Structural and Functional Analysis of Excised Skins and Human Reconstructed Epidermis with Confocal Raman Spectroscopy and in Microfluidic Diffusion Chambers.

Several ex vivo and in vitro skin models are available in the toolbox of dermatological and cosmetic research. Some of them are widely used in drug penetration testing. The excised skins show higher variability, while the in vitro skins provide more reproducible data. The aim of the current study was to compare the chemical composition of different skin models (excised rat skin, excised human skin and human-reconstructed epidermis) by measurement of ceramides, cholesterol, lactate, urea, protein and water at different depths of the tissues. The second goal was to compile a testing system, which includes a skin-on-a-chip diffusion setup and a confocal Raman spectroscopy for testing drug diffusion across the skin barrier and accumulation in the tissue models. A hydrophilic drug caffeine and the P-glycoprotein substrate quinidine were used in the study as topical cream formulations. The results indicate that although the transdermal diffusion of quinidine is lower, the skin accumulation was comparable for the two drugs. The various skin models showed different chemical compositions. The human skin was abundant in ceramides and cholesterol, while the reconstructed skin contained less water and more urea and protein. Based on these results, it can be concluded that skin-on-a-chip and confocal Raman microspectroscopy are suitable for testing drug penetration and distribution at different skin layers within an exposition window. Furthermore, obese human skin should be treated with caution for skin absorption testing due to its unbalanced composition.

Novel aspects of Raman spectroscopy in skin research.

The analytical technology of Raman spectroscopy has an almost 100-year history. During this period, many modifications and developments happened in the method like discovery of laser, improvements in optical elements and sensitivity of spectrometer and also more advanced light detection systems. Many types of the innovative techniques appeared (e.g. Transmittance Raman spectroscopy, Coherent Raman Scattering microscopy, Surface-Enhanced Raman scattering and Confocal Raman spectroscopy/microscopy). This review article gives a short description about these different Raman techniques and their possible applications. Then, a short statistical part is coming about the appearance of Raman spectroscopy in the scientific literature from the beginnings to these days. The third part of the paper shows the main application options of the technique (especially confocal Raman spectroscopy) in skin research, including skin composition analysis, drug penetration monitoring and analysis, diagnostic utilizations in dermatology and cosmeto-scientific applications. At the end, the possible role of artificial intelligence in Raman data analysis and the regulatory aspect of these techniques in dermatology are briefly summarized. For the future of Raman Spectroscopy, increasing clinical relevance and in vivo applications can be predicted with spreading of non-destructive methods and appearance with the most advanced instruments with rapid analysis time.

Characterization and ex vivo evaluation of excised skin samples as substitutes for human dermal barrier in pharmaceutical and dermatological studies.

BACKGROUND: Excised animal and human skins are frequently used in permeability testing in pharmaceutical research. Several factors exist that may have influence on the results. In the current study some of the skin parameters that may affect drug permeability were analysed for human, mouse, rat and pig skin. MATERIALS AND METHODS: Classic biophysical skin parameters were measured (e.g. pH, hydration, permittivity, transepidermal water loss). Physiological characteristics of the skins were also analysed by confocal Raman spectroscopy, scanning electron microscopy and two-photon microscopy. RESULTS: Based on biophysical testing, skin barrier function was damaged in psoriatic mouse skin and in marketed pig skin. Hydration and pH values were similar among the species, but freezing and thawing reduced the water content of the skins and shifted the surface pH to acidic. Aging reduced hydration and permittivity, resulting in impaired barrier function. Mechanical sensitization used in permeability studies resulted in proportional thinning of dead epidermis. DISCUSSION: Results indicate that depending on the scientific question it should be considered whether fresh or frozen tissue is used, and for certain purposes rodent skins are well usable. The structure of the skin tissue (ceramide, cholesterol, keratin, natural moisturizing factor or urea) is similar in rats and mice, but due to the higher skin thickness the lipid distribution is different in porcine skin. Psoriasis led to irregular chemical composition of the skin. CONCLUSION: A comprehensive evaluation of skin samples of four species was performed. The biophysical and microscopic observations should be considered when selecting drug penetration models and experimental conditions.

Drug Delivery through the Psoriatic Epidermal Barrier-A "Skin-On-A-Chip" Permeability Study and Ex Vivo Optical Imaging.

Psoriasis is a chronic inflammatory disease with unmet medical needs. To clarify potential therapeutic targets, different animal models have been developed. In the current study, imiquimod-induced psoriasiform dermatitis was used for monitoring the changes in skin thickness, transepidermal water loss, body weight, blood perfusion and drug permeability for a topical cream formulation of caffeine, both in wild type and in knock out mice. Morphological characterization of control and diseased tissues was performed by scanning electron microscopy and two-photon microscopy. The chemically induced psoriatic group showed increased skin permeability for the model drug during disease progression. In wild type and TRPA1 KO mice, however, enhanced skin thickness and hyperkeratosis blocked further increase of drug penetration at the late phase (96 h). These results indicate that topical drug therapy can be more effective in early phases of plaque development, when skin thickness is lower. Although paracellular connections (tight junctions) are looser in the advanced phase, hyperkeratosis blocks drug delivery through the transappendageal routes. Novel drug formulations may have the potency for effective drug delivery across the epidermal barrier even in the advanced phase. For development of more effective topical drugs, further research is proposed to explore drug penetration both in healthy and diseased conditions.

