Investigation of nanoparticle dispersibility and stability based on TiO2 analysis by SMLS, DLS, and SEM

Dispersibility and stability of nanoparticles (NPs) in the biological medium are playing a major role on their size, fate, transport, delivered dose to cells, and consequently to their toxicological response in vitro. However, these parameters are frequently confused and therefore very poorly characterized. Here, we critically discuss the definition and characterization of dispersibility and stability of NPs for in vitro studies. We provide a methodology based on static multiple light scattering (SMLS), dynamic light scattering (DLS), and scanning electron microscopy (SEM) measurements to characterize the NP dispersibility and stability in complex biological media. This methodology was applied to one pristine and two food-grade TiO2 NPs dispersed in commonly used cell culture media supplemented with various BSA concentrations. Dispersibility was characterized by measuring the NP size and homogeneity after applying an optimized dispersion protocol. Colloidal, gravitational, and macroscopic stabilities were distinguished by measuring the NP zeta potential, settling velocity, and Turbiscan stability index (TSI), respectively. This approach allowed to monitor in real time the NP stability instead of predictions based on initial assays such as size and zeta potential. The results also proved that (i) best NP dispersibility does not ensure the best stability, (ii) NP colloidally stable does not imply their gravitational stability in the biological medium, and (iii) TSI is a more reliable measurand of colloidal stability compared to zeta potential.