Nanoscale metal-organic frameworks for mitochondria-targeted radiotherapy-radiodynamic therapy

Selective delivery of photosensitizers to mitochondria of cancer cells can enhance the efficacy of photodynamic therapy (PDT). Though cationic Ru-based photosensitizers accumulate in mitochondria, they require excitation with less penetrating short-wavelength photons, limiting their application in PDT. We recently discovered X-ray based cancer therapy by nanoscale metal–organic frameworks (nMOFs) via enhancing radiotherapy (RT) and enabling radiodynamic therapy (RDT). Herein we report Hf-DBB-Ru as a mitochondria-targeted nMOF for RT-RDT. Constructed from Ru-based photosensitizers, the cationic framework exhibits strong mitochondria-targeting property. Upon X-ray irradiation, Hf-DBB-Ru efficiently generates hydroxyl radicals from the Hf6 SBUs and singlet oxygen from the DBB-Ru photosensitizers to lead to RT-RDT effects. Mitochondria-targeted RT-RDT depolarizes the mitochondrial membrane to initiate apoptosis of cancer cells, leading to significant regression of colorectal tumors in mouse models. Our work establishes an effective strategy to selectively target mitochondria with cationic nMOFs for enhanced cancer therapy via RT-RDT with low doses of deeply penetrating X-rays.

of the IFEFFIT package. Fits were performed in R space, with a k-weight of 3 for Ru samples. Refinement was performed by optimizing an amplitude factor S0 2 and energy shift ΔE0 which are common to all paths, in addition to parameters for bond length (ΔR) and Debye-Waller factor (σ 2 ). The fitting models for H2DBB-Ru and Hf-DBB-Ru were based on the crystal structure OPELOL obtained from the Cambridge Crystallographic Database.

Hydroxyl radical produced by Fenton reaction
Hf-DBA, and Hf-DBB-Ru were suspended in water at equivalent Hf concentrations of 20 μM. Aqueous solutions of 5 μM APF, 50 μM FeCl2, and various H2O2 concentration of 0, 400, 800, 1600, 3200, and 6400 μM were separately prepared. 100 μL of nanoparticle suspension and aqueous solution of APF, FeCl2, and H2O2 were added to a 96-well plate and the fluorescence signals were immediately collected with a Xenogen IVIS 200 imaging system (Xenogen, USA). Nanoparticle-free aqueous solutions of APF, FeCl2, and H2O2 served as controls.

Cellular uptake
The cellular uptake of Hf-DBA or Hf-DBB-Ru was evaluated in MC38 cells. Cells were seeded on 6-well plate at 1×10 6 /well and further cultured for 12 h. Particles were added to the cells at a Hf concentration of 20 µM. After incubation of 1, 2, 4 and 8 hours, the cells were collected and the cell numbers were counted by a hemocytometer. Cells were washed with PBS at least three times and then digested with 1% hydrofluoric acid and concentrated nitric acid in a microwave reactor and the Hf concentrations were determined by ICP-MS. Results were expressed as the amount of Hf (nmol) per 10 5 cells.
For the comparison of cellular uptake of Hf-DBA and Hf-DBA-R, MC38 cells were seeded on 6-well plate at 1×10 6 /well and further cultured for 12 h. Hf-DBA nMOFs with or without rhodamine labeling were added to the cells at a Hf concentration of 20 µM. After incubation of 4 hours, the cells were collected and the numbers of cell were counted by a hemocytometer. Cells were washed with PBS for at least three times then digested with 1% hydrofluoric acid and concentrated nitric acid in a microwave reactor (CEM, USA) and the Hf concentrations were determined by ICP-MS (Agilent, USA). Results were expressed as the amount of Hf (ng) per 10 5 cells.

Time-dependent Co-localization
MC38 cells were cultured in 35 mm tissue culture dishes overnight and incubated with Hf-DBA-R or Hf-DBB-Ru at an equivalent dose of 20 µM for 1, 2, 4, and 8 h. Cellular nuclei and mitochondria were labeled with hoechest 33258 and rhodamine 123, respectively. After washing with PBS, treated MC38 cells at each time point were then co-stained with Hoechest 33342 and Rhodamine 123 for 10 min at room temperature. Cells were then fixed with 4% paraformaldehyde at 4 ℃ for 30 min. The slides were then washed with PBS and observed under CLSM. Pearson' s coefficient was quantified using the Co-localization Analysis plugin for ImageJ.

Morphological Change Observation
MC38 cells seeded on cover slides in 35 mm tissue culture dishes overnight. Hf-DBA, H2DBB-Ru or Hf-DBB-Ru was added to the cells at an equivalent dose of 20 μM. Cells incubated with PBS served as a control. After incubation of 4 hours, cells were irradiated upon X-ray irradiation (250 kVp, 15 mA, 1 mm Cu filter) at a dose of 0 or 2 Gy. After 72-hour incubation, slides were washed with PBS and directly observed and the bright field images were obtained using a cutoff filter transmitting light >590 nm.

Respiratory Chain Activity Assay
Respiratory chain activity was assayed with (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). MC38 cells seeded on cover slides in in 35 mm tissue culture dishes overnight. Hf-DBA, H2DBB-Ru or Hf-DBB-Ru was added to the cells at an equivalent dose of 20 μM. Cells incubated with PBS served as a control. After incubation of 4 hours, cells were irradiated upon X-ray irradiation (250 kVp, 15 mA, 1 mm Cu filter) at a dose of 0 or 2 Gy. After 24-hour incubation, MTT was added with concentration of 0.1 mg/mL and stained for 20 min at room temperature. The slides were then washed with PBS and observed under the stereomicroscope.  M upon X-ray irradiation at 0 (-) or 2 (+) Gy dose were stained with JC-1 4 h after irradiation.
Green fluorescence indicates the monomerization of JC-1, suggesting the decrease of mitochondria membrane potential. From top to bottom: dark control and corresponding bright field images; X-ray irradiated groups and corresponding bright field images. From left to right: PBS control, Hf-DBA, H2DBB-Ru, or Hf-DBB-Ru, respectively. Scale bar = 50 μm. The images were obtained with one repetition to afford similar results.