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High dispersity of carbon nanotubes diminishes immunotoxicity in spleen.

Lee S, Khang D, Kim SH - Int J Nanomedicine (2015)

Bottom Line: For cytotoxicity of swCNTs, MTT assay, reactive oxygen species production, superoxide dismutase activity, cellular uptake, and confocal microscopy were used in macrophages.In short, less-dispersed swCNTs caused cytotoxicity in macrophages and abnormalities in immune organs such as spleen, whereas highly dispersed swCNTs did not result in immunotoxicity.Our findings clarified the effective immunotoxicological factors of swCNTs by increasing dispersity of swCNTs and provided useful guidelines for the effective use of nanomaterials.

View Article: PubMed Central - PubMed

Affiliation: CMRI, Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea.

ABSTRACT

Background: From the various physiochemical material properties, the chemical functionalization order of single-walled carbon nanotubes (swCNTs) has not been considered as a critical factor for modulating immunological responses and toxicological aspects in drug delivery applications. Although most nanomaterials, including carbon nanotubes, are specifically accumulated in spleen, few studies have focused on spleen immunotoxicity. For this reason, this study demonstrated that the dispersity of swCNTs significantly influenced immunotoxicity in vitro and in vivo.

Materials and methods: For cytotoxicity of swCNTs, MTT assay, reactive oxygen species production, superoxide dismutase activity, cellular uptake, and confocal microscopy were used in macrophages. In the in vivo study, female BALB/c mice were intravenously administered with 1 mg/kg/day of swCNTs for 2 weeks. The body weight, organ weight, hematological change, reverse-transcription polymerase chain reaction, and lymphocyte population were evaluated.

Results: Different orders of chemical functionalization of swCNTs controlled immunotoxicity. In short, less-dispersed swCNTs caused cytotoxicity in macrophages and abnormalities in immune organs such as spleen, whereas highly dispersed swCNTs did not result in immunotoxicity.

Conclusion: This study clarified that increasing carboxyl groups on swCNTs significantly mitigated immunotoxicity in vitro and in vivo. Our findings clarified the effective immunotoxicological factors of swCNTs by increasing dispersity of swCNTs and provided useful guidelines for the effective use of nanomaterials.

No MeSH data available.


Related in: MedlinePlus

Effects of dispersed single-walled carbon nanotubes on cytotoxicity and level of cellular uptake in macrophages.Notes: (A) Cells (2×104 cells/well in 96-well plates) were treated with various concentrations of dispersed swCNTs. After 24 hours of treatment, cell viability was determined using the MTT assay. Hydrogen peroxide (500 μM) was used as a positive control. Cell viability was determined by the relative absorbance compared to control. (B, C) Cells (2×105 cells/well in 4-well plates) were treated with 1 μg/mL swCNTs for 24 hours. After treatment with swCNTs, the cells were stained with F-actin (green) and DAPI (blue). Images showed intracellular location of swCNTs (black). The fluorescence was visualized using (B) densitometric analysis and (C) confocal microscopy (×400). The results are presented as mean ± SE of three independent experiments. *P<0.05 significantly different from control.Abbreviations: CON, control; DAPI, 4′,6-diamidino-2-phenylindole; MTT, 3(4, 5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; SE, standard error; swCNT, single-walled carbon nanotube; XPS, X-ray photoelectron spectroscopy.
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f2-ijn-10-2697: Effects of dispersed single-walled carbon nanotubes on cytotoxicity and level of cellular uptake in macrophages.Notes: (A) Cells (2×104 cells/well in 96-well plates) were treated with various concentrations of dispersed swCNTs. After 24 hours of treatment, cell viability was determined using the MTT assay. Hydrogen peroxide (500 μM) was used as a positive control. Cell viability was determined by the relative absorbance compared to control. (B, C) Cells (2×105 cells/well in 4-well plates) were treated with 1 μg/mL swCNTs for 24 hours. After treatment with swCNTs, the cells were stained with F-actin (green) and DAPI (blue). Images showed intracellular location of swCNTs (black). The fluorescence was visualized using (B) densitometric analysis and (C) confocal microscopy (×400). The results are presented as mean ± SE of three independent experiments. *P<0.05 significantly different from control.Abbreviations: CON, control; DAPI, 4′,6-diamidino-2-phenylindole; MTT, 3(4, 5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; SE, standard error; swCNT, single-walled carbon nanotube; XPS, X-ray photoelectron spectroscopy.

Mentions: Viability of macrophages (J774A.1) was analyzed using MTT assay to investigate whether differently dispersed swCNTs induce different levels of cytotoxicity after 24 hours exposure (Figure 2A). The COOH-max (highly dispersed) particles were less toxic than the COOH-low and COOH-mid particles and showed the lowest cytotoxicity toward macrophages. Hydrogen peroxide was used as a positive control. In addition, we observed the cellular uptake of dispersed swCNTs by densitometric analysis (Figure 2B) and confocal microscopy (Figure 2C). Cells were treated with each dispersed swCNT (1 μg/mL) for 24 hours and stained with F-actin-FITC and DAPI for actin and nuclei, respectively. Confocal microscopy showed that differently dispersed swCNTs were similarly translocated into the cytoplasm (Figures 2C and S2). These results suggest that each dispersed swCNT (COOH-low, -mid, and -high) induced different cytotoxicities, whereas differently dispersed swCNTs were similarly translocated into the cytoplasm after cellular uptake.


