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Background  Several properties of multi-walled carbon nanotubes (MWCNT) have the potential to affect their bioactivity. This study examined the  in vitro  and  in vivo  outcomes of the influence of diameter, length, purification and carboxylation ( in vitro  testing only) of MWCNT.    Methods  Three original ‘as received’ MWCNT that varied in size (diameter and length) were purified and functionalized by carboxylation. The resulting MWCNT were characterized and examined for cytotoxicity and inflammasome activation  in vitro  using THP-1 cells and primary alveolar macrophages from C57BL/6 mice. Oropharyngeal aspiration administration was used to deliver original MWCNT and  in vivo  bioactivity and lung retention was examined at 1 and 7 days.    Results  Studies with THP-1 macrophages demonstrated that increased length or diameter corresponded with increased bioactivity as measured by inflammasome activation. Purification had little effect on the original MWCNT, and functionalization completely eliminated bioactivity. Similar results were obtained using alveolar macrophages isolated from C57BL/6 mice. The  in vivo  studies demonstrated that all three original MWCNT caused similar neutrophil influx at one day, but increasing length or diameter resulted in the lavaged cells to release more inflammatory cytokines (IL-6, TNF-α, and IL-1β)  ex vivo . Seven-day histology revealed that, consistent with the  in vitro  results, increasing width or length of MWCNT caused more severe pathology with the longest MWCNT causing the most severe inflammation. In addition, the same two larger MWCNT were retained more in the lung at 7 days.    Conclusions  Taken together, the results indicated that  in vitro  and  in vivo  bioactivity of MWCNT increased with diameter and length. Purification had no significant modifying effect from the original MWCNT. Functionalization by carboxylation completely eliminated the bioactive potential of the MWCNT regardless of size in  in vitro  testing.

Effect of MWCNT size, carboxylation, and purification on in vitro and in vivo toxicity, inflammation and lung pathology