Chemicals or reagents
K3[Fe(CN)6], polyvinylpyrrolidone K30, ammonium hydroxide, tetraethyl orthosilicate (TEOS), 3,3',5,5'-tetramethylbenzidine (TMB), and nano-Fe3O4 (99.0% metals basis, d=20 nm) were purchased from Aladdin Chemistry, Co. Ltd (Shanghai, China). Cetyltrimethylammonium bromide (CTAB), Iron Assay Kit, and apoptosis inhibitor Ac-DEVD-CHO were obtained from Sigma-Aldrich (St. Louis, MO, USA). Calcium nitrate tetrahydrate, Vitamin C (Vc), and ethylene diamine tetraacetic acid (EDTA) were procured from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). The ROS Assay Kit, GSH and GSSG Assay Kit, and BCA Protein Assay Kit were obtained from Beyotime (Shanghai, China). PBS buffer (pH 7.4), dimethyl sulfoxide (DMSO), DMEM were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS), and trypsin were purchased from Invitrogen (Carlsbad, CA, USA). CCK-8 and Liproxstatin-1 (Lip-1) were bought from APExBIO (Houston, TX, USA). Deferoxamine (DFO) was purchased from MedChem Express (Monmouth Junction, NJ, USA). Malondialdehyde (MDA) assay kit was purchased from Solarbio (Beijing, China). Antibodies used in this work were ordered from Abmart (Shanghai, China). RIPA lysis buffer,BCA Protein Assay Kit, 12% SDS-PAGE gels, nitrocellulose membrane, 5% BSA, and ECL were purchased from Servicebio (Nanjing, China).
The human tongue squamous cell carcinoma line (CAL27 and HN6), human immortalized keratinocytes (HACAT), macrophages (RAW264.7) were purchased from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). All cells were cultured in DMEM medium containing 10% FBS and 100 U/mL penicillin-streptomycin in 5% CO2 and 37°C constant temperature and humidity incubator. When the cells were co-cultured with UPM to study their antitumor properties, the pH of the medium was adjusted to 6.5 with hydrochloric acid (HCl) to simulate the mildly acidic environment of TME.
Synthesis and characterization of UPM
The MCSNs were synthesized by template method,28-29 and the USPBNPs were synthesized by a single precursor synthesis technique.26 Then, the USPBNPs were successfully loaded into MCSNs by mixing-coupling method to synthesize UPM.
Briefly, 0.0275 g K3[Fe(CN)6] and 0.75 g polyvinyl pyrrolidone were dissolved in 10 mL 75% ethanol containing 0.01 M HCl and then cooled at room temperature after a water bath at 80°C for 3 h. The obtained mixture was purified, washed and dried, then added distilled water to prepare the USPBNPs solution by ultrasonic dispersion. Then, using CTAB as the template, dissolving 12 mL of ammonium hydroxide in 600 mL deionized water, adding 31.21 g of calcium nitrate tetrahydrate and 30 mL of TEOS, and calcined at 550°C for 2 h to obtain MCSNs. Then, the UPM was prepared by mixed coupling of USPBNPs and MCSNs with a mass ratio of 1:50.
The morphology and size of the UPM were characterized by SEM (JSM-7900F, JEOL, Japan) and TEM (JEM-2100, JEOL, Japan). The crystal structure of USPBNPs in UPM were investigated by high-resolution TEM (HRTEM). The composition and distribution of elements in UPM were analyzed by EDS element mapping (JEM-ARM200F, JEOL, Japan). Then, the prepared UPM powder was sealed in a dry environment for 365 d or dissolved in PBS solution and stood for 24 h to observe the change of the UPM solution.
