Catalase-like metal–organic framework nanoparticles to enhance radiotherapy in hypoxic cancer and prevent cancer recurrence

Tumor hypoxia typically occurs inside a solid tumor with an inadequate oxygen supply, sharply reducing the therapeutic efficiency of radiotherapy and significantly increasing the risk of local tumor recurrence.

S3 incubation in an oven at 80 °C for 2 h. Thereafter, 6 mL of free DMF was mixed with the solution mentioned above for another 24 h. Finally, after being cooled to room temperature, the final product of TCPP-Hf was obtained by centrifugation, followed by washing with DMF, triethylamine/ethanol (v/v = 1/20) and ethanol three times each. For the synthesis of MnTCPP-Hf,  and MnCl 2 (20.15 mg) in DMF were refluxed for one hour under an argon atmosphere. After being cooled to room temperature, the dark-green solid product was collected by centrifugation and washed with DMF, ethanol and water three times each. respectively. The testing solution was put into a water bath under magnetic stirring slowly. At given time, a small amount of degradation solution was taken out for ICP-AES test. The leaching test of S4 Mn in saline or HEPES buffer solution: the MnTCPP-Hf-FA MOF NPs were added into a dialysis bag with cut-off molecular weight (MV) of 500 Da and dialyzed against saline or HEPES buffer solution under magnetic stirring. At given time, a small amount of solution was taken out for ICP-AES test.
Thereafter, 150 μL MTT solution (0.5 mg/mL) was added to each well. After 4 h of treatment, the MTT solution was discarded, and 150 μL of DMSO was added to dissolve crystals. Finally, the absorbance was measured at 490 nm using an RT 6000 microplate reader.
Optimization of FA. B16-F10 cells were plated on 35-mm glass-bottomed dishes for 24 h. A series of MnTCPP-Hf-FA (20 µM, TCPP equiv) containing various FA was then incubated with the above cells in culture medium in 5% CO 2 at 37 °C for 2 h. Next, the cells were washed with PBS three times before trypsinization. Thereafter, the cell pellet was collected by centrifugation and solubilized in digesting solutions (V HClO4 /V HCl /V HNO3 = 2/1/3) overnight. For inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis, the above solution was diluted by Milli-Q water to 10 mL, and the concentration of Hf 4+ was detected in each sample. Additionally, the cellular uptake was also tested by confocal laser scanning microscopy (CLSM). After cells were incubated with different nanoparticles (MnTCPP-Hf (20 µM, TCPP equiv) and MnTCPP-Hf-FA (20 µM, TCPP equiv) S6 containing FA (2.5 µmol) for 2 h, confocal images were captured with 405 nm excitation and collected with the range of 600 nm to 700 nm.

Detection of H 2 O 2 and O 2 in cells. The cells were first incubated with different nanoparticles
(TCPP-Hf-FA (20 µM, TCPP equiv) and MnTCPP-Hf-FA (20 µM, TCPP equiv)) for 12 h in 5% CO 2 at 37 °C, followed by treatment with 150 µM H 2 O 2 for 1 h. Thereafter, the cells were washed with PBS three times and further incubated for another 4 h. Finally, the laser confocal images were captured by CLSM under Qcy7-H 2 O 2 labeled with cells (λ ex = 488 nm and λ em = 600 -700 nm).
Moreover, the detection of intracellular O 2 was also carried out. The cells were first incubated with different nanoparticles for 6 h at 37 °C in 5% CO 2 and then were washed with PBS three times.
HIF-1α immunostaining and western blotting. The cells were first incubated with different nanoparticles (TCPP-Hf-FA (20 µM, TCPP equiv) and MnTCPP-Hf-F (20 µM, TCPP equiv)) for 12 h at 37 °C in 5% CO 2 , and then, certain cells were subjected to anaerobic condition for 6 h. After fixation with 4% paraformaldehyde for 20 min, the cells were stained with antibodies against HIF-1α (ab190197; Abcam, Cambridge, MA, USA). The cell fluorescence was analyzed by CLSM with the excitation of 488 nm. Moreover, the content of HIF-1α was also quantified by western blotting. The cell samples were prepared according to the mentioned methods above and were monitored by enhanced chemiluminescence using the Gel Doc system.

Detection of ROS in cells.
The cells were first incubated with different nanoparticles (TCPP-Hf-FA (20 µM, TCPP equiv) and MnTCPP-Hf-FA (20 µM, TCPP equiv)) for 12 h in 5% CO 2 at 37 °C, and S7 then, certain cells were subjected to anaerobic condition for 6 h. After labeling with DCFH-DA for 20 min, the cells were subjected to X-ray irradiation of 4 Gy. Finally, the confocal images were immediately obtained by CLSM with 488-nm excitation. In addition, the kinds of ROS generation by MnTCPP-Hf-FA under X-ray irradiation were also clarified in vitro/in vivo. Methylene blue (MB) and coumarin were chosen as fluorescence indicator to selectively trap •OH in chemical and intracellular systems, respectively. Moreover, the 1 O 2 generation of MnTCPP-Hf-FA upon X-ray irradiation were determined by 1,3-Diphenylisobenzofuran (DPBF) and singlet oxygen sensor green (SOSG) in chemical and intracellular systems, respectively. DNA damage evaluation. DNA damage was detected using the γ-H2AX Phosphorylation Assay Kit.
Briefly, the cells were first incubated with different nanoparticles (TCPP-Hf-FA (20 µM, TCPP equiv) and MnTCPP-Hf-FA (20 µM, TCPP equiv)) for 6 h in 5% CO 2 at 37 °C, and then, certain cells were subjected to anaerobic condition for 12 h. After irradiation with 4 Gy of X-rays, the cells were further incubated for 4 h. Next, the cells were fixed with 4% paraformaldehyde for 20 min and were eventually stained with FITC-conjugated anti-phospho-histone γ-H2AX (Ser139). Finally, an imaging flow cytometer (Amnis Corporation) was applied to record the cell images with an excitation of 488 nm for FITC. IDEAS ® image analysis software (Amnis) was used to analyze the images.

