Micron-scale assessment of molecular lipid organization in human stratum corneum using microprobe X-ray diffraction

Abstract

Lipid and protein components of the stratum corneum (SC) are organized in complex supramolecular arrangements. Exploring spatial relations between various possible substructures is important for understanding the barrier function of this uppermost layer of epidermis. Here, we report the first study where micro-focus X-ray scattering was used for assessing fine structural variations of the human skin barrier with micrometer resolution. We found that the scattering profiles were unchanged when scanning in the direction parallel to the SC surface. Furthermore, small-angle scattering profiles did not change as a function of depth in the SC, confirming that the lipid lamellar spacings remained the same throughout the SC. However, the wide-angle scattering data showed that the orthorhombic phase was more abundant in the middle layers of the SC, whereas the hexagonal phase dominated in the surface layers both at the external and the lowest part of the SC; i.e., the lipids were most tightly packed in the middle region of the SC. Taken together, our results demonstrate that microprobe X-ray diffraction provides abundant information about spatial variations of the SC lipid structure and thus may be a promising tool for assessing the effects of topical formulations on the barrier function of skin. Lipid and protein components of the stratum corneum (SC) are organized in complex supramolecular arrangements. Exploring spatial relations between various possible substructures is important for understanding the barrier function of this uppermost layer of epidermis. Here, we report the first study where micro-focus X-ray scattering was used for assessing fine structural variations of the human skin barrier with micrometer resolution. We found that the scattering profiles were unchanged when scanning in the direction parallel to the SC surface. Furthermore, small-angle scattering profiles did not change as a function of depth in the SC, confirming that the lipid lamellar spacings remained the same throughout the SC. However, the wide-angle scattering data showed that the orthorhombic phase was more abundant in the middle layers of the SC, whereas the hexagonal phase dominated in the surface layers both at the external and the lowest part of the SC; i.e., the lipids were most tightly packed in the middle region of the SC. Taken together, our results demonstrate that microprobe X-ray diffraction provides abundant information about spatial variations of the SC lipid structure and thus may be a promising tool for assessing the effects of topical formulations on the barrier function of skin. The outermost layer of epidermis, the stratum corneum (SC), consists of keratin-rich corneocytes surrounded by lipid matrix. Despite the fact that they only represent around 10% of the total mass of the SC, the intercellular lipids play a major role in the barrier function of the skin. For example, lipid removal leads to a loss of the water diffusion barrier (1Berthaud F. Boncheva M. Correlation between the properties of the lipid matrix and the degrees of integrity and cohesion in healthy human stratum corneum.Exp. Dermatol. 2011; 20: 255-262Crossref PubMed Scopus (25) Google Scholar). The barrier function results from the complex and specific architecture of the inter-corneocyte lipid matrix (2Madison K.C. Barrier function of the skin: “la raison d'être” of the epidermis.J. Invest. Dermatol. 2003; 121: 231-241Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar) composed of a mixture of ceramides, cholesterol, and long-chain fatty acids. Ultrastructural studies, using electron microscopy and diffraction techniques, have revealed that these lipids are organized in stacked bilayers that are predominantly parallel to the skin surface (3Madison K.C. Swartzendruber D.C. Wertz P.W. Downing D.T. Presence of intact intercellular lipid lamellae in the upper layers of the stratum corneum.J. Invest. Dermatol. 1987; 88: 714-718Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 4Garson J.C. Doucet J. Leveque J.L. Tsoucaris G. Oriented structure in human stratum corneum revealed by X-ray diffraction.J. Invest. Dermatol. 