Toxicology 269 (2010) 41–53 Contents lists available at ScienceDirect Toxicology journal homepage: www.elsevier.com/locate/toxicol Effects of water-soluble functionalized multi-walled carbon nanotubes examined by different cytotoxicity methods in human astrocyte D384 and lung A549 cells T. Coccini a,∗ , E. Roda b , D.A. Sarigiannis c , P. Mustarelli d , E. Quartarone d , A. Profumo e , L. Manzo a,b a Salvatore Maugeri Foundation IRCCS, Toxicology Division, Pavia, Italy b University of Pavia, Department of Internal Medicine and Therapeutics, Toxicology Division, Pavia, Italy c European Commission Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy d University of Pavia, Department of Physical Chemistry, Pavia, Italy e University of Pavia, Department of General Chemistry, Pavia, Italy article info Article history: Received 22 October 2009 Received in revised form 29 December 2009 Accepted 5 January 2010 Available online 14 January 2010 Keywords: In vitro Nanomaterials Viability tests Carboxyl functionalized MWCNTs Amine functionalized MWCNTs abstract The widespread projected use of functionalized carbon nanotubes (CNTs) makes it important to under- stand 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-NH 2 and less functionalized CNTs (MW-COOH and MW-NH 2 ) were tested in comparison with pristine MWCNTs, carbon black (CB) and sil- ica (SiO 2 ) 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-NH 2 ) caused moderate loss of cell viability at doses ≥100 g/ml being apparently less cytotoxic than SiO 2 . In preparations treated with CB or the other nanotube types (pristine MWCNTs, MW-COOH and the less functionalized amino-substituted MW-NH 2 ) 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 (1 g/ml). The latter nanomaterials were difficult to disperse show- ing higher aggregate ranges and tendency to agglomerate in bundle-like form in cell cultures. In contrast, hf-MW-NH 2 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 com- pared 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 dispersibil- ity 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. © 2010 Elsevier Ireland Ltd. All rights reserved. Abbreviations: MWCNTs, commercial multi-walled CNTs (“MWCNTs” as pro- duced/pristine); hf-MW-NH2, laboratory-made highly functionalized nanotubes, amine-containing high amino groups content; MW-COOH, laboratory-made less functionalized CNTs, carboxyl functionalized; MW-NH2, laboratory-made less func- tionalized CNTs, functionalized amine-containing amino groups content; CB, carbon black; SiO2, silica nanoparticles. ∗ Corresponding author at: Toxicology Division, Research Center for Occupational and Environmental Toxicology, Salvatore Maugeri Foundation Medical Center IRCCS, Via Maugeri 10, 27100 Pavia, Italy. Tel.: +39 0382 592416; fax: +39 0382 24605. E-mail address: teresa.coccini@fsm.it (T. Coccini). 1. Introduction Carbon nanotubes (CNTs) possess unique electrical, mechani- cal, and thermal properties, with potential wide applications in the electronics, computer, aerospace, and other industries. CNTs are also being developed for a range of biomedical applications including miniaturized biosensors, targeted drug delivery, and tis- sue engineering (Kateb et al., 2007; Veetil and Ye, 2009). Wider application spectrum of CNTs in medicine can be expected due to recently developed functionalization schemes enabling the imple- mentation of new functions that cannot otherwise be acquired by nonfunctionalized nanotubes (Shvedova et al., 2009). Functional- ized CNTs were shown to cross cell membrane and deliver attached cargos into cells, properties that could be exploited in applica- 0300-483X/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2010.01.005