High dispersity of carbon nanotubes diminishes immunotoxicity in spleen.

Lee S, Khang D, Kim SH - Int J Nanomedicine (2015)

Effects of dispersed single-walled carbon nanotubes on cytotoxicity and level of cellular uptake in macrophages.Notes: (A) Cells (2×104 cells/well in 96-well plates) were treated with various concentrations of dispersed swCNTs. After 24 hours of treatment, cell viability was determined using the MTT assay. Hydrogen peroxide (500 μM) was used as a positive control. Cell viability was determined by the relative absorbance compared to control. (B, C) Cells (2×105 cells/well in 4-well plates) were treated with 1 μg/mL swCNTs for 24 hours. After treatment with swCNTs, the cells were stained with F-actin (green) and DAPI (blue). Images showed intracellular location of swCNTs (black). The fluorescence was visualized using (B) densitometric analysis and (C) confocal microscopy (×400). The results are presented as mean ± SE of three independent experiments. *P<0.05 significantly different from control.Abbreviations: CON, control; DAPI, 4′,6-diamidino-2-phenylindole; MTT, 3(4, 5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; SE, standard error; swCNT, single-walled carbon nanotube; XPS, X-ray photoelectron spectroscopy.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
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f2-ijn-10-2697: Effects of dispersed single-walled carbon nanotubes on cytotoxicity and level of cellular uptake in macrophages.Notes: (A) Cells (2×104 cells/well in 96-well plates) were treated with various concentrations of dispersed swCNTs. After 24 hours of treatment, cell viability was determined using the MTT assay. Hydrogen peroxide (500 μM) was used as a positive control. Cell viability was determined by the relative absorbance compared to control. (B, C) Cells (2×105 cells/well in 4-well plates) were treated with 1 μg/mL swCNTs for 24 hours. After treatment with swCNTs, the cells were stained with F-actin (green) and DAPI (blue). Images showed intracellular location of swCNTs (black). The fluorescence was visualized using (B) densitometric analysis and (C) confocal microscopy (×400). The results are presented as mean ± SE of three independent experiments. *P<0.05 significantly different from control.Abbreviations: CON, control; DAPI, 4′,6-diamidino-2-phenylindole; MTT, 3(4, 5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; SE, standard error; swCNT, single-walled carbon nanotube; XPS, X-ray photoelectron spectroscopy.
Mentions: Viability of macrophages (J774A.1) was analyzed using MTT assay to investigate whether differently dispersed swCNTs induce different levels of cytotoxicity after 24 hours exposure (Figure 2A). The COOH-max (highly dispersed) particles were less toxic than the COOH-low and COOH-mid particles and showed the lowest cytotoxicity toward macrophages. Hydrogen peroxide was used as a positive control. In addition, we observed the cellular uptake of dispersed swCNTs by densitometric analysis (Figure 2B) and confocal microscopy (Figure 2C). Cells were treated with each dispersed swCNT (1 μg/mL) for 24 hours and stained with F-actin-FITC and DAPI for actin and nuclei, respectively. Confocal microscopy showed that differently dispersed swCNTs were similarly translocated into the cytoplasm (Figures 2C and S2). These results suggest that each dispersed swCNT (COOH-low, -mid, and -high) induced different cytotoxicities, whereas differently dispersed swCNTs were similarly translocated into the cytoplasm after cellular uptake.

Bottom Line: For cytotoxicity of swCNTs, MTT assay, reactive oxygen species production, superoxide dismutase activity, cellular uptake, and confocal microscopy were used in macrophages.In short, less-dispersed swCNTs caused cytotoxicity in macrophages and abnormalities in immune organs such as spleen, whereas highly dispersed swCNTs did not result in immunotoxicity.Our findings clarified the effective immunotoxicological factors of swCNTs by increasing dispersity of swCNTs and provided useful guidelines for the effective use of nanomaterials.

View Article: PubMed Central - PubMed

Affiliation: CMRI, Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea.

ABSTRACT

Background: From the various physiochemical material properties, the chemical functionalization order of single-walled carbon nanotubes (swCNTs) has not been considered as a critical factor for modulating immunological responses and toxicological aspects in drug delivery applications. Although most nanomaterials, including carbon nanotubes, are specifically accumulated in spleen, few studies have focused on spleen immunotoxicity. For this reason, this study demonstrated that the dispersity of swCNTs significantly influenced immunotoxicity in vitro and in vivo.

Materials and methods: For cytotoxicity of swCNTs, MTT assay, reactive oxygen species production, superoxide dismutase activity, cellular uptake, and confocal microscopy were used in macrophages. In the in vivo study, female BALB/c mice were intravenously administered with 1 mg/kg/day of swCNTs for 2 weeks. The body weight, organ weight, hematological change, reverse-transcription polymerase chain reaction, and lymphocyte population were evaluated.

Results: Different orders of chemical functionalization of swCNTs controlled immunotoxicity. In short, less-dispersed swCNTs caused cytotoxicity in macrophages and abnormalities in immune organs such as spleen, whereas highly dispersed swCNTs did not result in immunotoxicity.

Conclusion: This study clarified that increasing carboxyl groups on swCNTs significantly mitigated immunotoxicity in vitro and in vivo. Our findings clarified the effective immunotoxicological factors of swCNTs by increasing dispersity of swCNTs and provided useful guidelines for the effective use of nanomaterials.

No MeSH data available.


Related in: MedlinePlus