1991; 96: 43-49Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 5Iwai I. Han H. den Hollander L. Svensson S. Ofverstedt L.G. Anwar J. Brewer J. Bloksgaard M. Laloeuf A. Nosek D. et al.The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety.J. Invest. Dermatol. 2012; 132: 2215-2225Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Part of the lipids is covalently bound to the corneocytes whereas another part represents the “free lipids” removable by solvent extraction. X-ray diffraction is an excellent technique for studying the spatial organization of the intercellular matrix because the lipid molecules are highly ordered and oriented in a crystalline structure. This technique has provided a number of important results with regard to the SC structure. In particular, small-angle X-ray scattering (SAXS) gives information on the inter-bilayer distance in the multi-lamellar lipid structures, whereas the wide-angle X-ray scattering (WAXS) data contain information about the in-plane crystalline arrangement of lipids (lattice type). Most of the SAXS studies of human SC find reflections corresponding to ∼4.5 nm and ∼6.5 nm inter-bilayer spacings (4Garson J.C. Doucet J. Leveque J.L. Tsoucaris G. Oriented structure in human stratum corneum revealed by X-ray diffraction.J. Invest. Dermatol. 1991; 96: 43-49Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 6Bouwstra J.A. Gooris G.S. van der Spek J.A. Bras W. Structural investigations of human stratum corneum by small-angle X-ray scattering.J. Invest. Dermatol. 1991; 97: 1005-1012Abstract Full Text PDF PubMed Scopus (462) Google Scholar, 7Schreiner V. Gooris G.S. Pfeiffer S. Lanzendorfer G. Wenck H. Diembeck W. Proksch E. Bouwstra J. Barrier characteristics of different human skin types investigated with X-ray diffraction, lipid analysis, and electron microscopy imaging.J. Invest. Dermatol. 2000; 114: 654-660Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Some studies have also detected a reflection corresponding to a spacing of 11–13.5 nm (6Bouwstra J.A. Gooris G.S. van der Spek J.A. Bras W. Structural investigations of human stratum corneum by small-angle X-ray scattering.J. Invest. Dermatol. 1991; 97: 1005-1012Abstract Full Text PDF PubMed Scopus (462) Google Scholar, 8Cocera M. Rodriguez G. Rubio L. Barbosa-Barros L. Seny-Cases N. Cladera J. Sabes M. Fauth F. de la Maza A. Lopez O. Characterisation of skin states by non-crystalline diffraction.Soft Matter. 2011; 7: 8605-8611Crossref Scopus (15) Google Scholar), referred to as the “long periodicity phase” (6Bouwstra J.A. Gooris G.S. van der Spek J.A. Bras W. Structural investigations of human stratum corneum by small-angle X-ray scattering.J. Invest. Dermatol. 1991; 97: 1005-1012Abstract Full Text PDF PubMed Scopus (462) Google Scholar). These data indicate that the SC lipid matrix has a complex organization into multiple sublayers or even phase-separated domains with different lamellar repeat distances. Similar complexity is found at the molecular level using WAXS, where two sets of peaks are found, corresponding to orthorhombic (peaks at 0.41 and 0.37 nm) and hexagonal (peak at 0.41 nm) packing of the lipid chains. In a of lipids in a or to the corresponding to an distance of These the of and spatial arrangement of these different X-ray scattering studies highly complex where of different substructures are to For example, is not different types of organization in the same region of the SC or in different at different or these we SAXS and with micrometer possible to structural at different or different in the same SC We used this to study of human SC that were studies, were at the of data or at and types of were and lipid removal by a solvent skin from were in corneum was using a (4Garson J.C. Doucet J. Leveque J.L. Tsoucaris G. Oriented structure in human stratum corneum revealed by X-ray diffraction.J. Invest. Dermatol. 1991; 96: 43-49Abstract Full Text PDF PubMed Scopus (130) Google Scholar). was and the was from by skin into water was by at The SC was and at and The SC is a of SC in 7Schreiner V. Gooris G.S. Pfeiffer S. Lanzendorfer G. Wenck H. Diembeck W. Proksch E. Bouwstra J. Barrier characteristics of different human skin types investigated with X-ray diffraction, lipid analysis, and electron microscopy imaging.J. Invest. Dermatol. 2000; 114: 654-660Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 8Cocera M. Rodriguez G. Rubio L. Barbosa-Barros L. Seny-Cases N. Cladera J. Sabes M. Fauth F. de la Maza A. Lopez O. Characterisation of skin states by non-crystalline diffraction.Soft Matter. 