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Langmuir 2000, 16, 4808-4812
Thermodynamic and Structural Aspects of Internal Wool Lipids Jordi Fonollosa,*,† Meritxell Martı´,† Alfons de la Maza,† Manel Sabe´s,‡ Jose´ Luis Parra,† and Luisa Coderch† Centro de Investigacio´ n y Desarrollo, CID, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain, and Facultat de Medicina, Universitat Auto` noma de Barcelona, Edifici M, 08193 Bellaterra (Barcelona) Spain Received August 10, 1999. In Final Form: December 13, 1999 Thermotropic physicochemical techniques such as differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and electron paramagnetic resonance (EPR) were applied to liposomes made up of internal wool lipids. These vesicles can be regarded as a model of wool membranes. Differences in fluidity obtained from EPR allow us to deduce a high rigidity of the polar external section of the vesicle in contrast to the high fluidity of the hydrophobic internal section, which accounts for the thermotropic properties of the liposome. The coincidence of the thermotropic changes obtained with the EPR, FTIR, and DSC techniques allows us to infer a reversible phase transition temperature in the range of 30-40 °C, indicating a transition from a mixture of hexagonal gel-liquid crystal state to a single liquid crystal state.
1. Introduction It is generally accepted that cell membrane lipids of wool govern the permeability of the fiber, which is essential in its dyeing and diffusion properties. Wool fiber is histologically made up of cuticle and cortical cells held together by the cell membrane complex which forms the only continuous phase in keratin fiber. The influence of this minor component, especially the lipids, on the physical, chemical, and mechanical properties of wool fibers is well-known.1 The internal wool lipid material (IWL), which accounts for only about 1.5% of total fiber weight, consists of three major lipid classes: sterols, free fatty acids, and polar lipids. The sterols consist mainly of cholesterol, desmosterol, and sterol esters. The main free fatty acids are stearic, palmitic, oleic, and 18-methyleicosanoic. The polar lipids consist predominantly of ceramides, cholesterol sulfate, and glycosphingolipids, only traces of phospholipids being detected.2-4 This lipid composition bears considerable resemblance to the one found in membranes from other keratinized tissues such as skin stratum corneum or human hair. The thermotropic phase behavior and structural parameters of lipids constituting the intercellular cement of human stratum corneum have been investigated over the past decade. Studies based on differential scanning calorimetry (DSC), infrared spectroscopy, or X-ray diffraction analysis can provide important insight into the function of these lipids and their role in the interactions of the stratum corneum with xenobiotic chemicals.5-8 * To whom correspondence may be addressed. Phone number: (34)934006179.FAXnumber: (34)932045904.E-mail:
[email protected]. † Centro de Investigacio ´ n y Desarrollo, CID, CSIC. ‡ Universitat Auto ` noma de Barcelona. (1) Leeder, J. D. Wool Sci. Rev. 1986, 63, 3. (2) Rivett, D. E. Wool Sci. Rev. 1991, 67, 1. (3) Ko¨rner, A.; Ho¨cker, H.; Rivett, D. E. Fresenius’ J. Anal. Chem. 1992, 344, 501. (4) Coderch, L.; de la Maza, A.; Soriano, C.; Erra, P.; Parra, J. L. J. Am. Oil Chem. Soc. 1995, 72, 715. (5) Golden, G. M.; Guzek, D. B.; Harris, R. R.; McKie, J. E.; Potts, R. O. J. Invest. Dermatol. 1986, 86, 255. (6) Bouwstra, J. A.; De Vries, M. A.; Gooris, G. S.; Bras, W.; Brussee, J.; Ponec, M. J. Controlled Release 1991, 15, 209.
Liposomes made up of stratum corneum lipids have been widely used as a model of skin-lipid bilayer, including possible interaction mechanisms between liposomes and several compounds, especially surfactants9,10 and retarders and enhancers.11-13 Changes in the lipid order have been related to the modification of their fluidity. This could be associated with the role of this lipid structure in skin permeation. In the case of wool, despite the advances made in characterizing the lipid composition of the cell membrane complex of wool, little progress has been made in furthering our understanding of structure, arrangement, and thermotropic behavior of these components. The cell membrane complex has different regions according to their dyeability. There are two resistant membranes, two unstained layers called the β-layers, and a dark-stained central layer, the δ layer. The β layers, which are generally believed to be made up of lipids, are assumed to form a bilayer structure. In fact, the bilayer-forming capability of internal wool lipids and some physicochemical properties have been studied in an attempt to improve our understanding of the lipid structure. Internal wool lipids were shown to form stable liposomes and also to be critical in the diffusion properties of the wool fibers,14,15 but not much work has been done on the study of their thermotropic behavior. This work deals with liposome formation as a model of a wool lipid membrane and with their thermotropic (7) Gay, C. L.; Guy, R. H.; Golden, G. M.; Mak, V. H. K.; Francocur, M. L. J. Invest. Dermatol. 1994, 103, 233. (8) Bonte´, F.; Pinguet, P.; Saunois, A.; Meybeck, A.; Beugin, S.; Ollivon, M.; Lesieur, S. Lipids 1997, 32, 653. (9) De la Maza, A.; Manich, A. M.; Coderch, L.; Baucells, J.; Parra, J. L. Colloid Surf., A 1996, 113, 259. (10) De la Maza, A.; Parra, J. L. Langmuir 1996, 12, 6218. (11) Hadgraft, J.; Peck, J.; Williams, D. G.; Pugh W. J.; Allan, G. Int. J. Pharm. 1996, 141, 17. (12) Yoneto, K.; Li, S. K.; Higuchi, W. I.; Jiskoot W.; Herron, J. N. J. Pharm. Sci. 1996, 85, 511. (13) Suhonen, T. M.; Pirskanen, L.; Ra¨isa¨nen, M.; Kosonen, K.; Rytting, J. H.; Paronen, P.; Urtti, A. J. Controlled Release 1997, 43, 251. (14) Ko¨rner, A.; Petrovic, S.; Ho¨cker, H. Textile Res. J. 1995, 65, 56. (15) Coderch, L.; de la Maza, A.; Pinazo, A.; Parra, J. L. J. Am. Oil Chem. Soc. 1996, 73, 1713.
