Chemicals and agents
Calcium chloride anhydrous (CaCl2, AR), hydrogen peroxide (H2O2, 30 wt%), ammonia solution (NH3·H2O, 25 wt%), N, N-dimethylformamide (DMF, AR), and ethanol anhydrous were purchased from Sinopharm Chemical Reagent Co. Ltd. Cobalt (II) acetate tetrahydrate ((CH3COO)2Co·4H2O, 99.9%) and 1,1’-Ferrocenedicarboxylic acid (Fc(COOH)2, > 98%) were purchased from Aladdin-Reagent Co. Ltd. Polyvinyl pyrrolidone (PVP, K30), Fluo-4 AM and Cell Count Kit-8 (CCK-8) were purchased from Meilunbio Co. Ltd. 3, 3’, 5, 5’-Tetramethylbenzidine (TMB), 5, 5-Dimethyl-1-pyrroline N-oxide (DMPO), 2’,7’-Dichlorofluorescin diacetate (DCFH-DA, ≥ 97%) were purchased from Sigma-Aldrich. Horseradish peroxidase (HRP) was purchased from Hefei Bomei Biotechnology. The ECL western blotting system was purchased from Beyotime Biotechnology. Primary and secondary antibodies were obtained from Abcam. Calcein AM and propidium iodide (PI) probes were obtained from Dojindo.
Scanning electron microscopy (SEM) images was obtained by field-emission scanning electron microscopy (FESEM, Phenom Pharos). Transmission electron microscopy (TEM) images was obtained by transmission electron microscopy (TEM, HITACHI HT-7700; X-MAXn65 T). The zeta potential and dynamic light scattering were measured by zetasizer (0.3–10,000 nm, Zetasizer Nano-ZS Malvern). The X-ray diffraction pattern was obtained by X-ray diffractometer with Cu Kα radiation (XRD, X’pert PRO MPD). The chemical composition and valence states of different elements were measured by X-ray photoelectron spectroscopy (XPS, AXIS Supra). The UV–visible absorbance was measured by UV–vis spectrophotometer (Shimadzu, UV-2600). Hydroxyl radical signal was monitored by electron spin resonance spectroscopy (EPR, Bruker A300). CCK-8 and intracellular H2O2 content were recorded using a microplate reader. The fluorescence images of Live and Dead, intracellular reactive oxygen species (ROS) and Ca2+ ions were obtained by an inverted fluorescent microscope (Nexcope, The USA).
Synthesis of CaO2@Co-ferrocene
CaO2 nanoparticles were prepared according to previous procedures with the following protocol: CaCl2 (0.1 g) and PVP (0.35 g) were dissolved in 15 mL ethanol. Subsequently, NH3·H2O (0.8 M, 1 mL) was added under continuously stirring. Then, H2O2 (1 M, 0.2 mL) was added dropwise to the mixture using a syringe pump. Finally, CaO2 nanoparticles were collected by centrifugation at 12,000 rpm for 10 min and washed three times with ethanol. The product was stored in 5 mL ethanol for further use. CaO2@Co-ferrocene was prepared via in situ assembly. Typically, CaO2 (5.3 mg) and PVP (160 mg) were dissolved into 18 mL DMF under ultrasound treatment. Afterwards, (CH3COO)2Co·4H2O (0.1 M, 0.2 mL) and Fc(COOH)2 (0.1 M, 0.2 mL) were added. After stirring at 80 °C for 4 h, the product was obtained by centrifugation and washed several times with DMF and ethanol, respectively.
In brief, CaO2@Co-ferrocene (0.5 mg, or CaO2 containing the same amount of Ca2+) was dissolved in 0.5 mL acetate buffer solution (ABS) at pH 5 or 7. Then, 20 μL supernatant was collected at the different time interval. HRP (1 U/mL, 300 μL) and phosphate buffer solution (pH = 5.8, 1.68 mL) were added. After 10 min’ reaction, TMB (2 mM, 300 μL) was added and the characteristic absorbance of ox-TMB at 371 nm was recorded to quantify the H2O2 concentration according to the standard curve.
3, 3’, 5, 5’-Tetramethylbenzidine (TMB) can be oxidized to the oxidation state of TMB by ·OH produced by CaO2@Co-ferrocene under acidic condition, which shows a characteristic UV–vis absorbance at 655 nm. In brief, different concentrations of CaO2@Co-ferrocene and TMB (8 mM, 300 μL) were added in ABS at different pH (5 and 7). The UV–vis absorption of various samples was recorded by UV–vis spectrophotometer. Subsequently, the reaction kinetic curve was monitored under a UV–vis spectrophotometer. The generation of ·OH was further identified by electron spin resonance (ESR) spectroscopy with 5, 5-Dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. In addition, CaO2@Co-ferrocene (1 mg, or CaO2 with the same amount of Ca2+) was dissolved in ABS (initial pH = 5, 2 mL). pH value was recorded with a real-time pH meter.
