We developed a dispersal method for multi-walled carbon nanotubes (MWCNTs) that allows quantitative assessment of dispersion on pro-fibrogenic responses in tissue culture cells as well as in mouse lung. Here we demonstrate that the dispersal of as-prepared (AP), purified (PD), and carboxylated (COOH) MWCNTs by bovine serum albumin (BSA) and dipalmitoylphosphatidylcholine (DPPC) influences TGF-β1, PDGF-AA and IL-1β production in vitro and in vivo. These biomarkers were chosen based on their synergy in promoting fibrogenesis and cellular communication in the epithelial-mesenchymal cell trophic unit in the lung. The effect of dispersal was most noticeable in AP- and PD-MWCNTs, which are more hydrophobic and unstable in aqueous buffers than hydrophilic COOH-MWCNTs. Well-dispersed AP- and PD-MWCNTs were readily taken up by BEAS-2B, THP-1 cells and alveolar macrophages (AM), and induced more prominent TGF-β1 and IL-1β production in vitro as well as TGF-β1, IL-1β and PDGF-AA production in vivo than non-dispersed tubes. Moreover, there was good agreement between the pro-fibrogenic responses in vitro and in vivo as well as the ability of dispersed tubes to generate granulomatous inflammation and fibrosis in airways. Tube dispersal also elicited more robust IL-1β production in THP-1 cells. While COOH-MWCNTs were poorly taken up in BEAS-2B and induced little TGF-β1 production, they were bio-processed by AM and induced less prominent collagen deposition at sites of non-granulomatous inflammation in the alveolar region. Taken together, these results indicate that the dispersal state of MWCNTs affects pro-fibrogenic cellular responses that correlate with the extent of pulmonary fibrosis and are of potential use to predict pulmonary toxicity.

In vivo studies have demonstrated that the state of dispersion of carbon nanotubes (CNT) 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 multi-walled carbon nanotubes (MWCNT) 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) MWCNT 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 MWCNT, providing steric and electrosteric hindrance 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-MWCNT in epithelial growth medium (BEGM). While dispersion state did not affect cytotoxicity, improved dispersion of AP- and PD-MWCNT 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 MWCNT when conducting mechanistic studies on the effects of dispersion on tissue culture cells.

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity.Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability.Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells.Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β.Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

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The colloidal behavior of aqueous dispersions of functionalized multiwall carbon nanotubes (F-CNTS) formed via carboxylation and polymer wrapping with polyvinyl pyrrolidone (PVP) is presented. The presence of polymer on the nanotube surface provided steric stabilization, and the aggregation behavior of the colloidal system was quite different from its covalently functionalized analog. Based on hydrophobicity index, particle size distribution, zeta potential as well as the aggregation kinetics studied using time-resolved dynamic light scattering, the PVP wrapped CNT was somewhat less prone to agglomeration. However, its long term stability was lower, and this was attributed to the partial unwrapping of the polyvinyl pyrrolidone layer on the CNT surface.