2011; 7: 8605-8611Crossref Scopus (15) Google Scholar, and I. N. N. of two domains in intercellular lipid matrix of stratum et PubMed Scopus Google Scholar). However, be about possible effects of the at to and possible from we X-ray scattering on SC that was to or The SAXS and profiles from were to on SC that was used in the study and In particular, the SAXS profiles the and nm and the profiles nm and nm as as the 0.41 nm and 0.37 nm The and variations were also to SC our did not in the X-ray data and these are of the skin surface. SC was by in a mixture for at This only of SC lipids using more may components of the SC and structure. of SC were with a were on two in a that the of was parallel to the of the SC The SC was to be by the of the in a The of to be was using a with with the X-ray and oriented of SC were for X-ray scattering were at and a of The were at the at the M. M. small-angle scattering and with 2000; Scopus Google Scholar). The from an and a was with an and to a or by a about was used to the scattering from the of the were on a with a used for the with a to The distance was using spacing an X-ray scattering were in the of scattering from to on a This SAXS and from nm periodicity to The data were two for the of the results and the However, the data from a to The were as The in was to of in the SAXS region and of in the The data of a of to the SC at in for with a of and were also to the of the structure at the micrometer The for was for a was to or in to data to the first were to was using the and profiles by of the as a function of were on a total of SC from at different of SC skin of and between and For the X-ray data in the to a of SC from a between and The and the are to The X-ray data from different and different revealed only variations in of scattering for example, the at and were more or in different were The in did not a of the scattering and a of in a of the SC. The is oriented with the SC surface parallel to the X-ray a is to information about the the of of lipid and protein the SC. The scattering at different the SC were with a of The of scattering is at the two the external and the because when scanning to the only a of the X-ray the In the analysis, scattering were to into variations of the scattering and to of the the was by in the wide-angle part of the where the lipid peaks are For the SAXS and the data are even they were on the same SAXS the and on the layer whereas and information about the in-plane molecular packing of lipids the small-angle scattering profiles for and stratum The to scanning the SC from the outermost layer to the SC is for to SC The of the for the is by variations of the SC of of the direction with the to the SC and the lipid The a of the scattering profiles on a to demonstrate the of the nm at the SC scattering data for and human stratum The to a depth in the middle part of the SC SC in the are as as two around and the at and in the The of the with where the of the 0.41 nm is for the the of the profiles of the and the are with the of of the from the The to the lipids by solvent extraction. that the profiles were not and to scattering of the profiles with the depth in the stratum The to scanning the SC from the outermost layer to the SC is for to SC of the of scattering of the 0.37 nm and the 0.41 nm of the packing in different SC that the profiles were not and the scattering is more from the middle of the SC to the scattering X-ray is the SC The SAXS are oriented in the is with lamellar predominantly oriented parallel to the SC surface. The of the SC the peaks to intercellular lipid lamellae (4Garson J.C. Doucet J. Leveque J.L. Tsoucaris G. Oriented structure in human stratum corneum revealed by X-ray diffraction.J. Invest. Dermatol. 1991; 96: 43-49Abstract Full Text PDF PubMed Scopus (130) Google Scholar). The major two a at to a lamellar repeat nm) and a at small-angle was detected at is by a a and was around is a of the at to cholesterol (6Bouwstra J.A. Gooris G.S. van der Spek J.A. Bras W. Structural investigations of human stratum corneum by small-angle X-ray scattering.J. Invest. Dermatol. 