Simulation of droplet-based microfluidic lab-on-a-chip applications

This paper was presented at the 3rd Micro and Nano Flows Conference (MNF2011), which was held at the Makedonia Palace Hotel, Thessaloniki in Greece. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, Aristotle University of Thessaloniki, University of Thessaly, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute.Miniaturization of biological and chemical assays in lab-on-a-chip systems is a highly topical field of research. Droplet-based microfluidic chips are types of these miniaturized systems. They expand the capability of assays with special features that are unreached by traditional workflows. In particular, small sample volumes, independent separated reaction units, high throughput, automation and parallelization of assays are prominent features of droplet-based microfluidic devices. Full custom centric design of droplet-based microfluidic lab-on-a-chip technology implicates a high system integration level and design complexity. Therefore advanced development methodologies are needed, comparable with the methods in electronic design automation. Our design and simulation toolkit meets these requirements for an agile and low-risk development of custom lab-on-a-chip devices. The system simulation approach enables a fast and precise prediction of complex microfluidic networks. This fact is confirmed by reference and benchmark experiments. The results show that the simulation correctly reproduces the experimental measurements.The German BMBF and the EU in the projects DiNaMiD, signature 0315591B and NoE Photonics4Life, Grant Agreement number: 224014

In vitro und in vivo Testung von Knorpelersatzmaterialien für chonrale und osteochondrale Defekte im Knie

Hyaliner Gelenkknorpel ist frei von Nerven, Blut und Lymphgefäßen und besitzt dadurch nur eine begrenzte Regenerationsfähigkeit. Die Zufuhr der Nährstoffe erfolgt ausschließlich per Diffusion durch die extrazelluläre Matrix (EZM) und/oder die Synovialflüssigkeit. Nicht behandelte Knorpeldefekte können daher im Langzeitverlauf zur Arthrose führen, die als Endpunkt einen vollständigen Gelenkersatz erfordert. Ziel der vorliegenden Arbeit war es, verschiedene Knorpelersatzmaterialien in vitro (bovines Knorpelstanzenmodell) und in vivo (Schaf) zu untersuchen. In der ersten Phase wurden die verschiedene Knorpelersatzmaterialien unter standardisierten Bedingungen für bis zu 12 Wochen in vitro kultiviert und anschließend biomechanisch, molekularbiologisch und histologisch untersucht. In der zweiten Phase wurde am Standort Jena das Knorpelersatzmaterial BioSeed®-C der Firma TTT in vivo untersucht. Die entstandenen Gewebsregenerate wurden nach 6 und 12 Monaten biomechanisch, molekularbiologisch und histologisch untersucht. Die gewonnenen Erkenntnisse sollen in die Entwicklung standardisierter Verfahren für die Prüfung von Tissue engineering (TE) Konstrukten für den orthopädisch-unfallchirurgischen Einsatz einfließen. Neben den in vitro und in vivo Versuchen wurde auch die Knorpeldickenvorhersage mittels Nahinfrarot-Spektroskopie (NIRS-B) ex vivo untersucht. Die vorliegende Arbeit liefert vielversprechende Erkenntnisse, um die Entwicklung standardisierter Verfahren für die Prüfung von TE Konstrukten für den orthopädisch-unfallchirurgischen Einsatz voran zu treiben. Die Modifizierung des etablierten in vitro Modells (z. B. für resorbierbaren Materialien) sowie weiterführende Untersuchungen zur Vorhersage von Knorpeldicke und Knorpeleigenschaften mittels der nicht invasiven Methode NIRS-B stellen interessante Aufgaben für zukünftige Studien dar