Cell migration and invasion assay.
(1) The cells were seeded into 60-mm dishes and were incubated at 37 °C under 5% CO 2 in RPMI 1640 for 12 h. The cells were further subjected to different conditions, and the dose of X-rays was 4 Gy. The monolayer was wounded using a 10-µL sterile pipette tip, and cell images were taken at time 0, 12, 24 and 36 h post-wounding. The area of wound healing was calculated using AJ-VERT software, and each experiment was performed in triplicate. The hypoxic conditions were applied in an incubator for 6 h. (2) Cell invasion assays were S8 conducted using Matrigel-coated invasion chambers with an 8-µm pore size in 24-well plates (BD Biosciences). B16-F10 cells were treated as mentioned method above; 2×10 4 cells were added into the upper compartment and further incubated for 24 h. After removing the noninvasive cells on the upper surface of the membrane, the invasive cells were fixed with 4% paraformaldehyde and were stained with 0.2% crystal violet before counting the number of invaded cells under a microscope.
Hypoxic conditions were applied in an incubator for 6 h.
Clonogenic assay. First, 8×10 3 cells were seeded in 35-mm dishes at 37 °C under 5% CO 2 for 12 h, and then some of them were placed in an incubator for 6 h. After incubation with various nanoparticles (20 µM, TCPP equiv) for another 12 h, the cells were subjected to 4 Gy X-ray irradiation. Next, the cells were incubated at 37 °C under 5% CO 2 for 10 days before being fixed with 4 % polysorbate and stained with crystal violet. Cell colonies were counted only if they contained more than 50 cells. The surviving fraction = (surviving colonies) / (cells seeded × plating efficiency). The mean surviving fraction was obtained from three replicates.

Tumor model establishment.
The B16-F10 melanoma model was used as an example to evaluate the therapeutic effect. To this end, 1×10 7 B16-F10 cells in 100 µL of serum-free RPMI 1640 medium were injected subcutaneously into the right axillary region of BALB/c mice. After the tumor size had reached approximately 80-100 mm 3 , the mice were used in subsequent experiments.

In vivo therapeutic experiments in xenograft tumor models.
First, the mice were divided into five different groups: (i) Control, (ii) MnTCPP-Hf-FA, (iii) X-ray, (iv) TCPP-Hf-FA+X-ray, and (v) MnTCPP-Hf-FA+X-ray. The MOF NPs were diluted in physiological saline to 5 mg/mL, and 50 µL was administered to each mouse by intravenous injection. Next, 4 Gy of X-ray was irradiated on the tumor site after injection for 12 h. The tumor sizes and body weights were measured every other day S9 for 14 days after treatment (tumor volume = W 2 ×L/2, W = width, L = length). The relative tumor volumes were calculated for each mouse as V/V 0 (V 0 was the tumor volume when the treatment was initiated. (2) H&E staining and TUNEL staining of tumor slides: 12 h after different treatments, the mice were sacrificed, and the tumors were harvested to use for hematoxylin and eosin (H&E) staining and TUNEL staining; H&E staining of five major organs (liver, lung, spleen, kidney, and heart): 7 days after different treatments, the mice were sacrificed, and five major organs (liver, lung, spleen, kidney, and heart) were harvested to use for H&E staining.
In vivo hemocompatibility. In vivo hemocompatibility of MnTCPP-Hf-FA was evaluated using serum biochemistry and routine blood test using BALB/c mice. The MOF NPs were diluted in physiological saline to 5 mg/mL, and 50 µL was administered to each mouse by intravenous injection. After 0.5, 1 and 7days feeding, mice were anesthetized to collect blood. Blood supernatant harvested via centrifugation was used to analyze the serum biochemistry parameters. Two hepatic function indicators (ALT, AST), two kidney function indicators (BUN, CR) were measured. Heparin Tumor recurrence prevention. B16-F10 melanoma cells were first injected subcutaneously into the right axillary region. When the tumor volumes reached ∼300 mm 3 in approximately 10 days, the tumors were removed through surgery, leaving residual microtumors. Next, the mice were divided into five different groups: (i) Control, (ii) MnTCPP-Hf-FA, (iii) X-ray, (iv) TCPP-Hf-FA+X-ray, and (v) MnTCPP-Hf-FA+X-ray. The MOF NPs were diluted in physiological saline to 5 mg/mL, S10 and 50 µL was administered to each mouse by intravenous injection. Then, 12 h postinjection, the tumor site was irradiated with 4 Gy of X-rays. The body weights were measured every other day after treatment.