Toxicity determinants of multi-walled carbon nanotubes: The relationship between functionalization and agglomeration

Abstract

The elucidation of toxicity determinants of multi-walled carbon nanotubes (MWCNT) is still incomplete. Functionalization with carboxyl groups is, however, commonly used to mitigate MWCNT toxicity, although the rationale for the mitigating effect has not been fully clarified yet. In this work, two optimized chemical vapor deposition methods were employed to obtain MWCNT of comparable length but different diameter, which were subsequently functionalized. For MWCNT of diameter larger than 40 nm, no detrimental effects on cell viability of macrophages were observed, while mild cytotoxicity was recorded for diameters between 15 and 40 nm, with a mitigating effect of functionalization. To investigate the factors responsible for the mitigation, we used the thinnest MWCNT preparation on different cell models, evaluating several endpoints, such as viability, production of nitric oxide (NO), expression of pro-inflammatory markers, the Trans-Epithelial Electrical Resistance (TEER), and clonogenic activity. Substantial mitigation of the changes caused by pristine MWCNT was observed not only with carboxyl- but also with amino-functionalized MWCNT, suggesting that negative or positive surface charge was not the main factor responsible for the effect. Instead, either functionalized preparation exhibited a stronger tendency to agglomerate that was strictly dependent on the presence of proteins. Moreover, we found that either carboxyl- or amino-functionalized MWCNT adsorbed a larger amount of serum proteins than pristine counterparts, with a distinctive pattern for each type of MWCNT. We propose, therefore, that the formation of larger agglomerates, dependent upon different protein coronae, contributes to mitigate the biological effects of functionalized MWCNT in protein-rich biological media.

Keywords: Agglomeration; Airway epithelium; BET, Brunauer, Emmett and Teller; BSA, Bovine Serum Albumin; CFE, colony forming efficiency; CNT, carbon nanotubes; CVD, carbon vapor deposition; Carbon nanotubes; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; EDS, energy dispersive X-ray spectrometry; FBS, Fetal Bovine Serum; FT-IR, Fourier transform infrared spectroscopy; Functionalization; Inflammation; MWCNT, multi-walled carbon nanotubes; Macrophages; NO, nitric oxide; Protein corona; SDS, sodium dodecyl sulphate; SDS-PAGE, SDS polyacrylamide gel electrophoresis; SSA, specific surface area; SWCNT, single-walled carbon nanotubes; TEER, Trans-Epithelial Electrical Resistance; TGA, thermogravimetric analysis; XRD, X-ray diffraction.

Graphical abstract

Fig. 1

TEM images of CNT1 (a), CNT2 (b), O-CNT2 (c), CNT3 (d), REF(e).

Fig. 2

SEM images of (A) CNT1, (B) CNT2 and (C) CNT3; (D) EDS analysis, (E) XRD diagram and (F) TGA graph of CNT3.

Fig. 3

FTIR spectra (A) and TGA graphs (B) of pristine MWCNT (REF), MWCNT–COOH (O-REF) and MWCNT-amino (A-REF) samples.

Fig. 4

The Suspension Stability Index of the three MWCNT preparations at 128 μg/mL was expressed as the % of the initial absorbance at the indicated times. The absorbance measurements were carried out as described in Section 2.4. (panel A) MWCNT were suspended in PBS (Ca- and Mg-free). (panel B) MWCNT were suspended in a solution of 0.05% BSA in PBS (Ca- and Mg-free). (panel C) MWCNT were suspended in growth medium (phenol-red free DMEM) supplemented with 10% FBS. The figure shows a representative experiment, repeated twice with comparable results.

Fig. 5

Protein adsorption on MWCNT. MWCNT preparations, dispersed in 0.05 wt% BSA, were incubated at the concentration of 128 μg/mL for 1 h in culture medium with or without 10% FBS. At the end of the incubation, the suspensions were centrifuged, and adsorbed proteins quantified (A) or separated and stained (B) as described in Section 2. For (A), data are means ± SD of 4 independent determinations. *p < 0.05, **p < 0.01 vs. REF. For (B) a representative experiment, performed twice with comparable results is shown.

Fig. 6

Cell viability of A549 cells exposed to MWCNT. Cells were treated with increasing doses of MWCNT (range dose 10–80 μg/cm² for REF, O-REF and A-REF; 80 μg/cm² for NM-401). After 48 h cell viability was determined with the resazurin assay. 0, control untreated cells. Data are means ± SD of 6 indipendent determinations obtained in two separate experiments. **p < 0.01, ***p < 0.001 vs. control, untreated cells (0); $$p < 0.01, $$$p < 0.001 vs. equivalent doses of REF.

Fig. 7

Colony forming efficiency of MWCNT-treated A549 cells. Cells were seeded at a concentration of 100 cells/plate and treated as described in Section 2.11. Data are means of 4 indipendent determinations. *p < 0.05, ***p < 0.001 vs. control, untreated cultures.

Fig. 8

Expression of pro-inflammatory markers in Raw264.7 exposed to MWCNT. (panels A and B) Nitrite concentration in cell culture medium of Raw264.7 cells exposed to MWCNT. Raw264.7 cells were treated with MWCNT (REF, O-REF and A-REF: range dose 20–80 μg/cm², NM-401: 80 μg/cm²). After 48 h (panel A) or 72 h (panel B), the nitrite concentration was determined in cell culture medium. Lipopolysaccharide (LPS, 10 ng/ml) was used as a positive control. Data are means ± SD of 8 independent determinations obtained in two experiments. (panels C, D and E) Expression of Nos2 (panel C), Ptgs2 (panel D), Il6 (panel E), and Il1b (panel F) in cells treated with MWCNT. Raw264.7 cells were treated with the indicated materials at the dose of 80 μg/cm². After 18 h mRNA was extracted and gene expression was analyzed with RT-PCR. Data are means of 2 independent determinations. **, ***p < 0.01, p < 0.001 vs. control, untreated cells. $, $$, $$$p < 0.05, p < 0.01, p < 0.001 vs. REF at the same dose.

Fig. 9

Contrast phase (top left) and confocal images (top right and bottom) of Raw264.7 cells exposed to REF (panel B), O-REF (panel C) and A-REF (panel D) MWCNT. Cells were treated with 80 μg/cm² of each MWCNT preparation for 24 h and labelled as described in Methods. The same representative fields are shown in contrast phase (left) and confocal microscopy (right). For each condition, a single horizontal confocal section is shown along with two orthogonal projections. White, MWCNT (reflection mode); blue, nuclei; red, cytoplasm. Bars = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 10

TEER and cell viability of Calu-3 cell monolayers. Calu-3 cells were seeded on permeable filters as described in Section 2.12. After a tight monolayer had formed, MWCNT (80 μg/cm²) were added to the apical compartment, and exposure prolonged for up to 12 days. At the indicated times, TEER was determined, as described under Methods. (A) TEER (% of control) recorded every 3d up to 12d. (B) TEER (% of control) at day 12. (C) Cell viability was assessed in the same wells used for TEER determinations with the resazurin assay. For all panels, data are means ± S.D of 4 independent determinations. *p < 0.05 vs. control, untreated cultures. The experiment was performed twice with comparable results.