1991; 97: 1005-1012Abstract Full Text PDF PubMed Scopus (462) Google Scholar) and the reflections of the these are on a that is to of the SC. This is to that for another F. Doucet J. of in by X-ray using the et PubMed Scopus Google Scholar). The scattering be by a function of scattering where the is of the of to F. Doucet J. of in by X-ray using the et PubMed Scopus Google Scholar). the thus of scattering peaks in the small-angle This is of the function not to of the or of the peaks The scattering the on a the The of the the was different from that of the corresponding to nm spacing was whereas the nm was This may be to intercellular lipids that were not by solvent The at nm) a at nm) of the layer lipid is because a part of the lipids is the same the nm may in fact be also in the in the of the at nm because of that the in is by the of this is the this lamellar spacing to or to the layers of the lipids in the intercellular that were of the lipids The profiles the scattering of the SC We the data at different in the SC on a micrometer The of SAXS profiles a to the SC surface of the did not only is for The lamellar is of the the major SAXS the peaks at and the at are at The of of the SAXS profiles is even more This that the arrangement of nm and nm sublayers in intact SC is throughout the SC the the profiles for the in the and of the to the are different from in the middle The of the at nm) to the two with a for of the the data are in the lamellar with the nm repeat distance are only in the middle of the SC and are by lipid in the of the SC. the solvent used is that of lipids only in the most to the i.e., the layers of the SC that are to the two For in the middle be in an where the lipid was for with and with of solvent we a of the nm in the scattering profiles to the we scanning in the direction parallel to the SC to that the molecular arrangement of in the SC was also of the SAXS and profiles with a were unchanged throughout the SC The corresponding scattering are in and The of the two at nm and around The is to lipid and is to protein of SC, in particular, the (4Garson J.C. Doucet J. Leveque J.L. Tsoucaris G. Oriented structure in human stratum corneum revealed by X-ray diffraction.J. Invest. Dermatol. 1991; 96: 43-49Abstract Full Text PDF PubMed Scopus (130) Google Scholar, J.A. Gooris G.S. van der Spek J.A. Bras W. of human stratum corneum as a function of and wide-angle X-ray diffraction J. Scopus Google Scholar). This repeat distance is to the molecular arrangement of in the for example, to the distance (4Garson J.C. Doucet J. Leveque J.L. Tsoucaris G. Oriented structure in human stratum corneum revealed by X-ray diffraction.J. Invest. Dermatol. 1991; 96: 43-49Abstract Full Text PDF PubMed Scopus (130) Google Scholar, J.A. Gooris G.S. van der Spek J.A. Bras W. of human stratum corneum as a function of and wide-angle X-ray diffraction J. Scopus Google Scholar, L. A. corneum and the and Invest. Dermatol. Full Text Full Text PDF PubMed Scopus (130) Google Scholar, Bouwstra J. X-ray diffraction of human stratum effects of and PubMed Scopus Google Scholar). The scattering corresponding to the nm spacing is the in the about in the and The be by of the to be oriented to the of i.e., parallel to the SC surface M. and function of the skin M. In and structure and Scholar). The around nm is a of the scattering from the structure of (4Garson J.C. Doucet J. Leveque J.L. Tsoucaris G. Oriented structure in human stratum corneum revealed by X-ray diffraction.J. Invest. Dermatol. 1991; 96: 43-49Abstract Full Text PDF PubMed Scopus (130) Google Scholar, J.A. Gooris G.S. van der Spek J.A. Bras W. of human stratum corneum as a function of and wide-angle X-ray diffraction J. Scopus Google Scholar, Bouwstra J. X-ray diffraction of human stratum effects of and PubMed Scopus Google Scholar) and the from the lipids by an distance of on the around nm are two lipid at nm and 0.37 nm The two are of a organization of lipid into hexagonal and orthorhombic and the of the the that the molecular are to the SC with the of the layers parallel to the SC The profiles the of the SC are in This scattering was in profiles at different in the SC with the scattering at the two of the SC to of the scattering when scanning to the The remained as as the of the nm the same the of the two corresponding to crystalline lipids was not The orthorhombic packing gives to two reflections corresponding to 0.41 nm and 0.37 whereas the hexagonal packing provides a at 0.41 We used this fact to the of the two lipid packing types by the of the reflections at 0.41 nm and 0.37 The of the of the