They were fixed in a fresh solution of 0.1?M sodium cacodylate containing 2.5% glutaraldehyde and 2% formaldehyde followed by a 2-h fixation at 4C with 2% osmium tetroxide in 50?mM sodium cacodylate (pH 7.2). a rapid intracellular release of zinc ions,31 thereby causing lysosomal and mitochondrial damage.32 Nagajyothi et?al. reported of an antioxidant effect of ZnO-NPs to be the mechanism involved in its anti-tumor properties,33 while other studies instead exhibited an increase in reactive oxygen species (ROS) inside of the cells.34 Regarding the effect of ZnO-NPs on HNSCC, the studies conducted so far proved a photocatalytic cell death of HNSCC malignancy cells when exposed to ZnO-NPs and UVA-1.23 The present study was conducted without UV-stimulation of ZnO-NPs and showed no reduction in tumor cell survival with increasing concentrations of ZnO-NPs. Thus, we could show that ZnO-NPs alone, without photo-stimulation, seem to have no anti-tumorigenic effects on HNSCC cell lines within the applied concentrations. As compared with the literature, the applied NP dosage was lower in this study than in Dimethylenastron other publications. Often, cytotoxic effects of ZnO-NPs occur at concentrations between 5 and 10?g/ml.35 Since we aimed to characterize the interactions of ZnO NPs with the tumortoxic agent Cetuximab, we chose a non-toxic concentration of OGN ZnO NPs and a toxic concentration of Cetuximab. Since with photoactivation even those non-toxic concentrations of ZnO-NP have been shown to reduce tumor cell viability (data not shown), we made the decision against photoactivation to prevent it from masking the desired antagonistic effect on cetuximab. However, a concentration of 1 1?g/ml would fit into the effective concentration range of photocatalytic cell killing of ZnO NPs. Thus, the chosen dosages are Dimethylenastron clinically relevant. Several studies have shown synergistic effects of UV-stimulated ZnO-NPs and different chemotherapeutic drugs on malignancy cells imaging of tumors.37,38 Cetuximab in particular has been conjugated to iron oxide nanoparticles to enhance delivery of the targeted therapy to the tumor cells via magnetic resonance imaging-guidance for glioblastoma37 and EGFR-overexpressing cell lines A431 and 32D/EGFR.39 However, to our knowledge no studies regarding interactions of nanoparticles with Cetuximab on tumor cells have been published so far. In the present study, Cetuximab showed a consecutive decrease in tumor cell survival with increasing concentrations, as was to be expected. In the presence of 1g/ml ZnO-NPs, the anti-proliferative effects of 1M Cetuximab were antagonized after 72?hours (for HLaC) and after 48 and 72?hours (for FaDu). These findings suggest a time- and dose-dependent antagonistic effect of ZnO-NPs and Cetuximab on HNSCC cell lines. The cytotoxic effect of Cetuximab at 0.1?M seemed too weak for an antagonistic effect of ZnO-NPs to show. Cetuximab at 10M, on the other hand, showed no antagonistic effects with ZnO-NPs either. An interference with the EGFR could be excluded as a mechanism of effects by fluorescence staining. Whether ZnO-NPs interfere with Cetuximab by binding to the Fab part of the molecule or mediate an antagonistic effect in another way remains unclear. Among different biomolecules, proteins are the most important factors which regulate biodistribution of NPs throughout the body as their binding can act as ligand that may favor NP internalization. ZnO NPs are able to bind proteins with important biologic functions, including immunoglobulins, lipoproteins and several others.40 Numerous factors such as electrostatic or hydrophobic interactions as well Dimethylenastron as specific chemical processes contribute to these interactions.41 This study must be seen as an example for the sensitive interactions of targeted drugs with nanomaterials. For future applications of nanotechnological therapeutic approaches, such interactions have to be taken into account. As shown in the present study, an exact definition of Dimethylenastron the relevant concentrations of both brokers is required. Further studies are warranted to elucidate these interactions. In conclusion, the present study could demonstrate an antagonizing effect of ZnO-NPs for Cetuximab-treated HNSCC cell lines at a certain range of concentrations. These effects seem to be time dependent as well as dose-dependent. Higher concentrations of Cetuximab could negate this effect. To further elucidate which mechanisms are involved in these findings and which implications they have for ZnO-NPs in oncologic research, further investigation is warranted. Material and methods Reagent preparation. ZnO-NPs with a diameter <100?nm and a.