ml lipid mixture (CER[AP]: CHOL: SA; 1:0.7:1 molar ratio, CHCl3/MeOH 15:1 (v/v)), spin coated at 4000 rpm on rectangular wafers without a PEI layer before (dark blue) and after incubation in water (light blue). The curves were shifted by a factor of 10 on the intensity axis for comparison reasons. The lower reflectivity curves displayed, represent a 2.5 mg/ml lipid mixture (CER[AP]: CHOL: TA; 1:0.7:1 molar ratio, CHCl3/MeOH 15:1 (v/v)), spin coated at 4000 rpm on rectangular wafers, pre-coated with a molecular linker layer of PEI, immediately after preparation (black line) and after application of excess water to the wafer surface for 4 hours (grey line).

Methanol content The ratio of the used solvents further influences the quality of the laid down lipid membranes since chloroform and methanol have different vapor pressures. Mixtures of both with a ratio of 1:2 (v/v) resulted in an inhomogeneous recrystallization of the lipids causing a low lipid order as indicated by few and broad peaks with low intensity. Furthermore, the preparation of the films was not reproducible. Better results were achieved with a spin coating solution with chloroform/methanol ratio of 15:1 (v/v), with reflectivity curves with many analyzable features (see also Figure 3).

Reproducibility Taking all previous findings into account, samples can be prepared in a reproducible manner, as proven by Figure 4. The calculated repeat distances of (43.06 ± 1.31) Å and (42.95 ± 1.28) Å for sample 1 and 2, respectively, agree within errors. The total sample thickness is (258.9 ± 1.2) Å for sample 1 and (212.0 ± 2.2) Å for sample 2, revealing a number of 6 bilayers in case of sample 1 and 5 bilayers in case of sample 2. The small variation in the total number of bilayers -


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by one lipid lamella- can be attributed to the low concentration of lipids available during the spin coating. Besides that, the lipid arrangement in the two oligolamellar films is identical.

Figure 4: Reflectivity curves of samples 1 (black line) and 2 (grey line) prepared by identical protocol: 3.5 mg/ml lipid mixtures (CER[AP]: CHOL: SA; 1:0.7:1 molar ratio; CHCl3/MeOH 15:1 (v/v)) were spin coated on disc-shaped wafers. A PEI linker layer was present in each case.

Cholesterol fraction We varied the molar ratio of CHOL in the lipid mixtures from 0.7 to 0.3 and found that the number of fringes in the recorded reflectivity curves of the as-prepared films, their shape as well as their intensity did not change considerably. In contrast the positions of the Kiessig fringes and the positions of the Bragg peaks shifted to higher Q values with increasing CHOL molar ratio. (data not shown), indicating a decreased bilayer thickness. A similar effect is described in the literature.32 Here, at temperatures below the main phase transition, higher CHOL amounts added to the gel phase of the lipids induce an increased membrane fluidization, which in turn results in a decreased membrane thickness.


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Annealing Annealing is a commonly used procedure for sample preparation of complex SC lipid multilayer systems. As shown by Schroeter et al.,15 annealing is an adequate method to improve the lipid lamellar orientation in a multilayer arrangement. It leads to a lower mosaicity, indicated by a reduced width of the respective Bragg peaks. Additionally, and due to the improved orientation, the intensity of the Bragg peaks is increased. Since our lipid model is prepared by recrystallization from solution in a mixture of two solvents with different vapor pressures, the asprepared lipid coating might not be in the state of equilibrium. The subsequent annealing process enables the system to achieve a state closer to the well-ordered lipid organization in SC. We applied a heating/cooling cycle to a spin coated sample with lipid ratio of CER[AP]: CHOL: SA of 1:0.3:1. The as-prepared sample showed well defined Kiessig fringes (Figure 5a, black curve), which allowed for a calculation of the total sample thickness of (215.3 ± 6.3) Å. The repeat distance of a single bilayer was (45.42 ± 0.66) Å, in line with the repeat distance reported for comparable mixtures.14,

32

In this phase of the lipid film, named phase A, all 3 lipid

components are expected to be present. After annealing the reflectivity curve (grey line) still showed Kiessig fringes and Bragg peaks. However, the high maxima and low minima of the defined peaks before annealing converge in a less defined reflectivity curve with broader peaks of lower intensity. Furthermore, the overall sample thickness decreased to (191.5 ± 5.1) Å as a result of a decreased membrane thickness of (40.52 ± 0.52) Å. It is known, that the L-enantiomer of CER[AP] can take a V-shaped conformation with a repeat distance of about 37 Å after exposure to a heating/cooling cycle.37 In combination with the untilted D-CER[AP], SA and CHOL a lamellar repeat distance of about 40 Å could result. The additional energy introduced into the system by the annealing process, would induce a tilt of the lipids with respect to the


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membrane normal and the increased lipid mobility would cause a less ordered lipid arrangement in the end. In consequence, the expected effect of a distinct increase of lipid order, as observed in multilayer models, does not emerge in our oligolamellar SC model.