reflections at 0.41 nm and 0.37 nm with to depth in a In the this is about in the middle the two that the orthorhombic phase is more abundant in the middle part of the SC at the the 0.37 nm that is to the orthorhombic phase the two of the SC. to spatial of the we found that the at nm was in fact into two corresponding to the of nm and nm to the This the of two crystalline orthorhombic and the be for the two J.A. Gooris G.S. van der Spek J.A. Bras W. of human stratum corneum as a function of and wide-angle X-ray diffraction J. Scopus Google Scholar, Bouwstra J.A. diffraction provides information on human stratum corneum lipid organization in to depth and Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). is for the reflections of the two crystalline to be The major of lipid in the is the of the 0.37 nm and in the of the SC with a in the The at 0.41 nm and the part of the around nm are a from the lipids and the part of lipids that not by solvent extraction. The SC layers of corneocytes and intercellular lipid lamellae A. in to stratum corneum structure and lipid Invest. Dermatol. Full Text PDF PubMed Scopus Google Scholar), the number of layers on The of lipid and structural arrangement as a function of depth in the SC has using electron E. of stratum corneum lipid a for electron Invest. Dermatol. Full Text Full Text PDF PubMed Scopus (25) Google Scholar), electron diffraction and microscopy Bouwstra J.A. diffraction provides information on human stratum corneum lipid organization in to depth and Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar), and D. of stratum corneum barrier function in by Invest. Dermatol. Full Text PDF PubMed Scopus Google Scholar). and have also used to the in the of the G. and of molecular and structure human Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). these studies used to different layers of the SC. results to the of our the first to the structural variations in the intact SC at at the of data The SAXS at different in the SC is to for human SC (4Garson J.C. Doucet J. Leveque J.L. Tsoucaris G. Oriented structure in human stratum corneum revealed by X-ray diffraction.J. Invest. Dermatol. 1991; 96: 43-49Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 6Bouwstra J.A. Gooris G.S. van der Spek J.A. Bras W. Structural investigations of human stratum corneum by small-angle X-ray scattering.J. Invest. Dermatol. 1991; 97: 1005-1012Abstract Full Text PDF PubMed Scopus (462) Google Scholar, 8Cocera M. Rodriguez G. Rubio L. Barbosa-Barros L. Seny-Cases N. Cladera J. Sabes M. Fauth F. de la Maza A. Lopez O. Characterisation of skin states by non-crystalline diffraction.Soft Matter. 2011; 7: 8605-8611Crossref Scopus (15) Google Scholar) and not to a lamellar has a the at around to nm corresponding to the of a lipid sublayers that are to be for the nm and nm) by SAXS I. Han H. den Hollander L. Svensson S. Ofverstedt L.G. Anwar J. Brewer J. Bloksgaard M. Laloeuf A. Nosek D. et al.The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety.J. Invest. Dermatol. 2012; 132: 2215-2225Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, J. G. Gooris G. H. M. of the skin barrier PubMed Scopus (118) Google Scholar, L. barrier structure and the phase Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar, of skin stratum corneum diffraction for of J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). For example, these data be by a of molecular lipid arrangement by et I. Han H. den Hollander L. Svensson S. Ofverstedt L.G. Anwar J. Brewer J. Bloksgaard M. Laloeuf A. Nosek D. et al.The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety.J. Invest. Dermatol. 2012; 132: 2215-2225Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). on microscopy and they that the intercellular lipids are organized in nm repeat of nm and nm This electron with the arrangement where ceramides are in with to the in the layer and the fatty to the fatty in the layer on the that two fatty layers the nm and two layers the nm be in the nm and the long-chain fatty be in in the nm to packing between the fatty and the long-chain fatty of This not the of the are to be for arrangement of the SC Some have in fact that the of the nm scattering of SC is to the of the V. Gooris G.S. Pfeiffer S. Lanzendorfer G. Wenck H. Diembeck W. Proksch E. Bouwstra J. Barrier characteristics of different human skin types investigated with X-ray diffraction, lipid analysis, and electron microscopy imaging.J. Invest. Dermatol. 