Zeta Potential

Electrophoretic mobility measurements of particle suspensions were performed by phase analysis light scattering on a Zetasizer Nano ZS using disposable zeta cells. Electrophoretic mobility of particles (measured voltage of 50.3 V) was converted into zeta potentials by the Dispersion Technology Software 7.11 using the Smoluchowski approximation, and a viscosity of 1.0031 cP and dielectric constant of 80.4 for the dispersant. Measurements were taken as three independent replicates.

Food grade TiO₂ when imaged by TEM showed typical dry particle sizes of 100–200 nm, albeit with some smaller and larger sizes also present (shown in Supporting Information Additional file 1). When suspended in water or tissue culture media (TCM), at 5 μg/ml, mean ± SD zeta potentials, assessed by electrophoretic light scattering, were −20 mV ± 1 and −8.7 mV ± 0.2, respectively. In the same experiment, the aquated size of the suspended particles in TCM was also assessed by DLS (Supporting Information Additional file 1A) and the Z average (i.e., intensity‐weighted mean diameters derived from Cumulants analysis) was 300 nm. Sizing was re‐examined at 3 h, since particle suspensions are generally more reliably stable when the zeta potential is either above 30 mV or below −30 mV 23. Moreover, the re‐analysis at 3 h in TCM showed that size distribution remained relatively unaltered (Z average 339 nm; data not shown). At double the concentration in TCM (10 μg/ml TiO₂), the Z average was 356 nm at 3 h and relative particle distribution remained similar to the other conditions.

Increases in particle size from the dry to aquated state, and then by a further 13–19% depending on concentration during three hours in TCM, were unsurprising due to the anticipated formation of a corona (e.g., hydration shell and interactions between the particle surface and TCM components such as protein) as well as a degree of agglomeration due to particle–particle interactions in solution 24.

DLS relies upon Brownian motion of nonsedimenting particles. Thus, while it is the most appropriate single technique for the analyses described above, it is still possible to “miss” (a) microparticles due to their sedimentation or (b) the true breadth of polydispersity in the nonsedimenting fraction due to masking of small nanoparticle signals by large nanoparticle signals (extent of light scattering by a given particle type is proportional to d ⁶, where d = diameter). In a population of particles, the smallest and largest fractions make up the “tails” of the distribution, by definition. Thus, for their analysis, techniques are generally chosen that can emphasize any signal from these tails thereby facilitating their detection. Hence, SLS, which commonly employs a stirred dispersion unit to prevent sedimentation and for which data are presented as “volume %,” enables a low number of larger particles to be detected in a high number of smaller particles because the former are maintained in suspension the output shows overall volume. Nanoparticle tracking analysis (NTA), in contrast, is best equipped to characterize polydisperse populations as it tracks particles individually and thus small particles are not “hidden” by the presence of larger, more light scattering particles. However, tracking is carried out on a horizontal focal plain and, as a consequence this technique under‐represents larger, sedimenting particle since these can drop out of the focal plain.

Here, SLS equipped with a dispersion unit, did reveal a less numerous second population of larger aggregates (ca. 1–10 µm; Supporting Information Additional file 1B) which were not easily observed by DLS, and skewed the average detected particles to larger sizes (circa 500 nm) as expected. Additionally, as anticipated, NTA revealed a dominant particle size consistent with the DLS data but also showed a second smaller peak of particles <100 nm in diameter (Supporting Information Additional file 1C), as previously reported for pigment grade TiO₂ 25.

In summary, the most numerically frequent (as determined by NTA) aquated particle size of the TiO₂ that is experienced by cells cultured in suspension is 150–300 nm but there is a range of sizes in suspension from <100 to circa 10,000 nm, with the larger sizes, which are not at all numerous, sedimenting out. In this way, experiments represented cell exposure to the natural size variance of food grade TiO₂.