2.3. In Vitro Mammalian Cell Culture Biological Test

Cell culture and reagents. KB cell line (ATCC^® CCL17TM) was purchased by the American Type Culture Collection. Cells were grown in minimal essential Eagle’s medium (EMEM, Sigma) supplemented with 10% heath inactivated fetal bovine serum (FBS, Sigma), 100 units/mL penicillin and 100 µg/mL streptomycin (Sigma) and maintained at 37 °C under a 5% CO₂ atmosphere. Cells were periodically tested for mycoplasma infection.

All the NCs solutions were freshly prepared from 1 mg/mL ethanol stock solutions. The ZnO NCs were bath sonicated at 40 kHz, 100% power for 10 min before being added to the culture medium and then immediately used for treating cells.

Cytotoxicity tests. 1.5 × 10³ cells/well were seeded onto 96-well plastic culture plates (Corning^® 96 Well TC-Treated Microplates) and incubated at 37 °C, 5% CO₂. After 24 h the cell medium was replaced with fresh medium containing ZnO-st-NH₂ or ZnO-mw-NH₂ at different concentrations (10, 15, 20, 25 µg/mL). After 24 h of incubation, cell proliferation was assessed by measuring cell metabolic activity through the WST-1 cell proliferation assay. 10 µL of the WST-1 reagent (Roche) were added to each well and after 2 h incubation in the dark at 37 °C, 5% CO₂, the formazan absorbance was measured at 490 nm by the Multiskan GO microplate spectrophotometer (Thermofisher Scientific) using a 620 nm reference. Control values (without treatments) were set at 100% viable and all values were expressed as a percentage of the control. The inhibitory concentration 50% (IC50) of the ZnO-mw-NH₂ was determined online using the IC50 calculator tool of the AAT Bioquest webpage [27].

Cell internalization. NCs internalization in KB cells was measured using a Guava Easycyte 6-2L flow cytometer (Merck Millipore). 1 × 10⁵ cells/well were seeded onto a 6-well plate (Corning^® Costar^® TC-Treated) with complete cell culture medium 24 h prior the assay. Then, cells were treated with 10 µg/mL of ZnO-st-NH₂ or ZnO-mw-NH₂ labeled with ATTO633-NHS ester dye (Thermofisher). A control well, containing the untreated cells, was instead filled with fresh medium without NCs. After 24 h incubation, cells were rinsed twice with phosphate buffered saline (PBS), trypsinized and centrifuged at 130 g for 5 min. Cell pellets were re-suspended in 1 mL PBS and immediately analysed with the flow cytometer; 10,000 gated events were considered for the analysis excluding cellular debris, characterized by low FSC (forward scatter) and SSC (Side scatter). Results were reported as the percentage of fluorescence positive events, characterized by a shift in fluorescence intensity compared to untreated cells. This evaluation was performed by Guava InCyte Software (Merck Millipore, Darmstadt, Germany). Independent experiments were performed 3 times. The different NCs internalization was compared using a t test and when equal variance test failed, Welch’s test was assumed instead of Student’s t test.

Statistical analysis. All experiments were done at least in triplicate and the results were presented as mean ± SEM (standard error of mean). The experimental data were analysed using Sigmaplot software version 14, demo version (Systat Software Inc., San Jose, California, CA, USA). t test and One Way Analysis of Variance were executed, where equal variances were not assumed, the results of Welch’s test were presented. When the normality test failed, Mann–Whitney rank sum test was run. Differences were considered significant at p value < 0.05.