Bioassay-guided development and in vitro characterization of lectin-drug conjugates for the targeted intravesical therapy of bladder cancer

Im Rahmen der vorliegenden Arbeit wurde ein Delivery-System entwickelt, das, basierend auf dem Konzept des aktiven Drug-Targetings, zur Verbesserung der adjuvanten Therapie des nicht-muskelinvasiven Harnblasenkarzinoms oder anderer intravesikal zugänglicher Erkrankungen beitragen soll. Das entwickelte Prodrug-System besteht aus fluoreszenzmarkiertem BSA (Bovines Serum Albumin), das über Divinylsulfon kovalent mit WGA (Wheat Germ Agglutinin) verknüpft wurde, welches als Targeting-Protein fungieren soll. Das Hauptaugenmerkt zunächst auf einer Optimierung der Kopplungs- und Reinigungsprozedur, wobei das Endprodukt zellkulturgestützt evaluiert wurde. Dabei erwies sich die Verwendung eines 20- fachen molaren Überschusses an WGA bezogen auf BSA als am geeignetsten. Um ein reines Produkt zu garantieren, wurden für die weitere Charakterisierung nach der Aufreinigung nur die ersten 10-12 Fraktionen des erhaltenen Peaks gepoolt. Unter den gewählten Bedingungen wurde so ein Konjugatgemisch mit durchschnittlichem Modifikationsgrad von 3-5 Molekülen WGA pro Molekül fBSA erhalten. Diese Parameter konnten dabei auch mit Hilfe alternativer Analyseverfahren (GEMMA, Acrylamid-Gelelektrophorese) bestätigt werden. Anhand von Zellstudien wurde nachgewiesen, dass das Konjugat zur spezifischen Bindung an die Zelloberfläche befähigt ist und in weiterer Folge relativ rasch von den Zellen internalisiert wird. Das konnte auch durch Fluoreszenzemissionsmessung vor und nach Behandlung mit Monensin untermauert werden. Um die Vermutung, dass WGA und somit auch das Kopplungsprodukt mit höherer Präferenz an maligne Zellen bindet, zu überprüfen, wurden konzentrationsabhängige Bindungsstudien neben SV-HUC-1 (Zellen ohne maligen Hintergrund) zusätzlich auch an 5637 (Tumorgrad 2) und HT1376 (Tumorgrad 3) durchgeführt. Hierbei konnte gezeigt werden, dass das Konjugat im höheren Konzentrationsbereich tatsächlich eine verstärkte Tendenz zur Bindung an Zellen mit malignem Hintergrund aufweist Außerdem wurden Konjugate mit PNA (Peanut Agglutinin) als Lektin entwickelt und deren Bindungsspezifität überprüft. Die RFI-Werte der Zelloberflächenbindung von fBSA/PNA-Konjugaten sind verglichen mit jenen der fBSA/WGA-Konjugate insgesamt sehr gering, was eventuell auf eine mangelnde Kopplungseffizienz im Fall von PNA hindeuten könnte. Zusätzlich wude fHSA (fluoreszenzmarkiertes humanes Serum Albumin) an WGA (Wheat Germ Agglutinin) gekoppelt. Allerdeings erwieß sich HSA als weniger homogen wie BSA. Die Feinjustierung der Konjugataufreinigung erschien deshalb mit dem homogeneren fBSA einfacher, überdies bietet der Einsatz von kostengünstigerem fBSA einen ökonomischen Vorteil Die Ergebnisse der vorliegenden Diplomarbeit belegen, dass fBSA/WGA-Konjugate einen ersten Schritt in Richtung Verbesserung der adjuvanten Therapie des nicht-muskelinvasiven Harnblasenkarzinoms beruhend auf dem Konzept des aktiven Drug-Targetings darstellen könnten. Hinsichtlich der Spezifität der Bindung und deren Aufnahme in maligne Zellen sind weitere Untersuchungen in ArbeitThe structural and compositional characteristics of the urothelial tissue account for a problematically low bioavailability in intravesical therapy, and call for the implementation of penetration-promoting delivery modalities to improve treatment impact. This study presents the first biorecognitive approach for intravesical drug delivery, based on the specific interaction between lectins and the corresponding carbohydrates at the cell membrane. Fluorescence-labeled bovine serum albumin (fBSA) or human serum albumin (fHSA) was covalently coupled to wheat germ agglutinin (WGA) to yield a model prodrug (fBSA/WGA conjugate) for the characterization of cytoadhesive and cytoinvasive potential in cell culture models. Coupling protocol and purification procedure were optimized and validated by a comparative screening in bioassays and physicochemical analysis of molecular weight. Preliminary studies demonstrated the general applicability of the coupling procedure to lectins of different carbohydrate specificity. Binding and uptake by human urothelial cells of non-malignant origin (SV-HUC-1) were compared to that by cells deriving from grade 2 (5637) or grade 3 (HT-1376) urothelial carcinoma. In contrast to unmodified fBSA, WGA-conjugates were effectively internalized (>50%) and processed to acidic compartments. Specificity of the conjugate-cell interaction was validated via competitive inhibition with the complementary carbohydrate. Intracellular accumulation was followed microscopically and verified by monensin-induced de-quenching. Utilizing receptor-mediated endocytotic pathways, glycotargeted delivery platforms may thus provide a potent tool for the establishing of novel therapy concepts for intravesical application