Figure 5: Reflectivity curves of films from lipid mixtures containing CER[AP]: CHOL and SA before (black lines) and after annealing (grey lines). The reflectivity curves taken after annealing were shifted by a factor of 10 on the intensity axis. A PEI linker layer was used in each case. a) Oligolamellar SC model from 3.5 mg/ml lipid mixture (1:0.3:1 molar ratio; CHCl3/MeOH 15:1 (v/v)), spin coated on disc-shaped wafers. b) Multilamellar SC model from 10 mg lipid mixture (1:0.7:1 molar ratio; CHCl3/MeOH 2:1 (v/v)), air-brushed on a disc-shaped wafer at 50 °C.

In contrast to the oligolamellar SC lipid film, the multilamellar SC lipid film (multi-SC-model) is so thick that the Kiessig fringes in its reflectivity curve cannot be resolved anymore. Only the Bragg peaks remain (Figure 5). Before annealing these peaks, especially at high Q, are very weak (Figure 5b; black line), and indicate a poorly ordered powder-like sample. The Bragg peaks describe a multilayer sample composed of 3 different phases (Table 2). One of these phases (D), with a repeat distance of (34.28 ± 0.09) Å, can be assigned to phase-separated crystalline CHOL


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monohydrate.32,

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The other two phases with d = (44.82 ± 0.11) Å and d = (40.04 ± 0.13) Å

correspond to phase A and C, containing the lipids of the mixture but with different conformational states of the CER[AP]. Whereas under dry conditions in phase A the fully extended conformation of CER is energetically preferred,14 phase C comprises large amounts of V-shaped conformers. By the applied annealing, a fourth phase (B) with d = (42.26 ± 0.03) Å occurs. This phase can be assigned to phase-separated SA. Former studies revealed a SA monolayer thickness of about 21 Å, hence an expected d-spacing of the order of ૛ ∗ ૛૚ Å = ૝૛ Å .39, 40 It is known that in a SC lipid mixture a phase-separation of CERs and free fatty acids can occur.41 The phase-separation process was potentially enforced by the energy put into the system by annealing. Phases C and D remain unchanged, while the lamellar repeat distance of phase A increases to (46.57 ± 0.06) Å upon annealing. As described in the literature, the fully extended CER molecules in a dry environment change their conformation by a chain flip to the hairpin conformation, when exposed to humidity, higher than 60 %.14 Consequently the humidity of 100 % applied during annealing generated membranes with a higher percentage of CER molecules in hairpin conformation. The ceramides in hairpin conformation would accommodate within the same membrane lamella, rather than spanning to adjacent lamellae and thus would be responsible for the observed increase in membrane thickness. On basis of the recorded datasets, however, we can only speculate on the composition of the different lamellar phases. To prove one of these theories, further studies are necessary. Despite these uncertainties, it is clear, that the annealing procedure applied to the multilamellar SC model enhances the lipid ordering, indicated by the sharp Bragg peaks with high intensities (grey line) after annealing, while application of the same procedure to the oligolamellar SC model does not.


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Table 2: Calculated repeat distances, d, and total thickness, t, of the lipid mixtures depicted in Figure 5 before and after annealing. In case of the multilamellar sample, different lamellar distances occur and a possible composition is given in each case.

Oligo-SC-model

Multi-SC-model

Phase

before annealing

after annealing

before annealing

after annealing

possible composition

A

45.42 ± 0.66

-

44.82 ± 0.11

46.57 ± 0.06

CER[AP] + CHOL + SA

B

-

-

-

42.26 ± 0.03

SA

C

-

40.52 ± 0.52

40.04 ± 0.13

39.90 ± 0.01

CER[AP] + CHOL + SA

D

-

-

34.28 ± 0.09

34.13 ± 0.02

CHOL

215.3 ± 6.3

182.8 ± 2.3

-

-

d (Å)

t (Å)

In conclusion, the application of an annealing procedure has converse effects on the two SC models. Whereas in case of multilamellar samples the lipid order increases, it decreases in case of oligolamellar samples. The origin of these effects might be in the different preparation methods in each case. The preparation of the multi-SC-model involves spraying of the dissolved lipids on the wafer surface several times, such as to pile up thousands of relatively disordered bilayers on the silicon support. During subsequent annealing, these disordered lipid layers have a great potential to aim for a more ordered alignment. On the contrary, spin coating yields highly ordered thin layers from the very beginning26 with little potential to increase ordering during annealing.