Photoaktivierbare Kinaseinhibitoren

Photochemistry and kinase inhibitor research have both independently been of major scientific interest in the last decades. The objective of this study was therefore to combine both research fields by the development of photoactivatable kinase inhibitors. Herein, the fascinating possibilities of photochemistry were applied on a unique group of pharmacological agents. This study reports on photoactivatable prodrugs of VEGFR inhibitors and the approved BRAF inhibitor vemurafenib. The latter shows impressive outcomes in the personalized treatment of malignant melanoma, but side effects and drug resistance limit therapeutic results. In order to conquer these limitations and to create powerful biological tools for kinase and cancer research, so-called caged prodrugs were developed. Photoremovable protecting groups (PPGs) were thus implemented at crucial pharmacophoric functionalities of the inhibitors in order to diminish their biological effects. This approach provides control over the activation of molecules triggered by ultraviolet light. First, the hinge binder moiety of vemurafenib, a 7-azaindole, was investigated concerning its properties as a leaving group in the photolysis reaction. Several PPGs were then examined with regard to chemical accessibility and release characteristics. Next, photoactivatable prodrugs of vemurafenib and VEGFR inhibitors, a diarylmaleimide and a carbazole, were designed and synthesized. Their photochemical features were subsequently investigated. In vitro evaluation proved the loss-of-function of the prodrugs and the light-dependent recovery of efficacy in kinase and cellular assays. The approved VEGFR inhibitor axitinib was furthermore investigated as a photoswitchable kinase inhibitor. Based on molecular modeling, the (Z)-isomer should be significantly less active toward the targets of (E)-axitinib. It was therefore explored if the inhibitory effect of axitinib could be turned on and off triggered by irradiation. The switching properties of the photoinduced E-Z isomerization were thus examined. Interestingly, the formation of a (Z)-axitinib tautomer was observed. In vitro analyses demonstrated the decreased efficacy of the (Z)-isomer at an enzymatic level. These results, however, did not translate into cell proliferation assays leaving the paradoxically more active (Z)-isomer for future investigation. In conclusion, the first photoactivatable vemurafenib and VEGFR inhibitor prodrugs have been synthesized and characterized in the course of this study. A decreased potency of the (Z)-stereoisomer of axitinib has been demonstrated in enzymatic assays. The applicability of axitinib as a photoswitch remains to be confirmed in further biological assays.Photochemie und die Entwicklung von Kinaseinhibitoren stehen seit Jahrzehnten unabhängig voneinander im Fokus der Wissenschaft. Das Ziel dieser Arbeit war daher, beide Forschungsgebiete durch die Entwicklung photoaktivierbarer Kinaseinhibitoren miteinander zu verknüpfen. Die faszinierenden Möglichkeiten der Photochemie wurden dabei auf eine einzigartige Gruppe pharmakologischer Substanzen angewendet. Diese Arbeit berichtet über photoaktivierbare Prodrugs von VEGFR-Inhibitoren sowie des zugelassenen BRAF-Inhibitors Vemurafenib. Dieser zeigt beeindruckende Erfolge in der personalisierten Behandlung des malignen Melanoms, jedoch limitiert durch Nebenwirkungen und Resistenzen. Um diesen Limitierungen zu begegnen und um leistungsfähige Werkzeuge für die Kinase- und Krebsforschung bereitzustellen, wurden sogenannte caged prodrugs entwickelt. Dazu wurden photoabspaltbare Schutzgruppen (PPGs) an zentralen pharmakophoren Gruppen der Inhibitoren angebracht, um diese biologisch zu inaktivieren. Dies erlaubt die Kontrolle über die Aktivierung von Molekülen durch ultraviolettes Licht. Zunächst wurde der hinge-binder des Vemurafenibs, das 7-Azaindol, hinsichtlich seiner Eigenschaft als Abgangsgruppe in der Photolyse-Reaktion näher untersucht. Verschiedene PPGs wurden bezüglich chemischer Zugänglichkeit und Freisetzung gegenübergestellt. Anschließend wurden caged prodrugs des Vemurafenibs sowie Inhibitoren aus der Klasse der Diarylmaleimide bzw. Carbazole designt und synthetisiert. Ihre photochemischen Eigenschaften wurden untersucht. In vitro-Experimente bestätigten den Wirkungsverlust der Prodrugs sowie die lichtabhängige Wiederherstellung der Wirkung in Kinase- und Zellassays. Ferner wurde der zugelassene VEGFR-Inhibitor Axitinib als photoschaltbarer Kinaseinhibitor untersucht. Molecular modeling legte nahe, dass das (Z)-Stereoisomer schwächer wirksam sei als das (E)-Isomer. Daher wurde untersucht, ob die Wirkung von Axitinib durch Bestrahlung reversibel geschaltet werden kann. Interessanterweise konnte dabei die Bildung eines Tautomers des (Z)-Isomers beobachtet werden. In vitro-Studien zeigten eine Verringerung des hemmenden Effekts des (Z)-Isomers auf enzymatischer Ebene. Diese Ergebnisse übertrugen sich allerdings nicht auf Zell-Proliferations-Assays. Die hierin paradoxerweise gesteigerte Wirkung des (Z)-Isomers sollte daher noch eingehender untersucht werden. Im Rahmen dieser Arbeit wurden die ersten photoaktivierbaren Vemurafenib- und VEGFR-Inhibitor-Prodrugs synthetisiert und charakterisiert. Die verringerte Wirkung des Axitinib-(Z)-Stereoisomers wurde in enzymatischen Assays nachgewiesen. Die Anwendbarkeit als Photoschalter muss jedoch noch in weiteren biologischen Testsystemen bestätigt werden

