Effects of water-soluble functionalized multi-walled carbon nanotubes examined by different cytotoxicity methods in human astrocyte D384 and lung A549 cells

The widespread projected use of functionalized carbon nanotubes (CNTs) makes it important to understand their potential harmful effects. Two cell culture systems, human A549 pneumocytes and D384 astrocytoma cells, were used to assess cytotoxicity of multi-walled CNTs (MWCNTs) with varying degrees of functionalization. Laboratory-made highly functionalized hf-MW-NH₂ and less functionalized CNTs (MW-COOH and MW-NH₂) were tested in comparison with pristine MWCNTs, carbon black (CB) and silica (SiO₂) by MTT assay and calcein/propidium iodide (PI) staining. Purity and physicochemical properties of the test nanomaterials were also determined.In both MTT and calcein/PI assays, highly functionalized CNTs (hf-MW-NH₂) caused moderate loss of cell viability at doses ≥100μg/ml being apparently less cytotoxic than SiO₂.In preparations treated with CB or the other nanotube types (pristine MWCNTs, MW-COOH and the less functionalized amino-substituted MW-NH₂) the calcein/PI test indicated no loss of cell viability, whereas MTT assay apparently showed apparent cytotoxic response, occurring not dose-dependently at exceedingly low CNT concentrations (1μg/ml). The latter nanomaterials were difficult to disperse showing higher aggregate ranges and tendency to agglomerate in bundle-like form in cell cultures. In contrast, hf-MW-NH₂ were water soluble and easily dispersible in medium; they presented lower aggregate size range as well as considerably lower length to diameter ratios and low tendency to form aggregates compared to the other CNTs tested. The MTT data may reflect a false positive cytotoxicity signal possibly due to non-specific CNT interaction with cell culture components.Thus, these properties obtained by chemical functionalization, such as water solubility, high dispersibility and low agglomeration tendency were relevant factors in modulating cytotoxicity.This study indicates that properties obtained by chemical functionalization, such as water solubility, high dispersibility and low agglomeration tendency are relevant factors in modulating cytotoxicity of CNTs.