Multi-walled carbon nanotubes (MWCNTs) are hollow fibre-like nanomaterials which are being investigated for use in drug delivery and as biosensors. However, due to their structural similarity to asbestos fibres, inhaled MWCNTs may elicit similar adverse health effects such as fibrosis and mesothelioma. Animal studies have suggested that this may be possible, however there is currently limited human data.
We hypothesised that the pro-fibrotic potential of MWCNTs would be determined by their physicochemical properties i.e length and concentration of impurities. We exposed primary human alveolar type II epithelial (ATII) cells and pulmonary fibroblasts to 30nm diameter CNTs of increasing length (0.2–2µm, 3–5µm and 10–30µm) and increasing purity (49%, 69% and >97%) for up to 96 hours. Oxidative stress, TGFβ release, soluble collagen release, and cell proliferation were measured in fibroblasts and release of VEGF, MCP-1, TGFβ and surfactant proteins (SP) A and D were measured in ATII cells.
MWCNTs induced oxidative stress in fibroblasts within 4h and a significant dose-dependent cell proliferation after 96h (P<0.05) that was not affected by MWCNT length or purity. Furthermore, there was a significant, dose-dependent, 4–7-fold increase in release of collagen, exceeding what could be accounted for by proliferation alone (P<0.05). There was a trend towards shorter and less pure CNTs inducing greater collagen release (P<0.1). Release of VEGF and MCP-1 from ATII cells was not induced by CNTs. However, TGFβ, SP-A and SP-D were released by ATII cells and were found bound to MWCNTs. More SP-A bound to the 0.6–2µm MWCNTs compared to the longer MWCNTs; the converse was true for SP-D. In addition the >97% pure MWCNTs bound more surfactant protein than the lower purity MWCNTs. Significantly more TGFβ was bound to the 10–30µm MWCNTs compared to shorter MWCNTs.
Our results demonstrate that MWCNTs can induce pro-fibrotic responses in primary human fibroblasts. Furthermore, our unique discovery of binding of TGFβ and surfactant proteins to MWCNTS suggests that this could exacerbate the fibrotic response if MWCNTs translocate across the epithelial barrier, due to the “Trojan horse” effect of MWCNTs delivering these mediators to the interstitium.