Quantitation of cell-associated carbon nanotubes: selective binding and accumulation of carboxylated carbon nanotubes by macrophages

Abstract

To understand the influence of carboxylation on the interaction of carbon nanotubes with cells, the amount of pristine multi-walled carbon nanotubes (P-MWNTs) or carboxylated multi-walled carbon nanotubes (C-MWNTs) coated with Pluronic^® F-108 that were accumulated by macrophages was measured by quantifying CNTs extracted from cells. Mouse RAW 264.7 macrophages and differentiated human THP-1 (dTHP-1) macrophages accumulated 80–100 times more C-MWNTs than P-MWNTs during a 24-h exposure at 37 °C. The accumulation of C-MWNTs by RAW 264.7 cells was not lethal; however, phagocytosis was impaired as subsequent uptake of polystyrene beads was reduced after a 20-h exposure to C-MWNTs. The selective accumulation of C-MWNTs suggested that there might be receptors on macrophages that bind C-MWNTs. The binding of C-MWNTs to macrophages was measured as a function of concentration at 4 °C in the absence of serum to minimize the potential interference by serum proteins or temperature-dependent uptake processes. The result was that the cells bound 8.7 times more C-MWNTs than P-MWNTs, consistent with the selective accumulation of C-MWNTs at 37 °C. In addition, serum strongly antagonized the binding of C-MWTS to macrophages, suggesting that serum contained inhibitors of binding. Moreover, inhibitors of class A scavenger receptor (SR-As) reduced the binding of C-MWNTs by about 50%, suggesting that SR-As contribute to the binding and endocytosis of C-MWNTs in macrophages but that other receptors may also be involved. Altogether, the evidence supports the hypothesis that macrophages contain binding sites selective for C-MWNTs that facilitate the high accumulation of C-MWNTs compared to P-MWNTs.

Keywords:

• Nanotoxicology
• carbon nanotube
• macrophages

Acknowledgments

The authors are grateful to the Undergraduate Research Fund of the University of Texas at Dallas School of Natural Sciences & Mathematics for support of undergraduate students. The authors also are grateful to Dr. Carole Mikoryak for reading the manuscript and to Dr. Vasanth Murali for early work on Raman microscopy that was not included in the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The authors thank the National Institute for Environmental Health Sciences [Grant R15-ES023666; P.P., R.D.], the National Cancer Institute [Grant R15-CA152917; R.D., P.P.], and the University of Arizona/Semiconductor Research Corporation Engineering Research Center for Environmentally Benign Semiconductor Manufacturing [Grant ERC425-048; R.D., P.P., R.W.] for supporting this work.