# Size Effect of Silver Nanoparticles Derived from Olive Mill Wastewater in THP-1 Cell Lines
## 3 Results and Discussion
DLS acquisitions were in line with the data acquired by TEM regarding the size of NPs; the hydrodynamic radius were 31 \(\pm\) 6 nm and 50 \(\pm\) 8 nm for the smaller and bigger AgNPs, respectively. The NPs surface charge of \(-\)40 \(\pm\) 3 mV and \(-\)35 \(\pm\) 4 mV were also measured (Table 2). Following the characterization of the NPs, we moved to understanding their potential effects on the THP1 differentiated in the primary macrophage (M0) phenotype after PMA incubation. The M0 macrophages can be polarized in M1 or M2 phenotypes: they control the inflammation phenomena in different ways; the M1 are pro-inflammatory, the M2 are anti-inflammatory cells [20; 21; 22]. The THP-1 were incubated with the two sizes of AgNPs at two different concentrations, namely 3 \(\upmu\)M and 5 \(\upmu\)M, for 24 h and 48 h, and the internalization amount of Ag was analyzed by elemental analysis. As shown in Figure 4a, the incubation with the two sizes of AgNPs did not induce evident differences in Ag uptake at either 24 h or 48 h. Subsequently, we concluded that the uptake in cells was independent from the size and time of incubation. The THP-1 viability assay (Figure 4b) showed viability reduction, although it was never below 25%. The effect was more evident when the NPs with smaller size were used compared to the 50 nm AgNPs. In particular, at the higher concentration tested, the 30 nm AgNPs induced a reduction of cell viability (25%), whereas the 50 nm AgNPs were responsible for about 20% of cell mortality after 48 h. To monitor the immune response onset, we evaluated the activation of the cytokines, proteins involved in inflammation control and activating immune cells [23]. IL-6 activates the acute phase response and hematopoiesis and it is synthesized by the immune cells, fibroblasts, and endothelial cells [24]. Additionally, IL-8, a neutrophil chemo-attractant, is produced by neutrophils, endothelial cells, fibroblasts and monocytes [25]. IL-5 is a cytokine secreted by type 2 T helper lymphocytes, mast cells and dendritic cells. Its main function is to stimulate the activation of B lymphocytes, mainly inducing them to produce IgA antibodies. Interleukin-5 also acts as a mediator in the activation process of eosinophilic white blood cells [26]. Finally, TNF-\(\upalpha\) is an inflammatory cytokine secreted by macrophages and monocytes during acute inflammation [27]. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Samples** & **DLS** & **Zeta Potential** \\ \hline AgNPs (30 nm) & 31 \(\pm\) 6 nm & \(-\)22 mV \\ \hline AgNPs (50 nm) & 50 \(\pm\) 8 nm & \(-\)28 mV \\ \hline \end{tabular}
\end{table} 
Table 2: Characterization of AgNPs (30 nm and 50 nm) in water by DLS and \(\upzeta\)-potential (mV) measurements. Figure 3: UV–vis (**a**) and FTIR spectra (**b**) of pristine OMWW. Next, we used an ELISA assay to quantify the amount of IL-6, IL-8, IL-5 and TNF-\(\alpha\) produced by THP-1 following interaction with the NPs after 24 h and 48 h of exposure. Generally, we observed an increase of the IL-6 amount both for the 30 nm AgNPs and the 50 nm AgNPs, but, similar to the viability assay, we found a high amount of IL-6 production after incubation with the 30 nm AgNPs compared to the NPs with the larger size (Figure 4c). Specifically, an amount of c.a. 142 pg/mL versus 134 pg/mL was measured using the concentration of 5 \(\upmu\)M for 30 nm AgNPs and 50 nm AgNPs, respectively. A similar trend was observed when we performed the IL-8, IL-5 and TNF-cassays. Additionally, in these cases, the greater secreted quantity observed was related to the smaller AgNPs (Figure 4d-f). Finally, the translocation of Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-\(\kappa\)B) was assessed by fluorescent analysis.