Microscopy can be used to detect the influence of excipients on the extracellular lipid matrix as well as on the TJ. Light microscopy combined with immunodetection is usually employed to follow changes in expression and distribution of protein components such as TJ molecules. Immunofluorescent studies performed on frozen tissue sections allow the evaluation of “native” tissue, not fixed chemically and not exposed to solvents during the preparative methods. When combined with exploration in confocal laser scanning microscopy, where virtual optical sections are recorded within the immunolabeled tissue, this approach is very useful to evaluate TJ topography in 3D reconstructions (Brandner et al. 2002; Kubo et al. 2009). Immunostaining of dewaxed skin sections, following the standard histology protocol involving aldehyde fixation and paraffin embedding, should be interpreted with care when the SC is the compartment of interest. Indeed, solvents used during tissue dehydration and then during dewaxing efficiently remove intercorneocyte lipid matrix thus rendering impossible observation of excipient-induced changes in this localization. On the other hand, lipid removal may be beneficial for studies of TJ antigens persisting in the SC, as it improves accessibility of the latter to adequate antibodies.

Electron microscopy approach gives further insight into the structural composition of the SC barrier elements. Its “standard” transmission electron microscopy version, consisting of aldehyde fixation followed by osmium tetroxide post-fixation and resin embedding, is less suitable for studies of the SC barrier, essentially for the same reasons as the “standard” light microscopy. Although osmium tetroxide is able to partially preserve lipid bilayers from extraction during the subsequent dehydration procedures, visualization of the fine lamellar structures, within the extracellular spaces of SC, is not possible with this technique. Ruthenium tetroxide post-fixation solves this problem; however, incubation with this highly oxidizing reagent results in significant deterioration of the ultrastructure of living cells (Swartzendruber et al. 1995). Low-temperature embedding of samples in partially hydrophilic acrylate resins also preserves, to some extent, lipids from extraction by solvents used during the dehydration phase. However, it is not compatible with osmium or ruthenium tetroxide post-fixation because polymerization takes place under ultraviolet radiation which poorly penetrates tissue blocks turned black by the fixatives (Haftek et al. 1998). Acrylate-embedded samples are widely used for on-section immunolabeling with colloidal gold, including that of TJ (Haftek et al. 2011). Efficiency of ultrastructural immunolabeling can be further improved by the use of non-resin-embedded tissue ultra-cryosections (Igawa et al. 2011). Transmission electron microscopy is also used for observation of freeze-fracture samples, with or without immunolabeling (Corcuff et al. 2002). In this approach, the snap-frozen tissue is broken into pieces and the resulting fracture profile is covered with metal atoms. After removal of the underlying tissue, the metallic replica is examined with an electron microscope. Exposed fragments of the plasma membranes, where the split occurs preferentially, may show the presence of transmembrane molecules, such as TJ proteins (Kurasawa et al. 2009). The latter are arranged in a characteristic crisscrossed linear pattern and their TJ origin may be confirmed using immunogold labeling of the replicas with specific antibodies. Also, freeze-fracture studies efficiently help to investigate the lipid arrangement within the SC extracellular spaces (Van Hal et al. 1996).

Scanning electron microscopy methods appear to be less suited for SC barrier studies because of the magnification range, which is much lower than in the transmission variant, and due to accessibility limited to the surface of a sample. Nevertheless, recent technological improvements allowing examination in partial vacuum with signals obtained with secondary and retro-diffused electrons herald new applications using tape-stripped and immunolabeled SC [Haftek 2013, unpublished data].

Vibrational spectroscopies are powerful nondestructive techniques that detect characteristic vibrational energy levels of a molecule. Infrared and Raman spectra contain detailed information about the structures and interactions of molecular classes of interest. Several studies have reported the potential of FTIR and Raman spectroscopies for the characterization of the molecular and supramolecular organization of lipids and to follow changes in the lipid chain conformational order. More particularly, these techniques have already been used to characterize phase transitions of the SC lipids (Moore et al. 1997; Lawson et al. 1998; Ponec et al. 2000; Wartewig and Neubert 2007) of related model systems (Bouwstra et al. 2002) and to study polar and nonpolar lipid–lipid interactions (Corbe et al. 2007).

