Toxicity determinants of multi-walled carbon nanotubes: The relationship between functionalization and agglomeration

The elucidation of toxicity determinants of multi-walled carbon nanotubes (MWCNT) is still incomplete. Functionalization with carboxyl groups is, however, commonly used to mitigate MWCNT toxicity, although the rationale for the mitigating effect has not been fully clarified yet. In this work, two optimized chemical vapor deposition methods were employed to obtain MWCNT of comparable length but different diameter, which were subsequently functionalized. For MWCNT of diameter larger than 40 nm, no detrimental effects on cell viability of macrophages were observed, while mild cytotoxicity was recorded for diameters between 15 and 40 nm, with a mitigating effect of functionalization. To investigate the factors responsible for the mitigation, we used the thinnest MWCNT preparation on different cell models, evaluating several endpoints, such as viability, production of nitric oxide (NO), expression of pro-inflammatory markers, the Trans-Epithelial Electrical Resistance (TEER), and clonogenic activity. Substantial mitigation of the changes caused by pristine MWCNT was observed not only with carboxyl- but also with amino-functionalized MWCNT, suggesting that negative or positive surface charge was not the main factor responsible for the effect. Instead, either functionalized preparation exhibited a stronger tendency to agglomerate that was strictly dependent on the presence of proteins. Moreover, we found that either carboxyl- or amino-functionalized MWCNT adsorbed a larger amount of serum proteins than pristine counterparts, with a distinctive pattern for each type of MWCNT. We propose, therefore, that the formation of larger agglomerates, dependent upon different protein coronae, contributes to mitigate the biological effects of functionalized MWCNT in protein-rich biological media.