Excessive delivery of nanostructured matter to submersed cells caused by rapid gravitational settling

With very few exceptions, previous nanotoxicity studies implicitly involved the assumption that the techniques developed for risk assessment of hazardous chemical substances can be applied in unchanged form to explore cell response in NP laden media. This misleading approach has the consequence that the actual dose of exposure is ill defined or, more often, completely unknown. Here the effect of gravitational settling on the dose of exposure was explored for commercially available engineered nanostructured matter (nanopowder). Micrometer sized aggregates abundantly present in all nanopowders were fractured as much as possible by probe-type sonication in water or cell culture media. The morphology of cracked aggregates was studied by scanning electron microscopy. Size distributions were determined by dynamic light scattering (DLS). Possible pitfalls encountered in using DLS were documented. Absorbance measurements and optical microscopy served to monitor the rate of gravitational settling on time sales ranging from minutes up to several days. The sonicated particles settled rapidly in all liquid media. At the well bottom, they exhibited intense lateral (two-dimensional) Brownian-like motion, which allowed them to travel large distances. Taken together, the probability for particle-cell contact may be enhanced by a factor of more than 1000 compared to the commonly advocated picture. The very high levels of exposure can give rise to overload effects which are often misinterpreted as evidence of cytotoxicity. To identify the true toxic potential of NPs, future studies must account for these phenomena. It is also argued that stable dispersions of NPs are not required in nanotoxicity studies.