Monitoring mitochondrial viscosity with anticancer phosphorescent Ir(III) complexes via two-photon lifetime imaging

Precise quantitative measurement of viscosity at the subcellular level presents great challenges. Twophoton phosphorescence lifetime imaging microscopy (TPPLIM) can reflect micro-environmental changes of a chromophore in a quantitative manner. Phosphorescent iridium complexes are potential TPPLIM probes due to their rich photophysical properties including environment-sensitive long-lifetime emission and high two-photon absorption (TPA) properties. In this work, a series of iridium(III) complexes containing rotatable groups are developed as mitochondria-targeting anticancer agents and quantitative viscosity probes. Among them, Ir6 ([Ir(ppy-CHO)(2)(dppe)]PF6; ppy-CHO: 4-(2-pyridyl) benzaldehyde; dppe: cis-1,2-bis(diphenylphosphino) ethene) shows satisfactory TPA properties and long lifetimes (up to 1 ms). The emission intensities and lifetimes of Ir6 are viscosity-dependent, which is mainly attributed to the configurational changes in the diphosphine ligand as proved by 1H NMR spectra. Ir6 displays potent cytotoxicity, and mechanism investigations show that it can accumulate in mitochondria and induce apoptotic cell death. Moreover, Ir6 can induce mitochondrial dysfunction and monitor the changes in mitochondrial viscosity simultaneously in a real-time and quantitative manner via TPPLIM. Upon Ir6 treatment, a time-dependent increase in viscosity and heterogeneity is observed along with the loss of membrane potential in mitochondria. In summary, our work shows that multifunctional phosphorescent metal complexes can induce and precisely detect microenvironmental changes simultaneously at the subcellular level using TPPLIM, which may deepen the understanding of the cell death mechanisms induced by these metallocompounds.