Current views on non-invasive in vivo determination of physiological parameters of the stratum corneum using confocal Raman microspectroscopy (original) (raw)
Related papers
Biomedical Optics Express
Confocal Raman microscopy has a number of advantages in investigating the human stratum corneum (SC) in vivo and ex vivo. The penetration profiles of xenobiotics in the SC, as well as depth profiles of the physiological parameters of the SC, such as the concentration of water depending on the strength of hydrogen bonds, total water concentration, the hydrogen bonding state of water molecules, concentration of intercellular lipids, the lamellar and lateral packing order of intercellular lipids, the concentration of natural moisturizing factor molecules, carotenoids, and the secondary and tertiary structure properties of keratin are well investigated. To consider the depth-dependent Raman signal attenuation, in most cases a normalization procedure is needed, which uses the main SC's protein keratin-related Raman peaks, based on the assumption that keratin is homogeneously distributed in the SC. We found that this assumption is not accurate for the bottom part of the SC, where the water concentration is considerably increased, thus, reducing the presence of keratin. Our results demonstrate that the bottom part of the SC depth profile should be multiplied by 0.94 in average in order to match this non-homogeneity, which result in a decrease of the uncorrected values in these depths. The correctly normalized depth profiles of the concentration of lipids, water, natural moisturizing factor and carotenoids are presented in this work. The obtained results should be taken into consideration in future skin research using confocal Raman microscopy.
European Journal of Pharmaceutical Sciences, 2013
The stratum corneum is a strong barrier that must be overcome to achieve successful transdermal delivery of a pharmaceutical agent. Many strategies have been developed to enhance the permeation through this barrier. Traditionally, drug penetration through the stratum corneum is evaluated by employing tape-stripping protocols and measuring the content of the analyte. Although effective, this method cannot provide a detailed information regarding the penetration pathways. To address this issue various microscopic techniques have been employed. Raman microscopy offers the advantage of label free imaging and provides spectral information regarding the chemical integrity of the drug as well as the tissue. In this paper we present a relatively simple method to obtain XZ-Raman profiles of human stratum corneum using confocal Raman microscopy on intact full thickness skin biopsies. The spectral datasets were analysed using a spectral unmixing algorithm. The spectral information obtained, highlights the different components of the tissue and the presence of drug. We present Raman images of untreated skin and diffusion patterns for deuterated water and beta-carotene after Franz-cell diffusion experiment.
Scientific Reports
The secondary and tertiary structure of keratin and natural moisturizing factor (NMF) are of great importance regarding the water regulating functions in the stratum corneum (SC). In this in vivo study, the depth-dependent keratin conformation and its relationship to the hydrogen bonding states of water and its content in the SC, are investigated using confocal Raman microscopy. Based on the obtained depth-profiles for the β-sheet/α-helix ratio, the stability of disulphide bonds, the amount of cysteine forming disulphide bonds, the buried/exposed tyrosine and the folding/unfolding states of keratin, a "three layer model" of the SC, regarding the keratin-water-NMF interaction is proposed. At the uppermost layers (30-0% SC depth), the keratin filaments are highly folded, entailing limited water binding sites, and NMF is mostly responsible for binding water. At the intermediate layers (70-30% SC depth), the keratin filaments are unfolded, have the most water binding sites and are prone to swelling. At the bottom layers (100-80% SC depth), the water binding sites are already occupied with water and cannot swell substantially. The hydrogen bonding states of water molecules can only be explained by considering both, the molecular structure of keratin and the contribution of NMF as a holistic system. Keratin is one of the abundant proteins found in the mammalian epidermis. Corneocytes of the stratum corneum (SC), the horny cells of human epidermis, are continuously proliferating from the stratum granulosum (SG) towards the skin surface 1,2 , contain a lot of the fibrous keratin 3 which is embedded in a water-lipid matrix 4 , and is almost homogenously distributed throughout the SC of healthy skin 5. However, this can be different in the diseased skin 6. The SC has been recognised as the skin barrier, which prevents exogenous substances from penetrating into the skin and also for water evaporation out of the body 7-9. The barrier function of the SC is primarily maintained by the lateral packing order of intercellular lipids (ICL) 10 , which is distributed non-homogeneously in the SC 11,12 and can vary age-dependently 13. Keratin filaments are recognised as a major factor for maintaining the mechanical skeleton of the corneocytes and, together with corneodesmosomes, the durability of the SC. They participate in the regulation of the water diffusion process, and the occlusion-based swelling effect 14-18. Meanwhile, the distribution of bound/unbound water in the SC and the trans-epidermal water loss have been important issues in dermatology and cosmetology 15,17-20. Proteins such as keratin are described on different levels of biomolecular structure. The primary structure is related to the order of amino acids in keratin chains. The secondary structure characterises the coiled, β-sheet and β-turns and random coil structures of proteins by the hydrogen bonds between N-H and C=O bonds. The tertiary structure of keratin describes the folding/unfolding structure of proteins, caused by the interaction of side-chains of keratin chains, e.g. disulphide bonds of cysteine, hydrogen bonds of tyrosine and other possible electrostatic forces between the side-chains of protein. Since most of the water is present in the corneocytes, rather than in the lamellas of ICL 21 , and the corneocytes largely consist of keratin filaments and hygroscopic natural moisturizing factor (NMF) (Fig. 1a), the secondary and tertiary structures of keratin are important for the water regulation in the SC. The structure of keratin depends on its extensive intra-and inter-molecular
Direct Observation of Domains in Model Stratum Corneum Lipid Mixtures by Raman Microspectroscopy
Biophysical Journal, 2001
Several studies on intact and model stratum corneum (SC), the top layer of the epidermis, have suggested the presence of crystalline domains. In the present work, we used micro-Raman mapping to detect lipid domains in model lipid mixtures formed by an equimolar mixture of ceramides, cholesterol, and palmitic acid, the three main lipid species of SC. We were able to determine the spatial distribution of the three compounds individually based on the systematic analysis of band areas. As a control, we studied freeze-dried lipid mixtures, and the Raman microspectroscopy reported faithfully the homogeneous distribution of the three compounds. Spectral mapping was then performed on hydrated equimolar mixtures carefully annealed. In this case, clear phase separations were observed. Domains enriched in cholesterol, ceramides, or palmitic acid with a size of a few tens of square microns were detected. These findings constitute the first direct evidence of the formation of heterogeneous domains in the SC lipid models in a bulk phase. Raman microspectroscopy is an innovative approach to characterize the conditions leading to the formation of domains and provides new insights into the understanding of the skin barrier.
