Conformational Changes in Stratum Corneum Lipids by Effect of

pubs.acs.org/Langmuir © 2009 American Chemical Society

Conformational Changes in Stratum Corneum Lipids by Effect of Bicellar Systems )

Gelen Rodrı´ guez,*,† Lucyanna Barbosa-Barros,† Laia Rubio,† Mercedes C
ocera,‡ Avencia Dı´ ez,§ Joan Estelrich, Ramon Pons,† Jaume Caelles,† Alfonso De la Maza,† and Olga L
opez† Departament de Tecnologia Quı´mica i de Tensioactius, Institut de Quı´mica Avanc-ada de Catalunya (IQAC), Consejo Superior de Investigaciones Cientı´ficas (CSIC), C/ Jordi Girona 18-26, 08034 Barcelona, Spain, ‡BM16, European Synchrotron Radiation Facility, Grenoble, France, §Servicio de Espectrofotometrı´a, IQAC-CSIC, C/ Jordi Girona 18-26, 08034 Barcelona, Spain, and Departamento de Fisicoquı´mica, Facultad de Farmacia, Universidad de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain )

†

Received April 21, 2009. Revised Manuscript Received June 17, 2009 Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was applied to study the effects of the bicelles formed by dimyristoyl-glycero-phosphocholine (DMPC) and dihexanoyl-glycero-phosphocholine (DHPC) in porcine stratum corneum (SC) in vitro. A comparison of skin samples treated and untreated with bicelles at different temperatures was carried out. The analysis of variations after treatment in the position of the symmetric CH2 stretching, CH2 scissoring, and CH2 rocking vibrations reported important information about the effect of bicelles on the skin. Bicellar systems caused a phase transition from the gel or solid state to the liquid crystalline state in the lipid conformation of SC, reflecting the major order-disorder transition from hexagonally packed to disordered chains. Grazing incidence small and wide X-ray scattering (GISAXS and GIWAXS) techniques confirmed this effect of bicelles on the SC. These results are probably related to with the permeabilizing effect previously described for the DMPC/DHPC bicelles.

Introduction Bicelles are discoidal aggregates constituted by long and short chain amphiphiles in water.1 These aggregates are used as membrane models providing an excellent support to study membrane associated proteins. Additionally, recent works propose the use of phospholipid bicelles for dermatological applications owing to their small size, suitable enough for passing through the skin, and their composition, consisting completely of lipids. These studies demonstrated that the effect of bicelles on the skin barrier depend on different compositional variables, working as permeabilizing agents of the skin or as reinforcing agents of the lipid structures present in this tissue.2,3 Despite the potential applicability of former results, knowledge about the mechanism that induces these specific effects is still lacking. The main function of the skin is to serve as a physical barrier at the interface between the body and the environment. This barrier is provided by the outermost layer of the epidermis, the stratum corneum (SC), which consists in anucleated cells (corneocytes) surrounded by lipid layers formed mainly by of ceramides, cholesterol, and fatty acids.4-6 The structural organization of this lipid matrix has been widely studied;7,8 however, consensus *To whom correspondence should be addressed. E-mail: gelen.rodriguez@ iiqab.csic.es. Phone: 34-93 400 61 00. Fax: 34-93 204 59 04. (1) Sanders, C. R.; Hare, B. J.; Howard, K. P.; Prestegard, J. H. Prog. NMR Spectrosc. 1994, 26, 421–444. (2) Barbosa-Barros, L.; Barba, C.; C
ocera, M.; Coderch, L.; L
opez-Iglesias, C.; de la Maza, A.; L
opez, O. Int. J. Pharm. 2008, 352, 263–272. (3) Barbosa-Barros, L.; de la Maza, A.; Estelrich, J.; Linares, A. M.; Feliz, M.; Walther, P.; Pons, R.; L
opez, O. Langmuir 2008, 24, 5700–5706. (4) Schaefer, H.; Redelmeier, T. E., Skin Barrier: Principles in Percutaneous Penetration; Karger: Basel, Switzerland, 1996; pp 55-58. (5) L
opez, O.; C
ocera, M.; L
opez-Iglesias, C.; Walter, P.; Coderch, L.; Parra, J. L.; de la Maza, A. Langmuir 2002, 18, 7002–7008. (6) Wertz, P. W.; Downing, D. T. J. Lipid Res. 1983, 24(6), 759–765. (7) L
opez, O.; C
ocera, M.; P.W., W.; L
opez-Iglesias, C.; de la Maza, A. Biochim. Biophys. Acta 2007, 1768, 521–529. (8) Elias, P. M.; Feingold, K. R. Semin. Dermatol. 1992, 11(2), 176–182. (9) Plasencia, I.; Norl
en, L.; Bagatolli, L. A. Biophys. J. 2007, 93, 3142–3155.