pH measurement and iron release
10 mg of UPM was immersed in 10 mL of double-distilled water (ddH2O) at 37˚C and the pH value was measured by pH meter (PH818, XIMA, China) every 30 min. In addition, the cumulative releases of total iron, Fe2+ and Fe3+ per hour were determined by Iron Assay Kit. The iron release rate was obtained by the ratio of the measured total iron content of UPM to the theoretical value of USPBNPs. The conversion rate of Fe3+ to Fe2+ in UPM was obtained by the ratio of the measured Fe2+ and Fe3+ contents of UPM to the theoretical value of USPBNPs.61
POD and CAT activity measurement
TMB was used for a substrate to study the intrinsic peroxidase (POD) activity.62 Firstly, different pH buffers (pH 2, 3.05, 4.02, 5.01, 6.2, 7.4, 8.01, 8.98, 10.1, and 11.02) were prepared, and TMB (3.2 mM in DMSO), H2O2 (5 mM), and UPM (0.2 μg/mL) were added. The reaction was performed at 650 nm after 1 min by microplate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA) determined. Secondly, PBS, MCSNs (1 mg/mL), USPBNPs (20 μg/mL), and UPM (20 μg/mL) were added into disodium hydrogen phosphate-citrate buffer (pH 4.0) containing TMB (3.2 mM) and H2O2 (5 mM), respectively, and their relative enzyme activities were measured. Then, 20 μg/mL UPM or 400 μg/mL Fe3O4 was added to a disodium hydrogen phosphate-citrate buffer (pH 4.0) containing TMB (3.2 mM in DMSO) and H2O2 (5 mM). The full wavelength between 550-750 nm was detected by microplate reader. Finally, a kinetic analysis of the enzyme was performed, that is, the Michaelis-Menten equation was evaluated.63 The enzyme activity of UPM on H2O2 concentration dependence was studied by using different concentrations (0, 1.25, 2.5, 5, 10, 20, 40, and 80 mM) of H2O2. And according to Beer-Lambert Law, A=εbc (A is the absorbance, ε is the molar absorbance coefficient, b is the thickness of the liquid pool, and c is the solution concentration, where ε652 nm=39 mM-1cm-1),64 and the velocity concentration curve was drawn. The Michaelis−Menten equation V=Vmax[S]/(Km+[S]) was used to fit the initial velocity and substrate concentration, [S] is the substrate concentration, V is the initial velocity, the maximum velocity is Vmax, and the Michaelis constant is Km. Then the Lineweaver-Burk equation [1/V=(Km/Vmax)(1/[S])+1/Vmax] was derived.
Furthermore, the catalase (CAT) activity was determined by the Aebi method in the presence of H2O2.65 In brief, 0.2 mg/mL UPM was added into containing PBS buffer (pH 7.4) and H2O2 (50 mM). The consumption of H2O2 was determined by microplate reader at 240 nm after 15 min.
Biocompatibility in vitro
Non-tumor cell lines (HACAT and RAW264.7) with a cell density of 2×104 per well were seeded in a 96-well plate. After 24 h, the cells adhered to the wall, and took out original medium. Experiment 1: 100 μL fresh culture medium containing PBS, 1 mg/mL MCSNs, 20 μg/mL USPBNPs or 20 μg/mL UPM was added and incubated at 37°C and 5% CO2 for 24 h. Then, the medium was removed and 100 μL of basic medium supplement with 10% CCK-8 solution was added to per well and incultured in a cell culture environment without light for 2 h. The absorbance of the tested solution at 450 nm was measured to evaluate the toxicity of UPM to cells. Experiment 2: 100 μL fresh culture medium containing different concentrations of UPM (0, 5, 10, 20, 40, and 80 μg/mL) was added and incubated at 37°C and 5% CO2 for 24 h. Then the relative cell viability was detected and calculated according to the CCK-8 method.
Antitumor effect in vitro
The OSCC cell lines CAL27 and HN6 were cultured and seeded into a 96-well plate. After 24 h of cultivation. Experiment 1: 100 μL fresh culture medium containing PBS, 1mg/mL MCSNs, 20 μg/mL USPBNPs or 20 μg/mL UPM was added and incubated for 24 h, then relative cell viability was measured and calculated by CCK-8 method. Experiment 2: 100 μL fresh culture medium containing different concentrations of UPM (0, 5, 10, 20, 40, and 80 μg/mL) was added and incubated for 24 h, then relative cell viability was measured and calculated by CCK-8 method.
