The suspension was washed and centrifuged two times using cold PBS to remove all traces of ethanol. Cells were suspended in 100 μl PBS, and 10 μl RNase A solution was added. The tubes were incubated at 37°C for 30 min. An equal volume (110 μl) of propidium iodide (PI) was added to each tube and incubated at 4°C for at least 30 min. The tubes were diluted using 280 μl PBS and measured by flow cytometry (FC500Mel, Beckman Coulter Ltd., Brea, CA, USA). Statistical analysis The data were expressed as mean ± SD of three independent experiments. SPSS 16.0 software

was used for the statistical analysis. Results The evaluation of nanomaterials buy Trichostatin A is based on their size, shape, and distribution. Size distribution was assessed using a Malvern instrument. Figure 1 shows representative transmission electron microscopy images of ZnO NPs. The results show the average particle diameter of ZnO NPs: 26.21 ± 11.14 nm (A), 62.42 ± 9.18 nm (B), and 90.81 ± 8.89 nm

(C). Figure 1D shows the ranges from 15 to 30 nm for a nanosphere, Figure 1E from 30 to 70 nm for a nanorod, and Figure 1 F from 60 to 100 nm for a nanorod. Figure 1 Microscopy characterizations of ZnO NPs. TEM images of an average (A) 26-nm ZnO NP, (B) 62-nm ZnO NP, check details and (C) 90-nm NP. Ranges (D) from 15 to 30 nm for a nanosphere, (E) from 30 to 70 nm for a nanorod, and (F) from 60 to 100 nm for a nanorod. TEM scale bars: (A) 50 nm, (B) 100 nm, and (C) 200 nm. To assess the cell activity, the intracellular dose of formazan was quantified. Three different sizes of NPs were tested over a 12-, 24-, and 36-h exposure. As shown in Figure 2, the MTT results demonstrated that higher concentrations and longer incubation times generated more serious cytotoxicity. It was observed that the cell activity is statistically significantly Amrubicin different between the concentrations of 12.5 and 50 μg/ml for 24 h. For the data regarding the exposure to 26-nm ZnO NPs for 12 h,

the percentage (%) MTT reduction (relative to control) of Caco-2 cells observed at concentrations of 25 and 50 μg/ml was 41.02% and 91.3%, respectively. The percentage of reduction was 25.3% and 58.1% after exposure to 62-nm ZnO NPs, and reduction was 42.11% and 90.7% after exposure to 90-nm ZnO NPs (Figure 2A). The 24-h value was chosen to confirm the viability and accessibility of the cells and taken as the appropriate time for the following test system [18–20]. The relevant IC50 values on Caco-2 cells were 15.55 ± 1.19 μg/ml, 22.84 ± 1.36 μg/ml, and 18.57 ± 1.27 μg/ml. Figure 2 Cytotoxicity of ZnO NPs on Caco-2 cells. MTT assay. Cell viability of Caco-2 cells treated with different concentrations of different-sized ZnO NPs at different times. Exposure to ZnO NPs for (A) 12 h, (B) 24 h, and (C) 36 h. The data are presented as the mean ± SD of three independent MI-503 experiments (n = 5).