Systemic and immunotoxicity of pristine and PEGylated multi-walled carbon nanotubes in an intravenous 28 days repeated dose toxicity study

Abstract

The numerous increasing use of carbon nanotubes (CNTs) derived from nanotechnology has raised concerns about their biosafety and potential toxicity. CNTs cause immunologic dysfunction and limit the application of CNTs in biomedicine. The immunological responses induced by pristine multi-walled carbon nanotubes (p-MWCNTs) and PEGylated multi-walled carbon nanotubes (MWCNTs-PEG) on BALB/c mice via an intravenous administration were investigated. The results reflect that the p-MWCNTs induced significant increases in spleen, thymus, and lung weight. Mice treated with p-MWCNTs showed altered lymphocyte populations (CD3⁺, CD4⁺, CD8⁺, and CD19⁺) in peripheral blood and increased serum IgM and IgG levels, and splenic macrophage ultrastructure indicated mitochondria swelling. p-MWCNTs inhibited humoral and cellular immunity function and were associated with decreased immune responses against sheep erythrocytes and serum hemolysis level. Natural killer (NK) activity was not modified by two types of MWCNTs. In comparison with two types of MWCNTs, for a same dose, p-MWCNTs caused higher levels of inflammation and immunosuppression than MWCNTs-PEG. The results of immunological function suggested that after intravenous administration with p-MWCNTs caused more damage to systemic immunity than MWCNTs-PEG. Here, we demonstrated that a surface functional modification on MWCNTs reduces their immune perturbations in vivo. The chemistry-modified MWCNTs change their preferred immune response in vivo and reduce the immunotoxicity of p-MWCNTs.

Keywords: BALB/c mice; immunosuppression; immunotoxicity; multi-walled carbon nanotubes; surface-functionalized.

Figure 1

Transmission electron microscopy image of pristine and functionalized MWCNTs. Notes: (A) p-MWCNTs and (B) MWCNTs-PEG. Abbreviations: p-MWCNTs, pristine multi-walled carbon nanotubes; MWCNTs-PEG, PEGylated multi-walled carbon nanotubes.

Figure 2

Gross observation of hypertrophy of inguinal lymph nodes (A), and congestive and edematous changes of the lung (B), liver (C), and spleen (D). Abbreviations: PBST, phosphate buffer containing 0.5% Tween-80; MWCNTs-PEG, PEGylated multi-walled carbon nanotubes; MWCNTs, multi-walled carbon nanotubes.

Figure 3

Organ coefficient of mice after 28 days of intravenous treatment with p-MWCNTs and MWCNTs-PEG. Notes: Data represent the mean ± SD of six mice. *Values significantly different from control results (P<0.05). Abbreviations: p-MWCNTs, pristine multi-walled carbon nanotubes; MWCNTs-PEG, PEGylated multi-walled carbon nanotubes; SD, standard deviation; PBST, phosphate buffer containing 0.5% Tween-80.

Figure 4

Microscopic images showing the histopathological changes in tissues samples (with a magnification of expansion ×200). Notes: (A) p-MWCNTs aggregated in the tail vein of the injection site and numerous cells became black after engulfing the injected MWCNTs; (B) lung pathological lesions in a dose-dependent manner after intravenous administration of high and medium doses of p-MWCNTs; (C) livers showed some loss of sinusoid space and hydropic degeneration with minor fatty change at the highest p-MWCNTs concentration. Abbreviations: p-MWCNTs, pristine multi-walled carbon nanotubes; MWCNTs, multi-walled carbon nanotubes; PBST, phosphate buffer containing 0.5% Tween-80.

Figure 5

Transmission electron microscope micrographs from the spleen after two types of MWCNTs administration and the control group. Notes: Splenocyte in the control group contained round nucleus with homogeneous chromatin (A); splenocyte from the 0.5 mg/kg p-MWCNT group indicates mitochondria swelling (B), such as nucleus chromatin condensation and vacuolization in splenocyte. Details of the macrophage cytoplasm showing internalized MWCNT are presented in image. Abbreviations: p-MWCNTs, pristine multi-walled carbon nanotubes; MWCNTs, multi-walled carbon nanotubes; PBST, phosphate buffer containing 0.5% Tween-80.

Figure 6

Effects of p-MWCNTs and MWCNTs-PEG on the secretion of IgG (A) and IgM (B) in the serum. Notes: Data represent the mean ± SD of six mice. *Values significantly different from control results (P<0.05). Abbreviations: SD, standard deviation; MWCNTs, multi-walled carbon nanotubes; MWCNTs-PEG, PEGylated multi-walled carbon nanotubes; PBST, phosphate buffer containing 0.5% Tween-80.