Bioresponsive theranostics for activity on demand delivery of Vascular Endothelial Growth Factor (VEGF)
Final Report Abstract
To minimize unintended effects, it is important to develop new methods for delivering medication that can activate the desired effect only at the target site when needed. Our project targeted a dualstimuli approach for activating VEGF-A165, which involved deactivating it through PEGylation in the first place (prodrug principle) and then re-activating it by de-PEGylation through stimuli such as elevated ROS-levels and overexpressed MMP. This stimulus was supposed to cleavage off the PEG moieties and reactivate VEGF-A165 bioactivity. The Meinel group successfully established the recombinant expression of the hrVEGF-A165 protein and confirmed its activity qualitatively and quantitatively in in vitro experiments. However, we were unable to obtain any VEGF-A165 variants with inserted unnatural amino acids, which were the prerequisite of our proposed approach targeting the stapling of VEGF-A165 with ROS-responsive molecules. Therefore, we explored N-terminal PEGylation of hrVEGF-A165 as an alternative and tested it for activity reduction (in the aforementioned prodrug sense). Although assessments from the VEGF-A165 crystal structure would have suggested that blocking the N-terminus with bulky PEG molecules should have impaired the bioactivity of the resulting bioconjugates, we did not observe a significant reduction of bioactivity after PEGylation. However, isolated mono-PEGylated and di-PEGylated VEGF-A165 species showed an increase in hydrodynamic radius, and pointed towards a new strategy of VEGF-A165 bioconjugates which may have potential in treatments of e.g., ischemic cardiovascular disorders. Another objective, targeted by the Marder group, was to introduce dual-gated (H2O2 and MMP) linker structures to re-activate the PEGylated hrVEGF-A165 by unleashing the VEGF-A165 protein as needed. We synthesized a compound with a distinct distance of the perfluoro units and tested it for conjugation with a model protein containing two cysteins with the desired distance. The compound was entirely insoluble in water and the perfluoroarylation was unsuccessful. In order to establish the perfluoroarylation in the absence of suitable linkers, the Meinel group synthesized peptides forming an alpha helix with cysteines at defined positions, and a perfluorinated diazobenzene linker. The perfluoroalkylation was successful and the conformation of the resulting bioconjugate could be controlled by light. Therefore, the Meinel group successfully demonstrated the stapling with control molecules (diazobenzenes) for light-controlled conformations of proteins and peptides. Furthermore, as of the insoluble nature of the dual-gated (H2O2 and MMP) linkers from the Marder group, both groups (Meinel & Marder) together synthesized phenylboronate H2O2-sensitive molecules. Promising results were obtained from preliminary experiments with these molecules for drug release in presence of H2O2. These linkers from an interesting starting point to leverage small chemical drugs and biologics by introducing a ROS-gated activity on demand functionality. The results of the study are currently compiled into a manuscript and final experiments are being conducted.
Publications
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Synthesis of Highly Functionalizable Symmetrically and Unsymmetrically Substituted Triarylboranes from Bench‐Stable Boron Precursors. Chemistry – A European Journal, 27(35), 9094-9101.
Ferger, Matthias; Berger, Sarina M.; Rauch, Florian; Schönitz, Markus; Rühe, Jessica; Krebs, Johannes; Friedrich, Alexandra & Marder, Todd B.
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Synthetic Approaches to Triarylboranes from 1885 to 2020. Chemistry – A European Journal, 27(24), 7043-7058.
Berger, Sarina M.; Ferger, Matthias & Marder, Todd B.
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Applications of triarylborane materials in cell imaging and sensing of bio-relevant molecules such as DNA, RNA, and proteins. Materials Horizons, 9(1), 112-120.
Berger, Sarina M. & Marder, Todd B.
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Amber Light Control of Peptide Secondary Structure by a Perfluoroaromatic Azobenzene Photoswitch. ChemBioChem, 24(5)
Cataldi, Eleonora; Raschig, Martina; Gutmann, Marcus; Geppert, Patrick T.; Ruopp, Matthias; Schock, Marvin; Gerwe, Hubert; Bertermann, Rüdiger; Meinel, Lorenz; Finze, Maik; Nowak‐Król, Agnieszka; Decker, Michael & Lühmann, Tessa
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PEGylation of Human Vascular Endothelial Growth Factor. ACS Biomaterials Science & Engineering, 10(1), 149-155.
Wolkersdorfer, Alena Maria; Jugovic, Isabelle; Scheller, Lena; Gutmann, Marcus; Hahn, Lukas; Diessner, Joachim; Lühmann, Tessa & Meinel, Lorenz
