Time-dependent degradation of carbon nanotubes correlates with decreased reactive oxygen species generation in macrophages

Abstract

Introduction and objective:

With the increase in carbon nanotube-based products on the commercial market, public concern regarding the possible toxicity of these nanomaterials has attracted much attention. Although previous studies found no obvious toxicity related to carbon nanotubes (CNTs), their safety has not been established because long-term evaluation is still needed. In vitro assays are used to understand the toxicity of nanomaterials. However, the data published so far were generated in short-term assays in which cells are continuously exposed to CNTs. Therefore, the objective of this study is to quantitatively assess the relative long-term cytotoxicity and degradation of CNTs after uptake by macrophages.

Methods:

We used macrophage cell line of RAW 264.7 and primary rat Kupffer cells to investigate macrophage uptake of CNTs as well as their quantity changes up to a relatively late time point after uptake (7 days) by measuring optical absorbance in the near infrared region and Raman spectra of CNTs in the cell lysates. The time-dependent cytotoxicity was evaluated by measuring reactive oxygen species (ROS), glutathione, cell viability, and caspase 3/7 activity in 1–7 days.

Results:

CNTs were degraded by approximately 25–30% within first 4 days after uptake; however, and no additional degradation occurred for the remainder of the 7-day test period. Generation of ROS by macrophages decreased as CNT degradation occurred, returning to control levels by Day 7. In the meantime, the glutathione level gradually recovered over time. There were no changes in cell viability or caspase 3/7 activation during CNT degradation.

Conclusion:

These results confirm that degradation of CNTs by macrophages is associated with ROS generation. The data also suggest that CNT cytotoxicity decreases as they are degraded.

Keywords:

• carbon nanomaterials
• biodegradation
• cytotoxicity
• cellular uptake

Acknowledgment

This work was supported in part by a KAKENHI Grant-in-Aid for Scientific Research B from the Japan Society for the Promotion of Science (17H02742) and by the Zeon Corporation.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

Characteristics of SG-CNT dispersion

To characterize the SG-CNT dispersion, we determined particle sizes of SG-CNT dispersion by dynamic light scattering (DLS) (FPAR-1000, Otsuka Electronics, Osaka, Japan). The result showed that the particles measured 70–150 nm (Figure S1A). SG-CNTs dispersed in BSA solution were observed under a TEM (Topcon EM-002B, Tokyo, Japan). For TEM observation, the SG-CNT dispersion was dropped onto a TEM grid with a carbon film coating. The lengths and diameters of the SG-CNTs bundles were estimated from TEM image (Figure S1B) to be at 100–400 nm and 3–7 nm, respectively.

Biodegradation of oxidized SG-CNTs (ox-SG-CNTs) in kupffer cells

As-grown SG-CNTs (24.5 mg) were oxidized by 48 mL of concentrated sulfuric acid: nitric acid at a ratio of 3:1 at 70 °C for 40 min, after diluting the solution with deionized water, the SG-CNTs were filtered and washed with pure water for five times until the pH of the filtrate was neutral. Then the obtained SG-CNTs were indicated as ox-SG-CNTs. These ox-SG-CNTs dispersed well in water after with a homogenizer (VC-750, Sonics & Materials, Newtown, CT, USA) for 10 min.

Kupffer cells were incubated with ox-SG-CNTs (50 µg/mL) for 24 hrs and washed (Day 0). The degradation protocol is the same as SG-CNTs mentioned in the method part. Because Kupffer cells don’t proliferate, a long-time observation was carried out up to 26 days. After uptake of ox-SG-CNT, we can found that the cells became black color representing ox-SG-CNTs inside the kupffer cells. The black color of cells was faded after uptake for 5 days and 15 days, suggesting that the ox-SG-CNTs were degraded by the kupffer cells (Figure S2A). Quantitative measurement by using optical absorbance at 750 nm showed that about 50% of ox-SG-CNTs were degraded from Day 0 to Day 5. The amount of ox-SG-CNTs did not significantly decrease further to Day 26 (Figure S2B).