Photodynamic immunotherapy of Ruthenium(II) polypyridyl complexes: Application in the treatment of colorectal Cancer

In this study, a new ligand 2-(4-(1H-imidazol-1-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (IPIP) was synthesized and reacted with cis-[Ru(phen)₂Cl₂]·2H₂O and cis-[Ru(dip)₂Cl₂]·2H₂O to form two new Ru(II) complexes [Ru(phen)₂(IPIP)](PF₆)₂ (Ru1a) and [Ru(dip)₂(IPIP)](PF₆)₂ (Ru1b). The cytotoxicity of Ru1a and Ru1b against both cancer and normal cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The results revealed that Ru1a exhibits moderate and Ru1b shows no cytotoxic activity in the dark. However, after 1 h of irradiation, Ru1a exhibited significant cytotoxicity against HCT116 cells, while Ru1b still showed no cytotoxic activity toward the selected cancer cells. Further investigation via cell uptake, mitochondrial localization, mitochondrial membrane potential, cytochrome C release, and western blotting techniques confirmed that Ru1a induced apoptosis in HCT116 cells via the mitochondrial pathway. RNA-sequence assay showcases that Ru1a caused cell death through ferroptosis. The levels of lipid peroxidation product of malondialdehyde (MDA) were elevated, while the expression of GPX4 was reduced. These findings affirmed that Ru1a promotes cell death through ferroptosis. Antitumor in vivo confirmed that Ru1a exerts anticancer activity in the tumor microenvironment (TME) by inducing immunogenic cell death and activating immune responses to enhance CD8⁺ T cells, thereby directly killing colorectal cancer cells.