# Size Effect of Silver Nanoparticles Derived from Olive Mill Wastewater in THP-1 Cell Lines
## 3 Results and Discussion
This factor is physiologically localized in the cytoplasm and it translocate to the nucleus when an inflammation phenomenon  
Figure 4: **(a)** Uptake of the AgNPs ( \(\upmu\)M and 5 \(\upmu\)M), 30 nm and 50 nm in THP-1 cell lines for 24 h and 48 h. Control cells were represented by cells without NP treatment (data not shown) Data are the mean \(\pm\) SD performing three independent experiments, and statistically significant for exposed cells vs. control cells with a \(p\) value \(<\) 0.05 (\(<\)0.05 *). **(b)** Viability of THP-1 cell lines after 24 h and 48 h of incubation with 3 \(\upmu\)M and 5 \(\upmu\)M of 30 nm and 50 nm AgNPs. The viability data of cells incubated with NPs was normalized to non-treated cells. The positive control (P) was represented by cells treated with 5% DMSO (data not shown). Data reported as the mean \(\pm\) SD from three independent experiments are considered statistically significant, compared with the control (n = 8) for \(p\) value \(<\) 0.05 (\(<\)0.05 *). **(c)** IL-6 (**d), IL-8 (**e), IL-5 (**f), TNF-\(\alpha\), amount expressed as pg/mL. The experiment consisted of cells treated with AgNPs (30 nm and 50 nm) at two concentrations (3 \(\upmu\)M and 5 \(\upmu\)M) for 24 h and 48 h. The cytokine amount was revealed in supernatants from the untreated cells and cells exposed to NPs by an ELISA assay. The results are reported as the mean \(\pm\) standard deviation of three independent experiments for * \(p<\) 0.05 compared to the control for 24 h and 48 h.  
occurs activating the cytokines production and transcription of DNA [28]. The evaluation of NF-\(\kappa\)B was important to corroborate the previous experiments. The translocation of NF-\(\kappa\)B was assessed by confocal analysis (Figure 5). Since the higher concentration showed stronger effects in the cells, we followed the exposure to the 5 \(\upmu\)M of AgNPs (30 nm and 50 nm) after 48 h in the THP-1 cells. In the control cells, the NF-\(\kappa\)B was localized in the cytoplasm; this evidence suggested that not all the inflammation pathways were activated (Figure 5a). Conversely, the exposure to the AgNPs induced an evident merge between the green florescence of the NF-\(\kappa\)B and DAPI (the nuclei label). This effect was clearer in the cells exposed to the smaller size of NPs (Figure 4b,c) compared with the others (Figure 4c,d). The quantitative analysis (Figure 4e) showed a rise of the co-localization percentage, measured by the Pearson coefficient, especially when the THP-1 cells were exposed to the 30 nm AgNPs. Hence, at the highest concentration tested, the 30 nm AgNPs triggered a 34% of NF-\(\kappa\)B translocation compared to the 50 nm AgNPs (24%). Figure 5: Confocal images of non-treated macrophages (M0) (**a**) and M0 incubated for 48 h with the  
3 \(\upmu\)M and 5 \(\upmu\)M of 30 nm AgNPs (**b**,**c**) and 50 nm AgNPs (**d**,**e**). Cells were fixed with formaldehyde and stained. The nuclei visualization was carried out by the use of Dapi (blue), the actin fibers with CellMask(tm) Deep Red (red) and NF-\(\upkappa\)B with NF-\(\upkappa\)B p65 Antibody (F-6) FITC (green signal). The co-localization analysis was carried out considering the merge of the fluorescence intensity related to blue (nuclei) and green (NF-\(\upkappa\)B) due to the NF-\(\upkappa\)B translocation from the cytoplasm to the nuclei (**f**). The data were expressed as the mean \(\pm\)SD and they were statistically significant for \({}^{*}p<0.5\).