## 5 Discussion  
In the current study, cultivation of ZnO NP under standard conditions was compared to cell cultivation with human plasma. The cell proliferation was checked by cell counting. Cells in the plasma group showed a significant increase in proliferation compared to cultivation with DMEM-EM. \begin{table}
\begin{tabular}{c c c} \hline \hline \multicolumn{2}{c}{**Protein and Zn\({}^{2+}\) Concentration in Plasma and DMEM-EM**} \\ \hline ZnO (\(\mu\)g/mL) & DMEM-EM ng/mL & Plasma ng/mL \\
0 & 0 & 9.9 \\
10 & 121.9 & 122.2 \\
15 & 130.7 & 170.4 \\
20 & 150.5 & 242.4 \\
25 & 171.1 & 396.5 \\ Protein \(\mu\)g/\(\mu\)L & DMEM-EM & Plasma \\  & 10.6 & 54.4 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The amount of soluble zinc ions in DMEM-EM and plasma independent of the different ZnO-NP concentrations. Furthermore, the protein concentration is pictured in the table as well. Figure 7: The comet assay was used in order to evaluate genotoxic events after hMSCs’ exposure to ZnO-NPs. hMSCs cultivated in DMEM-EM showed a significant enhancement of DNA damage at 15 \(\mu\)g/mL (\(p\)-value \(<\) 0.05) compared to the cultivation in plasma. Statistical analysis was performed as described above. MMS served as a positive control. * indicates the statically significant changes. The cell count was not performed as a toxicological evaluation, but to show the significant difference between the two media types with respect to cell proliferation. We demonstrated significant differences in genotoxicity and cytotoxicity. While standard cultivation with DMEM-EM showed dose-dependent DNA damage and toxicity at 15 \(\upmu\)g/mL of ZnO-NPs, no such effects were found in the cultivation of cells with plasma and exposure to 25 \(\upmu\)g/mL of ZnO-NPs. Interestingly, NPs were frequently seen intracellularly in both the DMEM-EM group and in the plasma group. However, in the plasma group a lower quantity of NPs was seen. [PERSON] et al. stated that exocytosis or the release of NPs occurs through vesicle-dependent release, non-vesicle-dependent release and lysosomal secretion [16]. It is obvious that there must be a correlation between toxicity of particles and their intracellular retention. A possible explanation of the differences of intracellular NP quantities could be a higher release of particles from the cells in the plasma group. In the plasma group, the concentration of zinc ions was more than double. The question arises as to whether the NPs were not taken up by the cells or were internalized but quickly released again. Since TEM was only performed at one specific time point, this question could not be answered through our investigation. However, it seems to be more likely that the major difference is due to NPs' uptake. Consequently, it has to be clarified why NPs are taken up less by the cells in the plasma group. Differences seen in DLS could explain our findings. Particle agglomerates were larger in the plasma group, so cellular uptake will consequently be decreased. As a limitation to this study, we have to state, that size distribution has a wide range in both groups. Therefore, a mixture of different sized particle eczema rates cannot be considered a homogeneous sample. However, particles pretreated according to the standard protocol may show such inhomogeneous agglomeration. For adequate toxicological evaluations, a high grade of dispersion has to be intended. There are some studies discussing the mechanisms of endo and exocytosis of NP into cells. According to [PERSON] et al., various endocytosis pathways can be differentiated, including phagocytosis, macroproteinocytosis, clathrin-mediated endocytosis and non-clathrin-mediated endocytosis, the latter with internalization via caveolae [17]. Furthermore, in a previous study the authors hinted the importance of size in particle entry [17; 18]. [PERSON] and colleagues confirmed this hypothesis. They evaluated the size-dependent particle endocytosis and found that gold NPs with the size \(<\)50 nm showed the highest cellular uptake [19].