- Email: [email protected]
Structural alterations of fully hydrated human stratum corneum
ARTICLE IN PRESS
Physica B 350 (2004) e603–e606
Structural alterations of fully hydrated human stratum corneum G.Ch. Charalambopouloua,*, Th.A. Steriotisa, Th. Haussb, A.K. Stubosa, N.K. Kanellopoulosa a
National Center for Scientific Research ‘‘Demokritos’’, Ag. Paraskevi Attikis, Athens 15310, Greece b Hahn-Meitner-Institut, BENSC, Glienicker Strasse 100, Berlin D-14109, Germany
Abstract The diffusional barrier function of skin is associated with the superficial epidermal layer, the stratum corneum, a highly complex biomembrane consisting of a staggered corneocyte arrangement in a lipid lamellar continuum. One of the key elements for stratum corneum barrier function is its hydration state. In the present work, the membrane neutron diffraction method is employed to reveal important stratum corneum structural changes that emanate upon water uptake. Increasing stratum corneum water content was observed to lead reversibly to the progressive disruption of the highly ordered lipid configuration and the distortion of the system’s barrier function. r 2004 Elsevier B.V. All rights reserved. Keywords: Stratum corneum; Hydration; Lamellar structure; Neutron diffraction
1. Introduction The skin is a highly complex organ whose primary function is to protect the body against physical, chemical or pathogen factors. Its barrier function resides in the superficial epidermal layer, the stratum corneum (SC), a compositionally and morphologically unique biomembrane [1]. SC forms a staggered arrangement of compressed keratin-filled corneocytes embedded in a lipid continuum, filled with a multilamellar bilayer structure, composed primarily of cholesterol, fatty acids, sphingolipids and ceramides. One of the key *Corresponding author. Fax: +30-210-6511766. Email-address: [email protected] (G.Ch. Charalambopoulou).
elements for SC barrier function is its hydration state. Water, a natural constituent of SC, affects its plasticity, while acting as a penetration enhancer. Nevertheless, the actual mechanism of water–SC interaction is yet unresolved. A wide range of methods has been employed for this purpose, leading quite often to contradictory conclusions about the effect of water on lipid organization [2,3]. In the present work, the membrane neutron diffraction method, which is advantageous for the study of biological structures, was employed aiming to reveal important SC structural changes that emanate upon water uptake. Some preliminary neutron diffraction studies performed by the authors [4] showed that excess hydration damages the intercellular region architecture. It was the primary aim of the present work to further
0921-4526/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.physb.2004.03.161
ARTICLE IN PRESS e604
G.Ch. Charalambopoulou et al. / Physica B 350 (2004) e603–e606
investigate those observations and prove that this behaviour is a general trend and not a result of biological variability.
2. Experimental The SC samples used were separated from human abdomen skin, obtained after cosmetic surgery and processed the same day. After removing the subcutaneous fat, the skin was dermatomed to a thickness of B300 mm and then incubated overnight at 4 C and for 1 h at 37 C with its dermal side on Whatman paper soaked with a solution of 0.1% w/v trypsin in 0.15 M Phosphate Buffer Saline. The SC was then peeled from the epidermis, equilibrated at 37 C and stored over silica gel. The diffraction measurements were carried out on the V1 Membrane Diffractometer at the Berlin Neutron Scattering Center (Hahn-Meitner Institute). The biological variability of SC was tackled by using epidermis samples originating from five donors. All samples were exposed initially to vapour and then excess D2O conditions (after measuring the vapour equilibrated sample, liquid D2O was added in situ in the sample cell and a new run was performed with the same experimental set-up) for time periods up to 24 h. A special, gas tight Al cell which could accommodate a stack of two or three SC layers, was used. After aligning the sample parallel to the beam, y22y scans from 0 to 20 were performed.
Fig. 1. The diffraction curves of human stratum corneum at various excess D2O exposure times (intensities are in arbitrary units).
3. Results and discussion All SC samples equilibrated over D2O vapours, exhibited a diffraction peak at Q ¼ 1:1 nm1 (curves a, Figs. 1 and 2), indicating that the SC intercellular ultrastructure consists of domains built up of a 5.7 nm unit cell that reflects the size of one lipid bilayer, in good agreement with literature data [5,6]. The scattering pattern altered significantly after loading the sample cell with liquid D2O. The increased D2O concentration improved the signal of the sample, in terms of intensity, but most importantly the position of the
Fig. 2. The recovery of stratum corneum from prolonged hydration.
