Development of polymeric theranostics using bioorthogonal chemistry

Abstract

Despite significant effort directed towards the development of new platforms for cancer diagnosis and treatment, it still remains as the second major leading cause of death worldwide. The efficacy of existing therapeutic systems is limited by challenges such as determining the actual drug release profile in vivo, as well as reducing side-effects for an increased standard of care for patients. Consequently, there is an unmet need to develop approaches that allow a better understanding of the pharmacokinetic/pharmacodynamic behaviour of nanomedicines and their therapeutic cargo. The use of bioorthogonal chemistry is a versatile and promising tool to address these challenges, as it can be tailored to develop theranostic systems where the drug release is controlled by the addition of an exogenous molecule, providing more direct control over drug release and minimising off-target effects. The primary objective of this thesis was to perform “click-to-release” bioorthogonal trans-cyclooctene (tCO) and tetrazine (Tz) reaction-based prodrug activation of polymer-drug conjugates by developing a modular and controlled theranostic system that can quantitatively assess site-specific drug activation and deposition from a nanocarrier, in particular, a hyperbranched polymer (HBP). The exploitation of quantitative imaging using positron emission tomography (PET) together with pre-targeted bioorthogonal chemistries in this system, provides an effective means to assess in real-time the exact amount of active drug administered at precise sites in living species. For successful “click-to-release” pro-drug activation, it is crucial to develop a suitable tetrazine reaction partner (activator) capable of remaining in the bloodstream long enough for the click reaction to occur, but which would clear fast enough to avoid interference from unreacted molecules during imaging and subsequent quantitation of drug activation. In Chapter 2, a suitable 64Cu radiolabelled tetrazine probe ([64Cu]Tz-PEG4-NOTA) was synthesised that showed rapid pharmacokinetics in healthy mice when tested in PET/CT. Given the rapid blood clearance profile of the molecule, it was ideal for further investigation in the study. Chapter 3 is dedicated to the development of a “stealthy” poly(ethylene glycol) (PEG)-based HBP nanocarrier that can offer high functional density while minimising an immunogenic response. A HBP having BOC-protected amine functional end groups was synthesised through reversible addition-fragmentation chain-transfer polymerization. The polymer was then modified post-polymerisation to cleave the trithiocarbonate end groups, followed by BOC deprotection to afford terminal amine groups. As a proof-of-concept study, the HBP was attached to a commercially-available tCO to develop a HBP-tCO conjugate, along with HBP-cis-cyclooctene (cCO) as control. Upon reaction with tetrazine, HBP-tCO displayed a rapid and successful click reaction, in contrast to HBP-cCO where no reaction was observed. Chapter 4 investigates the construction of a tCO molecule that can facilitate self-immolative drug release upon reaction with tetrazine. A detailed synthesis of the suitable tCO and control cCO molecules are reported, followed by the synthesis of the final polymer-drug conjugates of HBP-tCO-DOX and HBP-cCO-DOX. In vitro drug release studies of polymer-drug conjugates with Tz demonstrated an instantaneous doxorubicin (DOX) release from HBP-tCO-DOX, in contrast to the HBP-cCO-DOX where insignificant DOX release was observed. These systems were further validated by in vitro studies on two cell lines, employing two targeting agents; anti-PEG/anti-TAG72 bispecific antibody (BsAb) against MCF7 breast cancer cells that over-express the non-internalising TAG72 receptor, and anti-PEG/anti-EGFR BsAb against MDA-MB-468 breast cancer cells that over-express the internalising EGF receptor. The in vitro cellular cytotoxicity evaluation demonstrated significantly lower toxicity of HBP-tCO-DOX and HBP-cCO-DOX conjugates compared to the free drug DOX. However, when both cell lines exposed to polymer-drug conjugates were incubated with Tz, the HBP-tCO-DOX treated cells displayed a significant reduction in cell viability as opposed to HBP-cCO-DOX treated cells, suggesting the release of DOX upon Tz-tCO reaction.Following extensive in vitro validation in cells, the in vivo performance of the system is explored in Chapter 5 to demonstrate the “click-to-release” activation of the HBP-drug conjugate in mice. Despite the absence of any significant difference between in vitro cell results, the in vivo results in mice demonstrated a clear difference between the two receptor targets, indicating the advantage of using a non-internalising receptor (TAG72) as the in vivo target in pre-targeting. Significantly higher radioactivity was observed in tumours with tCO compared to cCO, together with a higher tumour-to-background signal ratio in MCF7 tumour-bearing mice, and was further confirmed via confocal microscopy. In MDA-MB-468 mice, a lower tumour accumulation was observed with tCO and was not significantly higher than cCO. However, in both cases, the radioactivity accumulation in the liver and spleen were commensurately low resulting in less non-specific drug release, thereby indicating a clear benefit of this approach by reducing off-target toxicity. Finally, the quantification of DOX release in the tumours was performed based on the PET/CT signal for the accumulation of [64Cu]Tz-PEG4-NOTA. In summary, this thesis demonstrates the successful development of a new HBP-based ‟click-to-release” theranostic system for quantitative therapeutic delivery, with improved specificity in drug delivery profile compared to conventional nanomedicines.