The pulmonary toxicity of carboxylated or aminated multi-walled carbon nanotubes in mice is determined by the prior purification method

Abstract

Background: Inhalation of multi-walled carbon nanotubes (MWCNTs) poses a potential risk to human health. In order to safeguard workers and consumers, the toxic properties of MWCNTs need to be identified. Functionalization has been shown to either decrease or increase MWCNT-related pulmonary injury, depending on the type of modification. We, therefore, investigated both acute and chronic pulmonary toxicity of a library of MWCNTs derived from a common pristine parent compound (NC7000).

Methods: MWCNTs were thermally or chemically purified and subsequently surface functionalized by carboxylation or amination. To evaluate pulmonary toxicity, male C57BL6 mice were dosed via oropharyngeal aspiration with either 1.6 or 4 mg/kg of each MWCNT type. Mitsui-7 MWCNT was used as a positive control. Necropsy was performed at days 3 and 60 post-exposure to collect bronchoalveolar lavage fluid (BALF) and lungs.

Results: At day 3 all MWCNTs increased the number of neutrophils in BALF. Chemical purification had a greater effect on pro-inflammatory cytokines (IL-1β, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-β1). At day 60, thermally purified, carboxylated MWCNTs had the strongest effect on lymphocyte numbers in BALF. Thermally purified MWCNTs caused the greatest increase in LDH and total protein in BALF. Furthermore, the thermally purified and carboxyl- or amine-functionalized MWCNTs caused the greatest number of granulomatous lesions in the lungs. The physicochemical characteristics mainly associated with increased toxicity of the thermally purified derivatives were decreased surface defects and decreased amorphous content as indicated by Raman spectroscopy.

Conclusions: These data demonstrate that the purification method is an important determinant of lung toxicity induced by carboxyl- and amine-functionalized MWCNTs.

Keywords: Carbon nanotubes; Fibrosis; Functionalization; Lung injury; Purification.

Fig. 1

Library of MWCNTs used in the present study. a Illustration showing derivation of carboxyl and amine functionalized MWCNTs after purification by heat or acid treatment. b TEM images of the NC7000 MWCNT samples. Magnification bars are indicated

Fig. 2

Inflammatory cell total counts and differentials in BAL fluid at 3 or 60 days after exposure to a low or high dose of purified and functionalized MWCNTs. a Average cell counts (based on 3 representative photomicrographs). b Macrophages (per 500 cells). c Neutrophils (per 500 cells). d Lymphocytes (per 500 cells). Statistically significant difference to the vehicle control: *** P < 0.001, ** P < 0.01, * P < 0.05 or to the NC7000 parent MWCNT sample: ^^^ P < 0.001, ^^ P < 0.01, ^ P < 0.05

Fig. 3

Lactate dehydrogenase (LDH) activity and total protein in BAL fluid at 3 or 60 days after exposure to a low and high dose of purified and functionalized MWCNTs. a LDH activity. b Total protein. Statistically significant difference to the vehicle control: *** P < 0.001, ** P < 0.01, * P < 0.05, the parent NC7000 sample: ^^^ P < 0.001, ^^ P < 0.01, or to the chemically-purified sample CP7000: ### P < 0.001, # P < 0.05

Fig. 4

Pro-inflammatory cytokines in the BAL fluid at 3 or 60 days after exposure to a low and high dose of purified and functionalized MWCNTs measured by ELISA. a IL-1β. b IL-6. c CXCL1. Statistically significant difference to the vehicle control: *** P < 0.001, ** P < 0.01, * P < 0.05, or between CP7000 and CP-NH2 (* P < 0.05) as indicated by the bar

Fig. 5

Pro-fibrotic cytokines in the BAL fluid at 3 or 60 days after exposure to a low and high dose of purified and functionalized MWCNTs measured by ELISA. a CCL2. b OPN. c TGF-β1. Statistically significant difference to the vehicle control: *** P < 0.001, ** P < 0.01, * P < 0.05, the parent NC7000 MWCNT sample: ^^^ P < 0.001, ^^ P < 0.01, or the chemically-purified CP7000 sample: ### P < 0.001, ## P < 0.01

Fig. 6

Histopathology at 60 days after exposure to a high dose of purified and subsequently functionalized MWCNTs. a Representative photomicrographs taken at 40x magnification of Gomori’s trichrome-stained lung sections. Red arrows indicate granulomatous lesions in lung tissue. Dotted lines with arrows linking panels indicate derivation of the purified or functionalized MWCNTs with the parent pristine NC7000 MWCNT sample. b Results of quantitative morphometry showing numbers of granulomatous lesions (left graph) and size (area) of granulomatous lesions (right graph). N = 4 mice per group/treatment. Statistically significant difference to the vehicle control: *P < 0.05, ***P < 0.001, or to the chemically purified CP7000 sample: ## P < 0.01 compared to CP7000

Fig. 7

Diagram showing selected biological endpoints at 3 days (a) and 60 days (b) post-exposure to the different MWCNTs samples used in this study. Increasing distance away from the center indicates increasing biological response. In general, chemically purified MWCNTs and functionalized derivatives caused a greater increase in pro-inflammatory chemokines at 3 days post-exposure, while thermally purified MWCNTs and functionalized derivatives caused a greater increase in pro-fibrotic cytokines (OPN, CCL2) at 3 days and greater pulmonary toxicity (LDH, total protein), BAL lymphocytes, and numbers of granulomas at 60 days post-exposure