10.1021/la991084w CCC: $19.00 © 2000 American Chemical Society Published on Web 04/15/2000
Thermodynamics of Internal Wool Lipids
physicochemical properties. We used the electron paramagnetic resonance (EPR) technique to monitor the fluidity and the arrangement of the liposomic bilayer and the Fourier transformed infrared spectroscopy (FTIR) technique to characterize the domain organization and the conformation order of the lipid molecules building the bilayer. Furthermore, we applied quasielastic light scattering (QELS) to determine size distribution and also differential scanning calorimetry (DSC) to obtain thermal transition temperature. Many of these physical techniques have been extensively used in characterizing phospholipid vesicle systems,16,17 and recently they have also been applied to stratum corneum lipid systems;18-20 however, they have not been applied to wool lipid systems. Advances in the knowledge of internal wool lipid arrangement will help us to understand the behavior of this minor fraction, which is so important in wool permeation. 2. Experimental Section 2.1. Materials. Raw industrially scoured Spanish Merino wool supplied by CORCOY S.A. (Terrassa, Spain) was used to obtain the internal wool lipids. Prior to the extraction, wool was equilibrated in a conditioned room (20 °C, 60% relative humidity). All chemicals were of analytical grade, and the standards used were supplied by SIGMA Co. (St. Louis, MO) in the case of Ceramides type III and cholesterol sulfate and by Fluka (Buchs, Switzerland) in the case of cholesteryl-palmitate, palmitic acid, and cholesterol. Spin labels 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA) were also purchased from SIGMA Co. Lipoid S-100, whose main component is soybean phosphatidylcholine (>95%) was obtained from LIPOID GmbH (Ludwigshafen, Germany). All these chemicals were stored in chloroform/ methanol (2:1) under freezing temperatures until their use. Tris(hydroxymethyl)aminomethane (TRIS buffer) supplied by MERCK (Darmstadt, Germany) was prepared as 5 mM TRIS buffer adjusted to pH 7.40 with HCl, containing 100 mM of NaCl. 2.2. Methods. 2.2.1. Preparation and Characterization of Liposomes. The internal lipids were Soxhlet extracted from cleaned wool (12 g) with chloroform/methanol azeotrope (360 mL, 79:21 v/v) for 5 h. The lipid extracts were concentrated down to 10 mL under a stream of dry nitrogen and stored in 2/1 chloroform/methanol at 6 °C. Aliquots were dried and weighed, and the lipid percentage extractions were quantified.4,21 Quantitative analysis was performed using thin-layer chromatography (TLC) coupled to an automated ionization detection (FID) system (Iatroscan MK-5, IATRON LAB. INC. Tokyo, Japan) with a sample spotter SES 3202/IS-01 (SES GmbH, Nieder-Olm, Germany) using an optimized analytical procedure.15 Samples of 1 mL of IWL solution (10 mg/mL chloroform/ methanol 2:1) were taken to dryness in culture tubes with a stream of nitrogen. Buffer containing 100 mM NaCl and 5 mM TRIS was added (1 mL) to provide a final lipid concentration of approximately 10 mg/mL. Suspensions were then sonicated in a sonicator Labsonic 1510 (B. BRAUN, Melsungen, Germany) at 100 W with a thermostated bath Ultraterm 6000383 (SELECTA, Barcelona, Spain) at a temperature of 65 °C for about 15 min until the suspensions became homogeneous. The preparations were then annealed at the same temperature for 30 min and incubated at 37 °C under nitrogen atmosphere. Phosphatidylcholine (PC) liposomes were prepared with Lipoid S-100 using the same methodology described above to achieve the same final lipid concentration of 10 mg/mL in TRIS buffer. (16) Nagumo, A.; Sato, Y.; Suzuki, Y. Chem. Pharm. Bull. 1991, 39, 3071. (17) Constanzo, R.; De Paoli, T.; Ihlo, J. E.; Hager, A. A.; Farach, H. A.; Poole, C. P.; Knight, J. M. Spectrochim. Acta 1994, 50A (2), 203. (18) Hou, S. Y. E.; Rehfeld, S. J.; Plachy, W. Z. Adv. Lipid Res. 1991, 24, 141. (19) Blume, A.; Jansen, M.; Ghycsy, M.; Gareiss, J. Int. J. Pharm. 1993, 99, 219. (20) Hatfield, R. M.; Fung, L. W. M. Biophys. J. 1995, 68, 196. (21) Gale, D. J.; Logan R. I.; Rivett, D. E. Textile Res. J. 1987, 57, 539.
Langmuir, Vol. 16, No. 11, 2000 4809 Mean vesicle size distribution and polydispersity indexes of the liposomes were determined using a photon correlator spectrometer, Malvern Autosizer 4700c PS/MV (MALVERN, Malvern, U.K.), by particle number measurement at 37 °C with a lecture angle of 90°. 2.2.2. Fourier Transformed Infrared Spectroscopy. In IR measurements, IWL liposomes (performed as described above at concentration about 28 mg/mL in TRIS buffer) and buffer references were placed in CaF2 windows with a 15 µm Teflon spacing. IR spectra were recorded on a Mattson Polaris Fourier transform spectrophotometer (MATTSON INS. GmbH, Stuttgart, Germany). To minimize water interference, a continuous dry air flow (
