Periodic Reporting for period 1 - NIRCOThera (Spatiotemporal, near-infrared light controlled carbon monoxide delivery for cancer immunotherapy) Reporting period: 2018-03-01 to 2020-02-29 Summary of the context and overall objectives of the project The success of advanced cancer diagnosis and treatment relies on the localized enrichment of drugs or imaging agents at tumour sites. The technologies enable targeted delivery and on-demand activation represent an excellent solution to the issue in the battle of cancer. The tumour-targeted delivery and on-demand activation of prodrugs could enhance the concentration of active drugs selectively at tumour sites while sparing the exposure of toxic drugs to normal tissues, alleviating the adverse drug effects. There are several methodologies for on-demand activation. Among them, the light-responsive release, especially with near-infrared light of better tissue penetration depth and biocompatibility, provides a high degree of spatiotemporal precision. Also, bioorthogonal click-to-release reactions have been rapidly emerging in recent years because of its bioorthogonality and controlled bond-cleavage. The objective of this research is to develop a novel cargo delivery avenue, enabling controlled prodrug release, e.g. the immunomodulatory molecule carbon monoxide (CO) and the chemotherapy drug doxorubicin, exclusively at tumour sites with spatiotemporal precision. The strategy proposed consists of single-walled carbon nanotubes (SWCNTs) and the prodrug cargos. The strategy will enable spatiotemporal control over prodrug activation via integrating the tumour-targeting property of SWCNTs, the stability and non-toxicity of the small molecule prodrugs and the on-demand activation of prodrugs at tumour sites. Besides, the lipid functionalized SWCNTs have shown biocompatibility, ultrahigh tumour uptake and relatively fast clearance and excretion from the health tissues, which impart the platform promising for in vivo application. The biocompatibility of the nanotool, the stability of the prodrug and the active drug release profile would be evaluated, followed by its dose- and time-dependent therapeutic potential on tumour treatment. We expect that this novel on-demand prodrug activation platform would enable significant improvements to the existing cancer treatment regimes in the near future. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far At the beginning of the fellowship, we aimed to develop a nanotool integrates single-walled carbon nanotubes (SWCNTs), PEGylated phospholipids and CO-releasing molecules (CORMs) to enable tumour-specific delivery and NIR-programmed CO release with spatiotemporal precision at tumour sites. Unfortunately, the construction of CORMs@SWCNTs conjugates that consist of SWCNTs, DSPE-PEG-ligand and CORMs prodrug could not be achieved due to the instability of the organometallic compounds under the conditions to functionalize the SWCNTs. As an alternative, we sought to explore other methodologies to achieve the activation of CO-prodrug and generalize the method to other prodrugs. We came up with a pretargeted strategy combining SWCNTs with IEDDA click-to-release bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO). The strategy comprises two bioorthogonal reagents that are administered in two steps: tetrazine modified SWCNTs (TZ@SWCNTs) and the TCO-carbamate containing molecules, a chemotherapeutic prodrug or diagnostic probe. We also designed and synthesized a TCO-caged NIR fluorogenic probe tHCA for in-depth real-time tumour imaging in living mice with low background signal ideal for imaging-guided tumour surgery.With this strategy, we have successfully realized selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells with no need of washing. We also demonstrated the bioorthogonal controlled tHCA turn-on NIR fluorescence imaging in the living mice xenograft model with high tumour-to-liver ratio, making it the first bioorthogonal fluorogenic probe actualized for in vivo tumour imaging. Based on the successful establishment of the two-step pretargeted strategy with SWCNTs and the IEDDA click-to-release reaction, we are applying this methodology for organic CO-prodrug delivery and bioorthogonal-controlled cancer immunotherapy. The bioorthogonal reaction between cyclopentadienone and cyclooctynes (e.g. BCN) are chosen to serve the purpose. This work is in progress and the bioorthogonal controlled CO release is currently being tested.The outcome of the project has been presented in 3 international academic conferences and introduced to general public during Cambridge Open Day. As to the submission of the report, 3 original articles from the work implemented during the project have been submitted to international journals. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This work developed a tumour-pretargeted activation strategy enables localized enrichment of active drug/probe with tumour specificity and spatiotemporal precision by integrating SWCNTs and tetrazine/TCO bioorthogonal cleavage reaction. In this work, we also demonstrated the first use of a bioorthogonally applicable fluorogenic NIR probe tHCA for turn-on real-time imaging in living mice xenograft tumour model, making it a promising candidate for image-guided cancer surgery. We expect the outcome of this work could contribute to meet unmet needs for efficient cancer treatment with minimal adverse effects.With the support from this project, the researcher received high level of training in the stimulating environment of Cambridge, which enhanced her potential and her career prospects in the field of cancer diagnostics and treatment. To disseminate the research results, the researcher attended conferences and presented her work in the 2019 Cancer Nanotechnology Gordon Research Conference in the United States and the 2020 International Symposium on Chemical Biology in Switzerland, which contributed to the communication among scientists across the globe. Furthermore, this is an interdisciplinary work of nanomaterials, bioorthogonal chemistry, prodrug design and synthesis, cell biology and cancer diagnosis and treatment. In the course of this project, we built close collaboration with Dr. João Conde in Instituto de Medicina Molecular (iMM), Universidade de Lisboa (recently moved to NOVA Medical School), an expert in cancer nanomedicine, which promoted the transfer of different skills and knowledges and enhanced the collaboration of excellent scientists from different countries within Europe. Spatiotemporal-controlled activation of prodrug/probe for tumour treatment
