Non-invasive depth profile imaging of the stratum corneum using confocal Raman microscopy: First insights into the method

Abstract

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.

Introduction

Trans-dermal drug delivery has received considerable attention over the past decade. Apart from being used in the treatment of skin diseases, trans-dermal delivery can be a good alternative to deliver drugs systemically, as skin offers a large surface area to which drugs can be applied. The non-invasive nature of the application, high patient compliance, controlled release kinetics of the drug etc. are some of the advantages, which make transdermal application an attractive route for drug administration. A major hurdle in achieving this is, however, the low permeability of the skin and especially of the stratum corneum (Walters and Brain, 2002, Walters and Roberts, 2002). One of the most important functions of the skin is to protect the organism against detrimental effects of the environment; for this purpose stratum corneum has evolved into a very tight barrier, which has proven difficult to overcome. First generation transdermal delivery systems such as creams, gels and patches have been successfully employed to deliver drugs such as ibuprofen (Ibuleve®), fentanyl (Duragesic®), nicotine (Nicotinell®). However application of these systems has only been successful for a limited number of drugs; traditional delivery systems do not readily cross the stratum corneum and skin permeation was found to be dependent upon the drug’s physiochemical properties like lipophilicity, molecular weight and molecular shape (Grice et al., 2010). Recently, 2nd and 3rd generation delivery systems were developed involving modification of the barrier properties of the stratum corneum by means of chemical penetration enhancers, iontophoresis, microneedles, laser ablation etc. (Prausnitz and Langer, 2008).

The stratum corneum is a 10–20 μm thick tightly packed layer of flattened cells, called corneocytes, originating from keratinocytes in the stratum basale, i.e., the bottom layer of the epidermis. Intercellular spaces are filled with laterally packed lamellar lipid domains (Bouwstra et al., 1991) and play an essential role in the barrier function but also provide a pathway through which drug molecules may diffuse during the transdermal delivery. The traditional method of studying skin penetration is by tape stripping, where layers of SC are progressively removed and the drug is quantified by analytical methods such as UV spectroscopy, fluorescence spectroscopy, and radioactivity measurement (Breternitz et al., 2007, Pinkus, 1951). Although a very powerful technique, tape stripping is an invasive technique. In addition, tape stripping is not an ideal technique for studying penetration pathways of the drug within the SC over several time points. Microscopic techniques such as confocal laser scanning microscopy (Alvarez-Roman et al., 2004, Fink-Puches et al., 1995, Schatzlein and Cevc, 1998, Verma et al., 2003a), two-photon fluorescence microscopy (Carrer et al., 2008, Schenke-Layland et al., 2006, van den Bergh et al., 1999, Yu et al., 2003) have been exploited for studying the detailed spatial distribution in the SC. A major drawback of both techniques is that they rely on a fluorescent signal limiting the method to fluorescent or fluorescently labelled drugs. Also the attachment of a fluorescent label to the drug might affect its diffusion profile.

Raman microscopy is commonly used to study the biological composition of especially diseased tissues and has been applied to tissue biopsies of virtually every organ (Krafft et al., 2009). Raman microscopy as well as the complementary infra-red (IR) microscopy have been utilised to image skin cross sections. Raman spectroscopy has also been employed to study the penetration of caffeine (Franzen et al., 2012) and anti-oxidants (Uragami et al., 2012). Confocal Raman microscopy provides a useful depth profiling technique as it allows label free imaging (Baia et al., 2002, Schmitt et al., 2003). The obtained spectral information allows imaging the distribution of substances or tissue features based on their chemical composition. Apart from penetration depth, the spectral information could be useful to identify chemical integrity (Zhang et al., 2007), hydration state (Chrit et al., 2007), oxidative stress (Ermakov et al., 2004) and the ability to individually track various ingredients of a formulation (Xiao et al., 2005). In Raman microscopy, the penetration depth of the excitation light strongly depends on the wavelength of the employed laser. Best penetration depths for biological samples are usually achieved in the near infra-red (NIR). Shorter wavelengths are scattered stronger, the intensity of the scattered light being proportional to the fourth power of the frequency. For longer wavelengths the absorption of water becomes a major problem. In this study, we employed a laser excitation source of 785 nm at constant laser intensity for imaging stratum corneum and the upper layers of the viable epidermis.

Information on intradermal water distribution is relevant as water is a natural carrier for hydrophilic compounds. Beta-carotene is of interest for dermatological applications as it has been suggested to reduce oxidative stresses in the skin related to ultraviolet and infra-red irradiation (Darvin et al., 2011, Stahl and Sies, 2012). Treatment with beta-carotene has also been shown to reduce skin carcinogenesis and is also indicated in the treatment of erythropoietic protoporphyria (Bayerl, 2008). Beta-carotene is often chosen as an example derivative of the carotenoid family, because it exhibits interesting resonance Raman properties. Due to the resonance effects the sensitivity of the Raman measurements is enhanced by several orders of magnitude. Measurements including quantification of β-carotene using resonance Raman effects are well established (Darvin et al., 2012, Ermakov and Gellermann, 2012). Our study provides a relatively simple methodology to non-invasively image the SC for distribution of deuterated water and beta-carotene, following transdermal application.