FTIR was used to obtain information on the lateral lipid organization and conformational ordering of the lipids (Janssens et al. 2012). FTIR spectroscopy can be used to investigate the biophysical alterations taking place in the lipid bilayer after treatment with penetration enhancers by studying the vibrational modes of its components. Different types of vibrations were monitored, the CH₂ symmetric stretching vibration and second derivatives of the scissoring bandwidth being the most informative for lateral organization (Boncheva et al. 2008; Damien and Boncheva 2010). For instance, the CH₂ symmetric and asymmetric stretching frequencies (υsym CH₂ and υasym CH₂) near 2,850 and 2,920 cm⁻¹, respectively, are primarily sensitive to lipid chain conformational order. A low (~2,848 cm⁻¹) wave number of the CH₂ symmetric stretching vibrations indicates the presence of a highly ordered lipid organization (either hexagonal or orthorhombic), while a high (2,853 cm⁻¹) wave number indicates the disordered liquid phase (Moore et al. 1997). However, a low average bandwidth of the scissoring vibrations represents a reduction of lipids in an orthorhombic organization and thus a less dense lipid organization (Janssens et al. 2012). Extraction of the SC lipids with solvents results in reduction of the CH₂ stretching absorbance. Indeed, the decrease in CH₂ stretching bandwidths accompanied by a decrease in CH₂ stretching band intensity suggests an overall extraction of SC lipids (Levang et al. 1999). The CH₂ rocking frequencies (710–735 cm⁻¹) are much more sensitive than the changes in CH₂ symmetric frequencies to the aforementioned hexagonal–orthorhombic phase transition. At temperatures below 40 °C, there are two distinct bands at approximately 720 and 729 cm⁻¹. The latter is a reliable marker of an orthorhombic phase in the SC lipids (Pensack et al. 2006).

DSC is a thermoanalytical technique measuring heat capacity of a sample. It allows thermal analysis of an isolated SC and is used to evaluate the interaction of excipients with the skin.

Alterations of the characteristic temperatures of the endothermic peaks can be observed, relative to disorganization of the lamellar structure of lipids and to dehydration or denaturation of the proteins. DSC is less sensitive to phase transitions, presumably because of relatively low enthalpy changes resulting from the transition, whereas FTIR is able to detect them with relative ease.

Raman spectroscopy reveals the spectral features specific to the modes of action of the penetration enhancers. These features can be simultaneously used as direct vibrational descriptors of the supramolecular organization, the polar interactions, and the conformational order of the CERs and as a descriptor of the penetration enhancer activity in drug delivery. Raman spectroscopy can be used to monitor not only the evolution of the lateral packing and the polar interactions due to penetration enhancer activity but also the intra-chain conformational order and the chain-end conformers, such as resulting from the thermotropic behavior of ceramides (Tfayli et al. 2010). Such information cannot be obtained with FTIR spectroscopy.

In this method, a primary X-ray beam, emitted by a source, is partially scattered by the structures present in a studied sample. When applied to SC, the obtained diffraction patterns give information about organization of the intercellular lipids. Position and intensity of the observed peaks are measured and interpreted, in order to evaluate the action of excipient on lipid organization (Bouwstra and Ponec 2006).

Molecular biology approach is required for the comprehension of viable cell responses. As such, it concerns exclusively studies of the living epidermal layers and not the SC. However, study of RNA gives an idea on the transcription rate of a specific gene and this, in turn, indicates possible repercussions on the subsequent SC formation and function. For example, quantification of mRNA coding for TJ proteins is possible using real-time polymerase chain reaction (RT PCR). Using this technique, one can monitor the presence of mRNA of various TJ proteins in keratinocyte cultures with different knockdown genes while testing permeability of the expressed TJ (Kirschner et al. 2013). Already in 2008, Yamamoto et al. have performed a posttranscriptional gene silencing of different TJ proteins to study the impact of these proteins on the formation of the SC barrier (Yamamoto et al. 2008). Keratinocytes were transfected with small interferent RNA (si-RNA) targeting either claudin-1 mRNA or occludin mRNA. It turns out that transfected cultures were not able to develop a functional barrier. Using molecular biology techniques, it would be possible to detect variations of the keratinocyte gene activity that excipients may engender when applied on the skin. Hybridization in situ, on tissue sections, is an approach combining structural information with mRNA detection. It is, thus, particularly well suited for studies of locally induced changes following topical treatments.

Biochemistry assays are commonly used to analyze proteins as well as lipids extracted from a tissue.

Immunoblotting technique can detect the presence of a given protein. In fact, the presence of the mRNA in the cell cytoplasm is insufficient to predict the presence of the corresponding protein due to the complexity of the translation machinery. Western blot is a reliable qualitative and quantitative technique applicable to TJ protein studies (Kurasawa et al. 2009; Kirschner et al. 2013).