ARTICLE IN PRESS G.Ch. Charalambopoulou et al. / Physica B 350 (2004) e603–e606
main peak was slightly shifted to smaller Q values implying that a small increase in spacing occurs due to increased hydration. Indeed moving from 100% RH to excess D2O conditions (curve b, Fig. 1) results in an increase of the repeated characteristic distance from 5.7 nm (Q ¼ 1:1 nm1) to 6.2 nm (Q ¼ 1:0 nm1). In an effort to monitor the evolution of the structural change induced by excess hydration, a series of subsequent scans was performed (Fig. 1). In this way, a trend towards a progressive increase of the total intensity, a slight transposition of the main peak to lower Q and a sustained deformation of the scattering pattern with exposure time, was revealed, demonstrating that the alteration of SC intercellular morphology due to overhydration exposure is most likely an existing mechanism. Ultimately, after 22 h of excess D2O exposure, a smooth curve was obtained. As the process under investigation is D2O sorption, and should therefore evolve in a relaxation mode, the disappearance of the diffraction peak should rather be attributed to a framework collapse (i.e. an extensive rupture of the lipid organisation) and not to a sudden D2O uptake, which would lead to a dramatic increase of the lamellar spacing beyond the instrumental limits. Due to the large difference between the scattering length density of D2O and the lipids, the increased deuterium content enhances the contrast between the two phases, and thus the obtained signal is attributed to alternating D2O layers. The sustained increase of the scattering pattern’s total intensity and the small shift of the main peak’s position implies that this behaviour is most likely associated with the swelling of the bilayer regions which occurs as a result of the increase in the thickness of the D2O layers that form between the adjacent polar lipid headgroups, contrary to earlier X-ray diffraction results [7] which reported that almost no water is present in the headgroup region. As water exposure prolongs, distinct water domains emanate in the intercellular space, greatly affecting the stability of the lamellae packing configuration which can turn into a disorganised, less cohesive domain with increased fluidity, explaining the generally accepted function of water as a very effective natural transdermal penetration enhancer. This mechanism is also supported by
e605
other studies which report water phase separations with ‘‘water pools’’, vesicle-like structures or lipid delamination in the intercellular space [8,9]. The water-induced disorder of the intercellular architecture was proved to be reversible to a significant extent. The experimental procedure described above (exposure to D2O vapour atmosphere—curve a, Fig. 2—and subsequently in situ immersion in D2O) was repeated for a new sample. After verifying that a smooth pattern (curve b, Fig. 2) was obtained after the 24 h excess hydration of the sample, this was left for sufficient time (B48 h) to re-equilibrate in D2O vapours before running a new measurement, which revealed that the removal of the destabilising water allowed the re-assembly of the lipid bilayers into a lamellar configuration since the main peak was partly recovered (curve c, Fig. 2). This remarkable observation is in accordance with the tendency of native skin to restore its biophysical properties after its prolonged exposure to water (during bathing, swimming, etc.).
4. Conclusions In the present work, the membrane neutron diffraction method is employed in order to elucidate the evolution of the response of SC intercellular architecture to excess hydration conditions. The increase of the sample’s water content progressively disrupts the highly ordered lipid lamellae, leading therefore to a degradation of the barrier function of the system. Nevertheless the process is proven to be rather reversible.
Acknowledgements The authors thank Dr. Joke Bouwstra (Leiden University, The Netherlands) for providing the SC samples. Support by BENSC, Hahn-Meitner Institute (Berlin, Germany) and EC (TMR Contract HPRI-CT-2001-00138, PROTOP Contract EVK3CT-2002-30004) is also acknowledged.
ARTICLE IN PRESS e606
G.Ch. Charalambopoulou et al. / Physica B 350 (2004) e603–e606
References [1] [2] [3] [4]
P.M. Elias, J. Invest. Dermatol. 80 (1983) 44. V.M. Mak, et al., Pharm. Res. 8 (1991) 1064. A. Alonso, et al., Biochim. Biophys. Acta 1237 (1995) 6. G.Ch. Charalambopoulou, et al., Appl. Phys. A 74s (2002) 1245.
[5] J.A. Bouwstra, et al., J. Lipid. Res. 36 (1995) 685. [6] G.Ch. Charalambopoulou, et al., Physica B 276–278 (2000) 530. [7] J.A. Bouwstra, et al., Int. J. Pharm. 84 (1992) 205. [8] D.A. Van Hal, et al., J. Invest. Dermatol. 106 (1996) 89. [9] R.R. Warner, et al., J. Invest. Dermatol. 120 (2003) 275.
