Preparation of Au nanoparticles supported on metal oxides (Au/MOx)
Materials
TiO2 (AEROXIDE P25) was supplied from Nippon Aerosil Co., Ltd. ZrO2 (RC-100) and CeO2 (high purity fine cerium oxide) were purchased from Daiichi Kigenso Kagaku Kogyo Co., Ltd. Tetrachloroauric acid (HAuCl4·4H2O) and dimethyl gold(III)(acetylacetonate), Me2Au(acac), were purchased from Tanaka Kikinzoku Kogyo K.K. and Tri Chemical Laboratories Inc., respectively. Au/ZrO2 and Au/CeO2 prepared by deposition–precipitation were purchased from Haruta Gold Inc. All reagents and materials were used as received.
Characterization of Au/MOx
Average particle size of metal oxides after the deposition of Au NPs were measured by laser diffraction/scattering particle size distribution analyzer using a HORIBA, Partica LA-950V2. Specific surface area of metal oxides was estimated from the N2 adsorption measurement was carried out using a SHIMADZU, Tristar 3000. The samples were pretreated at 200 °C for 2 h under vacuum, and then the N2 adsorption isotherms were obtained at − 196 °C. The Brunauer–Emmet–Teller (BET) method was used to calculate the specific surface area. Loading amount of Au on metal oxides were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Thermo Fisher Scientific, iCAP6500) or atomic absorption spectrometry (AAS, SHIMADZU, AA-6200). The samples were treated in aqua regia to dissolve Au NPs, filtered, and then diluted to measure. Average size of Au NPs supported on metal oxides were estimated by high-angle annular dark-field scanning transmission electron microscopic (HAADF-STEM) observation using a JEOL, JEM-3200FS.
Preparation of Au/MOx
Au NPs with two different mean diameters (3 and 8 nm) supported on pristine TiO2 NPs were prepared, termed Au3/TiO2 and Au8/TiO2, respectively. Au3/TiO2 was prepared by deposition–precipitation3. An aqueous solution of HAuCl4 (1 mM, 50 mL) was warmed to 70 °C, and the pH was adjusted to 6 by adding NaOH aqueous solution. TiO2 (1.0 g) was added to the solution and the suspension was stirred at 70 °C for 1 h. The suspension was centrifuged, washed with water at 40 °C for five times, and then filtered. The resultant powder was dried in air at 100 °C overnight and calcined at 300 °C for 4 h to reduce Au(III) to Au(0). Au/ZrO2 and Au/CeO2 were also prepared by deposition–precipitation as well as Au3/TiO2. Au8/TiO2 was prepared by solid grinding5. TiO2 (1.0 g) and Me2Au(acac) (15 mg) were ground in an agate mortar in air at room temperature for 20 min. The mixture was calcined at 300 °C for 2 h to give Au8/TiO2.
Physicochemical properties of the obtained Au/MOx are summarized in Table 1. The primary particle sizes of Au/TiO2, Au/ZrO2, and Au/CeO2 were 26 nm, 11 nm, and 7.3 nm respectively, estimated from their specific surface areas of the MOx NPs. The secondary particle sizes of Au/TiO2, Au/ZrO2, and Au/CeO2 were 9.4 µm, 3.6 µm, and 2.4 µm, respectively, determined by light scattering. The actual Au loadings for Au3/TiO2, Au8/TiO2, Au/ZrO2, and Au/CeO2 were 0.96, 1.18, 0.97, and 0.96 wt%, respectively. The mean diameters of Au NPs were estimated by HAADF-STEM observation to be 3.3 ± 1.4 nm for Au3/TiO2, 7.8 ± 2.7 nm for Au8/TiO2, and 3.3 ± 1.8 nm for Au/ZrO2 (Fig. 1). In the case of Au/CeO2, it was difficult to observe Au NPs because Au was mostly deposited as single atoms and the atomic number of Ce is close to that of Au.
The different NPs were evaluated for LPS contamination (Limulus Amebocyte Lysate test), and the observed absorbance at 405 nm was below detection limits demonstrating no LPS contamination.
Cells isolation and incubation with nanoparticles
Peritoneal macrophages and macrophages from the RAW 264.7 cell line were used in the different experiments.
Mice peritoneal macrophages were prepared as follows. C57BL/6 mice were injected intraperitoneally with 1 ml of 3% Brewer thioglycollate (Sigma-Aldrich, B2551). After 96 h, peritoneal cells were harvested by lavage with 0.67% phosphate buffered saline (PBS). After a soft centrifugation, cells were maintained in Dulbecco’s modified Eagle medium (DMEM) 4.5 g l−1 glucose supplemented with 10% fetal calf serum (FCS)and 1% penicillin and 1% streptomycin. Then mice peritoneal macrophagic cells were exposed for 6–48 h to 1–100 µg ml−1 particles of Au/TiO2, Au/ZrO2, Au/CeO2, and MOx alone.
The RAW 264.7 cells were cultured using a medium having the same composition as above.
Nanoparticle interference with the signal was tested in all of the colorimetric assays. No interference was observed.
Cellular viability
The viability of cells was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) assay, and the quantification of the release of lactate dehydrogenase (LDH).
Inflammatory responses
Inflammatory response was evaluated by the quantification of cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β in cell supernatant, measured by enzyme-linked immunosorbent assay (ELISA).
Measurement of oxidative stress by DCFH‐DA assay
Endogenous ROS were quantified by oxidation of 2′,7′-dichlorofluorescin diacetate (DCFH‐DA) into 2′,7′-dichlorofluorescin (Sigma, Saint Quentin Fallavier, France). Briefly, cells were cultivated in six‐well culture plates and treated with 50 µg ml−1 NPs. Cells were also treated with 250 µM H2O2 as a positive control (data not shown). Cells were incubated with 20 µM DCFH‐DA for 30 min at 37 °C and fluorescence recorded for 90 min. Results were expressed as the mean ratio of fluorescence recorded every 15 min during the 90 min period.
Measurement of TNF-α and IL-1β adsorption to NPs
Adsorption of TNF-α and IL-1β adsorption to NPs was performed by ELISA as described previously described29.
Statistical analysis
JASP software (version 0.11.1, https://jasp-stats.org/) was used to analyze quantitative data. Non-parametric ANOVA was used to compare multiple groups. Paired comparisons with Mann–Whitney test were performed if the differences using ANOVA were statistically significantly different. Data were presented as mean values ± standard error of the mean (SEM), and results were considered statistically significant if p < 0.05.
Study approval for animal experiments
The Institutional Animal Care and Use Committee approved experimental procedures on mice (APAFIS authorization #14914-2018042515599016).
All methods were carried out in accordance with relevant guidelines and regulations and are reported in accordance with ARRIVE guidelines for the reporting of animal experiments.
