The dispersion protocol used to administer nanomaterials (NMs) in in vitro cellular tests might affect their toxicity. For this reason, several dispersion procedures have been proposed to harmonize the toxicological methods, allowing for the comparison of the data that were obtained by different laboratories. At the same time, several techniques and methods are available to monitor the identity of the NMs in the cell media. However, while the characterization of suspensions of engineered NMs having narrow size distribution may be easily performed, the description of aggregated NMs forming polydispersions is still challenging. In the present study, sub-micrometric/nanometric TiO₂, SiO₂, and CeO₂ were dispersed in cell media by using two different dispersion protocols, with and without albumin (0.5%) and with different sonication procedures. Dynamic Light Scattering (DLS) was used to characterize NMs in stock solutions and culture media. Pitfalls that affect DLS measurements were identified and, guidance on a critical analysis of the results provided. The NMs were then tested for their cytotoxicity (LDH leakage) toward murine macrophages (RAW 264.7) and PMA-activated human monocytes (THP-1). As markers of pro-inflammatory response, nitric oxide (NO) and cytokine IL-1β production were measured on RAW 264.7 and THP-1 cells, respectively. The pre-treatment with albumin added to a strong sonication treatment increases the stability and homogeneity of the suspensions of nanometric samples, but not of the submicrometric-samples. Nevertheless, while TiO₂ and CeO₂ were non-cytotoxic in any conditions, differences in cytotoxicity, NO, and IL-1β releases were found for the SiO₂, depending upon the protocol. Overall, the results suggest that there is no one-fits-all method valid for all NMs, since each class of NMs respond differently. The definition of validated procedures and parameters for the selection of the most appropriate method of dispersion for each class of NM appears to be a more efficacious strategy for the harmonization of the dispersion protocols.

Background: Oral exposure to titanium dioxide (TiO₂) is common since it is widely used in food and pharmaceutical products. Concern on the safety of this substance has been recently raised, due to the presence of an ultrafine fraction in food-grade TiO₂. Discrepancy exists among data reported in in vitro and in vivo studies on intestinal acute/chronic toxicity of TiO₂. This might be due to the different biological identity of TiO₂ in traditional in vitro test by respect in vivo conditions. Methods: One food-grade TiO₂ and two nanometric TiO₂ samples were treated with a simulated human digestive dystem (SHDS) in order to investigate the bio-transformation occurring to the particles once ingested in term of size distribution (Dynamic Light Scattering—DLS-, Flow Particle Imaging, Asymmetric Flow Field Flow Fractionation-AF4-) and surface modification (Electrophoretic Light Scattering—ELS-, Electron Paramagnetic Resonance Spectroscopy—EPR-). The effect of SHDS on the cyto-, genotoxicity and potential to induce oxidative stress towards human colorectal carcinoma HCT116 cells was also assessed. Results: Aggregation as a consequence of the high ionic strength of the gastric and intestinal simulated fluids was observed, together with the formation of a partially irreversible bio-corona containing phosphate ions and proteins. Such bio-corona led to a partial masking of the TiO₂ particles surface and reactivity. Pristine and treated TiO₂ nanoparticles showed comparable acute toxicity and genotoxicity toward HCT116 cells, whereas a small decrease of the induction of oxidative stress after treatment was observed. Conclusions: Overall the results underline the importance of SHDS as a tool to improve the predictive power of in vitro tests towards intestinal nanomaterial toxicity.