Background: Titanium dioxide (TiO₂) nanomaterials have considerable beneficial uses as photocatalysts and solar cells. It has been established for many years that pigment-grade TiO₂ (200 nm sphere) is relatively inert when internalized into a biological model system (in vivo or in vitro). For this reason, TiO₂ nanomaterials are considered an attractive alternative in applications where biological exposures will occur. Unfortunately, metal oxides on the nanoscale (one dimension < 100 nm) may or may not exhibit the same toxic potential as the original material. A further complicating issue is the effect of modifying or engineering of the nanomaterial to be structurally and geometrically different from the original material. Results: TiO₂ nanospheres, short (< 5 μm) and long (> 15 μm) nanobelts were synthesized, characterized and tested for biological activity using primary murine alveolar macrophages and in vivo in mice. This study demonstrates that alteration of anatase TiO₂ nanomaterial into a fibre structure of greater than 15 μm creates a highly toxic particle and initiates an inflammatory response by alveolar macrophages. These fibre-shaped nanomaterials induced inflammasome activation and release of inflammatory cytokines through a cathepsin B-mediated mechanism. Consequently, long TiO₂ nanobelts interact with lung macrophages in a manner very similar to asbestos or silica. Conclusions: These observations suggest that any modification of a nanomaterial, resulting in a wire, fibre, belt or tube, be tested for pathogenic potential. As this study demonstrates, toxicity and pathogenic potential change dramatically as the shape of the material is altered into one that a phagocytic cell has difficulty processing, resulting in lysosomal disruption.

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.

Functionalization has been shown to alter toxicity of multi-walled carbon nanotube (MWCNT) in several studies. This study varied the degree of functionalization (viz., amount of MWCNT surface carboxylation) to define the relationship between the extent of carboxylation and effects in a variety of in vitro cell models and short-term ex vivo/in vivo particle exposures. Studies with vitamin D₃ plus phorbol ester transformed THP-1 macrophages demonstrated that functionalization, regardless of amount, corresponded with profoundly decreased NLRP3 inflammasome activation. However, all MWCNT variants were slightly toxic in this model. Alternatively, studies with A549 epithelial cells showed some varied effects. For example, IL-33 and TNF-α release were related to varying amounts of functionalization. For in vivo particle exposures, autophagy of alveolar macrophages, measured using green fluorescent protein (GFP)- fused-LC3 transgenic mice, increased for all MWCNT tested three days after exposure, but, by Day 7, autophagy was clearly dependent on the amount of carboxylation. The instilled source MWCNT continued to produce cellular injury in alveolar macrophages over seven days. In contrast, the more functionalized MWCNT initially showed similar effects, but reduced over time. Dark-field imaging showed the more functionalized MWCNTs were distributed more uniformly throughout the lung and not isolated to macrophages. Taken together, the results indicated that in vitro and in vivo bioactivity of MWCNT decreased with increased carboxylation. Functionalization by carboxylation eliminated the bioactive potential of the MWCNT in the exposure models tested. The observation that maximally functionalized MWCNT distribute more freely throughout the lung with the absence of cellular damage, and extended deposition, may establish a practical use for these particles as a safer alternative for unmodified MWCNT.