Tio2 nps

TiO₂-NPs (<25 nm; purity ≥99.7%; anatase) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The particles were prepared in dilution water (294.0 mg/L CaCl₂ · 2H₂O; 123.3 mg/L MgSO₄ · 7H₂O; 63.0 mg/L NaHCO₃; 5.5 mg/L KCl [28 ]) by vortexing the suspension ten times for 10 s followed by sonication for 30 min in a bath-type sonicator (35-kHz frequency, Fisherbrand FB 11010, Shanghai, China) to break down agglomerates and ensure a uniform suspension. Particle characterization of the TiO₂-NPs suspension sample was examined by a transmission electron microscope (TEM; JEM-2010FEF, JEOL, Akishima-shi, Japan) (Figure 1).
Bisphenol A (BPA; C₁₅H₁₆O₂, purity >99.5%) was purchased from the Chemical Agent Co. (Tianjin, China). A BPA stock solution was prepared in an aqueous solution that contained ethanol as a co-solvent. The final concentration of ethanol was less than 1%, and our previous evaluations had demonstrated no adverse biological effect at this concentration. The stock solution was then diluted to specific test concentrations with the dilution solvent.

TiO₂NPs were purchased from Sigma-Aldrich (particle size <25 nm, 637254, St. Louis, MO, USA). We quantified the morphology and size of TiO₂NPs using transmission electron microscopy (JEM-1210, JEOL, Tokyo, Japan) and scanning electron microscopy (Zeiss EVO-MA10; Carl Zeiss Meditec AG, Jena, Germany) at accelerating voltages of 200 kV and 15 kV, respectively. The specific surface area of TiO₂NPs was measured by nitrogen absorption methods based on the multipoint BET method (ASAP2020; Micromeritics, Norcross, GA, USA). The hydrodynamic size and zeta potential of TiO₂NPs were determined by ELS-8000 (Otsuka Electronic, Tokyo, Japan). The purity of TiO₂NPs used in the experiment was determined by energy-dispersive X-ray spectroscopy (Rayny EDX-700, Shimadzu, Kyoto, Japan). The endotoxin levels in thee TiO₂NP suspension were determined using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, USA). After completion of treatment procedures, lung tissue was harvested, weighed, and digested overnight with concentrated nitric acid, and the resultant samples were analyzed for elemental TiO₂NPs using ICP-MS (Perkin Elmer, Waltham, MA, USA).

A well-dispersed suspension of both the anatase and rutile forms of TiO₂-NPs (80/20) purchased from Sigma-Aldrich Chem (product #700347, Switzerland) was used for this experiment. TiO₂-NPs at 10 μg/ml dilution were dispersed in ultrapure water and ultrasonicated for 30 min. A drop of suspension was then placed on Formvar-coated copper grids and fully drained. The grids were allowed to air dry. The visualisation of the size and shape of the TiO₂-NPs was achieved using a JEM 1200 EX II transmission electron microscope (JEOL Ltd., Japan). The size distribution of the TiO₂-NPs included the intensity-weighted average diameter of the all-size population (Z-average). The polydispersity index was analysed by dynamic light scattering measurement using a Nano-ZS (Sysmex Co., Kobe, Japan).