Physica B 350 (2004) e603–e606
Structural alterations of fully hydrated human stratum corneum G.Ch. Charalambopouloua,*, Th.A. Steriotisa, Th. Haussb, A.K. Stubosa, N.K. Kanellopoulosa a
National Center for Scientific Research ‘‘Demokritos’’, Ag. Paraskevi Attikis, Athens 15310, Greece b Hahn-Meitner-Institut, BENSC, Glienicker Strasse 100, Berlin D-14109, Germany
Abstract The diffusional barrier function of skin is associated with the superficial epidermal layer, the stratum corneum, a highly complex biomembrane consisting of a staggered corneocyte arrangement in a lipid lamellar continuum. One of the key elements for stratum corneum barrier function is its hydration state. In the present work, the membrane neutron diffraction method is employed to reveal important stratum corneum structural changes that emanate upon water uptake. Increasing stratum corneum water content was observed to lead reversibly to the progressive disruption of the highly ordered lipid configuration and the distortion of the system’s barrier function. r 2004 Elsevier B.V. All rights reserved. Keywords: Stratum corneum; Hydration; Lamellar structure; Neutron diffraction
1. Introduction The skin is a highly complex organ whose primary function is to protect the body against physical, chemical or pathogen factors. Its barrier function resides in the superficial epidermal layer, the stratum corneum (SC), a compositionally and morphologically unique biomembrane [1]. SC forms a staggered arrangement of compressed keratin-filled corneocytes embedded in a lipid continuum, filled with a multilamellar bilayer structure, composed primarily of cholesterol, fatty acids, sphingolipids and ceramides. One of the key *Corresponding author. Fax: +30-210-6511766. Email-address: [email protected] (G.Ch. Charalambopoulou).
elements for SC barrier function is its hydration state. Water, a natural constituent of SC, affects its plasticity, while acting as a penetration enhancer. Nevertheless, the actual mechanism of water–SC interaction is yet unresolved. A wide range of methods has been employed for this purpose, leading quite often to contradictory conclusions about the effect of water on lipid organization [2,3]. In the present work, the membrane neutron diffraction method, which is advantageous for the study of biological structures, was employed aiming to reveal important SC structural changes that emanate upon water uptake. Some preliminary neutron diffraction studies performed by the authors [4] showed that excess hydration damages the intercellular region architecture. It was the primary aim of the present work to further
0921-4526/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.physb.2004.03.161
ARTICLE IN PRESS e604
G.Ch. Charalambopoulou et al. / Physica B 350 (2004) e603–e606
investigate those observations and prove that this behaviour is a general trend and not a result of biological variability.
2. Experimental The SC samples used were separated from human abdomen skin, obtained after cosmetic surgery and processed the same day. After removing the subcutaneous fat, the skin was dermatomed to a thickness of B300 mm and then incubated overnight at 4 C and for 1 h at 37 C with its dermal side on Whatman paper soaked with a solution of 0.1% w/v trypsin in 0.15 M Phosphate Buffer Saline. The SC was then peeled from the epidermis, equilibrated at 37 C and stored over silica gel. The diffraction measurements were carried out on the V1 Membrane Diffractometer at the Berlin Neutron Scattering Center (Hahn-Meitner Institute). The biological variability of SC was tackled by using epidermis samples originating from five donors. All samples were exposed initially to vapour and then excess D2O conditions (after measuring the vapour equilibrated sample, liquid D2O was added in situ in the sample cell and a new run was performed with the same experimental set-up) for time periods up to 24 h. A special, gas tight Al cell which could accommodate a stack of two or three SC layers, was used. After aligning the sample parallel to the beam, y22y scans from 0 to 20 were performed.
Fig. 1. The diffraction curves of human stratum corneum at various excess D2O exposure times (intensities are in arbitrary units).
3. Results and discussion All SC samples equilibrated over D2O vapours, exhibited a diffraction peak at Q ¼ 1:1 nm1 (curves a, Figs. 1 and 2), indicating that the SC intercellular ultrastructure consists of domains built up of a 5.7 nm unit cell that reflects the size of one lipid bilayer, in good agreement with literature data [5,6]. The scattering pattern altered significantly after loading the sample cell with liquid D2O. The increased D2O concentration improved the signal of the sample, in terms of intensity, but most importantly the position of the
Fig. 2. The recovery of stratum corneum from prolonged hydration.
