Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo

There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals

Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum

Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR—partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400–1500 cm−1) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R2 = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R2 (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies

Monitoring Dermal Penetration and Permeation Kinetics of Topical Products; the Role of Raman Microspectroscopy

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

Modeling the human skin barrier - towards a better understanding of dermal absorption

Many drugs are presently delivered through the skin from products developed for topical and transdermal applications. Underpinning these technologies are the interactions between the drug, product and skin that define drug penetration, distribution, and elimination in and through the skin. Most work has been focused on modeling transport of drugs through the stratum corneum, the outermost skin layer widely recognized as presenting the rate-determining step for the penetration of most compounds. However, a growing body of literature is dedicated to considering the influence of the rest of the skin on drug penetration and distribution. In this article we review how our understanding of skin physiology and the experimentally observed mechanisms of transdermal drug transport inform the current models of drug penetration and distribution in the skin. Our focus is on models that have been developed to describe particular phenomena observed at particular sites of the skin, reflecting the most recent directions of investigation

Mathematical and pharmacokinetic modelling of epidermal and dermal transport processes

Topical delivery to the various regions of the skin and underlying tissues, transdermal drug delivery and dermal exposure to environmental chemicals are important areas of research. Mathematical models of epidermal and dermal transport, involving penetration of a solute through various layers of the skin, metabolism in the skin and its subsequent distribution and clearance into systemic circulation from underlying tissues, play an essential role in this research area and are reviewed in this work

Beyond stratum corneum

The stratum corneum is seen as the main physical barrier that prevents the entry of solutes into the skin and manages the egress of water and endogenous molecules from the body. It is the thin outermost layer of the skin consisting of corneocytes (flattened, closely packed, and interdigitated “dead” cells packed with keratin) embedded in a highly organized, dense lipid intercellular space (1,2). The skin also has other barrier and support properties including immunological, metabolic, cushioning, sensing, and temperature control. In addition, the blood and lymphatics in the skin enable the removal of absorbed and skin metabolized solutes into the systemic circulation for further detoxification and excretion. The other distinct skin layers involved in providing these functions are the viable epidermis, dermis, and hypodermis. In this chapter, we first review the morphology of the viable skin layers and the skin appendages. We then examine the role the substructures below the stratum corneum may play in the physical and metabolic barrier properties of the skin and in the removal of solutes absorbed into the skin

Cutaneous metabolism and active transport in transdermal drug delivery

As the largest organ of the human body, the skin provides an exceptional heterogeneous interface to protect the internal organs from various physical and chemical environmental insults by functioning as a physical, metabolic, and anti-UV barrier. It is well documented in the percutaneous penetration literature that the tightly packed lipid bilayer of the stratum corneum is efficacious in preventing exogenous compounds from entering the body (1). Most of the work done on the skin barrier focuses on the stratum corneum’s physical barrier property. However, the skin should not only be thought of as an inert barrier. It is also a chemically active barrier, with enzymes located in the viable epidermis (2) as well as in the extracellular spaces of the stratum corneum (3–5) and the dermis, in which the primary sites of metabolism are the skin appendages (2). Cutaneous transport proteins expressed in skin constitute a second part of the chemical barrier function of the skin. Keratinocytes are the sites of expression of these proteins, which can act as substrates for a variety of topically applied compounds. In this brief review we focus on the possible therapeutic and adverse effects that cutaneous enzymes and transporters have on drug penetration into the skin. We also focus on the predictive role of mathematical models in understanding cutaneous biotransformation

An investigation into the effect of freezing conditions on the barrier function of reconstructed human epidermis using Raman spectroscopy and percutaneous permeation

International audienceReconstructed human epidermis (RHE) is an emerging skin modelin pharmaceutical, toxicological and cosmetic sciences, yieldingscientific and ethical advantages. RHEs remain costly, however,due to consumables and time required for their culture and a shortshelf-life. Storing, i.e., freezing RHE could help reduce costs butlittle is known on the effects of freezing on the barrier function ofRHE. We studied such effects using commercial EpiSkin™ RHEstored at −20, −80 and −150 °C for 1 and 10 weeks. We acquiredintrinsic Raman spectra in the stratum corneum (SC) of the RHEsas well as spectra obtained following topical application ofresorcinol in an aqueous solution. In parallel, we quantified theeffects of freezing on the permeation kinetics of resorcinol fromtime-dependent permeation experiments. Principal componentanalyses discriminated the intrinsic SC spectra and the spectra ofresorcinol-containing RHEs, in each case on the basis of thefreezing conditions. Permeation of resorcinol through the frozenRHE increased 3- to 6-fold compared to fresh RHE, with thestrongest effect obtained from freezing at −20 °C for 10 weeks. Dueto the extensive optimization and standardization of EpiSkin™RHE, the effects observed in our work may be expected to be morepronounced with other RHEs