Scratch test
The migratory potential of tumor cells was evaluated by scratch test.66 CAL27 cells were plated in 6-well plates with a cell density of 1×106 in each well, and cultured for 48 h for complete cell fusion. A straight scratch was created at the bottom of 6-well plates at the center of the monolayer fusion cell with a 200 μL sterile pipette tip. After rinsing with PBS for 3 times, microscopic images were taken 0 h after scratching. Then the cells were incubated with 2 mL culture medium containing 1 mg/mL MCSNs, 20 μg/mL USPBNPs and 20 μg/ml UPM, and 2 mL culture medium with PBS as control. In the following 12 and 24 h, the migration of cells at the scratches was observed with a microscope and the microscopic images were captured at 100× magnification. The wound closure percentage was calculated by measuring the scratch area with ImageJ software: wound closure percentage = (scratch area at 0 h - scratch area at 12 or 24 h) / scratch area at 0 h*100%.
Transwell assay
The invasion potential of tumor cells was evaluated by Transwell assay.54,67 2×105 CAL27 cells (in 200 μL serum-free DMED) were seeded into the upper chamber of Transwell (3422, Corning, NY, USA) coated with matrigel, and the lower chamber was incubated with 800 μL containing 20% FBS DMEM for 36 h. Then the DMEM was removed, cells were fixed with 4% paraformaldehyde for 30 min and stained with crystal violet for 15 min. The non-penetrate cells and matrigel were removed with cotton swabs, the invasive cells were observed and photographed under a microscope, and the number of invaded cells was calculated, which represents the invasive ability of cells.
TEM images of CAL27 cells treated with UPM
The subcellular location of UPM and the morphological and mitochondria changes of cells treated with UPM were observed by TEM. Briefly, CAL27 cells (1×106 per well) were seeded in a 6-well plate and cultured overnight to make the cells adhere and grow. 2 mL fresh culture medium without or containing 20 μg/mL UPM per well was added. Then, each well was rinsed twice with PBS, cells were collected and fixed overnight with 2.5% glutaraldehyde at 4°C. The cells were postfixed in 1% w/v osmium tetraoxide after rinsing with PBS, dehydrated by ethanol, and finally encased in resin. The 70 nm ultrathin sections were double stained with uranyl acetate and lead citrate and observed using the TEM (JEM-1010, JEOL, Japan).
ROS and LPO assay
The intracellular ROS or LPO generation after UPM treatment was investigated by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) or C11-BODIPY581/591 fluorescence probe. Briefly, CAL27 cells were seeded on 6-well plates at a density of 5×105 per well and cultured overnight to obtain attachment. Then the fresh culture medium containing different concentrations of UPM (0, 5, 10, 20, and 40 μg/mL) was added into wells for 12 or 24 h incubation. The samples treated for 12 h were used for ROS detection, washed with PBS for 3 times, and then cultured in the dark at 37°C for 30 min with fluorescent probe DCFH-DA (10 μM). The treated cells were observed by CLSM (DMI6000B, Leica, Germany), or the cells were collected, and the fluorescence intensity of cells with excitation wavelength at 488 nm and emission wavelength at 525 nm was detected by flow cytometry (FACS Calibur, Becton-Dickinson, Franklin Lake, NJ, USA), and analyzed by FlowJo 10.8 software. The samples treated for 24 h were used for LPO detection. Cells were cultured with the C11-BODIPY581/591 (5 μM) probe in darkness for 30 min. The excitation at 488 or 565 nm, and emission at 510 or 591 nm to observe oxidized BODIPY or reduced BODIPY.
