Correspondence: Dr. Maike Windbergs, Department for Biopharmaceutics and Pharmaceutical Technology, Saarland University, Campus A4.1, 66123 Saarbruecken, Germany, Tel.: +49 681 302 2358, Fax: +49 681 302 4677, e-mail: email@example.com
In vitro testing of drugs with excised human skin is a valuable prerequisite for clinical studies. However, the analysis of excised human skin presents several obstacles. Ongoing drug diffusion, microbial growth and changes in hydration state influence the results of drug penetration studies. In this work, we evaluate freeze-drying as a preserving preparation method for skin samples to overcome these obstacles. We analyse excised human skin before and after freeze-drying and compare these results with human skin in vivo. Based on comprehensive thermal and spectroscopic analysis, we demonstrate comparability to in vivo conditions and exclude significant changes within the skin samples due to freeze-drying. Furthermore, we show that freeze-drying after skin incubation with drugs prevents growth of drug crystals on the skin surface due to drying effects. In conclusion, we introduce freeze-drying as a preserving preparation technique for in vitro testing of human skin.
For rational development of novel drug delivery strategies via the skin, in vitro testing of drug behaviour and penetration into excised human skin is a mandatory prerequisite for clinical studies. As the accessibility of drug penetration data within the different skin layers, apart from plasma concentration, is severely limited in human in vivo studies, detailed and spatially resolved information about the extent of drug penetration and kinetics can solely be gained by in vitro testing.
Even though the in vitro methodology is well established, the handling of excised human skin for in vitro testing reveals many obstacles [1-3]. Drug penetration studies generally follow strict time intervals for sampling. However, penetration within the tissue continues after termination of drug incubation, and thus time-consuming analysis can lead to misinterpretations of drug penetration kinetics . In addition, microbial growth facilitated by long-term experiments and changes in the hydration state of the sample influence the results. Based on this situation, there is a high demand for a technique that preserves the skin samples.
In this study, we evaluate freeze-drying as a preserving preparation technique for in vitro testing of human skin. To assure applicability of freeze-dried skin samples for in vitro analysis, the absence of structural changes within the skin tissue has to be verified in a comparison with human skin in vivo. Furthermore, detectability and the behaviour of drugs within the tissue have to be investigated.
For detailed information on experimental design, please see supporting information (Data S1).
To investigate thermal properties of excised skin samples and reveal potential changes in their structural organization, stratum corneum (SC) and heat separated epidermis (HSE) were analysed by differential scanning calorimetry before and after freeze-drying. Isolated SC (Fig. 1a) showed specific endothermic peaks in accordance with the literature . Freeze-dried samples exhibited more pronounced peaks with a slight temperature shift due to the absence of water in the tissue . Rehydration of the freeze-dried samples in controlled humidity restored the original thermogram peaks demonstrating this water effect (data not shown). As expected, thermograms of HSE did not exhibit any thermic events in the analysed temperature range, due to the low lipid concentration and a broad protein melting peak of keratin [7, 8].
Infrared (IR) spectroscopy as a valid technique to identify structural changes exposed no significant differences between human skin in vivo, fresh and freeze-dried HSE and SC, except for their water content (Fig. 1b,c). The water-related peak was embodied by the ν(OH) band around 3250 cm−1 [9, 10]. No additional peaks or peak shifts indicating chemical changes or conformational alterations could be observed for freeze-dried samples. The higher lipid content in SC compared to HSE could be visualized by higher intensities in the spectral region representing ν(CH) between 2800 cm−1 and 3060 cm−1 [11, 12].
Furthermore, Raman spectroscopy analysis as an upcoming analytical technique for skin research was used to demonstrate optical similarity between human skin in vivo, fresh and freeze-dried excised skin. Spectra from fresh and freeze-dried SC (Fig. 2a) and HSE samples (Fig. 2b) were compared to human skin in vivo.
To gain statistically valid information regarding the similarity of freeze-dried and fresh skin samples, a spectra subtraction analysis was performed. This included the subtraction of the intensity values of two spectra resulting in a graphical illustration of the spectral difference. The absolute area under the curve (AUC) of this plot is employed to quantify the discrepancy between the subtracted spectra.
To determine a zero value representing the intra-individual variability, three spectra recorded from the same skin sample on different positions were analysed. The result was a mean AUC of 70.7 cts/cm−1 with a 95% confidence interval of up to 94.6 cts/cm−1.
In the next step, the spectra of freeze-dried and fresh SC (Fig. 2c) and HSE (Fig. 2d), respectively, were subtracted as well. The determined AUCs for SC (65.1 cts/cm−1) and HSE (79.4 cts/cm−1) were both covered by the 95% confidence interval of the reference. Therefore, it can be concluded that the spectral difference between freeze-dried and fresh skin samples is not statistically different from the individual spectral variability.
Based on this comprehensive analysis using thermal as well as spectroscopic techniques, comparability to in vivo conditions can be proved and significant changes within the skin samples due to freeze-drying are excluded.
As a further issue, the uncontrolled crystallization of drugs in skin samples due to supersaturation during drying is addressed. Human skin samples, incubated in caffeine solution, were analysed by polarized light microscopy and confocal Raman microscopy directly after incubation and after freeze-drying. Already hours after incubation caffeine crystals appeared on the skin. Figure 2e depicts polarized-transmitted light microscopic pictures of skin samples freeze-dried and non-freeze-dried (I and II HSE, III and IV SC). Figure 2e I and III expose the presence of crystalline caffeine-hydrate in typical needle shape in skin samples. No such crystals were observed in samples freeze-dried immediately after incubation (Fig. 2e II and IV). Thus, freeze-drying preserves the molecular disperse distribution and avoids large crystal growth due to slow drying effects .
Figure 2f exhibits Raman spectra of crystalline caffeine as observed in incubated skin samples and caffeine in freeze-dried samples. The higher intensity of caffeine bands in crystalline state outshines the skin bands. In freeze-dried skin, the caffeine peak at 553 cm−1 [δ(O=C-N)]  is clearly detectable and not interfering with any skin-related peaks. This facilitates identification and quantification of drug inside the skin.
Freeze-drying has been successfully introduced as a suitable preserving preparation technique for in vitro testing of human skin. We highlight that optical similarity to human skin in vivo remains after excision and neither HSE nor SC samples are chemically affected by the freeze-drying procedure. Besides easier sample handling and prevention of microbial growth, the technique instantly preserves the drug diffusion status, thus avoiding drug crystallization and misinterpretation of penetration kinetics.
L. Franzen, M. Windbergs and L. Vidlářová performed the experiments and analysed the data. M. Windbergs and U.F. Schaefer designed the research study. K.-H. Kostka provided the human skin samples. L. Franzen and M. Windbergs wrote the manuscript.