2000; 114: 654-660Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). number of of lipid organization also have to scattering J. G. Gooris G. H. M. of the skin barrier PubMed Scopus (118) Google Scholar, L. barrier structure and the phase Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar, of skin stratum corneum diffraction for of J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, D.C. Wertz P.W. K.C. Downing D.T. of the intercellular lipid lamellae in stratum corneum.J. Invest. Dermatol. Full Text PDF PubMed Scopus Google Scholar, of the skin Google Scholar). The of this study is to possible variations in scattering profiles as a function of the the SC. We did not in the lamellar periodicity as a function of depth in the SC. The scattering with and nm reflections is in layers the SC. This is in with the electron microscopy studies of where the with the same spacing was throughout the layers A. J. L. of the as by microscopy of of human Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). The lamellar is of the the of the layers from stratum to stratum The lipid was not the SC. We found that the orthorhombic was more abundant in the middle layers of the SC and both the external and the packing of lipids the skin surface be to of the intercellular as corneocytes cohesion in the This also be by lipids at the skin these and lipids in the upper layers of SC lipids of lipid in the outermost SC layers has by D. of stratum corneum barrier function in by Invest. Dermatol. Full Text PDF PubMed Scopus Google Scholar). Furthermore, electron diffraction with SC layers have that hexagonal structure dominated in the region of the SC whereas the orthorhombic structure was in the the Bouwstra J.A. diffraction provides information on human stratum corneum lipid organization in to depth and Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). This with our results for the of the SC be to in Taken together, our results the of the microprobe X-ray diffraction technique to of SC structure in various This is in our to structural with or The effects are in the to the in the for different and the complexity of of the at the SC surface. the X-ray microprobe diffraction is to the structural variations in the protein of the SC as and supramolecular arrangement be from the and periodicity information in the X-ray In this we reflections at nm and nm to (4Garson J.C. Doucet J. Leveque J.L. Tsoucaris G. Oriented structure in human stratum corneum revealed by X-ray diffraction.J. Invest. Dermatol. 1991; 96: 43-49Abstract Full Text PDF PubMed Scopus (130) Google Scholar, Bouwstra J. X-ray diffraction of human stratum effects of and PubMed Scopus Google Scholar). For the SC we did not find variations of these However, of SC with to in the protein structure that are with this variations of the lipid and packing or have as a to the complex and properties of the SC For example, of the skin Google Scholar) the where crystalline and phase domains in the studies or the of or domains with different molecular organization of lipids I. N. N. of two domains in intercellular lipid matrix of stratum et PubMed Scopus Google Scholar, J.A. Gooris G.S. The lipid in human stratum corneum and Scholar). did not variations of the lamellar spacing or the lipid packing at the with the This the studies by microscopy of the intercellular throughout the layers A. J. L. of the as by microscopy of of human Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). In an to the of the lipids in we the profiles of from of SC The to the lipids that have by solvent extraction. consists of a to a phase with peaks at 0.41 nm and 0.37 Despite the of the the of lipids in the SC the lipids that have by solvent be from the of the two in the in the on the by of X-ray for of the in Google Scholar), the of lipids in to the lipids be at This that the of SC lipids are This the that the of intercellular lipids are in as as the data for SC S. A. Bouwstra J.A. E. D. of stratum corneum molecular by PubMed Scopus Google Scholar). This may be to lipid in our to possible of more In a from intercellular lipids be with an and lipid be to this The are to and the of the of the for with the X-ray data We and for the and for The of with the at is with small-angle X-ray scattering stratum corneum wide-angle X-ray scattering