5.1. Reagent and chemicals
CoNPs (<50nm, #7440-48-4), alpha-lipoic acid (#1077-28-7), and dichlorofluorescin diacetate (DCFH-DA) (#4091-99-0) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and trypsin-EDTA were procured from Gibco (Life Technologies, Paisley, UK). CCK-8 kits were obtained from Dojindo (Kumamoto, Japan). Annexin V-FITC (#AP101-100-AVF), Calcein AM/PI (#C2013FT& ST511) was purchased from Multi Sciences (Hanzhou, China). DMSO, GSH, and GSSG Assay Kit (#S0053) were procured from Beyotime (Shanghai, China). Iron Assay Kit (#ab83366), Anti-GPx4 antibody (#ab125066), Goat anti-rabbit IgG H&L (HRP) (ab205718), Goat anti-mouse IgG H&L (HRP) (ab205719), and β-actin antibody were obtained from Abcam Technology (Cambridge, UK).
5.2. Cell culture and preparation of stock solutions of CoNPs/ALA
Balb/3T3 cell line was procured from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in a DMEM medium containing 10% FBS, 50 U/ml penicillin, and 50 mg/ml streptomycin at 37˚C in the presence of 5% CO2. The cells were differentiated up to 6 days, and the cell medium of differentiating cells was replaced after every 2 days to ensure the availability of optimal nutrients and differentiation factors for the growth of cells. The cells were sub-cultured after every 3 to 4 days using 0.25% trypsin/1 mM EDTA solution and TNS.
The CoNPs were weighed the day before the experiment, sterilized at 180˚C for 4 h, and suspended in ultrapure water to prepare a stock solution of 6 mM. The freshly prepared stock solution was then ultrasonicated at room temperature at 40W for 30 min to minimize the particle aggregation and diluted with complete culture medium to achieve the required concentrations. The ultrasonicated fresh stock solution was continuously oscillated using a vortex shaker before adding to the sample well. The solution was distributed to various sample wells within 10 min because of the rapid precipitation of CoNPs（Figure S1, S2）. Alpha-lipoic acid of appropriate concentration was dissolved in DMSO. The final working concentration of DMSO was controlled below 0.1% to avoid its cytotoxicity.
5.3. CCK-8 cell viability assay
The cell viability assay was performed by employing the Cell Counting Kit-8 (CCK-8). The cells (1.5x104 cells/well) were seeded into 96-well plates and adhered for about 8 h before being exposed toward different concentrations of CoNPs (20-800 μM) for 8-24 h at 37˚C to explore the IC50 value. The cells cultured with DMEM media without any CoNPs treatment served as the control group. Further, the cells were incubated with ALA of various concentrations (50-1200 μM) for 24 h to evaluate its cytotoxicity. To investigate the effects of ALA on the viability of Balb/3T3 cells exposed to CoNPs, the cells were first exposed to 400 μM (IC50) of CoNPs and subsequently incubated with ALA (50-600 μM) for 24 h. After that, CCK-8 solution (10 μl) was added to each well of the plate for 1 h at 37˚C before the detection. The CCK-8 formazan product was measured at 450 nm using a microplate reader. The cell viability ratio was expressed as a percentage of the control experiment. Each experimental condition was repeated in triplicate.
5.4 Cell staining to observe live and dead cells
Cells were seeded in a 6-well plate and treated in the same manner as before. After 24 hours of exposure, aspirate the medium, add Calcein AM/PI stain and incubate at 37°C for 30 minutes. Observe the number of live and dead cells with a fluorescent inverted microscope(LEICA DMi8).
5.5. Detection of intracellular iron ion concentration
The cells were seeded into a 6-well tissue culture plate. When the cells were in the logarithmic growth phase, the cells were pre-treated with ALA (200 μM) for 3 h, followed by incubation with CoNPs (400 μM) for a total of 24 h. The cells were collected, washed with 1 ml phosphate-buffered saline (PBS), and centrifuged for 5 min (1000 rpm) to remove the supernatant. The cells were then resuspended in 0.5 ml PBS, and the cell concentration was calculated using a cell counter. Next, 1 x 106 cells were again centrifuged at 1000 rpm for 5 min, and all the supernatant was carefully aspirated. After that, 500 μl of lysis buffer was added to the cells and place on a shaker for 2 h to lyse them. The Iron Assay Kit was then employed according to the manufacturer's instructions to detect the intracellular iron(II) ion concentration and total iron by spectrophotometry.
5.6. Detection of intracellular &extracellular cobalt concentration
Cells were seeded in a 6-well plate and incubated. When the cells were in the logarithmic growth phase, aspirate the medium, 2 ml of medium containing CoNPs (400μM), and ALA (200μM) were added to each well. After 24 hours of incubation, 1 ml of the upper-medium was drawn before collecting the cells to measure the concentration of cobalt in the extracellular solution. Then, the medium was removed, and cells were washed with PBS 3 times, detached, and collected with centrifugation at 1000 rpm for 5 minutes. 2 mL of PBS was used to re-suspend the cells. After 5 mins, 10 uL of supernatant was extracted to check with a microscope to confirm there are no CoNPs in the solution. Next, 1 x 106 cells were again centrifuged at 1000 rpm for 5 min. After removing the supernatant, 500 μL of lysis buffer was added, followed by centrifugation at 12,000 rpm for 2 h. Finally, the supernatant was used to measure the cobalt concentration with an inductively coupled plasma mass spectrometer (ICP-MS, PerkinElmer NexION 350).