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about a unique model describing the lipid packing does not exist.9 Thus, several models have been proposed: In the “mosaic fluid model” described by Forslind,10,11 the lipids are organized in gel and liquid-crystalline domains. The “single gel phase model” by Norl
en12 claims an exclusively lipid gel phase organization and the “sandwich model” by Bouwstra13 proposes a central liquidcrystalline layer and a gel-crystalline structure located at both sides of this central layer. Other models such as the “armature reinforcement model” by Kiselev14 explains the structural alteration of the SC lipid matrix in water excess as a consequence of the transformation of the ceramide molecules from the fully extended to the hairpin conformation. These findings are in part based on studies using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray scattering techniques. Although the chemical composition of the SC is very heterogeneous, the exhaustive analysis of band positions by ATR-FTIR has reported relevant data on the lipid organization of native SC, allowing detection of the alkyl chain vibrations of the lipids and consequently to know the lipid conformational order and alkyl chain packing.15 Figure 1 shows schematically the chain conformation and the lateral chain packing in different phases. In the orthorhombic (OR) phase, the alkyl chains adopt all-trans conformation and are organized in a rectangular crystalline lattice. The neighboring molecular distance is not equal, being about 3.7 and 4.1 A˚.16 In the hexagonal (HEX) phase, the all-trans (10) Forslind, B. Acta Derm.-Venereol. 1994, 74, 1–6. (11) Forslind, B.; Engstroem, S.; Engblom, J.; Norl
en, L. J. Dermatol. Sci. 1997, 14, 115–125. (12) Norl
en, L. J. Invest. Dermatol. 2001, 117, 830–836. (13) Bouwstra, J. A.; Dubbelaar, F. E.; Gooris, G. S.; Ponec., M. Acta Derm.Venereol. 2000, 208(Suppl.), 23–30. (14) Kiselev, M. A.; Ryabova, N. Y.; Balagurov, A. M.; Dante, S.; Hauss, T.; Zbytovska, J.; Wartewig, S.; Neubert, R. H. H. Eur. Biophys. J. 2005, 34, 1030– 1040. (15) Boncheva, M.; Damien, F.; Normand, V. Biochim. Biophys. Acta 2008, 1778, 1344–1355. (16) de Jager, M. W.; Gooris, G. S.; Ponec, M.; Bouwstra, J. A. J. Lipid Res. 2005, 46, 2649–2656.

Published on Web 07/14/2009

DOI: 10.1021/la901410h

10595

Article

Rodrı´guez et al. washing process, sodium lauryl ether sulfate (SLES) solution at 0.5% w/v from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) was used. To isolate the stratum corneum from the full skin, trypsin (from porcine pancreas) from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and phosphate buffered saline tablets from Sigma Chemical CO. (St. Louis, MO) were used. Skin Preparation. For ATR-FTIR Experiments. Porcine skin was obtained from the back of experimental animals in the Department of Dermatology, University Hospital Clinic of Barcelona (Spain), 2-3 h after the animals were sacrificed. The bristles were removed carefully with an animal clipper and then the skin was washed with tap water. The excised skin was dermatomed to 500 ( 50 μm thickness (Dermatome GA630, Aesculap, Tuttlingen, Germany). Then, full skin containing dermis, epidermis, and SC was used to perform the experiments. Pieces were vacuum-packed and stored at 4 °C until use.

