Quantitative techniques for assessing and controlling the dispersion and biological effects of multiwalled carbon nanotubes in mammalian tissue culture cells

In vivo studies have demonstrated that the state of dispersion of carbon nanotubes (CNTs) plays an important role in generating adverse pulmonary effects. However, little has been done to develop reproducible and quantifiable dispersion techniques to conduct mechanistic studies in vitro. This study was to evaluate the dispersion of multiwalled carbon nanotubes (MWCNTs) in tissue culture media, with particular emphasis on understanding the forces that govern agglomeration and how to modify these forces. Quantitative techniques such as hydrophobicity index, suspension stability index, attachment efficiency, and dynamic light scattering were used to assess the effects of agglomeration and dispersion of as-prepared (AP), purified (PD), or carboxylated (COOH) MWCNTs on bronchial epithelial and fibroblast cell lines. We found that hydrophobicity is the major factor determining AP-and PD-MWCNT agglomeration in tissue culture media but that the ionic strength is the main factor determining COOH-MWCNT suspendability. Bovine serum albumin (BSA) was an effective dispersant for MWCNTs, providing steric and electrosteric hindrances that are capable of overcoming hydrophobic attachment and the electrostatic screening of double layer formation in ionic media. Thus, BSA was capable of stabilizing all tube versions. Dipalmitoylphosphatidylcholine (DPPC) provided additional stability for AP-MWCNTs in epithelial growth medium (BEGM). While the dispersion state did not affect cytotoxicity, improved dispersion of AP-and PD-MWCNTs increased TGF-β1 production in epithelial cells and fibroblast proliferation. In summary, we demonstrate how quantitative techniques can be used to assess the agglomeration state of MWCNTs when conducting mechanistic studies on the effects of dispersion on tissue culture cells.