Cell culture and treatment
Human hepatoma cell line HepaRG (Thermo Fisher Scientific) was used in this study. Cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS, Australia Origin, Gibco) and 1% Penicillin-Streptomycin-Glutamine solution (Gibco) in a humidified atmosphere of 5% CO2 at 37 °C. The cells were treated with increasing concentrations of [email protected] NR for 24 h or 72 h respectively, and the concentrations were determined in accordance with IC50 estimated by cell viability assay. To investigate the role of ROS in the genotoxicity, 1 mM N-Acetyl-L-cysteine (NAC, Sigma-Aldrich) was applied for 1 h prior to the treatment with [email protected] NR.
ATP cell growth/viability assay
The cells were seeded in a 96-well plate at a density of 5 × 103/well. After 24 h of incubation, the medium was aspirated and the cells were exposed to different concentrations of [email protected] NR for 24 h or 48 h, respectively. A broad spectrum of concentrations were prepared and four wells per treatment were performed in one treatment period. The cytotoxicity of [email protected] NR was examined by adenosine triphosphate (ATP) assay (CellTiter-Glo® 2.0 Assay, Promega), which measures the cellular metabolic activity by quantitating the amount of ATP, an important metabolism parameter in viable cells. The luminescent signals which reflect the amounts of viable cells, were detected using VICTOR Multilabel Plate Reader (2030-0050, PerkinElmer), and IC50 values were estimated as the concentration of [email protected] NR for half maximal viability by Prism 7 (GraphPad Prism 7, CA, USA). The viability ratio was calculated using the following equation:
Viability Ratio (%) = RLUsample/RLUvehicle ×100%
Where RLU is the relative light unit represented as the mean value of four wells, RLUvehicle represented cells not treated with nanorods, and RLUsample represented cells that were treated with different concentrations of [email protected] NR.
Concentration determination of silver and gold in cells
The cell samples were digested in nitric acid using the microwave digestion system. Following the digestion, the samples were prepared with a mixture containing 1% nitric acid and hydrochloric acid. The quantities of Ag and Au in the solutions were determined by ICP-MS (NexION300X, Perkin Elmer).TEM analysis was used to determine the presence of Au NR and [email protected] NR in the cell. The cell samples were fixed in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde for 2 h at 4 °C. The cell pellets were fixed and rinsed three times in phosphate buffer (pH 7.4), and post-fixed in 1% osmium tetroxide for 2 h at 4 °C. The samples were subsequently rinsed in distilled water three times, and dehydrated for 15 min in different concentrations of ethanol (50%, 70%, 90% and 100% ethanol, respectively) one after the other. Subsequently, propylene oxide at 1:1 and 1:3 dilutions was applied to the resin at 20 °C ~ 26 °C for 2 h. Polymerization was performed by graded heating at 35 °C for 16 h, 45 °C for 8 h, 55 °C for 14 h, and 65 °C for 48 h. Ultrathin sections were stained for 25 min with uranyl acetate and lead citrate, and analyzed by a transmission electron microscope (H-7650, HITACHI, Japan).
Conventional and modified comet assay
The cells were seeded in 12-well plates at densities of 2 × 105/well or 3 × 105/well for a 24 or 72 h treatment, respectively. Hydrogen peroxide (H2O2) at a concentration of 200 µmol was exposed to the cells as positive control for an hour. For each sample, two wells were prepared for both the conventional treatment and the formamidopyrimidine glycosylase (Fpg) treatment. Conventional comet assay was performed in alkaline conditions (pH > 13) as described previously(Wang et al., 2018)(Wang et al., 2018). For the Fpg treated wells, an additional Fpg treatment was applied before the DNA unwinding procedure, and the slides were immersed in an enzyme buffer (0.1 M KCl, 0.5 mM EDTA, 40 mM HEPES, 0.2 mg.mL− 1 BSA) three times for 5 min each. The Fpg (New England Biolabs, Inc. UK) was diluted at 1:50000 with enzyme buffer. 100 mL aliquots of the diluted enzyme were added to each gel on the microscope slides and incubated in a humidity chamber at 37 °C for 30 minutes. The remaining steps were the same as the conventional treatment. The comet assays were performed in triplicate. At least 50 cells per sample were independently scored using the Nikon Eclipse 80i fluorescent microscope (Nikon, Tokyo, Japan), while Komet 6.0 (Andor Technology, Belfast, UK) was used to analyze the medium value of percentage DNA in tail and Olive tail moment (OTM) of each sample.
Qualification of γ-H2AX foci by flow cytometry and high content screening
For the quantification using flow cytometry, cells were seeded in 12-well plates at densities of 2 × 105/well or 3 × 105/well for a 24 or 72 h treatment, respectively; while for the high content screening assay, cells were seeded in 96-well plates at densities of 6 × 103/well or 1 × 104/well for a 24 or 72 h treatment, respectively. As a positive control, 2 µM methyl methanesulfonate (MMS, Sigma-Aldrich) was applied in parallel to the cells for an hour. The cells were rinsed in tris buffered saline (TBS) and fixed with 4% paraformaldehyde for 15 min at room temperature. After washing with TBS, the cells were incubated with 50 µL ice-cold methanol for 30 min at -20 °C. The cells were further rinsed in TBS three times, and the blocking reagent (TBS containing 0.3% Triton X-100 and 10% goat serum) was applied for 1 h. The primary antibody (mouse anti-phospho-H2AX Ser139, Millipore) was diluted to 1:200 with blocking reagent, and incubated with the cells overnight at 4 °C. The plate was then again rinsed with TBS three times, and the secondary antibody (Alexa Fluor 488 goat anti-mouse, Life Technologies), diluted with the blocking reagent in 1:20 ratio, was added subsequently. The samples was kept in the dark at room temperature for 1 h, and 2 µg.mL− 1 (20 µL/well) DAPI (Invitrogen) was added to each well. The fluorescence was measured using a flow cytometry (FACSCalibur, BD Bioscience, NJ, USA) or High Content Analysis System (Operetta CLS, PerkinElmer). For the flow cytometry assay, data from at least 10,000 cells per group were analyzed, and the experiments were performed in triplicate; for high content analysis, 20 visual fields in each well and at least five wells in each group were analyzed.