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Attenuated Total Reflection Fourier Transform Infrared Spectroscopy IR measurements of oligolamellar samples show two distinct peaks at around 2920 cm-1 and 2850 cm-1 (Figure 6, grey line). They are characteristic for the symmetric and asymmetric CH2 stretching vibrations of the SC lipids, respectively. The position of these bands give detailed information on conformational order (trans/gauche ratio) and acyl chain packing. With an increasing amount of gauche conformers, the wavenumbers of both vibrations increase.42, 43 Such changes might be induced by interaction with an enhancer molecule. A third peak is visible at around 1700 cm-1. In this range, between 1800 cm-1 and 1500 cm-1, C=O stretching and N-H bending vibrations of the lipid mixture occur. Because of the complexity of the system, they cannot be assigned to one particular functional group. Applying penetration enhancers, these bands might also be affected or even new bands, characteristic for the enhancer molecule itself, might occur. At wavenumbers below 1400 cm-1, IR signals cannot be obtained, due to the low IR transmission of silicon in this spectral region. Oligolamellar and multilamellar samples show the symmetric and asymmetric CH2 stretching vibrations. In the case of the multi-SC-model, however, the peaks are much more defined (black line). Due to the higher amount of lipids in the multi-SC-model and hence a longer beam path through the sample, features at lower wavenumbers between 1800 cm-1 and 1500 cm-1 become observable.


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Figure 6: IR measurements against D2O of an airbrushed multi- (black line) and a spin coted oligolamellar (grey line) SC lipid model (CER[AP]: CHOL: SA; 1:0.7:1 molar ratio). A PEI linker layer was used in each case. Symmetric and asymmetric CH2 stretching vibrations occur at higher wavenumbers, C=O stretching and N-H bending vibrations at lower wavenumbers.

In conclusion, the most detailed information from IR measurements on SC lipid lamellar structures can be obtained using multilamellar lipid models. Here the peaks of the symmetric and asymmetric CH2 stretching vibrations are well pronounced and even at lower wavenumbers between 1800 and 1500 cm-1 many distinct signals occur. But, the investigation shows that also oligolamellar samples can be characterized by ATR-FTIR, resulting in a clear and analyzable signal. With respect to the prospective applications of hydrophilic penetration enhancers to the oligolamellar model system, it can be assumed, that occurring enhancer effects will be detectable.

SUMMARY In this study, we established a preparation method for a new SC lipid model containing CER[AP], CHOL and SA in an oligolamellar arrangement by spin coating. The method was


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adapted from a protocol applied successfully to single phospholipid linings.25, 26, 27 With respect to prospective studies on the influence of hydrophilic penetration enhancers or other drugs supplied from aqueous solution, integrity and stability of the solid-supported SC lipid model is essential and has been optimized. The new oligolamellar SC lipid model is reproducibly achieved by: (I)

Pre-coating the silicon support by a PEI molecular layer. This layer acts as an adsorption enhancer for the lipids.

(II)

Subsequent spin coating of the SC lipid model from a lipid solution (CER[AP]: CHOL: SA at 1:0.7:1 molar ratio) at a concentration of 3.5 mg/ml in chloroform:methanol 15:1 (v/v) at a rotation speed of 4000 rpm, immediately after step (I) and without intermediate drying.

(III)

Avoiding subsequent annealing procedures known from multilamellar SC lipid models.15

In comparison with the commonly used multilamellar assemblies the new oligolamellar SC model has several advantages: A.

The oligolamellar SC model is much closer to the native SC than the multilayer model.

B.

It is composed of one single phase, i.e. no phase separation occurs.

C.

Effects of drugs on the structure of the new oligo-SC-model can be assessed in full detail by X-ray or neutron reflectivity. Not only the lamellar spacing, but in addition the total thickness of the SC model and changes thereof can be monitored.


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D.

Potential conformational changes of the lipids in the olio-SC-model upon interaction with drugs stay assessable by ATR-FTIR spectroscopy despite the severe reduction in the number of lipid lamellae.

AUTHOR INFORMATION Corresponding Author * Prof. Dr. Dr. h.c. R. H. H. Neubert; Department of Pharmaceutics and Biopharmaceutics; Martin Luther University Halle-Wittenberg; Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germany; E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT The authors thank Evonik Industries AG (Essen, Germany) for providing the ceramide [AP]. Authors acknowledge granting of beam-time and financial support by Helmholtz-Zentrum Berlin (HZB). This work is funded by the Deutsche Forschungsgemeinschaft (Project: NE 427/30-1).

ABBREVIATIONS SC, stratum corneum; CER[AP], ceramide [AP]; CHOL, cholesterol; SA, stearic acid; FFA, free fatty acid; NR, neutron reflectivity; polyethyleneimine, PEI.


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TABLE OF CONTENTS GRAPHIC AND SYNOPSIS To mimic the native stratum corneum structure, a new oligolamellar lipid model was developed by a spin coating procedure. Here, the influence of different parameters was investigated during sample optimization. The resulting model is suitable for detailed investigations, especially of the influence of hydrophilic substances on the SC lipid organization.


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Preparation of a New Oligolamellar Stratum Corneum Lipid Model

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