Label-free, quantitative detection of drugs and nanocarriers in cells and skin

In dieser Arbeit wurden Wirkstoffe mithilfe von labelfreien Methoden ortsaufgelöst in biologischen Proben detektiert. Die Detektion erfolgte durch eine Simulation der gemessenen Spektren hyperspektraler Messungen im spektralen Bereich an der O 1s Kante (520-565 eV) und im mittleren Infrarotbereich (1190-1320 cm^-1 und 1600-1850 cm^-1) von behandelten Proben mithilfe einer numerischen Singulärwertzerlegung auf Basis von einzeln gemessenen Reinsubstanzspektren und Spektren der unbehandelten biologischen Spezies. Der erste Teil der Arbeit beinhaltet die labelfreie Detektion des Wirkstoffs Rapamycin und der redoxsensitiven Nanocarrier ex vivo topisch behandelter Humanhaut. Hierbei wurden in Epoxidharz eingebettete Proben mithilfe von Röntgenmikroskopie an der O 1s-Kante untersucht. Weitere Proben wurden mithilfe von photothermischer Expansionsspektroskopie (AFM-IR) untersucht. Es kann gezeigt werden, dass eine Vorbehandlung mit der Serinprotease Trypsin die Membranen der Corneocyten öffnet und der Wirkstoff intrazellulär in das Stratum Corneum transportiert werden kann. Der Wirkstoffnachweis erfolgte für diese Proben quantitativ. Es lässt sich auch die transportverstärkende Wirkung der Vaseline mit hoher Ortsauflösung beobachten. Beide genutzten Nanocarrier zeigen einen Transport des Wirkstoffs Rapamycin in die Haut. Langzeitbehandlungen zeigen eine Anreicherung der lipophilen Nanocarrier in den Lipidmembranen der Zellkerne der viablen Epidermis. Die Messung einer Kryoprobe mittels AFM-IR zeigt vergleichbare Ergebnisse zu den fixierten und eingebetteten Proben. Dies zeigt, dass die umfangreiche Probenvorbereitung fixierter Haut für die Röntgenmikroskopie keinen Einfluss auf die lokale Wirkstoffverteilung hat. Im zweiten Teil der Arbeit werden die Wirkstoffe Cetuximab und Docetaxel in SCC-25-Kollagen-Zellkulturen auf Basis von hyperspektralen AFM-IR-Messungen ortsaufgelöst und labelfrei nachgewiesen. Die Auswertung erfolgt ebenfalls auf Basis einer numerischen Singulärwertzerlegung mit den jeweiligen Spektren der Reinsubstanzen im mittleren Infrarotbereich zur Bestimmung der lokalen Wirkstoffverteilung. Die Gabe des Wirkstoffs Cetuximab erfolgt bei einer Zellkultur topisch auf den Tumorzellen und bei der anderen über das Nährmedium an der Seite der Kollagene, um eine systemische Behandlung zu simulieren. Es soll untersucht werden, ob eine topische Behandlung als Ergänzung oder Alternative zu einer systemischen dienen kann. Der Wirkstoff Cetuximab kann bei beiden Autragungsmethoden detekiert werden. Messungen mit hoher räumlicher Auflösung deuten darauf hin, dass eine zelluläre Aufnahme des Wirkstoffs stattfindet. Docetaxel kann in zwei Zellkulturen, welche mit unterschiedlichen Konzentrationen topisch behandelt wurden, labelfrei nachgewiesen werden.In this thesis, drugs were detected spatially resolved in biological samples using label-free methods. The detection was performed by simulating the experimental spectra taken in hyperspectral measurements in the spectral in the regime of O 1s edge (520-565 eV) and in the mid-infrared regime (1190-1320 cm^-1 and 1600-1850 cm^-1) using a numerical singular value decomposition based on individually measured reference spectra and spectra of the untreated biological species. The first part of this work involves label-free detection of the drug rapamycin and the redoxsensitive nanocarriers in topically treated human skin ex vivo. Here, samples embedded in epoxy resin were examined using scanning X-ray microscopy at the O 1s edge. Additionally the samples were examined using photothermal expansion spectroscopy (AFM-IR). It is shown that pretreatment with the serine protease trypsin opens the membranes of the corneocytes and the drug can be transported intracellularly into the stratum corneum. Quantitative drug detection was accomplished for these samples. The transport enhancing effect of petrolatum was also investigated with high spatial resolution. Redoxsensitive core-multishell nanocarriers show enhanced rapamycin penetration into the top skin layers. Long-term treatments show an accumulation of the lipophilic nanocarriers in the lipid membranes of the cell nuclei in the viable epidermis. Studies on a cryosample by AFM-IR shows comparable results to fixed and embedded skin samples. This supports the validity of extensive sample preparation protocols used along with scanning X-ray microscopy indicating that there is no influence on the local drug distribution. In the second part of this work, the drugs cetuximab and docetaxel are spatially resolved and label-free detected in SCC-25 collagen cell cultures based on hyperspectral AFM-IR measurements. Singular value decomposition is also used for determining the local drug distribution using reference spectra of the pure substances in the mid-infrared regime. The drug cetuximab is topically applied on a cellular tumor model to prove the efficacy of this approach. Alternatively, the drug is administered via the culture medium on the side of the collagen base of the model in the other to simulate a systemic treatment. The aim of this approach wasto investigate whether topical treatment can serve as an adjunct or alternative to the established systemic treatment. The results indicate that drug cetuximab can be detected by both systemic and topical treatment. High spatialresolution measurements indicate that cellular uptake of the drug takes place. Label-free detection of docetaxel was also accomplished in cell cultures that were topically treated with different drug concentrations

Probing the Penetration of Rapamycin by Scanning Transmission X-ray Microscopy

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

Controlled assembly of artificial 2D materials based on the transfer of oxo-functionalized graphene

Functionalized 2D materials have unique properties, but are currently not used for the assembly of van der Waals heterostructures. Here, we present the controlled transfer of artificially synthesized, polar and highly transparent oxo-functionalized graphene, which can decouple graphene layers

Appendicitis during the COVID-19 lockdown: results of a multicenter analysis in Germany

Purpose The COVID-19 pandemic has transformed medical care worldwide. General surgery has been affected in elective procedures, yet the implications for emergency surgery are unclear. The current study analyzes the effect of the COVID-19 lockdown in spring 2020 on appendicitis treatment in Germany. Methods Hospitals that provided emergency surgical care during the COVID-19 lockdown were invited to participate. All patients diagnosed with appendicitis during the lockdown period (10 weeks) and, as a comparison group, patients from the same period in 2019 were analyzed. Clinical and laboratory parameters, intraoperative and pathological findings, and postoperative outcomes were analyzed. Results A total of 1915 appendectomies from 41 surgical departments in Germany were included. Compared to 2019 the number of appendectomies decreased by 13.5% (1.027 to 888, p=0.003) during the first 2020 COVID-19 lockdown. The delay between the onset of symptoms and medical consultation was substantially longer in the COVID-19 risk group and for the elderly. The rate of complicated appendicitis increased (58.2 to 64.4%), while the absolute number of complicated appendicitis decreased from 597 to 569, (p=0.012). The rate of negative appendectomies decreased significantly (6.7 to 4.6%; p=0.012). Overall postoperative morbidity and mortality, however, did not change. Conclusion The COVID-19 lockdown had significant effects on abdominal emergency surgery in Germany. These seem to result from a stricter selection and a longer waiting time between the onset of symptoms and medical consultation for risk patients. However, the standard of emergency surgical care in Germany was maintained