In this project three organoruthenium precursor complexes and three cyclometallated complexes with strerically hindered ligands were synthesized and fully characterized, by standard techniques (NMR, mass spectrometry, UV-Vis spectroscopy, elemental analysis and X-ray for some of them). Photochemical and photophysical properties of the new complexes were also investigated.The photosubstitution of the monodentate ligand(s) by a solvent molecule was investigated by UV-vis and 1H NMR spectroscopy. The stability of the compounds was monitored in the dark and after light irradiation. This has been done using 1H NMR, UV-vis spectroscopy, and mass spectrometry. Cyclometallated compounds were stable in the dark up to 6 hours, while organoruthenium compounds were stable in the dark for at least 24 hours. When organoruthenium complexes were irradiated with white light, they substituted 1 or 2 ligands by solvent molecule, which makes them very good candidates for photoactivated chemotherapy (PACT).
The potential of new compounds to be used in photodynamic therapy (PDT) was further investigated using singlet oxygen measurements. Obtained results suggest that the PDT properties of the new complexes are low, which is expected for PACT complexes.
The antiproliferative activity of the ruthenium complexes on commercially available human cancer cell lines has been tested first in the dark and then upon visible light irradiation using a colorimetric SRB assay. The cytotoxicity was tested on A431 (skin) and A549 (lung) cancer cells for the organoruthenium complexes, and on A431 (skin) for cyclometallated compounds, in both normoxic (95% air and 5% CO2) and hypoxic (1% O2 and 5% CO2) conditions. The biological experiments showed that the organoruthenium compounds have a low dark cytotoxicity, with EC50-values above 100 µM, and a low phototoxicity. This indicates that either there was not enough photo-activation of these complexes in the irradiation conditions of the biological experiment (blue light λ = 455 nm), or that the photoactivated products were not cytotoxic.
The cytotoxicity of the cyclometallated compounds was overall very high, with effective concentrations (EC50) values around 0.5 µM and photoindexes around 2 in normoxic conditions, and around 1 for hypoxic condition. The low photo index comes from the toxicity in the dark, which is already quite high, because these compounds are highly hydrophobic. Overall the cyclometallated complexes have shown more potential for PACT than the organoruthenium complexes, but their phototoxicity index was still low. Their toxicity in the dark needs to be reduced. A possible way to do this would be to attach a hydrophilic group to the ligand that undergoes light-mediated photosubstitution. This would decrease the lipophilicity and hence cellular uptake and therefore decrease the toxicity before irradiation, without jeopardizing the light toxicity, for which the hydrophilic ligand would be detached. This strategy could not be investigated due to the early termination of the contract.
During the project various trainings were followed by the researcher. In May 2018 a course on lab safety and fire extinguishing were offered by the Faculty of Science. Trainings on physical and health hazards in the work area, along with applicable exposure control measures were provided. Within the research group the researcher followed 1 day training related to using a glove box for handling air sensitive compounds or reactions. Also, training on photosubstitution quantum yield and singlet oxygen measurement by members of the Prof. Bonnet group has been provided. Training on cancer cell cytotoxicity measurements and cell irradiation, using their specific training protocols, were organised in November and December 2018, also by members of the Bonnet group.
The results of this research will be disseminated via 2 scientific publications in peer-reviewed international journals such as Angewandte Chemie and Dalton Transaction. Some of results were presented in national conference CHAINS Dutch Chemistry meeting and HRSMC annual symposium. The supervisor Bonnet will also show the results of this work in international scientific conferences planned in 2020.