Effect of inhibitors on antitumor effect of UPM
CAL27 cells were inoculated on 96-well plates, cultured for 24 h, and the stock solution was removed. Add 100 μL 20 μg/mL UPM containing various inhibitors (antioxidant 100 μg/mL Vc and 10 mM NAC, iron chelator 200 μM DFO and 200 μM EDTA, ferroptosis inhibitor 10 μM Lip-1, apoptosis inhibitor 100 μM Ac-DEVD-CHO, was incubated for 24 h, and the relative cell viability was determined by CCK-8 method.
Intracellular iron ions and MDA determination
The content of Iron ions and the level of malondialdehyde (MDA) were detected using the corresponding kit. 68 In brief, CAL27 cells were treated with UPM for 24 h, then the extract was added and the cells were lysed by ultrasonic. Using Na2SO3 as an oxidant and 2-2’ dipyridyl as a chromogenic agent, the content of total iron ions in cells was determined at the maximum absorption peak 520 nm. Then part of the homogenate was added to thiobarbituric acid (TBA) and was placed in a bath at 100°C for 60 min. The absorbance was measured at 532 nm.
Intracellular GSH and GSSG ratio
CAL27, HACAT, and RAW264.7 cells were plated in a 6-well plate with a number of 1×106 cells per well, and cultured overnight to make the cells adhere and grow. Fresh culture medium without or containing 20 μg/mL UPM was added to per well and co-cultured for 24 h. Then, the GSH and GSSG Assay Kit was used in term of the manufacturer’s instructions, the GSH/GSSG level was determined by microplate reader at the absorbance of 412 nm.
Western blotting analysis
The cells were lysed with RIPA lysis buffer containing PMFS for 30 min. The supernatant was collected after high-speed centrifugation (12000 rpm, 15 min). The proteins were isolated with 12% SDS-PAGE gels, then the proteins were transferred to a nitrocellulose membrane. After blocking with 5% BSA for 1 h, the membrane was incubated overnight at 4°C with the primary antibodies. Then it was washed 3 times in Tris-buffer containing 0.1% Tween-20 (TBST) and incubated with the secondary antibodies for 1 h at 37°C. Finally, the protein bands were observed by ECL and fluorescent western blotting imaging systems.
Antitumor effect and biosafety in vivo
The animal experiments in this study were approved by the Animal Ethics Committee of Nanjing Medical University (IACUC No. 2112009), and all experiments were conducted under the principle of Animal Care and Use (ACU). The subcutaneous tumor models were established by using 5-week-old male Balb/c nude mice, which were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). Briefly, CAL27 cells (5×106 in 150 μL of PBS) were hypodermic injection into the right axillary of mice. When the tumor volume of nude mice grew to about 100 mm3, the mice were randomly divided into 3 groups (n=5). PBS, UPM (5 mg/kg), or UPM (5 mg/kg) + Lip-1 (0.5 μmol/kg) was injected intravenously every 3 d, and the body weight and tumor volume of mice were recorded. After 18 d of treatment, the tumor-bearing mice were euthanized, and tumors, blood samples and main organs (heart, liver, spleen, kidney and lung) of each group were collected. The tumor tissues were arranged, photographed and weighed according to the corresponding group. The tumor tissues were then fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, H&E staining or IHC staining with GPX4 and Caspase-3.
In vivo biocompatibility was then assessed. A whole blood sample with anticoagulant was tested hematologically using an automated analyzer (Sysmex, XN-10(B4), Kobe, Japan). Another blood sample without anticoagulant was set for 1h, centrifuged (3000 rpm, 10 min), and upper serum was collected. The samples were tested for ALT, AST, BUN and CREA levels by an automatic biochemical analyzer (HF-240, Han Fang Medical Instrument Co., Ltd., Jinan, China). Finally, the major organs of mice were evaluated by H&E staining.
Statistical analysis
All data analysis results were shown as mean ± standard deviation (SD) of at least three independent experiments. Differences between the two groups were compared using the Student’s t-test, and multi-group comparisons were made using a one-way analysis of variance (ANOVA). When P < 0.05, the difference was statistically significant.