Aggregation Reduces Subcellular Localization and Cytotoxicity of Single-Walled Carbon Nanotubes.

The non-covalent biomolecular functionalization of fluorescent single-walled carbon nanotubes (SWCNTs) has resulted in numerous in vitro and in vivo sensing and imaging applications due to many desirable optical properties. In these applications, it is generally presumed that pristine, singly dispersed SWCNTs interact with and enter live cells at the so-called nano-biointerface, for example, the cell membrane. Despite numerous fundamental studies published on this presumption, it is known that nanomaterials have the propensity to aggregate in protein-containing environments before ever contacting the nano-biointerface. Here, using DNA-functionalized SWCNTs with defined degrees of aggregation as well as near-infrared hyperspectral microscopy and toxicological assays, we show that despite equal rates of internalization, initially aggregated SWCNTs do not further accumulate within individual subcellular locations. In addition to subcellular accumulations, SWCNTs initially with a low degree of aggregation can induce significant deleterious effects in various long-term cytotoxicity and real-time proliferation assays, which are markedly different when compared to those of SWCNTs that are initially aggregated. These findings suggest the importance of the aggregation state as a critical component related to intracellular processing and toxicological response of engineered nanomaterials.