Despite all the effort that has been directed towards the development of new platforms for cancer diagnosis and treatment, cancer still remains the second major leading cause of death worldwide. Existing theranostic systems that combine both diagnosis and therapy together have many efficacy challenges associated with them, such as determining the actual drug release profile in vivo as well as reducing side-effects for increased standard of care.¹ Consequently, there is a significant need to develop approaches that allow for better understanding of in vivo pharmacokinetics of theranostic systems. 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 stimulus is controlled only by the addition of an exogenous molecule. This provides more direct control over in vivo drug release while at the same time minimising toxic side effects², thereby creating more effective drug delivery systems with better therapeutic design.

Herein, a ⁶⁴Cu radiolabelled tetrazine probe (tetrazine-PEG₄-NOTA) was synthesised and showed rapid pharmacokinetics in healthy mice when tested in positron emission tomography/computed tomography (PET/CT). A PEGylated hyperbranched polymer (HBP) tagged with bispecific antibodies as targeting ligands, and trans-cyclooctene (tCO) bearing the anti-cancer drug doxorubicin (DOX) was also synthesised, with a HBP-cis-cyclooctene (cCO)-DOX conjugate as the control. The in vitro reaction of the HBP-tCO-DOX conjugate with tetrazine showed rapid and instantaneous doxorubicin release when monitored by HPLC. A preliminary in vivo theranostic study was then carried out with mice bearing MCF7 breast cancer xenograft using PET/CT that showed successful in vivo click reaction of tetrazine with tCO and subsequent doxorubicin release. Ex vivo radioactivity calculations indicated a tumour to spleen ratio of 3.2:1 for HBP-tCO-DOX compared to the control ratio of 1.7:1 for HBP-cCO-DOX (24 h pre-targeting interval). A lower radioactivity accumulation in the liver and spleen was also observed, indicating the potential in reducing non-specific drug release through the pre-targeting approach. Conversely, the administration of a pre-reacted HBP-tCO-DOX and radiolabelled tetrazine showed higher liver and spleen uptake when compared to the tumour. These experiments show that the tetrazine-tCO click reaction not only facilitates pro-drug activation only at the tumour site but also acts as a means to quantify the amount of drug administered via PET/CT.