Comprehensive In Vivo Analysis of Keratin in the Human Stratum Corneum using Confocal Raman Microscopy

1. Comprehensive in vivo analysis of keratin in the human stratum corneum using confocal Raman microscopy Maxim E. Darvin, Johannes Schleusener, Chunsik Choe, Jürgen Lademann

2. In vivo confocal Raman microscopy River Diagnostics (Model 3510) Spatial (axial) resolution <5 µm Spectralresolution 2 cm-1 Excitation at 785 nm (400-2200 cm-1) Excitation at 671 nm (2200-4000 cm-1) (1-2) µm increment focus Corneocytethickness ≈1 µm Lipid layerthickness <0,1 µm Spatial axial resolution <5 µm Spectralresolution 2 cm-1 Darvin et al. J Biomed. Opt. 18(6), 061230, 2013

3. Raman spectra of human skin in vivo 38

4. The structure of a keratin filament and the types of side-chain reactions 1: the hydrogen bonds between N–H and C=O groups (corresponding to the Amide I Raman band around 1655 cm-1), 2: disulphide bonds between two cysteine side-chains (corresponding to the Raman band around 491 cm‑1 and 546 cm-1), 3: free cysteine side-chain that does not form disulphide bonds (corresponding to the Raman band around 700 cm-1), 4: buried tyrosine side-chains in keratin chains (corresponding to the Raman bands around 830 cm-1), 5: exposed tyrosine side-chains in keratin chains (corresponding to the Raman bands around 850 cm-1), 6, 7, 8: the hydrogen bound water molecules with the keratin chains, also showing the water-binding sites of keratin chains. Keratin isthemainprotein of the StratumCorneum Choe, Schleusener, Lademann, Darvin. Scientific Reports, 7: 15900, 2017

5. Protein structure Keratin isthemainprotein of theStratumCorneum(uppermosthornylayer of theskin) https://en.wikipedia.org/wiki/Protein_folding

6. Secondary structure of keratin in vivo There are two forms of secondary structure of keratin, α-helix and β-sheet. The α-helix keratin, a highly stable form of keratin, has a coiled-coil structure (determined by disulphide bonds) and less exposed (folded) side-chains, which does not efficiently interact with water and other biomolecules. The β-sheet keratin is softer than the α-helix and has a large number of exposed side-chains between the sheets. Water molecules can easily intercalate between the sheets of β-sheet keratin and bind with hydrogen bonds. (b) (a) Deconvolution of the Amide I band of human SC (a) and the depth profile of the β-sheet/α-helix keratin (b) in the human SC in vivo. The β-sheet/α-helix is determined by the 960 cm−1/938 cm−1 ratio (squares, black) and the (β-sheet + turns and random coils)/α-helix keratin forms ratio determined by the (1670 cm−1 + 1685 cm−1)/1655 cm−1 AUGCs ratio of the deconvoluted amide I band (cycles, red dotted). Mean ± standard deviation for 11 volunteers. / lower/higher possibility to bind water molecules. Choe, Schleusener, Lademann, Darvin. Scientific Reports, 7: 15900, 2017

7. Tertiary structure of keratin in vivo (a) (b) The depth-dependent stability of disulphide bonds and the amount of cysteine in keratin chains in the human SC in vivo. The stability in keratin is determined by the ratio of gauche-gauche-gauche conformation (474–508cm-1 AUC) to total disulphide bonds gauche-gauche-gauche + gauche-gauche-trans + trans-gauche-trans (474–578cm-1 AUC) (a). The amount of cysteine in keratin chains which form disulphide bonds determined by the C–S (690–712cm-1 AUC) / S–S (474–578cm-1 AUC) ratio (b). Mean ± standard deviation is shown for 11 volunteers. / lower/higher possibility to bind water molecules. Choe, Schleusener, Lademann, Darvin. Scientific Reports, 7: 15900, 2017

8. Tertiary structure of keratin in vivo / lower/higher possibility to bind water molecules Vyumvuhore et al. Analyst 138:4103-4111, 2013 Choe, Schleusener, Lademann, Darvin. Scientific Reports, 7: 15900, 2017

9. Tertiary structure of keratin in vivo (folding/unfolding states of keratin in the stratum corneum) The Raman peak at 2930 cm-1 originates from CH3 vibration of keratin chains and its position is sensitive to the exposed states of CH3 side-chains of keratin (tertiary structure), i.e., keratin´s folding/unfolding states. The unfolding state is associated with the enhanced exposure of CH3 side-chains to the surrounding molecules. moreunfoldedkeratin „Younger group“: 3M+4F, 23-34, mean 29 y.o. „Oldergroup“: 4F, 45-62, mean 50 y.o. / lower/higher possibility to bind water molecules morefoldedkeratin Choe, Schleusener, Lademann, Darvin. Scientific Reports, 7: 15900, 2017 Choe, Schleusener, Lademann, Darvin. Mechanisms of Ageing and Development, 172: 6-12, 2018

10. Other parameters of human stratum corneum determined in vivo using confocal Raman microscopy lower hydrogen bondingstate Natural moisturizing factor (NMF) Hydrogen bounding state of water higher hydrogen bondingstate / lower/higher possibility to bind water molecules NMF is determined using non-restricted classical least squares (CLS) regression using reference spectra of skin components Choe, Lademann, Darvin. Analyst 141: 6329-6337, 2016

11. Keratin-water-NMF interaction in the human stratum corneum Combining all presentedresultstogether, a „three-layermodel“ of human stratumcorneum in vivo regardingthekeratin-water-NMF interactionisproposed: 0-30% SC: non-swelling region 30-70% SC: highly-swelling region 70-100% SC: low-swelling region Choe, Schleusener, Lademann, Darvin. Scientific Reports, 7: 15900, 2017

12. Conclusions • The hydrogen bonding states of water molecules has maximum value in the 20-40% SC depth, which can only be explained by considering both, the molecular structure of keratin and the contribution of NMF as a holistic system. • Based on folding/unfolding properties of keratin, a “three layer model” of the SC, regarding the keratin-water-NMF interaction and SC swelling is proposed. • Confocal Raman microscopy is a well-suited method for non-invasive in vivo analysis of the skin barrier function, SC composition, and Lipids-Water-Keratin-NMF interaction.

13. Thank you for your attention