In vitro study
Human liver cell (7702), human umbilical vein endothelial cell (HUVEC) and murine breast cancer (4T1) cells were used for the in vitro study. The culture medium RPMI 1640 was supplemented with 10% fetal bovine serum (FBS) and the cells were cultured in an incubator with 5% CO2 at 37 °C.
Cell viability assay
7702 and HUVEC cells were seeded in 96-well plates (104 cells per well) and incubated for 12 h. Subsequently, CaO2@Co-ferrocene at desired concentrations were added and incubated for another 24 h. Finally, culture medium containing 10% CCK-8 was added to each well and the absorbance was detected by a microplate reader at 450 nm after incubation at 37 °C for another 1 h.
Intracellular antitumor performance
4T1 cells were seeded in 96-well plates (104 cells per well). After incubation for 12 h, the culture medium was replaced with fresh medium at different pH values. To simulate acidic tumor microenviroment, the pH of cell culture was regulated to 6.5. HCl solution (1 M, 15 μL) was added into the medium (980 μL). The acidic medium was added along with CaO2@Co-ferrocene. Then, CaO2@Co-ferrocene at desired concentrations were added and incubated for another 24 h. Finally, culture medium containing 10% CCK-8 was added to each well and the absorbance was detected by a microplate reader at 450 nm after incubation at 37 °C for another 1 h.
In addition, the live & dead cells staining was carried out using calcein AM/PI staining. After seeded in 6-well plate and cultured for 12 h, 4T1 cells were cultured with CaO2@Co-ferrocene at desired concentrations for 10 h. After treatments, cells were cultured with calcein AM and PI for 30 min. After staining, cells were washed with PBS for three times and further observed at 480 nm and 525 nm, respectively.
Colony efficiency assay
4T1 cells were seeded in 6-well plates (200 cells per well). After incubation for 12 h, the cells were cultured in fresh medium containing different concentrations of CaO2@Co-ferrocene for 10 days. Then, 4T1 cells were fixed in 10% formaldehyde after washed with PBS. Finally, 4T1 cells were stained by crystal violet for 20 min.
Examination of intracellular calcium, ROS and H2O
ROS was detected by a fluorescent probe, 2, 7- dichlorofluorescein diacetate (DCFH-DA). 4T1 cells were seeded in 6-well plates. After incubation for 12 h, the cells were cultured in fresh medium containing different concentrations of CaO2@Co-ferrocene for another 24 h. Then, the medium was discarded, and the cells were washed three times with PBS before the addition of DCFH-DA (2 mg/mL, 10 μL). After staining, cells were washed with PBS for three times and DCF fluorescence images were observed by an inverted fluorescent microscope at an excitation wavelength of 480 nm. To detect intracellular Ca2+ ions, Fluo-4 AM, was applied as a green fluorescent probe according to the above steps. After treatments with CaO2@Co-ferrocene (60 μg/mL), 4T1 cells were lysed by RIPA lysis buffer with 1% PMSF. Supernatant containing H2O2 was collected by centrifugation at 12,000 rpm. Then, hydrogen peroxide assay kit was added and the absorbance at 560 nm was recorded with microplate reader.
Western blot assay
After treatments with CaO2@Co-ferrocene, 4T1 cells were lysed by RIPA lysis buffer with 1% PMSF. The collected cells were further crushed with an ultrasonic probe. Supernatant containing proteins was collected by centrifugation at 12,000 rpm. After quantification, protein (50 μg) was loaded onto SDS-PAGE, further transferred to the PVDF membrane and blocked by 5% skim milk for 2 h. Then, the PVDF membrane was incubated with primary antibodies and secondary antibodies successively. After washing three times in Tris-buffered saline Tween buffer (TBST), the band intensity was measured.
In vivo study
Four-week-old female Balb/c mice were purchased from Shanghai Slac Laboratory Animal Co. Ltd. and used in compliance with guidelines of the Biological Resource Centre of the Agency for Science, Technology and Research, Zhejiang University. The tumor-bearing mouse model was built via injecting 50 μL PBS with 4T1 cells (1 × 106) into the right side back of each mouse. The mice were randomly distributed into four groups for in vivo experiments (5 mice per group) when the tumor volumes reached about 80 mm3 and intratumorally injected with different formulations: Group 1: PBS, Group 2: Co-Fc MOF, Group 3: 5 mg/kg CaO2@Co-Fc, Group 4: 15 mg/kg CaO2@Co-Fc. The tumor size (V) was calculated as follows: V = width2 × length/2. Body weight, tumor size and images were recorded every 2 days. After 2 weeks, tumors were collected for immunohistochemistry analysis after sacrifice.
The paraffin-embedded slices were deparaffinized in xylene and subsequently hydrated in serially diluted grades of ethanol. Endogenous peroxidase was blocked with 3% hydrogen peroxide, followed by antigen retrieval using a microwave oven under at pH = 6.0 (citrate buffer). Slices were incubated with ki67 overnight at 4 °C and then overlaid with secondary antibody for 20 min at room temperature. Finally, a diaminobenzidine tetrahydrochloride (DAB) working solution was applied and the slices were counterstained with hematoxylin.
Data were expressed as mean ± SD. Comparison analysis between groups was conducted by student’s test.
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