ARTICLE IN PRESS G.Ch. Charalambopoulou et al. / Physica B 350 (2004) e603–e606
main peak was slightly shifted to smaller Q values implying that a small increase in spacing occurs due to increased hydration. Indeed moving from 100% RH to excess D2O conditions (curve b, Fig. 1) results in an increase of the repeated characteristic distance from 5.7 nm (Q ¼ 1:1 nm1) to 6.2 nm (Q ¼ 1:0 nm1). In an effort to monitor the evolution of the structural change induced by excess hydration, a series of subsequent scans was performed (Fig. 1). In this way, a trend towards a progressive increase of the total intensity, a slight transposition of the main peak to lower Q and a sustained deformation of the scattering pattern with exposure time, was revealed, demonstrating that the alteration of SC intercellular morphology due to overhydration exposure is most likely an existing mechanism. Ultimately, after 22 h of excess D2O exposure, a smooth curve was obtained. As the process under investigation is D2O sorption, and should therefore evolve in a relaxation mode, the disappearance of the diffraction peak should rather be attributed to a framework collapse (i.e. an extensive rupture of the lipid organisation) and not to a sudden D2O uptake, which would lead to a dramatic increase of the lamellar spacing beyond the instrumental limits. Due to the large difference between the scattering length density of D2O and the lipids, the increased deuterium content enhances the contrast between the two phases, and thus the obtained signal is attributed to alternating D2O layers. The sustained increase of the scattering pattern’s total intensity and the small shift of the main peak’s position implies that this behaviour is most likely associated with the swelling of the bilayer regions which occurs as a result of the increase in the thickness of the D2O layers that form between the adjacent polar lipid headgroups, contrary to earlier X-ray diffraction results [7] which reported that almost no water is present in the headgroup region. As water exposure prolongs, distinct water domains emanate in the intercellular space, greatly affecting the stability of the lamellae packing configuration which can turn into a disorganised, less cohesive domain with increased fluidity, explaining the generally accepted function of water as a very effective natural transdermal penetration enhancer. This mechanism is also supported by
e605
other studies which report water phase separations with ‘‘water pools’’, vesicle-like structures or lipid delamination in the intercellular space [8,9]. The water-induced disorder of the intercellular architecture was proved to be reversible to a significant extent. The experimental procedure described above (exposure to D2O vapour atmosphere—curve a, Fig. 2—and subsequently in situ immersion in D2O) was repeated for a new sample. After verifying that a smooth pattern (curve b, Fig. 2) was obtained after the 24 h excess hydration of the sample, this was left for sufficient time (B48 h) to re-equilibrate in D2O vapours before running a new measurement, which revealed that the removal of the destabilising water allowed the re-assembly of the lipid bilayers into a lamellar configuration since the main peak was partly recovered (curve c, Fig. 2). This remarkable observation is in accordance with the tendency of native skin to restore its biophysical properties after its prolonged exposure to water (during bathing, swimming, etc.).
4. Conclusions In the present work, the membrane neutron diffraction method is employed in order to elucidate the evolution of the response of SC intercellular architecture to excess hydration conditions. The increase of the sample’s water content progressively disrupts the highly ordered lipid lamellae, leading therefore to a degradation of the barrier function of the system. Nevertheless the process is proven to be rather reversible.
Acknowledgements The authors thank Dr. Joke Bouwstra (Leiden University, The Netherlands) for providing the SC samples. Support by BENSC, Hahn-Meitner Institute (Berlin, Germany) and EC (TMR Contract HPRI-CT-2001-00138, PROTOP Contract EVK3CT-2002-30004) is also acknowledged.
ARTICLE IN PRESS e606
G.Ch. Charalambopoulou et al. / Physica B 350 (2004) e603–e606
References [1] [2] [3] [4]
P.M. Elias, J. Invest. Dermatol. 80 (1983) 44. V.M. Mak, et al., Pharm. Res. 8 (1991) 1064. A. Alonso, et al., Biochim. Biophys. Acta 1237 (1995) 6. G.Ch. Charalambopoulou, et al., Appl. Phys. A 74s (2002) 1245.
[5] J.A. Bouwstra, et al., J. Lipid. Res. 36 (1995) 685. [6] G.Ch. Charalambopoulou, et al., Physica B 276–278 (2000) 530. [7] J.A. Bouwstra, et al., Int. J. Pharm. 84 (1992) 205. [8] D.A. Van Hal, et al., J. Invest. Dermatol. 106 (1996) 89. [9] R.R. Warner, et al., J. Invest. Dermatol. 120 (2003) 275.