Light‐Driven Cascade Mitochondria‐to‐Nucleus Photosensitization in Cancer Cell Ablation

Abstract Nuclei and mitochondria are the only cellular organelles containing genes, which are specific targets for efficient cancer therapy. So far, several photosensitizers have been reported for mitochondria targeting, and another few have been reported for nuclei targeting. However, none have been reported for photosensitization in both mitochondria and nucleus, especially in cascade mode, which can significantly reduce the photosensitizers needed for maximal treatment effect. Herein, a light‐driven, mitochondria‐to‐nucleus cascade dual organelle cancer cell ablation strategy is reported. A functionalized iridium complex, named BT‐Ir, is designed as a photosensitizer, which targets mitochondria first for photosensitization and subsequently is translocated to a cell nucleus for continuous photodynamic cancer cell ablation. This strategy opens new opportunities for efficient photodynamic therapy.


Materials and instruments
All reagents and solvents (analytical grade) were used as received from commercial sources unless otherwise indicated. Solvents were purified by standard procedures. Scheme S1. Synthetic routes of the compounds.
After 4 h, the solution was cooled to room temperature, and then a 6-fold excess of NH 4 PF 6 was added under stirring, and the mixture was stirred for another 1 h. The mixture was filtered and evaporated to dryness under reduced pressure. The obtained solid was dissolved in CH 2 Cl 2 and purified by column chromatography on silica gel eluted with CH 2 Cl 2 /acetone. Compound S4 was obtained as orange solid (1.85g, yield 95%).

ROS detection in solution.
ABDA was used as a ROS indicator to detect the ROS generation in the aerated disodium hydrogen phosphate/citric acid buffer solutions. The ABDA stock solution was mixed with the [Ru(bpy) 3 ]Cl 2 , BT-Ir(C), and BT-Ir (10 μM) exposed to light irradiation at a power of 425 nm LED light 40 mW/cm 2 for different times. The decomposition of ABDA was monitored by the absorbance decrease at 378 nm.

Hydroxyl radical (OH • ) detection and Singlet oxygen detection.
The hydroxyl radical (OH • ) and singlet oxygen were detected according to the literature method. [5] DNA/RNA titration experiments. The original ligand and water were removed by PyMOL [6] , and the DNA/RNA was prepared by Accelrys Discovery Studio 2.5.5 [7] for docking studies.

BT-Ir
Ligand preparation: BT-Ir is optimized using DFT calculations by the Gaussian09 package [8] at B3LYP/6-31g (d, p) level. Using the optimized BT-Ir structure, the partial atomic charges were obtained by restrained electrostatic potential (RESP) [9] calculating with the Gaussian 09 package at the level of HF/6-31g*. After that, the docking calculations were conducted by the AutoDock 4.2 suite of programs [10] with a ligand flexible docking approach. The Lamarckian genetic algorithm [11] was chosen as the search protocol using the default parameters except for the number of GA runs (ga_run = 80) and the maximum number of energy evaluations (ga_num_evals = 25,000,000).
The docking model, with the 20 lowest docked free energy, was selected for further investigation in this article. The displaying images were rendered with PyMOL.
Cell lines and culture conditions.
The cells were cultured in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO 2 and 95% air. In each experiment, the cells treated with DMSO (1%, v/v) were used as the reference group.

Analysis of MMP.
For flow cytometry, A549 cells were seeded into 6 well plates and cultured for 48 h.

Statistical analysis.
All biological experiments were performed at least twice with triplicates in each experiment. Representative results were depicted in this report, and data were presented as means ± standard deviations (SD) with statistical significance.