Bioorthogonal activation and mitochondrial targeting of a near-infrared-emitting iridium(III) nitrone complex via cyclooctynylated phosphonium cations for enhanced cellular imaging and photodynamic therapy

In this work, we designed and synthesised a new cyclometallated iridium(III) nitrone complex [Ir(bpz)2(bpy-nitrone)](PF6) (1) (Hbpz = benzo[a]phenazine; bpy-nitrone = 4-((methyl(oxido)imino)methyl)-4’-methyl-2,2’-bipyridine) as a bioorthogonally activatable phototheranostic agent. Complex 1 displayed very weak emission and singlet oxygen (1O2) photosensitisation in solutions due to the quenching nitrone moiety. However, upon the strain-promoted alkyne–nitrone cycloaddition (SPANC) reaction with bicyclo[6.1.0]non-4-yne (BCN), which converted the nitrone unit to a non-quenching isoxazoline derivative, the complex exhibited a substantial increase in emission intensity in the near-infrared region and 1O2 generation efficiency. Given that mitochondria are a crucial target in cancer therapy, we prepared a series of BCN-functionalised phosphonium cations (BCN-Phos-n), each bearing different substituents, to serve as mitochondrial-targeting vectors for delivering complex 1 to the mitochondria via the bioorthogonal SPANC reaction. Notably, complex 1 exhibited more significant emission turn-on upon reaction with BCN-Phos-5 and BCN-Phos-6 (I/Io = 24.7 and 14.1, respectively), attributed to their increased hydrophobicity resulting from the methylation or methoxylation of the phenyl rings on the phosphonium cation. Live-cell confocal imaging and flow cytometric analyses revealed that complex 1 showed larger emission enhancement in HeLa cells pretreated with BCN-Phos-5 or BCN-Phos-6 compared to other BCN-Phos-n analogues. Co-staining experiments confirmed that the resultant luminescent isoxazoline cycloadducts predominantly accumulated in the mitochondria. Additionally, both dark and light-induced cytotoxicity of complex 1 increased upon pretreatment of the cells with BCN-Phos-5 or BCN-Phos-6. Our results demonstrate that the theranostic potential of transition metal nitrone complexes can be significantly enhanced via strategic structural manipulation of their bioorthogonal reaction partners.