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Modulation of apoptotic pathways of macrophages by surface-functionalized multi-walled carbon nanotubes.

Jiang Y, Zhang H, Wang Y, Chen M, Ye S, Hou Z, Ren L - PLoS ONE (2013)

Bottom Line: However, the potential adverse effects of surface-functionalized CNTs have not been well characterized.The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB.These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomaterials, College of Materials, Xiamen University, Xiamen, China.

ABSTRACT
Biomedical applications of carbon nanotubes (CNTs) often involve improving their hydrophilicity and dispersion in biological media by modifying them through noncovalent or covalent functionalization. However, the potential adverse effects of surface-functionalized CNTs have not been well characterized. In this study, we functionalized multi-walled CNTs (MWCNTs) via carboxylation, to produce MWCNTs-COOH, and via poly (ethylene glycol) linking, to produce MWCNTs-PEG. We used these functionalized MWCNTs to study the effect of surface functionalization on MWCNTs-induced toxicity to macrophages, and elucidate the underlying mechanisms of action. Our results revealed that MWCNTs-PEG were less cytotoxic and were associated with less apoptotic cell death of macrophages than MWCNTs-COOH. Additionally, MWCNTs-PEG induced less generation of reactive oxygen species (ROS) involving less activation of NADPH oxidase compared with MWCNTs-COOH, as evidenced by membrane translocation of p47(phox) and p67(phox) in macrophages. The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB. These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways. Our study thus provides new insights into the molecular basis for the surface properties affecting CNTs toxicity.

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MWCNTs-COOH and MWCNTs-PEG induced a mitochondria-associated death pathway in RAW 264.7 cells.(A, B) MWCNTs-COOH and MWCNTs-PEG induced the loss of ΔΨm in RAW 264.7 cells. Cells treated with or without 75 µg/mL of MWCNT samples for the indicated times were stained with the mitochondria-selective JC-1 dye, and then analyzed by flow cytometry and fluorescence microscope. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. *p<0.05 compared to control sample, #p<0.05 compared to MWCNTs-PEG. (C) Western blot analysis of the effects of MWCNTs-COOH and MWCNTs-PEG on translocation of cyto c, and expression of Bax and Bcl-2 in RAW 264.7 cells. Cells were treated with or without 75 µg/mL of MWCNT samples for 24 h. Data represent similar results from three independent experiments.
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pone-0065756-g006: MWCNTs-COOH and MWCNTs-PEG induced a mitochondria-associated death pathway in RAW 264.7 cells.(A, B) MWCNTs-COOH and MWCNTs-PEG induced the loss of ΔΨm in RAW 264.7 cells. Cells treated with or without 75 µg/mL of MWCNT samples for the indicated times were stained with the mitochondria-selective JC-1 dye, and then analyzed by flow cytometry and fluorescence microscope. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. *p<0.05 compared to control sample, #p<0.05 compared to MWCNTs-PEG. (C) Western blot analysis of the effects of MWCNTs-COOH and MWCNTs-PEG on translocation of cyto c, and expression of Bax and Bcl-2 in RAW 264.7 cells. Cells were treated with or without 75 µg/mL of MWCNT samples for 24 h. Data represent similar results from three independent experiments.

Mentions: Using JC-1 as a marker of ΔΨm, flow cytometry revealed that MWCNTs-COOH depolarized the level of ΔΨm in RAW 264.7 cells. The response occurred in a time-dependent manner, and a significant depletion of ΔΨm could be detected within 2 h of treatment (Figure 6A, B). Furthermore, western blot analysis showed that MWCNTs-COOH induced the release of cytochrome c from the mitochondria to the cytosol, suppressed the expression of pro-survival Bcl-2 proteins (such as Bcl-2), and increased the expression of pro-apoptosis Bcl-2 proteins (such as Bax) (Figure 6C, D). In contrast, MWCNTs-PEG had less effect on mitochondria-associated apoptotic components (Figure 6A-D).


