Abstract
Ruthenium(Ru)-based complexes have emerged as leading chemotherapeutic candidates with less potential adverse effects relatively to the approved Platinum(II) drug cisplatin or other platinum analogues. Strained Ru(II) polypyridyl complexes that exhibit an octahedral geometry, are particularly appealing as photoactivatable drugs that undergo ligand dissociation upon irradiation, leading to the formation of active photoproducts that are significantly more cytotoxic than the prodrugs. The present study explored the photochemotherapeutic potential of two target Ru(II) bipyridyl drugs, Ru(bipy)2dpphen(Cl2) or Ru1(Cl2) and Ru(bipy)2BC(Cl2) or Ru2(Cl2), in vitro and in vivo, as compared to cisplatin. Ru2(Cl2) was synthesized and characterized by spectroscopy, elemental analysis, nuclear magnetic resonance (NMR), high resolution-electrospray ionization-mass spectrometry (HR-ESI-MS) and matrix-assisted laser desorption ionization (MALDI). Upon exposure to blue light, both bipy and BC ligands were proven to dissociate, as confirmed by NMR and ESI-MS studies. Cell viability assays (WST-1) suggested that Ru2(Cl2) exhibited a cell line-dependent activity as photoactivated chemotherapy, with the highest response in melanoma cell lines, where the phototoxicity index (PI) was as high as 120-fold. The aquated photoproducts were found to be the active species, rather than the dissociating ligands, with higher cytotoxicity than cisplatin in all tested cell lines, including cisplatin-resistant HT-29 cells. Flow cytometric studies determined an apoptotic mode of cell death mediated by both the prodrug and the photoproducts. This was consistent with gel electrophoresis experiments which suggested that the interactions of Ru2(Cl2) with DNA and the possible damage incurred were not exclusively photoinduced. Cellular uptake was achieved via active transport, as measured by LC-MS/MS, which was validated as a quantification method in biological matrices, based on the USFDA validation guidelines for sensitivity, specificity, accuracy, precision, recovery and stability. Having consistently lower cytotoxicity in dark conditions, as well as selectivity of the photoproducts towards cancer cells, Ru1(Cl2) was selected for in vivo testing using a 7,12-Dimethylbenz[a]anthracene/12-O-Tetradecanoylphorbol 13-acetate skin carcinogenesis model. Ru1(Cl2) was well tolerated up to a dose of 1 mg.Kg-1; no significant changes in body weight, behaviour, or serum biochemistry were reported. Toxicity was only observed at higher bolus doses (1.5 mg.Kg-1) where death was recorded in some animals following shaking and seizure-like symptoms. Upon IP injection, high bioavailability of the drug was obtained in the plasma which favoured its accumulation in organs, particularly the highly perfused liver, kidneys and tumour tissues. Renal excretion was shown to be the primary mode of elimination. Efficacy studies demonstrated that Ru1(Cl2) exhibited a significant anticancer activity upon photoactivation in vivo, however, the response was similar when animals were treated with blue light, Ru1(Cl2) prodrug or sub-tumoral injection with Ru1(Cl2) photoproducts. Compared to cisplatin, all treatment strategies were more effective in reducing tumour volumes, incidence and malignant transformation, an effect that was partly mediated via caspase-3 independent apoptosis, as suggested by western blot analyses. Moreover, no toxicities were observed, as opposed to cisplatin which was shown to cause nephrotoxicity. In conclusion, both Ru1(Cl2) and Ru2(Cl2) proved to be promising as photochemotherapeutic drugs with Ru1(Cl2) having greater potential because of its low cytotoxicity in dark conditions, selectivity of the photoproducts, tolerability and effectiveness, in vivo.