Cytokinesis-block micronucleus cytome (CBMN-cyt) assay
CBMN-cyt was performed according to the procedure described by Fenech et al . Cells were seeded in 12-well plates at densities of 2 × 105/well or 3 × 105/well for a 24 or 72 h treatment, respectively. 0.2 µg.mL− 1 Mitomycin C (MMC, Tokyo Chemical Industry Co., Ltd. Japan) was exposed to the cells as positive control for 24 h. 3 µg.mL− 1 Cytochalasin B was applied after a 24 or 72 h treatment to block the cytokinesis process and the cells were harvested after 40 hours. The samples were stained with 5% Giemsa after hypotonicity with pre-warmed 0.075 mol.L− 1 KCl and fixation with a 3:1 mixture of methanol and acetic acid. Triplicate wells per group were prepared and at least 1,000 binucleate cells per well were examined.
Measurement of MDA, total GSH and SOD contents
The cells were cultured in 12-well plates at densities of 5 × 105/well or 3 × 105/well for a 24 or 72 h treatment, respectively. Subsequently, the cells were harvested and rinsed three times with phosphate buffer saline (PBS). The amounts of malondialdehyde (MDA) in the cell homogenates were determined using a thiobarbituric acid-based method (Nanjing Jiancheng Bio-engineering Institute, Nanjing, China). The amounts of total glutathione (GSH) and superoxide dismutase (SOD) were determined using the total glutathione quantification and SOD assay kits (Dojindo Molecular Technologies, Inc. Kumamoto, Japan), respectively. Optical densities (O.D) of each well was measured using VICTOR Multilabel Plate Reader (2030-0050, Perkin Elmer).
Flow cytometric analysis for cell cycle
The cells were cultured in 6-well plates at densities of 1 × 106/well or 5 × 105/well for a 24 or 72 h treatment, respectively, and were subsequently fixed with 70% ethanol at 4 °C overnight. The samples were rinsed with PBS three times, and stained with PI/Rnase staining buffer (BD Biosciences) for 15 min at room temperature. Cell populations under G0/G1, S and G2/M phase among 20,000 cells were determined by employing regions with FL2 area vs FL2 width. Analysis was done by flow cytometry (FACSCalibur, BD Bioscience, NJ, USA) and FlowJo (BD Bioscience), and the experiments were performed in triplicate.
Flow cytometric analysis of cell apoptosis
The cells were cultured in 6-well plates at densities of 1 × 106/well or 5 × 105/well for a 24 or 72 h treatment, respectively. They were subsequently rinsed twice with PBS, and diluted with 500 µL 1 × binding buffer (FITC Annexin V Apoptosis Detection Kit I, BD Bioscience) to adjust the suspension to around 1 × 106 cells/mL, and subsequently 100 µL dilution was mixed with 5 µl FITC Annexin V and 5 µl PI. The samples were stained at room temperature for 15 min, and at least 10,000 cells were analyzed to determine the cell population under early and late apoptosis by employing regions with FL1H vs FL2H using flow cytometry (FACSCalibur, BD Bioscience, NJ, USA) and FlowJo (BD Bioscience). The experiments were performed in triplicate.
Western blot analysis
The cells were cultured in a 75 cm2 flask at densities of 1 × 107/well and 6 × 106/well for a 24 and 72 h treatment, respectively. The cells were lysed with RIPA lysis buffer containing protease inhibitor (PMSF), and the concentration of proteins were determined using a BCA protein quantification kit (Beyotime Biotechnology, China). The concentrations of the samples were adjusted using RIPA lysis buffer prior to denaturation by heating at 95 ºC for 3 min. The protein samples were separated by electrophoresis on 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Millipore). The membranes were blocked with 5% skim milk for 30 min and incubated with primary p53 (SC-137174,Santa Cruz), p21 (SC-6246, Santa Cruz) and β-actin (sc-47778, Santa Cruz) and secondary antibodies goat anti-mouse IgG(H + L)-HRP(SE131, solabio)respectively. The expression levels of the target proteins in the samples were visualized using an enhanced chemiluminescence (ECL) method and analyzed by ImageJ system (National Institutes of Health).
The data was presented as the Mean ± SEM. One-way analysis of variance (ANOVA) were used to test statistical significance of differences among negative control and treated groups, followed by the Dunnett multiple comparison test using SPSS (version 22, IBM, Armonk, NY, USA), and data were considered statistically significant at P < 0.05.The figures were prepared using GraphPad Prism 7 for Windows (GraphPad Software, La Jolla, CA, USA).