Skin-on-a-Chip Technology for Testing Transdermal Drug Delivery-Starting Points and Recent Developments.

During the last decades, several technologies were developed for testing drug delivery through the dermal barrier. Investigation of drug penetration across the skin can be important in topical pharmaceutical formulations and also in cosmeto-science. The state-of- the-art in the field of skin diffusion measurements, different devices, and diffusion platforms used, are summarized in the introductory part of this review. Then the methodologies applied at Pázmány Péter Catholic University are shown in detail. The main testing platforms (Franz diffusion cells, skin-on-a-chip devices) and the major scientific projects (P-glycoprotein interaction in the skin; new skin equivalents for diffusion purposes) are also presented in one section. The main achievements of our research are briefly summarized: (1) new skin-on-a-chip microfluidic devices were validated as tools for drug penetration studies for the skin; (2) P-glycoprotein transport has an absorptive orientation in the skin; (3) skin samples cannot be used for transporter interaction studies after freezing and thawing; (4) penetration of hydrophilic model drugs is lower in aged than in young skin; (5) mechanical sensitization is needed for excised rodent and pig skins for drug absorption measurements. Our validated skin-on-a-chip platform is available for other research groups to use for testing and for utilizing it for different purposes.

Age-Related Inflammatory Balance Shift, Nasal Barrier Function, and Cerebro-Morphological Status in Healthy and Diseased Rodents.

Increased blood-brain barrier (BBB) permeability and extensive neuronal changes have been described earlier in both healthy and pathological aging like apolipoprotein B-100 (APOB-100) and amyloid precursor protein (APP)-presenilin-1 (PSEN1) transgenic mouse models. APOB-100 hypertriglyceridemic model is a useful tool to study the link between cerebrovascular pathology and neurodegeneration, while APP-PSEN1 humanized mouse is a model of Alzheimer's disease. The aim of the current study was to characterize the inflammatory changes in the brain with healthy aging and in neurodegeneration. Also, the cerebro-morphological and cognitive alterations have been investigated. The nose-to-brain delivery of a P-glycoprotein substrate model drug (quinidine) was monitored in the disease models and compared with the age-matched controls. Our results revealed an inflammatory balance shift in both the healthy aged and neurodegenerative models. In normal aging monocyte chemoattractant protein-1, stem cell factor and Rantes were highly upregulated indicating a stimulated leukocyte status. In APOB-100 mice, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB), and interleukin-17A (IL-17A) were induced (vascular reaction), while in APP-PSEN1 mice resistin, IL-17A and GM-CSF were mostly upregulated. The nasal drug absorption was similar in the brain and blood indicating the molecular bypass of the BBB. The learning and memory tests showed no difference in the cognitive performance of healthy aged and young animals. Based on these results, it can be concluded that various markers of chronic inflammation are present in healthy aged and diseased animals. In APOB-100 mice, a cerebro-ventricular dilation can also be observed. For development of proper anti-aging and neuroprotective compounds, further studies focusing on the above inflammatory targets are suggested.

Development and Evaluation of a Human Skin Equivalent in a Semiautomatic Microfluidic Diffusion Chamber.

There is an increasing demand for transdermal transport measurements to optimize topical drug formulations and to achieve proper penetration profile of cosmetic ingredients. Reflecting ethical concerns the use of both human and animal tissues is becoming more restricted. Therefore, the focus of dermal research is shifting towards in vitro assays. In the current proof-of-concept study a three-layer skin equivalent using human HaCaT keratinocytes, an electrospun polycaprolactone mesh and a collagen-I gel was compared to human excised skin samples. We measured the permeability of the samples for 2% caffeine cream using a miniaturized dynamic diffusion cell ("skin-on-a-chip" microfluidic device). Caffeine delivery exhibits similar transport kinetics through the artificial skin and the human tissue: after a rapid rise, a long-lasting high concentration steady state develops. This is markedly distinct from the kinetics measured when using cell-free constructs, where a shorter release was observable. These results imply that both the established skin equivalent and the microfluidic diffusion chamber can serve as a suitable base for further development of more complex tissue substitutes.

Development of Skin-On-A-Chip Platforms for Different Utilizations: Factors to Be Considered.

There is increasing interest in miniaturized technologies in diagnostics, therapeutic testing, and biomedicinal fundamental research. The same is true for the dermal studies in topical drug development, dermatological disease pathology testing, and cosmetic science. This review aims to collect the recent scientific literature and knowledge about the application of skin-on-a-chip technology in drug diffusion studies, in pharmacological and toxicological experiments, in wound healing, and in fields of cosmetic science (ageing or repair). The basic mathematical models are also presented in the article to predict physical phenomena, such as fluid movement, drug diffusion, and heat transfer taking place across the dermal layers in the chip using Computational Fluid Dynamics techniques. Soon, it can be envisioned that animal studies might be at least in part replaced with skin-on-a-chip technology leading to more reliable results close to study on humans. The new technology is a cost-effective alternative to traditional methods used in research institutes, university labs, and industry. With this article, the authors would like to call attention to a new investigational family of platforms to refresh the researchers' theranostics and preclinical, experimental toolbox.