**4. Conclusions**

In this work we report a new ZnO microwave-assisted solvothermal synthesis, optimized for biological uses. Homogeneous and spherical nanocrystals of 20 nm were obtained, with high reaction yields, and fully characterized from the physical-chemical point of view. By comparison with a chemically equivalent wet synthetic method we were able to evaluate their different biological behaviour, in terms of cytotoxicity and cell internalization in KB cancerous human cells.

X-ray diffraction, XPS and optical analysis demonstrated similar physical-chemical properties of the ZnO NCs obtained by the two different synthetic procedures, in terms of surface chemistry and electron band gap. However, we found substantial differences related to the hydrodynamic size, shelf-life stability (tendency to agglomerate in time) leading to a better reproducibility of biological test outcomes. Through a deep statistical analysis, it was in fact possible to estimate that the ZnO NCs, obtained via microwave synthesis, show more reproducible and reliable results.

These findings sugges<sup>t</sup> that not only different preparation methods, but also similar procedures that generate particles with the same surface chemistry could drive biological responses to different ways. In particular, the ability to control and to obtain narrow and reliable NPs size distributions and highly stable behaviour in solution can be considered crucial factors in drive reproducible results, i.e., cytotoxicity and cell internalization tests. In fact, minor changes within the same synthetic route can alter both the shape and the size distribution. In our case, we demonstrate how the proposed microwave procedure is highly effective to better control and optimize the ZnO NCs morphology and size. Furthermore, we prospectively welcome the use of nano-sized ZnO particles with surface modification. We actually demonstrated that amine-functionalized NCs possess improved optical properties, useful for further bio-imaging applications, and allow future biomolecules anchoring, i.e., drugs, proteins or targeting ligands against cancer cells.

**Author Contributions:** Conceptualization, N.G., T.L. and V.C.; Methodology, N.G., T.L., M.C., A.F. and V.C.; Validation, B.D., M.C., L.R., M.C., M.L. and A.C.; Resources, A.F., V.C.; Data Curation, N.G., T.L. and V.C.; Writing-Original Draft Preparation, N.G., T.L., A.C., M.C. and V.C.; Writing-Review & Editing, N.G., T.L., M.L., A.F. and V.C.; Supervision, N.G., T.L. and V.C.; Project Administration, V.C.; Funding Acquisition, V.C. All authors have given approval to the final version of the manuscript.

**Funding:** This work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (Grant Agreement No 678151–Project Acronym "TROJANANOHORSE"–ERC starting Grant).

**Acknowledgments:** Cristiano Di Benedetto is thankfully acknowledged for preliminary CTEM imaging. **Conflicts of Interest:** There are no conflicts to declare.
