Iron (II)-based metal-organic framework nanozyme for boosting tumor ferroptosis through inhibiting DNA damage repair and system Xc- | Journal of Nanobiotechnology
Materials
Polyvinylpyrrolidone (PVP, K40) was obtained from Sigma Aldrich. N, N-dimethylformamide (DMF), dithiodiglycolic acid, iron (II) chloride tetrahydrate, triethanolamine (TEA), ferrostatin-1, indocyanine green (ICG), and D-α-tocopherol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Actinomycin D (ActD) was obtained from MedChemExpress. DSPE-mPEG was acquired from Shanghai Yaye Gaoyuan Enterprise Management Co., Ltd. CCK8 kit and cell culture medium were received from YENSEN Technology Co., Ltd. 3-Methyladenine was bought from Shanghai Macklin Biochemical Co., Ltd. Necrostatin-1, Calcein-AM, propidium iodide (PI), BeyoClickTMEdU-555 Cell proliferation test kit, and Z-VAD-FMK were purchased from Beyotime Biochemical Co., Ltd. GSH assay kit and 1% TMB solution were purchased from Solarbio Science & Technology Co., Ltd. BBoxiProbe® HPF Hydroxyl Radical Probe was gained from Shanghai BestBio Biochemical Co., Ltd. Beta actin (β-actin) monoclonal antibody, LC3 polyclonal antibody, GPX4 monoclonal antibody, HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), HRP-conjugated Affinipure Goat Anti-Mice IgG, and SLC7A11/xCT antibody were got from Proteintech. 4-HNE antibody was obtained from Origo Biopharma Co., Ltd. FTH1 antibody and NCOA4 antibody were acquired from Shanghai absin Biochemical Co., Ltd. Liperfluo and Annexin V-FITC/PI apoptosis detection kit were gained from Dojindo Molecular Technologies, Inc. Fetal bovine serum (FBS) was got from PAN. All reagents were used as received.
Synthesis of FessMOF/ActD-PEG
FessMOF nanoparticles were synthesized at first according to a reported work with some modifications [27]. In detail, 300 mg PVP (K40) was thoroughly ultrasonically dispersed in 3 mL DMF. Then, dithioglycolic acid (52 µL, 100 mg/mL), FeCl2•4H2O (7 mg), triethanolamine (600 µL), and 10 mL of a mixed solution of DMF and anhydrous ethanol (the volumetric ratio of DMF and anhydrous ethanol is 5: 3) were added into the above PVP solution under stirring. After thoroughly mixed, the mixture was transferred to a Teflon-lined stainless-steel autoclave and kept at 150 °C for 12 h. The sediments were centrifuged (11,000 rpm, 15 min) and washed several times with water. Next, FessMOF nanoparticles were absorbed with DSPE-mPEG. FessMOF nanoparticles (1 mg) were mixed with DSPE-mPEG (1 mg) in 1 mL distilled water for 6 h at 4 °C. Afterward, FessMOF-PEG nanoparticles were obtained by centrifugation. Finally, FessMOF-PEG nanoparticles (1 mg) were added into the 1 mL ActD solution (0.5 ng/mL) overnight at 4 °C, and the final precipitates were washed with distilled water, obtaining the FessMOF/ActD-PEG nanoplatform. The loading efficiency of ActD in the FessMOF-PEG was calculated as (input ActD quantity – supernatant ActD quantity) / input ActD quantity × 100%. The final load rate of ActD is about 100%.
Characterization
Transmission electron microscopy (TEM) images were obtained by HT7700 Exalens transmission electron microscope. Zeta potential and dynamic light scattering (DLS) were measured on the Malvern Zetasizer Nano ZS (Malvern Instruments, Ltd., Worcestershire, UK). X-ray photoelectron spectroscopy (XPS) spectra were acquired by the Thermo SCIENTIFIC Nexsa a K-Alpha 1063 instrument (Thermo Fisher Scientific, USA). Fourier transform infrared spectroscopy (FTIR) spectra were tested on an FTIR spectrometer (ThermoNicolet IS50). UV-vis spectra were measured by UV-8000 S spectrophotometer. X-ray Diffraction (XRD) analysis of samples was conducted by a Rigaku Miniflex600 X-ray diffractometer.
Hemolysis experiment
Fresh blood was obtained from Balb/c mice through eyeballs. Red blood cells were centrifuged and washed 3 times with PBS solution. Then the red blood cells were diluted with 5 mL PBS. Preparation of positive control solution: 50 µL diluted mice blood + 50 µL 0.5% Triton-100; preparation of negative control solution: 50 µL diluted mice blood + 50 µL PBS solution; preparation of test group solutions: 50 µL diluted mice blood + different volumes of 1 mg/mL FessMOF-PEG. The final concentrations of FessMOF-PEG were 10, 20, 50, 100, 150, 200, 400, and 600 µg/mL. After incubation for 60 min at 37 °C on a shaker, the mixture was centrifugated at 3000 rpm for 3 min. Then the supernatant was placed in a 96-well plate, and the absorbance values were measured by a microplate reader (adjusted to 576 nm). The hemolysis rate (%) was measured by the hemolysis rate formula: hemolysis rate (%) = [(absorbance value of the test group – absorbance value of the negative control group) / (absorbance value of the positive control group – absorbance value of the negative control group)] × 100%.
