Additional file 1 of Agglomeration of titanium dioxide nanoparticles increases toxicological responses in vitro and in vivo

Additional file 1 : Table S1. Main parameters necessary to calculate the delivered dose in vitro for different the TiO2 suspensions. Figure S1. pH vs Zeta potential curves. 17 nm TiO2 (A) and 117 nm TiO2 (B). Figure S2. Scheme of the protocol for the preparation of SA and LA from TiO2 suspensions. To obtain small (SA) and large agglomerates (LA), 17 and 117 nm TiO2 were dispersed at different pH conditions, sonicated and stabilized with BSA 0.25%. The suspensions dispersed at pH 2 were readjusted to pH 7–7.5 using 0.1 M NaOH. Figure S3. Influence of TiO2 agglomeration on cytotoxicity in vitro. WST-1 and LDH assay were used to measure the cell metabolic activity in HBE (A), Caco2 (C) and THP-1 (E) and cell viability in HBE (B) Caco2 (D) and THP1 (F) after 24 h exposure to small (SA) and large agglomerates (LA) of 17 nm and 117 nm TiO2. Data are expressed as means ± SD from three independent experiments performed in triplicates. p < 0.001 (***) represents significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test). Figure S4. Influence of TiO2 agglomeration on total glutathione (GSH) in vitro. GSH depletion was measured as an indicator of oxidative stress in HBE (A,B) and Caco2 (C) cells after 24 h exposure to small (SA) and large agglomerates (LA) of 17 nm (A,C) or 117 nm TiO2 (B). Data are expressed as means ± SD from three independent experiments performed in duplicates. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represent significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test). Figure S5. Influence of TiO2 agglomeration on barrier integrity in epithelial monolayers in vitro. Trans-epithelial electrical resistance (TEER) was measured in HBE (A, B) and Caco2 (C) after 24 h exposure to small (SA) and large agglomerates (LA) of 17 nm (A) or 117 nm TiO2 (B, C). Data are expressed as means ± SD from three independent experiments performed in duplicates. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represent significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test). Figure S6. Influence of TiO2 agglomeration on cytokine release in vitro. TNF-α (A), IL-6 (B) and IL-β (C) levels were measured in the supernatant of the HBE (A,B) and THP-1 (C) after 24 h exposure to small (SA) and large agglomerates (LA) of 17 nm (A, C) or 117 nm TiO2 (B). Data are expressed as means ± SD from three independent experiments performed in duplicates. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represent significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test). Figure S7. Influence of TiO2 agglomeration on DNA damage in vitro. DNA damage was measured in HBE (A,C), Caco2 (B,D) and THP-1 (E) after 24 h exposure to small (SA) and large agglomerates (LA) of 17 nm (A,B) or 117 nm TiO2 (C, D,E). Data are expressed as means ± SD from three independent experiments performed in duplicates. P < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represent significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test). Figure S8. Influence of TiO2 agglomeration on in vivo toxicity in mice exposed via oropharyngeal aspiration. BAL lymphocytes (A), Ti persistence in lung tissues (B) and BALF LDH activity (C) measured after 3 d in mice aspirated with different doses of small (SA) and large agglomerates (LA). Data are expressed as means ± SD from 4 to 5 mice in each group. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represent significant difference compared to control (One-way ANOVA followed by Dunnett’s multiple comparison test). Figure S9. Intracellular uptake of TiO2 agglomerates by HBE cell cultures and cellular distribution. TEM images of control cells (A) and exposed to 50 μg/mL of TiO2 NPs for 24 h: 17 nm-SA (B), 17 nm-LA (C), 117 nm-SA (D) and 117 nm-LA (E). N -Nucleus; C-Cytoplasm. Some TiO2 agglomerates close to the nucleus induced arch like structures (indicated in red arrow).