In vivo skin characterization by confocal Raman microspectroscopy
2003
markdownabstract__Abstract__ Various areas of skin research depend on detailed knowledge of the molecular composition of skin and molecular structure of skin constituents. On a microscopic scale the skin is a highly heterogeneous tissue. Molecular composition and structure vary tremendously, depending on depth and location on the body, and may be affected by skin disorders and environmental factors such as sun exposure, seasonal variation, and cosmetic or medical treatments can influence the molecular properties of the skin. For many aspects of skin research or skin characterization, noninvasive methods are particularly welcome. This is partly because they cause less discomfort for the patient or volunteer, as the skin is not damaged, but more importantly because noninvasive methods enable investigation of the skin in its natural state, without affecting its integrity, morphology or molecular composition. Only noninvasive measurements can be performed repeatedly on the same skin are...
Sensitive Skin: Assessment of the Skin Barrier Using Confocal Raman Microspectroscopy
Skin pharmacology and physiology, 2017
Sensitive skin (SS), a frequently reported condition in the Western world, has been suggested to be underlined by an impaired skin barrier. The aim of this study was to investigate the skin barrier molecular composition in SS subjects using confocal Raman microspectroscopy (CRS), and to compare it with that of non-SS (NSS) individuals as well as atopic dermatitis (AD) and allergic rhinoconjunctivitis (AR) subjects, who frequently report SS. Subjects with SS (n = 29), NSS (n = 30), AD (n = 11), and AR (n = 27) were included. Stratum corneum (SC) thickness, water, ceramides/fatty acids, and natural moisturizing factor (NMF) were measured by CRS along with transepidermal water loss and capacitance on the ventral forearm, thenar, and cheek. Sebum levels were additionally measured on the forearm and cheek. No differences between SS and NSS subjects were found regarding SC thickness, water, and NMF content, yet a trend towards lower ceramides/fatty acids was observed in the cheek. Compare...
In vitro and in vivo Raman spectroscopy of human skin
Biospectroscopy, 1998
Noninvasive techniques that provide detailed information about molecular composition, structure, and interactions are crucial to further our understanding of the relation between skin disease and biochemical changes in the skin, as well as for the development of penetration enhancers for transdermal drug administration. In this study we present in vitro and in vivo Raman spectra of human skin. Using a Raman microspectrometer, in vitro spectra were obtained of thin cross sections of human skin. They provided insight into the molecular composition of different skin layers. Evidence was found for the existence of a large variation in lipid content of the stratum corneum. A simple experimental setup for in vivo confocal Raman microspectroscopy of the skin was developed. In vivo Raman spectra of the stratum corneum were obtained at different positions of the arm and hand of three volunteers. They provided evidence for differences in the concentration of natural moisturizing factor at the...
SSRN Electronic Journal, 2022
The study of human skin represents an important area of research and development in dermatology, toxicology, pharmacology and cosmetology, in order to assess the effects of exogenous agents, their interaction, their absorption mechanism, and/or their toxicity towards the different cutaneous structures. The processes can be parameterised by mathematical models of diffusion, of varying degrees of complexity, and are commonly measured by Franz cell diffusion, in vitro, and tape stripping, in vitro or in vivo, techniques which are recognised by regulatory bodies for commercialisation of dermally applied products. These techniques do not directly provide chemically specific measurement of the penetration and/or permeation of formulations in situ, however. Raman microspectroscopy provides a non-destructive, non-invasive and chemically specific methodology for in vitro, and in vivo investigations, in-situ, and can provide a powerful alternative to the current gold standard methods approved by regulatory bodies. This review provides an analysis of the current state of art of the field of monitoring dermal penetration and permeation kinetics of topical products, in vitro and in vivo, as well as the regulatory requirements of international guidelines governing them. It furthermore outlines developments in the analysis of skin using Raman microspectroscopy, towards the most recent demonstrations of quantitative monitoring of the penetration and permeation kinetics of topical products in situ, for in vitro and in vivo applications, before discussing the challenges and future perspectives of the field.