Cuo nanoparticles

Butyl(2-cyanoacrylate) (BCA) monomer (MW: 153 g/mol) (BLD Pharmatech Ltd., Shanghai, China), CuO nanoparticles (Sigma-Aldrich, Milan, Italy), with particle dimensions < 50 nm, and CPP CE90CPP enzymatically hydrolyzed casein (DMV International, Delhi, NY, USA) with a purity of 26% (w/w) were used.

The suspension of CuO nanoparticles (Sigma-Aldrich, particle size <50 nm, mean 30 nm) was prepared according to Manusadžianas et al. (2012) (link). Before fractionation, the cells were exposed to 100 mg/L nCuO suspensions for 30 min (three independent replicates). Preparation of algae, exposure conditions and Cu concentration measurements in cell fractions are described in detail in Manusadžianas et al. (2017) (link).

Cells from a human type II alveolar epithelial cell line (A549, obtained from the American Type Culture Collection, ATCC, Manassas, USA) were cultured in DMEM (Dulbeccos´s Minimal Essential Medium, Cat. No. 41965–039, Gibco^® Invitrogen) cell culture medium supplemented with 10% Fetal Bovine Serum (European grade, Biological Industries), 1 mM Sodium Puryvate (Gibco^® Life Technologies), 100 units mL^-1 Penicillin and 100 μg mL^-1 Streptomycin (Pen Strep, Gibco^® Life Technologies). The supplemented medium is denoted as DMEM⁺. The cells were cultured in cell culture flasks in a humidified (RH > 99%) CO₂-atmosphere (5%) at 37°C. The cells were seeded 24 h prior to each assay at concentrations of 0.08·10⁶, 0.04·10⁶ and 0.02·10⁶ cells cm^-2 for 4, 24 and 48 h exposure times, respectively, in order for the (control) cells to reach confluence in the end of each exposure. CuO nanoparticles (20–40 nm diameter, Sigma-Aldrich), dispersed in DMEM⁺ at concentrations of 20 or 40 μg cm^-2, were used as positive controls in all cellular assays.