COVID-19 Shutdown: die Sicht der Bürger*innen – Persönliche und gesellschaftliche Nachteile

Zielsetzung und Design: Die Umfrage (482 in Österreich lebende Personen) erhebt in Form von zwei offenen Fragen die Wahrnehmung von persönlichen und gesellschaftlichen Nachteilen in der Zeit des COVID-19 bedingten Shutdowns. Die Daten werden im Hinblick auf Inhalt und Häufigkeit für die Gesamtgruppe sowie geschichtet nach Alter und Geschlecht analysiert. Zielsetzung ist, die direkten sozialen, wirtschaftlichen, ökologischen und politischen Folgen der Krise bzw. der zur Bewältigung gesetzten Maßnahmen zu identifizieren, und für die wissenschaftliche und politische Auseinandersetzung mit Krisensituationen aufzubereiten. Erkenntnisse: Die Ergebnisse zeigen ein gewisses Auseinanderfallen der wahrgenommenen individuellen Nachtteile und der gesellschaftlichen Nachteile: Während als die drei wichtigsten persönlichen Nachteile das Social Distancing, der Alltag in der neuen Situation und die eingeschränkte (Bewegungs)freiheit genannt werden, wird auf gesell-schaftlicher Ebene primär der wirtschaftliche Nachteil, Social Distancing und der Bereich Politik | Medien | Gesellschaft genannt

Teaching an old pET new tricks: tuning of inclusion body formation and properties by a mixed feed system in E. coli

Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided during recombinant protein production, numerous biopharmaceutically important proteins are currently produced as IBs. To obtain correctly folded, soluble product, IBs have to be processed, namely, harvested, solubilized, and refolded. Several years ago, it was discovered that, depending on cultivation conditions and protein properties, IBs contain partially correctly folded protein structures, which makes IB processing more efficient. Here, we present a method of tailored induction of recombinant protein production in E. coli by a mixed feed system using glucose and lactose and its impact on IB formation. Our method allows tuning of IB amount, IB size, size distribution, and purity, which does not only facilitate IB processing, but is also crucial for potential direct applications of IBs as nanomaterials and biomaterials in regenerative medicine.COMET6676761

Digital health - digital 2D/3D printing of personalized medication

Without any doubt the young technology of additive manufacturing, including 2D and 3D printing, has been changing the world. A wide spectrum of objects in medical and pharmaceutical field can already be successfully printed. This ranges from a detailed figure in the micrometer regime to a full size residential house unit made from concrete [Sakin et al., 2017; Doherty et al., 2020]. In the pharmaceutical sector, additive manufacturing offers the option of medication customization [Pravin et al., 2018]. Integrating this new technology has several advantages over the current, well-established but outdated “one-size-fits-all” approach. Besides avoiding medication errors, printing tailored oral dosage forms is financially attractive for small scale on-demand production [Awad et al., 2018]. The development of dosage forms, production of sample batches and modification of samples can be done with little effort compared to generic powder-pressed pills which require heavy pharmaceutical machinery [Awad et al., 2018; Dachtler et al., 2020]. To further progress and to create the legal framework for the approval of additively manufactured drugs it requires a paradigm change of stakeholders who have the power to make decisions and changes [Horst et al., 2019]

Production strategies for active heme-containing peroxidases from E. coli inclusion bodies – a review