Real-time image processing for label-free enrichment of Actinobacteria cultivated in picolitre droplets

The majority of today's antimicrobial therapeutics is derived from secondary metabolites produced by Actinobacteria. While it is generally assumed that less than 1% of Actinobacteria species from soil habitats have been cultivated so far, classic screening approaches fail to supply new substances, often due to limited throughput and frequent rediscovery of already known strains. To overcome these restrictions, we implement high-throughput cultivation of soil-derived Actinobacteria in microfluidic pL-droplets by generating more than 600000 pure cultures per hour from a spore suspension that can subsequently be incubated for days to weeks. Moreover, we introduce triggered imaging with real-time image-based droplet classification as a novel universal method for pL-droplet sorting. Growth-dependent droplet sorting at frequencies above 100 Hz is performed for label-free enrichment and extraction of microcultures. The combination of both cultivation of Actinobacteria in pL-droplets and real-time detection of growing Actinobacteria has great potential in screening for yet unknown species as well as their undiscovered natural products

Single Application of Low-Dose, Hydroxyapatite-Bound BMP-2 or GDF-5 Induces Long-Term Bone Formation and Biomechanical Stabilization of a Bone Defect in a Senile Sheep Lumbar Osteopenia Model

Effects of hydroxyapatite (HA) particles with bone morphogenetic BMP-2 or GDF-5 were compared in sheep lumbar osteopenia; in vitro release in phosphate-buffered saline (PBS) or sheep serum was assessed by ELISA. Lumbar (L) vertebral bone defects (Ø 3.5 mm) were generated in aged, osteopenic female sheep ( n = 72; 9.00 ± 0.11 years; mean ± SEM). Treatment was: (a) HA particles (2.5 mg; L5); or (b) particles coated with BMP-2 (1 µg; 10 µg) or GDF-5 (5 µg; 50 µg; L4; all groups n = 6). Untouched vertebrae (L3) served as controls. Three and nine months post-therapy, bone formation was assessed by osteodensitometry, histomorphometry, and biomechanical testing. Cumulative 14-day BMP release was high in serum (76–100%), but max. 1.4% in PBS. In vivo induction of bone formation by HA particles with either growth factor was shown by: (i) significantly increased bone volume, trabecular and cortical thickness (overall increase HA + BMP vs. control close to the injection channel 71%, 110%, and 37%, respectively); (ii) partial significant effects for bone mineral density, bone formation, and compressive strength (increase 17%; 9 months; GDF-5). Treatment effects were not dose-dependent. Combined HA and BMPs (single low-dose) highly augment long-term bone formation and biomechanical stabilization in sheep lumbar osteopenia. Thus, carrier-bound BMP doses 20,000-fold to 1000-fold lower than previously applied appear suitable for spinal fusion/bone regeneration and improved treatment safety

Photoactivatable prodrugs of antimelanoma agent Vemurafenib

In this study, we report on novel photoactivatable caged prodrugs of vemurafenib. This kinase inhibitor was the first approved drug for the personalized treatment of BRAF-mutated melanoma and showed impressive results in clinical studies. However, the occurrence of severe side effects and drug resistance illustrates the urgent need for innovative therapeutic approaches. To conquer these limitations, we implemented photoremovable protecting groups into vemurafenib. In general, this caging concept provides spatial and temporal control over the activation of molecules triggered by ultraviolet light. Thus, higher inhibitor concentrations in tumor tissues might be reached with less systemic effects. Our study describes the first development of caged vemurafenib prodrugs useful as pharmacological tools. We investigated their photochemical characteristics and photoactivation. <i>In vitro</i> evaluation proved the intended loss-of-function and the light-dependent recovery of efficacy in kinase and cellular assays. The reported vemurafenib photo prodrugs represent a powerful biological tool for novel pharmacological approaches in cancer research