Lipid analysis may be performed on SC extracts using high performance thin layer chromatography (HPTLC) (Rissmann et al. 2008; Popa et al. 2012). Combined with sequential tape-stripping, HPTLC approach allows following lipid composition of intercellular spaces between corneocytes in the SC according to the sample’s depth (Popa et al. 2011).

One of the key parameters to monitor the skin barrier function is the transepidermal water loss, i.e., the rate of water evaporation through the epidermis, independent of sweating. TEWL provides a noninvasive method for assessing changes in the barrier properties of the SC. It can be considered as a determinant indicative of the functional state of the epidermal barrier in normal, pathological, and experimental conditions (Janssens et al. 2012). Measurements are performed in standardized ambient conditions according to the well-defined consensus guidelines (Pinnagoda et al. 1990).

Diffusion cell is helpful to study the influence of excipients on drug permeation and penetration. Indirectly, information is gained on the functional state of the treated skin barrier. Skin permeation is evaluated by measuring the steady-state permeation flux of the tested drug. An enhancement ratio can be determined in the presence of the studied excipient. The diffusion cell consists of the upper, i.e., “donor,” and the lower, i.e., “receptor,” chambers, separated by a tested skin. The epidermis faces the donor chamber where the tested products are applied, and the dermis faces the receptor chamber filled with a receptor fluid, where the drug or another tracer will be measured after permeation. Temperature is controlled throughout the experiment and should be maintained at in vivo skin conditions (32 °C). Ensuring that the skin used for testing maintains its physical integrity is an essential factor to the successful performance of permeation experiments, as specified in the test guideline OECD 428 (Lévêque et al. 1993). The guidance document recommends the measurement of TEWL, transepithelial electrical resistance, or the use of tritiated water as permeation markers.

TEER measurement can be used to detect the TJ functionality status. Usually the functionality of TJ is evaluated in vitro on monolayer of cultured cells or on reconstructed human epidermis. Functional TJ significantly increase the tissue impedance towards a very weak electrical current generated by ohmmeters, by virtue of limiting ion flux through the intercellular space (Kurasawa et al. 2009; Abdayem et al. 2011). In normal human skin, SC by itself is able to prevent the passage of the testing current. Therefore, to detect any variation of resistance due to TJ modulation, the SC has to be partially or completely removed. This is usually done by tape stripping. In the case of in vitro reconstructed epidermis, the extracellular lipid matrix is not perfectly organized (Ponec et al. 2000) and the SC is fully hydrated. This results in readable TEER measurements, as the electrical current is able to cross this imperfect SC barrier (Abdayem et al. 2011). For the detection of the effects of different excipients on the TJ barrier, reconstructed epidermis is cultured in plastic inserts equipped with a permeable polycarbonate bottom. This experimental setting allows positioning of the ohmmeter electrodes on both sides of the cultured tissue. Tested excipients can be added at different doses either topically, i.e., directly on the SC surface, or “systemically,” into the feeding medium penetrating the reconstructed epidermis from the bottom compartment. In case of “systemic” treatment, excipients are in direct interaction with the epidermal TJ and the SC remains intact. Topically applied agents have first to cross the SC barrier, before acting on TJ situated in the granular layer. Thus, TEER readings in the latter case reflect both TJ and SC permeability modulation.

Intact SC and functional epidermal TJ are able to block or to limit the passage of various molecules depending on their size and nature, e.g., lipophilicity, pH, and charge. Following the passage of a tracer applied at the surface of the skin or injected at the basal layers of the epidermis is another way to study the functional quality of the complementary SC and TJ barriers. For example, in the case of topical application of Lucifer yellow, detection of this hydrophilic fluorescent dye, with a confocal microscope, in the living layers of epidermis, may indicate the presence of poor SC barrier, either constitutive or induced (Kurasawa et al. 2009). Another widely used small molecular marker is biotin (433–666 Da) (Ding et al. 2011). After being injected to the dermis, the tracer diffuses through the intercellular spaces until it reaches the TJ barrier in the stratum granulosum. Immunohistochemical detection of the biotin on vertical skin sections reveals the sites where the diffusion has been stopped. These points are recognized as TJ because of their co-localization with anti-occludin staining (Furuse et al. 2002). Tracer penetration studies can also be performed at the ultrastructural level using electron-dense lanthanum salts. Ken Hashimoto was the first to describe TJ structures in human epidermis using, precisely, this approach (Hashimoto 1971).