Isolation of the SC for X-ray Scattering Experiments.

Figure 1. Scheme of the chain conformation (left column) and lateral chain packing (right column) of alkyl chain lipids in orthorhombic (OR), hexagonal (HEX), and liquid-crystalline (LIQ) phases, with molecular distances (scheme based on that reported by Pilgram et al.18).

alkyl chains are tilted with respect to the crystal plane and are organized in a less dense, hexagonal lattice, with a constant distance between neighboring molecules around 4.1 A˚.16 In the liquid (LIQ) phase, the chains exhibit a high degree of gauche isomerization and the lateral organization is entirely lost.15,17,18 In this phase, the distance between the molecules is not well-defined, although it has been reported as around 4.6 A˚.16 These three phases are easily discernible by IR spectroscopy analyzing different vibrational modes as asymmetric and symmetric CH2 stretching frequency that monitors lipid conformational order, and CH2 scissoring and rocking frequencies, which are sensible markers of lateral chain packing. X-ray scattering studies also reported on phases and organization of lipids.19 Whereas small-angle X-ray scattering provides information about the larger structural units, namely the repeat distance of the lipid lamellar phases; wide-angle X-ray scattering reports about the lateral packing of the lipids within the lamellae that informs about the aforementioned distance between neighboring molecules.20 This study seeks to clarify the molecular organization of the native SC lipids and to investigate the effect of bicelles over the lipid phase transitions in this tissue. Our results point to a lipid scenario formed exclusively by a gel phase organization at the skin physiological temperature, which evolves to a liquid crystalline packing after treatment with bicelles. The modification of the phase behavior of the SC by the bicelles seems to be the responsible for of the skin barrier modulation induced by bicelles.

Materials and Methods Chemicals. Bicelles were formed by 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1, 2-dihexanoyl-sn-Glycero3-phosphocholine (DHPC) purchased from Avanti Polar Lipids (Alabaster, AL). Purified water was obtained by an ultrapure water system, Milli-Q plus 185 (Millipore, Bedford, MA). In the (17) Mendelsohn, R.; Moore, D. J. Chem. Phys. Lipids 1998, 96, 141–157. (18) Pilgram, G. S. K.; Petl, A. M. E.; Koerten, H. K.; Bouwstra, J. A. Pharm. Res. 2000, 17, 796–802. (19) Pereira-Lachataignerais, J.; Pons, R.; Amenitsch, H.; Rappolt, M.; Sartori, B.; L
opez, O. Langmuir 2006, 22(12), 5256–5260. (20) Garson, J. C.; Doucet, J.; Leveque, J. L.; Tsoucaris, G. J. Invest. Dermatol. 1991, 96(1), 43–49.

10596 DOI: 10.1021/la901410h

Sections of fresh pig skin were heated with the dermal side in contact with a metal plate for 10 s at 80 °C, and the epidermis was scraped off in sheets. To isolate the SC the epidermal sheets were incubated for 2 h at 37 °C with the epidermal side in contact with a solution of 0.5% trypsin in phosphate-buffered saline at pH 7.4. After this time, the SC was washed with abundant Milli-Q water to remove the Trypsin.