ESR detection
For ESR detections, the PBS solution (pH 6.0) containing FessMOF-PEG (100 µg/mL) and 50 mM H2O2 were incubated for 15 min at room temperature. After that, the DMPO (100 mM) as the spin-trapping agent was added to the sbove mixture.
The POD-like activity of FessMOF-PEG
The POD-like activity of FessMOF-PEG was evaluated by the TMB colorimetric assay. In detail, 50 µg/mL FessMOF-PEG was dispersed in buffer solutions with different pH values (7.4, 6.0, and 4.5). Then, H2O2 solution (50 mmol/L) and 1% TMB solution were added. After 15 min, the absorbance values at 652 nm of various groups were immediately measured using a microplate reader (Thermo Fisher Scientific).
GSH consumption evaluation
FessMOF-PEG nanoparticles (50 µg/mL) were incubated with GSH (500 µg/mL) at 37 °C for 12 and 24 h. The GSH solution (500 µg/mL) without FessMOF-PEG nanoparticles as the control. GSH concentration was measured by a GSH detection kit.
Degradation of FessMOF-PEG
Fifty µg/mL FessMOF-PEG nanoparticles were dispersed in buffer solutions with different pH values (7.4, 6.0, and 4.5) with or without 500 µg/mL GSH solution. After slowly shaking for 24 h at 37 °C, the morphology of FessMOF-PEG nanoparticles was observed using transmission electron microscopy.
Cellular uptake
4T1 cells were seeded in 6-well plates at a density of 105 cells for 24 h and then co-incubated with 50 µg/mL FessMOF-PEG@FITC (FITC, 10 µg/mL) for different times (0, 1, 2, 4, 8, 12, and 24 h). Afterward, the fluorescence intensity of FITC in cells was assessed by flow cytometer (NovoCyte Flow Cytometer).
Cell viability
4T1 cells were seeded in 96-well plates with 104 cells per well for 24 h, and then treated with various treatment solutions, (a) PBS; (b) ActD (0.5 ng/mL); (c) FessMOF-PEG (50 µg/mL); (d) FessMOF/ActD-PEG (50 µg/mL, containing 50 µg/mL FessMOF-PEG and 0.5 ng/mL ActD) for 24 h. The CCK8 assay kit was used to evaluate cell viability. Each well of the 96-well plate received 10 µL of CCK-8 solution. After 1 h of incubation, the cell absorbance values at 450 nm were measured using a microplate reader.
Colony-forming assay
4T1 cells were seeded in 6-well plates at a density of 1000 cells per well and grew for 24 h. The cells were incubated with various treatment solutions for 24 h. The medium was changed every three days under the same treatment conditions. The co-incubation was terminated after 14 days when colonies were visible to the naked eye. Thereafter, the cells were washed three times with PBS, then fixed with 4% paraformaldehyde for 30 min, rinsed twice with running water, and stained for 30 min with crystal violet solution. Finally, the crystal violet solution was carefully washed away with running water, and the 6-well plates were inverted and naturally dried. Photographs were taken and videotapes were made.
•OH detection
4T1 cells were seeded in confocal dishes at a density of 104 cells per well and grew for 24 h. After 4T1 cells were incubated with various treatment solutions for 24 h. Diluted BBoxiProbe®O26 probe solution was added into confocal dishes. Then, the cells were washed twice with PBS and imaged by a confocal laser scanning microscopy (LSM780, Zeiss).
Liperfluo
For cellular LPO evaluation, 4T1 cells were incubated with various treatment solutions for 24 h. Afterward, the cell culture medium was replaced with 10 µmol/L liperfluo probe solution and cultured for 30 min at 37 °C. At last, the cells were imaged under a confocal laser scanning microscopy. Simultaneously, the fluorescence intensity was assessed by a flow cytometer.
Live/dead assay
After various treatments, 4T1 cells were co-incubated with Calcein-AM (2 µmol/L) and PI (5 µmol/L) staining reagents for 2 h and subsequently imaged by a confocal laser scanning microscopy.
GSH and MDA detection
4T1 cells were cultured in 6-well plates with a density of 106 cells per well and co-cultivated with (a) PBS; (b) ActD (0.5 ng/mL); (c) FessMOF-PEG (50 µg/mL); (d) FessMOF/ActD-PEG (50 µg/mL, containing FessMOF-PEG 50 µg/mL and ActD 0.5 ng/mL). After 24 h, the GSH and MDA content of each group were measured by the GSH/MDA detection kit according to the manufacturer’s instructions.
