Germer Gregor, Ohigashi Takuji, Yuzawa Hayato, Kosugi Nobuhiro, Flesch Roman, Rancan Fiorenza, Vogt Annika, Rühl Eckart
Physical Chemistry, Freie Universität Berlin, Arnimallee 22, Berlin 14195, Germany.
UVSOR Synchrotron Facility, Institute for Molecular Science, Okazaki 444-8585, Japan.
ACS Omega. 2021 Apr 28;6(18):12213-12222. doi: 10.1021/acsomega.1c01058. eCollection 2021 May 11.
Drug penetration in human skin ex vivo following a modification of skin barrier permeability is systematically investigated by scanning transmission X-ray microscopy. Element-selective excitation is used in the O 1s regime for probing quantitatively the penetration of topically applied rapamycin in different formulations with a spatial resolution reaching <75 nm. The data were analyzed by a comparison of two methods: (i) two-photon energies employing the Beer-Lambert law and (ii) a singular value decomposition approach making use of the full spectral information in each pixel of the X-ray micrographs. The latter approach yields local drug concentrations more reliably and sensitively probed than the former. The present results from both approaches indicate that rapamycin is not observed within the stratum corneum of nontreated skin ex vivo, providing evidence for the observation that this high-molecular-weight drug inefficiently penetrates intact skin. However, rapamycin is observed to penetrate more efficiently the stratum corneum when modifications of the skin barrier are induced by the topical pretreatment with the serine protease trypsin for variable time periods ranging from 2 to 16 h. After the longest exposure time to serine protease, the drug is even found in the viable epidermis. High-resolution micrographs indicate that the lipophilic drug preferably associates with corneocytes, while signals found in the intercellular lipid compartment were less pronounced. This result is discussed in comparison to previous work obtained from low-molecular-weight lipophilic drugs as well as polymer nanocarriers, which were found to penetrate the intact stratum corneum exclusively via the lipid layers between the corneocytes. Also, the role of the tight junction barrier in the stratum granulosum is briefly discussed with respect to modifications of the skin barrier induced by enhanced serine protease activity, a phenomenon of clinical relevance in a range of inflammatory skin disorders.
通过扫描透射X射线显微镜系统地研究了皮肤屏障通透性改变后药物在人离体皮肤中的渗透情况。在O 1s区域使用元素选择性激发，以定量探测不同制剂中局部应用的雷帕霉素的渗透情况，空间分辨率达到<75 nm。通过比较两种方法对数据进行分析：(i)采用比尔-朗伯定律的双光子能量法；(ii)利用X射线显微照片每个像素中的全光谱信息的奇异值分解方法。后一种方法比前一种方法能更可靠、更灵敏地探测局部药物浓度。两种方法的当前结果均表明，在离体未处理皮肤的角质层中未观察到雷帕霉素，这为这种高分子量药物不能有效穿透完整皮肤的观察提供了证据。然而，当用丝氨酸蛋白酶胰蛋白酶进行2至16小时不等的局部预处理诱导皮肤屏障改变时，观察到雷帕霉素能更有效地穿透角质层。在最长时间暴露于丝氨酸蛋白酶后，甚至在活表皮中发现了该药物。高分辨率显微照片表明，亲脂性药物优先与角质形成细胞结合，而在细胞间脂质区室中发现的信号则不那么明显。将该结果与先前从低分子量亲脂性药物以及聚合物纳米载体获得的研究结果进行了比较讨论，先前的研究发现它们仅通过角质形成细胞之间的脂质层穿透完整的角质层。此外，还简要讨论了颗粒层紧密连接屏障在丝氨酸蛋白酶活性增强引起的皮肤屏障改变中的作用，这一现象在一系列炎症性皮肤病中具有临床相关性。
