Dispersion of nanomaterials used in toxicological studies: a comparison of sonication approaches demonstrated on TiO₂ P25

Toxicological testing of nanomaterials often involves in vitro studies of nanoscale powders, which, therefore, need to be dispersed. Various dispersion approaches have been described in the literature, with the main differences between them being the sonication method, the medium, and the additives used. In the present study, five dispersion approaches were compared using titanium dioxide nanoparticles (Aeroxide TiO₂ P25). Three of the selected approaches were two- or multi-step processes consisting of dispersion in water followed by transfer to cell culture media, while the two other procedures include only the dispersion in water or in media. One approach was based on bath sonication, whereas the others used direct sonication through insertion of a probe into the sample. The main finding arising out of the comparison of the direct dispersion methods was that the specific energy input played a crucial role in determining the achievable particle size (133–182 nm). A low volume of liquid and a relatively low power in conjunction with a long pulsed dispersion time were found to be favorable for minimizing the side effects of ultrasonication like radical formation or material degradation. The resulting size in the cell culture media showed a strong dependence on the dilution method. Predilution with water before addition to the media prevented the agglomeration of the TiO₂ particles under physiological conditions. Direct dispersion in media resulted in the smallest dispersible size. This study succeeded in furthering the understanding of the dispersion process and yielded useful tips for avoiding pitfalls in the preparation of suspensions for toxicological experiments.