Many chemicals show skin penetration enhancing properties. Well-known examples are Azone^®, dimethylsulfoxide, alcohols, fatty acids such as oleic acid, surfactants, solvents, and terpenes. However, the first major penetration enhancer is water. The permeability of skin for hydrophilic molecules increases dramatically as skin is hydrated. Hydration by occlusion or exposition to high humidity level results in an increase of the SC water content from 13% (standard value) to near 400% of the dry tissue weight (Williams and Barry 2004). In the SC, the water can be bound to structural elements (natural moisturizing factor (NMF), functional groups) or free. The free water acts as a solvent for hydrophilic molecules in the SC, thus increasing the solubility of drugs in the tissue. Drug partition between the formulation and the SC is then modified, and the increase of solubility leads to higher transdermal fluxes of hydrophilic substances in hydrated skin. It is not clearly established whether water can enhance the skin penetration of lipophilic drugs. It has been proposed that swelling of the polar head group regions of the lipid bilayers by water could disrupt the lipid domains and cause higher permeability for lipophilic permeants such as steroids. However, several researchers have shown that water does not cause modification of the lipid bilayer packing (Bouwstra et al. 1991; Gay et al. 1994; Bouwstra et al. 1996; Bouwstra et al. 2003). van Hal et al. (1996) incubated human skin explants with a saline phosphate buffered solution for 24 h under occlusive or nonocclusive conditions. They proved that water penetrated in the corneocytes inducing their swelling, but also between the cells in the intercellular lipids region, mainly located in separated water pools. The images revealed these water pools and some vesicle-like structures occasionally found. However, the majority of the lipid bilayers exhibited a smooth fracture plane and no change in appearance. The amount of water in these smooth regions was low, and swelling of lamellae or lateral swelling of lipids was not observed.

Transepidermal water loss (TEWL) and electrical impedance (corneometry) measurements are two standard methods used in vivo to get information about the skin barrier integrity and the skin hydration level. Both methods are noninvasive and they can be considered as indirect measurements of the skin water content. CRM enables to monitor the water content in the tissue at various depths, enabling the water concentration profiles through the skin depth to be drawn (Caspers et al. 2000). The water content in the tissue can be determined from the ratio of Raman intensities of the OH stretch vibration of water at 3390 cm⁻¹ and the CH₃ stretch of protein at 2935 cm⁻¹. To improve the signal-to-noise ratio, intensities were integrated from two bands (I _OH: 3350–3550 cm⁻¹ and I _CH: 2910–2965 cm⁻¹). Caspers et al. (2000, 2001) proposed a quantification method to determine absolute water content by calibration of the ratio of the abovementioned areas using spectra of concentrated aqueous solutions of proteins. The profiles of water content can also be used to estimate the SC thickness, as it is well known that the water content increases sharply at the separation surface between SC and viable epidermis from about 13% in the SC to 60–70% in the viable epidermis. Several analysis methods used to determine the water depth profiles were proposed to determine the boundary between SC and viable epidermis (Caspers et al. 2000, 2003; van der Pol et al. 2007; Crowther et al. 2008; Egawa and Kajikawa 2009; Darlenski et al. 2009; Hancewicz et al. 2012). The values of water content in SC reported by various authors are rather scattered, which is mainly due to the intrinsic variability of different types of skin samples (Hancewicz et al. 2012), variation with respect to body site (Egawa et al. 2007), whether the skin is dry or freshly hydrated (Egawa and Kajikawa 2009; Hancewicz et al. 2012), and also may come from different CRM data treatments. Raman microspectroscopy data correlated with values obtained from other methods such as Karl Fisher analysis (Wu and Polefka 2008) or optical coherence microscopy for evaluating SC thickness (Crowther et al. 2008). Based on the same methodology, NMF content and depth profiles of NMF can also be determined. CRM is a useful tool for the measurements of skin hydration levels, moisturizing product efficacy, and the influence of skin hydration on drug penetration enhancement. Egawa and Kajikawa (2009) addressed by using CRM the effect of skin hydration by measuring water profile after applying water or steam on the forearm of volunteers. The incubation time of the skin with water and the water temperature were varied to study the influence of these parameters on water profile changes. Water content in the SC increased with increasing the duration of water application from 1 to 10 min. Heating the applied water from 22 to 32 °C also resulted in an increase of the penetration depth, penetrated amount, and holding time of water. Furthermore, application water-steam increased the water content in the upper part of the SC. However, this study revealed an increase of the water content by comparing the water profile before and after water application, but it was not possible to differentiate the endogenous water from that penetrated in the skin. Direct measurement of water penetration in the skin by CRM requires replacing water of natural isotopic abundance by deuterated water D₂O, which shows a Raman peak corresponding to the O-D stretching vibration at 2500 cm⁻¹, different from endogenous water and clearly distinguishable in the spectra (Förster et al. 2011b). By applying emulsions and surfactant solutions made with deuterated water, Förster et al. showed that water penetrated from the formulations into the SC with a profile similar to that of endogenous water. However, no significant differences were observed between D₂O ingress from all formulations, and no correlations could be found between water penetration and the penetration of trans-retinol present in the formulations.

Penetration of deuterated water in the SC after application of pure D₂O on skin sample under occlusive conditions was recently confirmed by Ashtikara (2013).

