## 5 Discussion
Furthermore, the shape of the particles play an important role as well. In a study performed by [PERSON] it was shown that ZnO-NPs had a higher toxicity than bulk ZnO in human lung epithelial cells [20]. Furthermore, the rod-shaped particles were much more toxic than the spherical ones, suggesting that both the size and shape of the NPs affect their cytotoxicity [20]. One possible cause could be that rod-shaped particles might have an enhanced contact with the cell membrane due to their configuration and this may lead to an enhanced particle incorporation into the cytoplasm. Besides the size and shape, the surface charge and chemistry of the NPs may influence cellular uptake, too. It was shown that positively charged NPs had a higher bioavailability in vitro and in vivo [21]. The uptake of the positively-charged particles is also enhanced compared to their negative counterparts [22]. [PERSON] could show that cellular uptake kinetics are controlled by surface chemical properties [23]. In the present study, there was no difference between the two groups in terms of physical and chemical properties. Thus, the corona and agglomeration of NPs may mainly influence the cellular uptake compared to the other parameters discussed above. Surely, the protein concentration in the cell culture fluid plays an important role in this context. In the plasma group, the concentration of the protein was understandably higher than the control group. In terms of endocytosis, there are many publications, whereas few studies are found in the literature dealing with exocytosis. However, this topic is very important since with the exocytosis the intracellular NPs concentration is regulated. In a previous study, we showed that NPs persisted in mesenchymal stem cells for at least 3 weeks, without being discharged [24]. Definitely, the assertion of this finding has its limitations, since the in vitro system is not comparable to the mechanism in vivo. Nevertheless, the question arises about the fate of the intracellular NPs. In our opinion, the size of the single particles and the agglomerations might play important roles, since small NPs have the ability to leave the cell much easier compared to the bigger ones. [PERSON] et al. showed that transferrin-coatedgold nanoparticles (Au NPs) with sizes of 14 nm were discharged much better than NPs with diameters of 50 nm and 74 nm [25]. In terms of exocytosis, the coating is a relevant factor, too. To substantiate this statement, [PERSON] and colleagues could show a significant affection of NPs exocytosis by altering their surface coating [26]. Furthermore, in an in vitro system the exocytosis also depends on the amount of extracellular NPs [27]. The authors are aware that the use of plasma alone cannot be representative based on the blood compositions. However, the results of the present study are distinctly different from the standards cultivation in DMEM. Therefore, there is a need for further studies evaluating toxicological aspects of NPs in human cells cultured with human plasma. Of course, human-particle interactions take not only place in the plasma but also in the cells of the aerodigestive tract, and on all bodily surfaces. This might be a relevant cause for the divergent results between in vitro and in vivo systems. To clarify and emphasize, our study does not put toxicological investigations of in vitro system with medium into question. Much more, these systems are tremendously necessary in order to clarify cellular mechanisms after NP contact. Conceptualization, A.S. and S.H. ; methodology, T.M., P.I., M.B. and S.D. ; software, T.M. ; validation, T.E.G., M.B. and S.H. ; formal analysis, T.E.G., A.S.; investigation, A.S., S.D. and S.H. ; resources, S.H. ; data curation, A.S. and S.H. ; writing--A.S. and S.H. ; writing--review and editing, R.H., S.H. ; visualization, A.S., P.I. and S.H. ; supervision, R.H., A.S. and S.H. ; project administration, T.M. This research received no external funding. The authors declare no conflict of interest.