Varian 700 es series spectrometer

NiO NPs -treated SCs were detached by trypsin/ethylenediaminetetraacetic acid (EDTA) (Lonza, Verviers, Belgium) at 37°C for 8 min, to promote the enzymatic reaction. After washing with 1 ml Hank’s balanced salt solution (HBSS) (Sigma-Aldrich Co., St. Louis, MO, USA), samples were centrifuged at 150×g for 6 min, the supernatant was removed, the pellets were freeze-dried, and accurately weighed. Samples were dissolved by treatment with 10 ml of a mixture of sulfuric acid (H₂SO₄), (97% Sigma-Aldrich Co., St. Louis, MO, USA)/nitric acid (HNO₃ 70%), (Sigma-Aldrich Co.,St. Louis, MO, USA) (2:1). After solubilization, the obtained solutions were diluted with the EDTA solution (1:10) prior Ni2+ content determination using a Varian 700-Es series spectrometer (Agilent, Milan, Italy) in triplicate. Calibration was performed diluting a Nickel nitric acid stock solution for ICP (Sigma Aldrich, Milan, Italy) to obtain Nickel standard solutions in the 1–15-mg/ml range. The Ni2+ uptake in SCs was calculated per unit weight of freeze-dried NiO NP-treated SCs and % of the total amount added and the error expressed as SEM.

TiO₂ NP-treated SCs were detached by trypsin/ethylenediaminetetraacetic acid (EDTA) (Lonza, Verviers, Belgium) at 37°C for 8 min, to promote the enzymatic reaction. After washing with 1 ml Hank’s balanced salt solution (HBSS) (Sigma-Aldrich Co., St. Louis, MO, USA), samples were centrifuged at 150×g for 6 min, the supernatant was removed, the pellets were freeze-dried, and accurately weighed. Samples were dissolved by treatment with 10 ml of a mixture of sulfuric acid (H₂SO₄), (97% Sigma-Aldrich Co., St. Louis, MO, USA)/nitric acid (HNO₃ 70%), (Sigma-Aldrich Co., St. Louis, MO, USA) (2:1). After solubilization, the obtained solutions were diluted with the EDTA solution (1:10) prior Ti⁴⁺ content determination using a Varian 700-Es series spectrometer (Agilent, Milan, Italy) in triplicate. Calibration was performed diluting a Ti⁴⁺ standard solution obtaining titanium solutions in the 1–15-μg/ml range. The Ti⁴⁺ content was expressed per unit weight of freeze-dried TiO₂ NP-treated SCs and % of the total amount added and the error expressed as SEM.

ICP-OES was performed to measure AuNPs uptake in cells, using a Varian 700-Es series spectrometer (Agilent, Milan, Italy). For this purpose, supernatants and pellets of BEAS-2B and A549 cells treated with AuNPs at 0.8 and 1.6 µg/cm² were collected after 3, 24, and 48 h of incubation. Cells were extensively washed with PBS to remove possible, not internalized AuNPs, and counted by a hemocytometer for cell number normalization purposes. The pellets were then extracted, adding 0.5 mL of a concentrated 1:3 HNO3-HCl solution at room temperature and bath sonicated for 10 min. After proper digestion time, the obtained solutions were diluted 20 times with water. The obtained extracted samples and supernatants were submitted directly to ICP-OES analysis. The equipment was calibrated with 1M HCl standard Au solutions, in the range 0.5–15 µg/mL.