Several studies aimed at explaining the penetration enhancement mechanism of dimethylsulfoxide (DMSO), one of the most common penetration enhancers. DMSO enhances the penetration of both hydrophilic and lipophilic drugs; its action in the skin is complex and remains largely an open question. Upon interaction with keratin, DMSO changes the keratin conformation, from α-helical to a β-sheet structure. DMSO can also interact with the polar head-groups of intercellular lipids, resulting in a perturbation of their packing. Finally, DMSO can alter the drug partition between the formulation and the skin layers due to its good solvent properties for a large number of molecules (Williams and Barry 2004).

Most studies on the behavior of SC lipids in skin have been carried out using IR spectroscopy. The poor signal-to-noise ratio in Raman spectroscopy has often limited its use for precise mechanistic investigation inside skin. Experiments using thin films of SC lipids allow devising what could be expected from measurements of skin. Tfayli et al. (2012c) studied the modification of the lipids’ conformational order, lateral packing, and polar interactions induced by three penetration enhancers. The Raman investigation was carried out on thin films of ceramides, the main lipid class of SC lipids, differing by their hydrocarbon chain length, polar head group, and the presence of a lateral group (ester or hydroxyl groups). The DMSO penetration enhancer affected mainly the polar interaction between lipid chains and the lateral packing of lipids, and its effect was dependent on the structure of the ceramides. For example, adding DMSO on ceramides IIIb resulted in the increase of the band at 1633 cm⁻¹ indicating weak H-bonds at the expense of the Amide I band at 1614 cm⁻¹ characteristic of strong H-bonds. DMSO also induced a slight change of the peak height ratio of the bands at 2850 cm⁻¹ (νCH₂ sym) and 2880 cm⁻¹ (νCH₂ asym) that reflected a looser lateral packing of all ceramides studied, indicating a decrease of compactness which could be associated with a higher permeability.

Finally, DMSO had a weak influence on the lipid chain conformations. Caspers et al. (2002) recorded the distribution of DMSO in the SC by CRM after application of an 80/20 mixture of DMSO and a 5% aqueous solution of propylene glycol. The mixture presented some characteristic bands, a doublet at 671 and 702 cm⁻¹ assigned to the ν(CSC) modes and a broad band at 1030–1048 cm⁻¹ assigned to ν(S = O) stretching. After application to the skin of volunteers, there was a shift of these bands, indicating the interaction of DMSO with water and possibly other components of SC. The SC depth profile was calculated as a function of time from the ratio of DMSO/proteins bands (ν(CSC)677 cm⁻¹/δ(CH₂)1450 cm⁻¹). They showed that most of the applied DMSO permeated through SC within 20 min and reached the viable epidermis where a small fraction was detected. Data collected for a longer time showed evidence of a progressive penetration of DMSO through the SC. DMSO could still be detected inside skin 72 h after application. Other conclusions about DMSO–lipids or DMSO–protein interactions could not be drawn during this study. However, in a study by Zhang et al. (2007b), reversible alteration of keratin secondary structure was shown using CRM when DMSO was applied in keratinocytes culture. They reported an appearance of β-sheet structures in the cellular keratin after treatment with DMSO; this change was reversible, and the original α-helix structure was recovered upon rehydration. This result was explained by strong complex formation between DMSO and water, which displaced the bound water that stabilizes the secondary structure of keratin.

Saar et al. (2010) used stimulated Raman scattering (SRS) microscopy for in vivo skin optical imaging in mice, first focusing on 3 vibration bands to image skin structure: lipids (CH₂ stretching, 2845 cm⁻¹), water (OH stretching, 3250 cm⁻¹), and proteins (CH₃ stretching, 2950 cm⁻¹). This allowed imaging the water, lipid, and protein distribution of the SC and the viable epidermis (Fig 13.2a, b image of the protein distribution in the SC and viable epidermis). The corneocytes and the intercellular spaces clearly appeared, as well as the sebaceous glands in the epidermis, which appeared both in lipids and water images, that is, when the incident laser frequency was tuned to the lipids (CH₂ stretching) or water (OH stretching) vibrations. The sebaceous gland appeared clear when the frequency was tuned to that of lipids vibration and dark when it was tuned to the water vibration. It was possible to image the ear of living mice by SRS frequency-tuned to the protein vibration (CH₃ stretching) (Fig. 13.2c). SRS was also used by the authors to explore the skin penetration of drugs and excipients (Saar et al. 2010, 2011). They explored DMSO penetration on human skin by SRS imaging using a specific vibration of deuterated DMSO-d6 at 2120 cm⁻¹ (2010). As shown in Fig. 13.2d, DMSO was found in the area surrounding the hair follicles, confirming the initial rapid penetration of DMSO into hair follicles. However, DMSO was not found to penetrate the hair itself.

A novel group of chemical penetration enhancers, iminosulfuranes, was studied by Song et al. (2005) on mouse and human skins. The objective was to design new penetration enhancers having high efficacy and less toxicity and irritation potency. The iminosulfurane enhancers were easily detected in the skin by their specific Raman peak at 670 cm⁻¹; its intensity was normalized to that of the amide I vibration band (1743 cm⁻¹). The results showed penetration of the enhancer into the SC to a depth of approximately 20 μm, the maximum content being found 5–10 μm below the skin surface. This new enhancer did not penetrate into the viable epidermis, which meant that it might bind to the protein and/or the lipids in the SC. Furthermore, its penetration enhancing activity was proved both for a lipophilic drug (hydrocortisone) and a hydrophilic one (caffeine). The penetration enhancement of caffeine was lower than that of hydrocortisone (Song et al. 2005).