Effect of multi-walled carbon nanotube surface modification on bioactivity in the C57BL/6 mouse model

Abstract

The current study tests the hypothesis that multi-walled carbon nanotubes (MWCNT) with different surface chemistries exhibit different bioactivity profiles in vivo. In addition, the study examined the potential contribution of the NLRP3 inflammasome in MWCNT-induced lung pathology. Unmodified (BMWCNT) and MWCNT that were surface functionalised with -COOH (FMWCNT), were instilled into C57BL/6 mice. The mice were then examined for biomarkers of inflammation and injury, as well as examined histologically for development of pulmonary disease as a function of dose and time. Biomarkers for pulmonary inflammation included cytokines, mediators and the presence of inflammatory cells (IL-1β, IL-18, IL-33, cathepsin B and neutrophils) and markers of injury (albumin and lactate dehydrogenase). The results show that surface modification by the addition of the -COOH group to the MWCNT, significantly reduced the bioactivity and pathogenicity. The results of this study also suggest that in vivo pathogenicity of the BMWCNT and FMWCNT correlates with activation of the NLRP3 inflammasome in the lung.

Figure 1

Comparison of inflammation induced by pharyngeal aspiration exposure to 0, 2.5, 10 and 40 µg/mouse of BMWCNT (A) and FMWCNT (B) at 1 and 7 days post-exposure. WLL PMNs were used as a marker of pulmonary inflammation. Values are given as means ± SE (n = 8). An asterisk (*) indicates that PMN influx for that group were significantly higher than control (p < 0.05).

Figure 2

Comparison of air/blood barrier injury induced by pharyngeal aspiration exposure to 0, 2.5, 10 and 40 µg/mouse of BMWCNT (A) or FMWCNT (B) at 1 and 7 days post-exposure. WLL fluid albumin concentrations were used as a marker of the air/blood barrier. Values are given as means ± SE (n = 8). An asterisk (*) indicates that albumin levels for that group were significantly higher than control (p < 0.05).

Figure 3

Comparison of cytotoxicity induced by pharyngeal aspiration exposure to 0, 2.5, 10 and 40 µg/mouse of BMWCNT (A) or FMWCNT (B) at 1 and 7 days post-exposure. WLL fluid LDH activities were used as a marker of cytotoxicity. Values are given as means ± SE (n = 8). An asterisk (*) indicates that LDH activity for that group was significantly higher than control (p < 0.05).

Figure 4

Comparison of inflammation (A), air/blood barrier injury (B) and cytotoxicity (C) induced by pharyngeal aspiration exposure to 40 µg/mouse of BMWCNT or FMWCNT at 1 and 7 days post-exposure. Values are given as means ± SE (n = 8). An asterisk (*) indicates that levels for that group were significantly higher than control (p < 0.05). A (+) indicates that BMWCNT elicited levels that were significantly higher than FMWCNT (p < 0.05).

Figure 5

Differential activation of the NLRP3 inflammasome by BMWCNT and FMWCNT. Mice were exposed by pharyngeal aspiration to 0 or 40 µg/mouse of either BMWCNT or FMWCNT and first WLL fluid was isolated at 1 day post-exposure. Cathepsin B activities (A) and concentrations of IL-1β (B), IL-18 (C) and IL-33 (D) were determined as described in the section “Materials and Methods”. Values are means ± SE (n = 8). An asterisk (*) indicates significant increase vs. control (p < 0.05). A (+) indicates that BMWCNT was significantly greater than FMWCNT (p < 0.05).

Figure 6

Enhanced dark field imaging of BMWCNT (A) and FMWCNT (B) in mouse lungs at 56 days post-exposure. Arrows indicate presence of MWCNT.

Figure 7

Photomicrograph from the lung of a mouse 56 days after a single aspiration exposure to BMWCNT. Mild alveolar septal fibrosis is in the wall of an alveolar duct and is associated with interstitial BMWCNT (solid arrow). Macrophages contain phagocytised BMWCNT (dashed arrows). The nucleus in one of the macrophages is undergoing karyolysis, which suggests cytotoxicity. Sirius red stain; bar = 10 µm.

Figure 8

Schematic of the NLRP3 inflammasome. In this study, the authors propose that the MWCNT being tested will act as danger signals to AMs, initiating an inflammatory cascade mediated through the NLRP3 inflammasome. In this schematic representation, MWCNT disrupt phagolysosomes, releasing cathepsin B and activating the NLRP3 inflammasome. Once activated, the NLRP3 inflammasome cleaves the proinflammatory mediators IL-1β and IL-18, which are subsequently released to initiate the inflammatory cascade.