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-hour 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.

The Importance of Evaluating the Lot-to-Lot Batch Consistency of Commercial Multi-Walled Carbon Nanotube Products

“…The sonication and centrifugation protocol developed in our previous works to prepare purified BSA-coated MWNT suspensions [ 50 , 51 ] was modified by using a ten-fold lower concentration of BSA (0.1 mg/mL) in the BSA working solution. Normally, this procedure results in a suspension containing 400–500 μg/mL of BSA-coated MWNTs, which was the case for each MWNT product except for the 2018-pMWNTs ( Table 2 ).…”

“…Normally, this procedure results in a suspension containing 400–500 μg/mL of BSA-coated MWNTs, which was the case for each MWNT product except for the 2018-pMWNTs ( Table 2 ). Zeta potential and DLS analyses were part of a quality control routine for the preparation of all MWNT suspensions, as previously described [ 50 , 51 , 54 ]. Table 2 shows that the zeta potentials for the BSA-cMWNTs in deionized water were slightly more negative than those for the BSA-pMWNTs, as expected; and, there were only minor differences in the particle size distributions of BSA-pMWNT and BSA-cMWNT suspensions indicating that the suspended MWNTs possessed similar dimensions with no evidence of major agglomeration.…”

“…The sonication and centrifugation protocol described in our previous works [ 50 , 51 ] was used with slight modifications to prepare purified BSA-coated MWNT suspensions. First, 10.0 mg of pMWNT or cMWNT powder was weighed into a pre-cleaned 20-mL glass vial and baked at 200 °C for 2 h to inactivate potential endotoxin contaminants [ 52 ].…”

“…Dynamic light scattering (DLS) and zeta potential analyses were used as part of a quality control routine for the preparation of all MWNT suspensions, as previously described [ 50 , 51 , 54 ]. The particle size distributions, in terms of hydrodynamic diameter, of the BSA-MWNT suspensions were determined by DLS.…”

“…The SDS-PAGE method with optical detection, recently validated by a large-area Raman scan technique [ 51 ], was used for quantifying pMWNTs extracted from RAW 264.7 cells. In brief, aliquots of known amounts of pMWNT standard suspensions, lysates of control cells, and lysates of cells treated with BSA-pMWNTs were mixed with 5% 2-mercaptoethanol, 10% glycerol, 62.5 mM Tris-HCl, and 2X-concentrated SDS sample loading buffer to a final concentration of 2% SDS, and boiled for 3 min.…”