Towards accelerated bioorthogonal drug activation in cancer cells upon independent double-targeting

Abstract

One of the main problems in the field of chemotherapy remains to be the off-target toxicity caused by cancer therapeutics. Many chemotherapeutics do not sufficiently differentiate between healthy cells and cancer cells and therefore damage healthy tissue too. A keyword in that context is drug targeting, which aims for the directed delivery and activation of drugs at the target zone.In general, there are two common drug targeting approaches: Strategies based on tumor-associated receptors (“active targeting”) or based on nanoparticles (“passive targeting”) for anticancer drug delivery. Both strategies have their advantages but also their limitations. Tumor-associated receptors are often also expressed on the surface of healthy cells, and due to their size and polarity, small-molecule ligands are renally excreted and thus mainly accumulate in the kidneys and bladder. Nanoparticles, on the other side, end up to a large part in the filtering organs, the liver and lungs. So, off-target toxicity remains a big challenge in both these targeting approaches.To tackle these limitations, we aim for the development of independent double-targeting as a new concept that combines established targeting strategies and takes advantage of the different pharmacokinetics of nanoparticles and small-molecule ligand conjugates. Due to the combination of bioorthogonal drug active-tion and different biodistribution profiles, we aim to substantially reduce off-target toxicity. The prodrug activation in this strategy is done via a bioorthogonal cleavage reaction between trans-cyclooctenes (TCOs) and tetrazines in a click-to-release approach.Based on preliminary work, confirming the double-targeting approach in cell experiments, the aim of this master thesis was to provide various chemical compounds for further investigation and improvement of the independent double-targeting approach.The main focus of this work was put on the eleven-step synthesis of a drug-TCO conjugate by implementing a superior TCO that shows outstanding properties regarding the efficiency of the bioorthogonal cleavage process. The synthesized TCO was successfully attached to a potent cytotoxin and modified with a C16-carbon chain as lipophilic anchor to facilitate and enhance nanoencapsu-lation. The formation of a dehydrated side-product could be prevented, and the yield was improved by examining different coupling conditions and reversing the order of synthetic steps. The synthesized compound was encapsu-lated in PLGA/PEG nanoparticles and provided for first stability experiments. In addition, three lipophilic C16-modified tetrazines were prepared and nanoencap-sulated. PLGA was conjugated to a fluorescent BODIPY-dye and used for nanoparticle synthesis to allow the monitoring of the nanoparticles in cell studies via fluorescence microscopy. In addition, a fluorescently labeled non-releasing TCO was prepared to enable the visualization of tetrazines by bioorthogonal imaging. A second TCO-drug conjugate was successfully prepared in order to compare the performances of different TCOs in this targeting strategy. The attachment of drug-TCO conjugates and tetrazines to small-molecule ligands will enable various approaches and detailed investigation of independent double-targeting and accelerated drug activation inside cells.