Studies toward polymeric nanoparticle encapsulation of novel aziridine DNA crosslinkers for the treatment of cancer

DNA alkylators have long been used as standard treatments in chemotherapy. Their mechanism of action relies on covalently tethering DNA strands together by inter-strand crosslinks (ICLs), triggering apoptosis. However, they can cause toxic side effects through covalent binding to off-target proteins. Their reactive nature also makes them prone to degradation under biological conditions. In contrast, targeted covalent inhibitors (TCIs) are a class of drugs that are designed to selectively direct covalent moieties on the drug to their biological targets. This thesis explores a series of potential anti-cancer TCIs that are designed to bind to DNA via the targeting ability of non-covalent interactions, followed by alkylation. The novel TCIs in this thesis contain two aziridine warheads, which are subtly less reactive compared to those found in many other DNA alkylators. Furthermore, this thesis investigates the use of polymeric nanoparticles as a drug delivery mechanism for the aziridine-containing TCIs. Chapter 2 of this thesis reviews the current literature on covalent drug delivery by nanoparticles. The bulk of this chapter has previously been published as the article: Weissberger, D.; Stenzel, M.; Hunter, L. Precious Cargo: The Role of Polymeric Nanoparticles in the Delivery of Covalent Drugs. Molecules 2024, 29(20), 4949. DOI: https://doi.org/10.3390/molecules29204949. This publication has been included as Chapter 2, unchanged and in its entirety. Chapter 3 describes the synthesis of two DNA minor-groove binders and the resynthesis of two DNA threading agents. Despite the mild reactivity of the aziridines, the synthesised compounds were found to have poor long-term storage stability. The compounds also generally suffered from solubility issues, however one promising candidate, compound 12, proved to perform better than the others and has been shown in other work to have excellent cytotoxicity in vitro. Chapter 4 focuses on preliminary investigations into the DNA binding mechanism of the most promising drug candidate, 12. Previous results have hinted that it can bind to the TpG site of telomere-like 5'–TTGG–3' and 5'–TTTGGG–3' DNA sequences. In this work, matrix assisted laser desorption/ionisation with time-of-flight mass spectrometry (MALDI-TOF MS) showed that compound 12 tends to primarily alkylate guanines in such sequences, but not the intended adenines, with the possible formation of a G-L-G inter-strand crosslink. However, extension of the aziridine side chains could allow the second aziridine to reach the adenine binding site. Chapter 5 attempts to improve the pharmacokinetics of compound 12 by encapsulation into polymeric nanoparticles. Among a number of other benefits, the matrix of the nanoparticle provides a physical barrier that protects the drug from the biological environment. To this end, polyoxazoline (POx) and polyethylene glycol–polylactic acid (PEG–PLA) block copolymers were successfully synthesised as candidates for delivering the novel aziridines. Bottom-up nanoparticle formation of these polymers was investigated by comparing a range of self-assembly techniques and solvents. The optimised formulation used PEG113–PLA56 for drug loading prepared via the antisolvent addition technique, using DMF as the dissolution solvent. Dynamic light scattering (DLS) analysis showed that these nanoparticles were micelle-sized (