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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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Cellular uptake and distribution of MWCNTs-COOH and MWCNTs-PEG.(A) Quantitative analysis of cellular uptake of MWCNTs-COOH and MWCNTs-PEG by RAW 264.7 cells as shown by flow cytometry using the light SSC parameter. Cells were treated with or without f-MWCNT samples for the indicated time and assayed for SSC intensity by flow cytometry. 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. (B) Intracellular distribution of MWCNTs-COOH and MWCNTs-PEG in RAW 264.7 cells viewed under a fluorescence microscope. Cells were incubated with or without FITC-labeled f-MWCNTs samples for 12 h, and then processed for CLSM examination.
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pone-0065756-g003: Cellular uptake and distribution of MWCNTs-COOH and MWCNTs-PEG.(A) Quantitative analysis of cellular uptake of MWCNTs-COOH and MWCNTs-PEG by RAW 264.7 cells as shown by flow cytometry using the light SSC parameter. Cells were treated with or without f-MWCNT samples for the indicated time and assayed for SSC intensity by flow cytometry. 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. (B) Intracellular distribution of MWCNTs-COOH and MWCNTs-PEG in RAW 264.7 cells viewed under a fluorescence microscope. Cells were incubated with or without FITC-labeled f-MWCNTs samples for 12 h, and then processed for CLSM examination.

Mentions: It has been proposed that the intracellular concentration of nanoparticles is reflected in the intensity of light SSC measured by flow cytometry [21]. Using this experimental approach, a time-dependent increase in the light SSC of cells was detected after treatment with MWCNTs-COOH, which began as early as 2 h (Figure 3A, B). In contrast, MWCNTs-PEG showed less intensity of light SSC following treatment (Figure 3A, B) compared with MWCNTs-COOH. Next, we studied the intracellular distribution of the f-MWCNTs in RAW 264.7 cells. Confocal laser scanning microscopy (CLSM) analysis using FITC-labeled f-MWCNTs revealed that the fluorescence signals were located mainly in the cytoplasm within 12 h of incubation, indicating the lack of f-MWCNTs translocating to the nucleus. Consistent with the intensity of SSC, MWCNTs-COOH showed stronger fluorescence compared with MWCNTs-PEG. The cell uptake and distribution of f-MWCNTs was directly confirmed using optical microscopy (Figure S1).


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)

Cellular uptake and distribution of MWCNTs-COOH and MWCNTs-PEG.(A) Quantitative analysis of cellular uptake of MWCNTs-COOH and MWCNTs-PEG by RAW 264.7 cells as shown by flow cytometry using the light SSC parameter. Cells were treated with or without f-MWCNT samples for the indicated time and assayed for SSC intensity by flow cytometry. 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. (B) Intracellular distribution of MWCNTs-COOH and MWCNTs-PEG in RAW 264.7 cells viewed under a fluorescence microscope. Cells were incubated with or without FITC-labeled f-MWCNTs samples for 12 h, and then processed for CLSM examination.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675050&req=5

pone-0065756-g003: Cellular uptake and distribution of MWCNTs-COOH and MWCNTs-PEG.(A) Quantitative analysis of cellular uptake of MWCNTs-COOH and MWCNTs-PEG by RAW 264.7 cells as shown by flow cytometry using the light SSC parameter. Cells were treated with or without f-MWCNT samples for the indicated time and assayed for SSC intensity by flow cytometry. 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. (B) Intracellular distribution of MWCNTs-COOH and MWCNTs-PEG in RAW 264.7 cells viewed under a fluorescence microscope. Cells were incubated with or without FITC-labeled f-MWCNTs samples for 12 h, and then processed for CLSM examination.
Mentions: It has been proposed that the intracellular concentration of nanoparticles is reflected in the intensity of light SSC measured by flow cytometry [21]. Using this experimental approach, a time-dependent increase in the light SSC of cells was detected after treatment with MWCNTs-COOH, which began as early as 2 h (Figure 3A, B). In contrast, MWCNTs-PEG showed less intensity of light SSC following treatment (Figure 3A, B) compared with MWCNTs-COOH. Next, we studied the intracellular distribution of the f-MWCNTs in RAW 264.7 cells. Confocal laser scanning microscopy (CLSM) analysis using FITC-labeled f-MWCNTs revealed that the fluorescence signals were located mainly in the cytoplasm within 12 h of incubation, indicating the lack of f-MWCNTs translocating to the nucleus. Consistent with the intensity of SSC, MWCNTs-COOH showed stronger fluorescence compared with MWCNTs-PEG. The cell uptake and distribution of f-MWCNTs was directly confirmed using optical microscopy (Figure S1).

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