Heme-containing peroxidases are frequently used in medical applications. However, these enzymes are still extracted from their native source, which leads to inadequate yields and a mixture of isoenzymes differing in glycosylation which limits subsequent enzyme applications. Thus, recombinant production of these enzymes in Escherichia coli is a reasonable alternative. Even though production yields are high, the product is frequently found as protein aggregates called inclusion bodies (IBs). These IBs have to be solubilized and laboriously refolded to obtain active enzyme. Unfortunately, refolding yields are still very low making the recombinant production of these enzymes in E. coli not competitive. Motivated by the high importance of that enzyme class, this review aims at providing a comprehensive summary of state-of-the-art strategies to obtain active peroxidases from IBs. Additionally, various refolding techniques, which have not yet been used for this enzyme class, are discussed to show alternative and potentially more efficient ways to obtain active peroxidases from E. coli

Controlled-Release from High-Loaded Reservoir-Type Systems—A Case Study of Ethylene-Vinyl Acetate and Progesterone

Reservoir systems (drug-loaded core surrounded by drug-free membrane) provide long-term controlled drug release. This is especially beneficial for drug delivery to specific body regions including the vagina. In this study, we investigated the potential of reservoir systems to provide high drug release rates over several weeks. The considered model system was an intra-vaginal ring (IVR) delivering progesterone (P4) in the mg/day range using ethylene-vinyl acetate (EVA) as release rate-controlling polymers. To circumvent the high material needs associated with IVR manufacturing, we implemented a small-scale screening procedure that predicts the drug release from IVRs. Formulations were designed based on the solubility and diffusivity of P4 in EVAs with varying vinyl acetate content. High in-vitro P4 release was achieved by (i) high P4 solubility in the core polymer; (ii) high P4 partition coefficient between the membrane and the core; and/or (iii) low membrane thicknesses. It was challenging for systems designed to release comparatively high fractions of P4 at early times to retain a constant drug release over a long time. P4 crystal dissolution in the core could not counterbalance drug diffusion through the membrane and drug crystal dissolution was found to be the rate-limiting step. Overall, high P4 release rates can be achieved from EVA-based reservoir system

The production of a recombinant tandem single chain fragment variable capable of binding prolamins triggering celiac disease

Abstract Background Celiac disease (CD) is one of the most common food-related chronic disorders. It is mediated by the dietary consumption of prolamins, which are storage proteins of different grains. So far, no therapy exists and patients are bound to maintain a lifelong diet to avoid symptoms and long-term complications. To support those patients we developed a tandem single chain Fragment variable (tscFv) acting as a neutralizing agent against prolamins. We recombinantly produced this molecule in E. coli, but mainly obtained misfolded product aggregates, so-called inclusion bodies, independent of the cultivation strategy we applied. Results In this study, we introduce this novel tscFv against CD and present our strategy of obtaining active product from inclusion bodies. The refolded tscFv shows binding capabilities towards all tested CD-triggering grains. Compared to a standard polyclonal anti-PT-gliadin-IgY, the tscFv displays a slightly reduced affinity towards digested gliadin, but an additional affinity towards prolamins of barley. Conclusion The high binding specificity of tscFv towards prolamin-containing grains makes this novel molecule a valuable candidate to support patients suffering from CD in the future

Novel Approach to Pharmaceutical 3D-Printing Omitting the Need for Filament—Investigation of Materials, Process, and Product Characteristics

The utilized 3D printhead employs an innovative hot-melt extrusion (HME) design approach being fed by drug-loaded polymer granules and making filament strands obsolete. Oscillatory rheology is a key tool for understanding the behavior of a polymer melt in extrusion processes. In this study, small amplitude shear oscillatory (SAOS) rheology was applied to investigate formulations of model antihypertensive drug Metoprolol Succinate (MSN) in two carrier polymers for pharmaceutical three-dimensional printing (3DP). For a standardized printing process, the feeding polymers viscosity results were correlated to their printability and a better understanding of the 3DP extrudability of a pharmaceutical formulation was developed. It was found that the printing temperature is of fundamental importance, although it is limited by process parameters and the decomposition of the active pharmaceutical ingredients (API). Material characterization including differential scanning calorimetry (DSC) and thermogravimetric analyses (TGA) of the formulations were performed to evaluate component miscibility and ensure thermal durability. To assure the development of a printing process eligible for approval, all print runs were investigated for uniformity of mass and uniformity of dosage in accordance with the European Pharmacopoeia (Ph. Eur.)