Modulation of apoptotic pathways of macrophages by surface-functionalized multi-walled carbon nanotubes.

Jiang Y, Zhang H, Wang Y, Chen M, Ye S, Hou Z, Ren L - PLoS ONE (2013)

MWCNTs-COOH and MWCNTs-PEG induced a mitochondria-associated death pathway in RAW 264.7 cells.(A, B) MWCNTs-COOH and MWCNTs-PEG induced the loss of ΔΨm in RAW 264.7 cells. Cells treated with or without 75 µg/mL of MWCNT samples for the indicated times were stained with the mitochondria-selective JC-1 dye, and then analyzed by flow cytometry and fluorescence microscope. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. *p<0.05 compared to control sample, #p<0.05 compared to MWCNTs-PEG. (C) Western blot analysis of the effects of MWCNTs-COOH and MWCNTs-PEG on translocation of cyto c, and expression of Bax and Bcl-2 in RAW 264.7 cells. Cells were treated with or without 75 µg/mL of MWCNT samples for 24 h. Data represent similar results from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675050&req=5

pone-0065756-g006: MWCNTs-COOH and MWCNTs-PEG induced a mitochondria-associated death pathway in RAW 264.7 cells.(A, B) MWCNTs-COOH and MWCNTs-PEG induced the loss of ΔΨm in RAW 264.7 cells. Cells treated with or without 75 µg/mL of MWCNT samples for the indicated times were stained with the mitochondria-selective JC-1 dye, and then analyzed by flow cytometry and fluorescence microscope. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. *p<0.05 compared to control sample, #p<0.05 compared to MWCNTs-PEG. (C) Western blot analysis of the effects of MWCNTs-COOH and MWCNTs-PEG on translocation of cyto c, and expression of Bax and Bcl-2 in RAW 264.7 cells. Cells were treated with or without 75 µg/mL of MWCNT samples for 24 h. Data represent similar results from three independent experiments.
Mentions: Using JC-1 as a marker of ΔΨm, flow cytometry revealed that MWCNTs-COOH depolarized the level of ΔΨm in RAW 264.7 cells. The response occurred in a time-dependent manner, and a significant depletion of ΔΨm could be detected within 2 h of treatment (Figure 6A, B). Furthermore, western blot analysis showed that MWCNTs-COOH induced the release of cytochrome c from the mitochondria to the cytosol, suppressed the expression of pro-survival Bcl-2 proteins (such as Bcl-2), and increased the expression of pro-apoptosis Bcl-2 proteins (such as Bax) (Figure 6C, D). In contrast, MWCNTs-PEG had less effect on mitochondria-associated apoptotic components (Figure 6A-D).

Bottom Line: However, the potential adverse effects of surface-functionalized CNTs have not been well characterized.The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB.These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomaterials, College of Materials, Xiamen University, Xiamen, China.

ABSTRACT
Biomedical applications of carbon nanotubes (CNTs) often involve improving their hydrophilicity and dispersion in biological media by modifying them through noncovalent or covalent functionalization. However, the potential adverse effects of surface-functionalized CNTs have not been well characterized. In this study, we functionalized multi-walled CNTs (MWCNTs) via carboxylation, to produce MWCNTs-COOH, and via poly (ethylene glycol) linking, to produce MWCNTs-PEG. We used these functionalized MWCNTs to study the effect of surface functionalization on MWCNTs-induced toxicity to macrophages, and elucidate the underlying mechanisms of action. Our results revealed that MWCNTs-PEG were less cytotoxic and were associated with less apoptotic cell death of macrophages than MWCNTs-COOH. Additionally, MWCNTs-PEG induced less generation of reactive oxygen species (ROS) involving less activation of NADPH oxidase compared with MWCNTs-COOH, as evidenced by membrane translocation of p47(phox) and p67(phox) in macrophages. The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB. These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways. Our study thus provides new insights into the molecular basis for the surface properties affecting CNTs toxicity.

Show MeSH
Related in: MedlinePlus