Preparation and Characterization of the Bicellar System. Samples were prepared by mixing appropriate amounts of DMPC and DHPC chloroform solutions to reach DMPC/DHPC molar ratio, q =2. This molar ratio was previously described for structures with about 20 nm of diameter.21 After mixing the components, the chloroform was removed with a rotary evaporator and the systems were hydrated with water to reach 20% (w/v) of total lipid concentration. Bicellar solutions were prepared by subjecting the sample to several cycles of sonication and freezing until sample became transparent.22 The systems were analyzed by dynamic light scattering (DLS) and maintained under refrigeration (≈4 °C) during 7 days. Dynamic Light Scattering. The hydrodynamic diameter (HD) and polydispersity index (PDI) were determined by means of DLS using a Zetasizer Nano ZS90 (Malvern Systems, Southborough, MA), which provides the size distribution curves. The DLS measures the Brownian motion of the particles and correlates this to the particle sizes. The relationship between the size of a particle and its speed due to Brownian motion is defined in the Stokes-Einstein equation: HD ¼ kT=3πηD where HD is the hydrodynamic diameter, D is the translational diffusion coefficient (m2/s), k is the Boltzmann’s constant (1.3806503  10-23 m2 kg s-2 k-1), T is the absolute temperature (K), and η is the viscosity (mPa 3 s). The particle sizes are determined by detection and analysis of scattered light when a 632 nm He/Ne laser beam incidents in the particles. The measurements were performed at experimental temperatures (32 °C, 37 °C, 45 °C). The interpretation of the data was made by considering the size distribution by intensity and by volume of light scattered. All data were obtained with the software provided by Malvern Instruments. Treatment of Skin Tissues with Bicelles. Three pieces of whole skin with dimensions of approximately 2 cm  4 cm were used to perform this experiment, which was performed by triplicate. One of the tissues was kept untreated (control sample), and the others were treated with the bicellar system. The treatment was carried out placing the skin pieces SC side up, over a thin layer of water (but not submerged), on a Petri plate at 37 °C controlled by (21) Marcotte, I.; Auger, M. Concept Magn. Res. 2005, 24, 17–37. (22) Soong, R.; Macdonald, P. M. Langmuir 2009, 25(1), 380–90.

Langmuir 2009, 25(18), 10595–10603

Rodrı´guez et al. a thermostat. Under these specific conditions, the samples were maintained as completely hydrated. Bicelles (40 μL) were applied with a spatula on the skin surface, and after 1 h, samples were washed with Milli-Q water, left to dry, and treated again. This procedure was repeated three times more. At the end of the treatment, one of the samples was washed with Mill-Q water and the other was first washed with SLES solution (at 0.5% w/v) and then with Milli-Q water. IR Experiments. Infrared spectra of the sample tissue were obtained using a 360-FTIR spectrophotometer Nicolet Avatar (Nicolet Instruments, Inc., Madison, WI) equipped with a 45° ZnSe thermal horizontal attenuated total reflection (ATR) crystal. All spectra were the average of 256 interferograms, collected within a period of 7 min at 2 cm-1 resolution, over the 4,000-700 cm-1 region. Although the FTIR had a data collection resolution of 2 cm-1, interpolation between points was reliable because the noise level was low and the reproducibility and precision of FTIR spectra was high. In fact, the constant typical error of our measurements was (0.18 cm-1. To collect the IR spectra, the skin sample was placed SC side down onto the ZnSe ATR crystal. To ensure reproducible contact between the sample and the crystal, a weight of 260 g was applied on to the samples. The spectra were collected at different temperatures: 32 °C (skin temperature), 37 °C (physiological temperature), and 45 °C, transition temperature described for SC lipids.9,23,24 The temperature was controlled by a temperature controller integrated in the ATR device and by an external thermostatic camera. The samples were placed on the equipment 30 min before collecting the spectra for temperature equilibration. Fitting Procedure. To resolve the IR peaks, a number of individual Gaussian peaks were calculated to fit a complex set of overlapping peaks in the original spectrum. The operation was performed on selected spectrum region using a linear baseline correction. The algorithm used to find peaks looks looked for minima in the Savitsky-Golay second derivative of the selected regions of the spectrum. A polynomial order of 3 was was used to calculate this derivative. The peak fitting is an automatic process where the peak center, height, and width are adjusted to produce a composite spectrum that matches the original (developed by Omnic program). All the peaks fitting were calculated with a standard error