5.7. Detection of cell necrosis and apoptosis ratio
To compare the ratio of apoptosis and necrosis among the cells in each group, the Annexin V-FITC assay kit was conducted according to the manufacturer's protocol. Briefly, after incubating the cells with respective treatments for a total of 24 has previously been described, they were carefully washed with PBS solution and collected. The cells were resuspended gently with PBS and counted using a cell counter. Next, 1 x 106 cells were centrifuged at 1000 g for 5 min, and the supernatant was discarded. The cell pellet was incubated with Annexin V-FITC and propidium iodide staining solution for 10 - 20 min at room temperature (20 - 25 °C) in the dark and then placed in an ice bath. A flow cytometer (Becton Dickinson flow cytometer) was employed for the detection of cell necrosis and apoptosis. The ratios of apoptosis and necrosis were calculated separately.
5.8. Determination of intracellular ROS
To assess the ROS generation in cells exposed to target concentrations of CoNPs and the protective effects of ALA, spectrofluorometry, and fluorescent microscopy were employed. For spectrofluorometry analysis, the cells (1 x 105/well) were seeded into 96-well tissue culture plates. After 8 h of adherence, the cells were incubated with CoNPs (400 μM) and ALA (200 μM) for 24 h. Following staining with DCFDA for 30 min, the cells were washed twice by 200 μl of PBS. The fluorescent intensity of the suspension was measured using a multi-well microplate reader (Varioskan LUX, Thermo Fisher, USA) at excitation and emission wavelengths of 485 nm and 525 nm, respectively. Values were expressed as the percentage of fluorescent intensity relative to the control wells. The intracellular fluorescence of cells was also measured using an upright fluorescent microscope equipped with a CCD camera (Nikon, Japan).
5.9. Lipid peroxidation (MDA) assay
The cells were treated as described earlier followed by centrifugation at 8000×g for 4 min to obtain the supernatant cell lysate, which was used for measuring lipid peroxidation (MDA) according to the manufacturer’s protocol. In brief, the samples and standards were incubated with a TBA solution at 100 ºC for 30 min. The mixture was cooled in an ice bath, followed by centrifugation at room temperature for 10 min. Next, 200 µl of the supernatant was placed into a 96-well tissue culture plate, and the absorbance of each sample was measured at 450 nm, 532 nm, and 600 nm. The calculation was performed using following formula: ΔA450 = A450measurement-A450blank, ΔA532 = A532measurement-A532blank, ΔA600=A600 measurement-A600blank.
5.10. GSH /GSSH measurement
The cells were exposed to CoNPs (400 μM) and ALA (200 μM) for 24 h. The blank group, positive control group (CoNPs, 400 μM), and a negative control group (ALA, 200 μM) were also established at the same time. After the exposure, the cells were washed with PBS twice, scraped off, suspended in PBS, and centrifuged at 1000×g. The cell pellet was then homogenized in 5 % 5-sulfosalicylic acid. Next. the suspension was lysed by freezing and thawing twice after 5 min centrifuged at 10,000×g for 10 min. Intracellular total GSH and GSSG concentrations were measured according to the supplier's protocol.
5.11. Western blot analysis to check the level of GPx4
The cells were treated as described above, collected, and washed twice with cold PBS, followed by lysing with RIPA buffer containing 1 mM phenyl sulfonyl fluoride on ice for around 30 min. The lysed cells were centrifuged at 12,000×g for 15 min to obtain the supernatant cell lysate, and protein concentrations were measured using the bicinchoninic acid (BCA) assay kit. The concentrated protein samples were subjected to 15% SDS polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane by a transfer apparatus at 300 mA for 1 h. The membranes were then blocked and incubated with primary rabbit antibody specific for GPx4 at 4˚C for overnight. After carefully washing with TBST, the membranes were incubated with HRP-conjugated secondary antibody for 2 h at room temperature. After that, the PVDF membranes were washed with TBST for four times, and the blot was developed using ECL reagent according to the manufacturer’s protocol.
5.12 Observation of cell structure changes by transmission electron microscope
The cells were treated as described above, harvested and washed twice with PBS, followed by centrifugation at 10,000×g for 5 minutes. Next, the excess PBS was aspirated, and glutaraldehyde was added for fixation. Finally, the cells were treated with osmic acid, ethanol, acetone, and other steps. The changes of cells were observed with a transmission electron microscope.
5.10. Statistical analysis
All the experiments were performed in triplicates, and the data were expressed as mean ± standard deviation (SD). All statistical analyses were performed by GraphPad Prism 8 software (La Jolla, California, USA) employing a one-way analysis of variance, followed by Dunnett’s test to evaluate the significance relative to the control experiment. Differences between groups were considered significant if the p-value is less than 0.05.