Comet electrophoresis experiment
The experiment was conducted according to the kit instructions (KGI Bio). In detail, the layer of 1% normal melting point gel was configured on the slides. Then, 10 µL of cells solution (about 104 cells) and 75 µL of 0.7% low melting point gel were mixed well and spread on the pretreated slides at 4°C for 10 min to allow the agarose to solidify. Afterward, the glued slides were lysed for 90 min in a freshly prepared lysis buffer. Subsequently, the slides were rinsed in PBS solution before being placed in an electrophoresis buffer, denatured for 30 min, and electrophoresed for 20 min (25 V, 300 mA). And then, neutralization was performed three times for 5 min each with 400 mmol/L tris buffer (pH 7.5). At last, each slide was stained for 20 min with 20 µL of PI staining solution and observed under a Zeiss fluorescence microscope.
Apoptosis assay
4T1 cells were pre-seeded in 6-well plates at a density of 105 cells per well. The cells were incubated with various treatment solutions for 24 h. Afterward, the adherent cells in 6-well plates were digested with trypsin, washed twice with pre-cooled PBS solution, and finally collected 5 × 105 cells in tubes. Each tube was incubated with 5 µL Annexin V -FITC and 10 µL PI staining solutions for 10 min at 4 °C. Detection on flow cytometer: upper right quadrant (Annexin V+/PI+, late apoptotic cells); lower right quadrant (Annexin V+/PI–, early apoptotic cells), upper left quadrant (Annexin V–/PI+, dead cells), and lower left quadrant (Annexin V–/PI–, live cells).
Cell cycle assay
The cultivated 4T1 cells were tagged with EdU. Then, cells were fixed and permeabilized with a BeyoClickTMEdU-555 kit. The results were analyzed by a flow cytometer.
Western blot
4T1 cells were pre-seeded in a 6-well culture plate. Cells were washed twice with PBS solution and then collected using cell lysate, PMSF, and protease inhibitor. The contents of protein were measured using the BCA protein concentration assay kit. According to the required sample volume, 5 × loading buffer was added into the protein solution at a ratio of 4: 1. Then the final protein solution was denatured at 100 ℃ for 10 min in a boiling water bath. The separation gel and concentrated gel were prepared, and the samples were loaded at 30 µg of protein volume, electrophoretically separated and transferred into the polyvinylidene fluoride (PVDF) membrane, and then closed by 5% skim milk solution at room temperature for 2 h. These samples were incubated with the corresponding primary antibody such as β-actin (1: 20,000, Proteintech), SLC7A11 (1: 1000, Origo), GPX4 (1: 1000, Proteintech), 4- HNE (1: 1000, Origo), LC3 (1: 1000, Proteintech), NCOA4 (1: 1000, Proteintech), FTH1 (1: 1000, Proteintech), and γ-H2AX (1: 1000, Proteintech) overnight at 4 °C. After that, these samples were cultivated with the secondary antibody (1: 10,000, Proteintech) for 2 h. At last, the membranes were visualized and processed with image J for grayscale values.
In vivo biodistribution
Female Balb/c mice (4 weeks) were purchased from Shanghai Slack Laboratory Animal Co., Ltd., and the experiments were implemented in accordance with protocols approved by the Animal Experimental Ethics Committee of Fujian Normal University.
4T1 tumor models were obtained by orthotopically injecting 5 × 106 4T1 cells in the breast fat pads of mice. When the tumor volume approached about 80 mm3, the eight tumor-bearing mice were randomly divided into two groups. Then, mice were intravenously injected with free ICG or FessMOF-PEG@ICG (containing 100 µg ICG per mouse). Subsequently, fluorescent signals of mice were acquired by an imaging spectrum system (PerkinElmer) at different time points (1, 2, 4, 8, 10, and 24 h). After injection for 24 h, mice were euthanized and tumors and major organs (heart, liver, spleen, lung, kidney, and brain) were collected. The ex vivo fluorescence images and intensities of tumors and major organs were recorded by the imaging system.
In vivo tumor therapy
4T1 cells (5 × 106) were orthotopically injected into the breast fat pads of female Balb/c mice. When the tumor volume reached approximately 100 mm3, these mice were injected intravenously with (a) PBS; (b) ActD (0.8 µg/kg); (c) FessMOF-PEG (8 mg/kg); (d) FessMOF/ActD-PEG (8 mg/kg, containing 8 mg/kg FessMOF-PEG and 0.8 µg/kg ActD). Afterward, the tumor volumes and body weights were recorded every other day. The tumor volumes (mm3) were measured according to V = ab2/2, in which a and b represent the length and width of the tumor, respectively. After 14 days, these mice were executed and their tumors and organs (heart, liver, spleen, lung, and kidney) were collected. Tumor slices were conducted with H&E staining and TUNEL staining. Slices of organs were stained with H&E for damage evaluation. In addition, blood solutions of treated mice were harvested for blood routine analysis including hemoglobin (HGB), red blood cells (RBC), lymphocytes (Lymph), and red blood cells (WBC). In the meanwhile, glutamic aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and UREA in plasmas were measured to evaluate the hepatic and renal functions.
Quantification and statistical analysis
Quantitative data were presented as mean ± standard error of the mean (SEM). Statistics were analyzed by one-way or two-way analysis of variance (ANOVA), followed by two-sided Student’s t-test using GraphPad Prism 5.0. Differences were considered significant when the p-value was less than or equal to 0.05. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.