Comparative in Vitro Cytotoxicity of Realistic Doses of Benchmark Multi-Walled Carbon Nanotubes towards Macrophages and Airway Epithelial Cells

Abstract

Multi-walled carbon nanotubes (MWCNT) have many outstanding physical and chemical properties that make them useful in many applications in nanotechnology. However, these properties are reported to be potentially harmful for the human body. The effects of low and realistic doses of three well-characterized preparations of MWCNT, obtained from the Joint Research Centre (JRC) (NM-400, NM-401, and NM-402), were assessed in two murine macrophage lines, Raw264.7, of peritoneal origin, and MH-S, derived from alveolar macrophages. Macrophage viability, evaluated with two distinct methods, was significantly lowered by NM-401 (needle-like, average length 4 μm, diameter 67 nm) with IC ₅₀ values of 10 μg/cm ², whereas NM-400 and NM-402 (tangled, average lengths 846–1372 nm, diameter 11 nm) had much smaller effects. In contrast, at 10 μg/cm ², NM-400 and NM-402 induced the M1 marker Nos2 and, consistently, a sizable accumulation of nitrites in the medium, whereas NM-401 had no significant effect. None of the MWCNT preparations induced the M2 marker Arg1. Phagocytic activity, assessed in Raw264.7 macrophages, was significantly reduced in cells exposed to NM-401, but not to NM-400 or NM-402. When tested on Calu-3 bronchial epithelial cell monolayers, the three MWCNT preparations did not affect cell viability, but decreased the trans-epithelial electrical resistance at the maximal dose tested (80 μg/cm ²), with the most evident effect detected for NM-401, even at 10 μg/cm ². In conclusion, among the possible structural determinants of the toxic effects exerted by MWCNT towards macrophages and airway epithelial cells, shape and length appear the most relevant at low, realistic doses.