Preparation and characterization of a PNA-Ir(III) conjugate for 1O2 production in PDT

Photodynamic Therapy (PDT) is a medical treatment based on the production of ROS through the excitation of a photosensitizing agent (PS). Among many different PS, cyclometalled Ir(III) complexes are particularly suitable for Type II PDT, in which the production of the toxic singlet oxygen (1O2) from its triplet ground state (3O2) is involved. The excitation of the PS to the singlet state (S1) is followed by intersystem crossing (ISC) to the triplet excited state (T1), where its energy transfers to 3O2. Those Ir(III) complexes are indeed characterized by accessible triplet excited states and high 1O2 quantum yields given by an excellent energy transfer process.[1] Therefore, we have successfully synthesised a novel Ir(III) cyclometalled complex (Ir-COOH) and carried out its conjugation with a model peptide nucleic acid (PNA) tetramer (Ir-PNA). Both have been characterized using UV-vis absorption and emission spectroscopy, demonstrating that the PNA chain influences only partially the photophysical properties of Ir-COOH. Moreover, we have investigated with confocal microscopy the cellular uptake and intracellular localization of the two compounds on HeLa cells: while Ir-COOH showed a homogeneous distribution in the cytoplasm, the Ir-PNA was contained in accumulations both in the cytoplasm as well as at the nuclear level. Remarkably, Ir-COOH and Ir-PNA have been excited using two-photon excitation at 720 nm, an important feature for a future possible in vivo use as PS. Finally, we tested the photo-production of 1O2 in cuvette exploiting the oxidation of DHN to Juglone as an indirect reporter for 1O2, showing a good quantum yield for 1O2 production (ΦΔ=0.44 for Ir-COOH and ΦΔ=0.55 for Ir-PNA), and in vitro on HeLa cells, demonstrating the photo-cytotoxicity of the two compounds (EC50=3.5 µM for Ir-COOH and EC50=18 µM for Ir-PNA) with the absence of significant cytotoxicity in the dark (EC50 >50 µM).