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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 serum WBC count and the spleen.Notes: Various swCNTs (1 mg/kg/day) were administered to mice by intravenous injection through the tail for 2 weeks. (A) At the end of animal treatment, the mice (n=10) were fasted overnight and euthanized using carbon dioxide. Serum was collected, and WBC count was obtained using a hemocytometer after lysis of RBCs. (B) Flow cytometry analysis of T lymphocyte (CD3+) populations. T-cell population comprised the absolute number of cells that expressed CD3+. (C) Splenocytes were isolated and cultured into 96-well plates containing Con A (10 μg/mL) for 72 hours. Cell proliferation is represented by the relative absorbance from Con A-stimulated control splenocytes. Gene expression of (D) IL-2, (E) IL-1β, and (F) TNF-α in the spleen was analyzed using real-time PCR. The results are presented as mean ± SE (n=10). *P<0.05 significantly different from control.Abbreviations: CON, control; Con A, Concanavalin A; IL, interleukin; PCR, polymerase chain reaction; RBC, red blood cell; SE, standard error; swCNT, single-walled carbon nanotube; TNF, tumor necrosis factor; WBC, white blood cell.
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f5-ijn-10-2697: Effects of dispersed single-walled carbon nanotubes on serum WBC count and the spleen.Notes: Various swCNTs (1 mg/kg/day) were administered to mice by intravenous injection through the tail for 2 weeks. (A) At the end of animal treatment, the mice (n=10) were fasted overnight and euthanized using carbon dioxide. Serum was collected, and WBC count was obtained using a hemocytometer after lysis of RBCs. (B) Flow cytometry analysis of T lymphocyte (CD3+) populations. T-cell population comprised the absolute number of cells that expressed CD3+. (C) Splenocytes were isolated and cultured into 96-well plates containing Con A (10 μg/mL) for 72 hours. Cell proliferation is represented by the relative absorbance from Con A-stimulated control splenocytes. Gene expression of (D) IL-2, (E) IL-1β, and (F) TNF-α in the spleen was analyzed using real-time PCR. The results are presented as mean ± SE (n=10). *P<0.05 significantly different from control.Abbreviations: CON, control; Con A, Concanavalin A; IL, interleukin; PCR, polymerase chain reaction; RBC, red blood cell; SE, standard error; swCNT, single-walled carbon nanotube; TNF, tumor necrosis factor; WBC, white blood cell.

Mentions: To confirm the cytotoxicity of differently dispersed swCNTs, an in vivo experiment was performed to analyze the immunotoxicity of dispersed swCNTs (1 mg/kg/day, administered by intravenous injection during 2 weeks). Neither mean body weight (Figure S4A) nor food and water consumption (data not shown) was altered by swCNTs treatment. No significant changes in tissue weights were detected in given administration (Figure S4B). However, the white blood cell (WBC) counts in the serum decreased significantly upon exposure to COOH-low particles, but remained at normal levels for COOH-max (Figure 5A). Because WBCs help defend the body against pathogen invasion, a change in the serum WBC count implies an alteration of the immune response. Furthermore, exposure to swCNTs altered the T lymphocyte population in the spleen (Figure 5B). The spleen, a reticuloendothelial system organ, is a part of the immune system that consists of phagocytic cells located in reticular connective tissue. The spleen is highly sensitive to damage by xenobiotics, and the lymphocyte population of the spleen is important in the immune response.36,37 Significant increases in both CD4+ and CD8+ T cells 1 day following intravenously injection of multiwall CNTs (mwCNTs) have been reported, as well as the continued elevation of CD4+ T cells through 7 days.38 As shown in Figure 5B, although the T lymphocyte population was increased by all dispersed swCNTs, the COOH-max produced a lesser effect on the T lymphocyte population than that produced by the COOH-low and COOH-mid. In addition, a splenocyte proliferation assay showed similar effects on the T lymphocyte population (Figure 5C). Other studies indicate good tolerability and no tissue accumulation for water-dispersible highly functionalized mwCNTs in mice. In this regard, a higher degree of functionalization leads to less retention of the nanotubes in tissues.39 Therefore, our results suggest that less-dispersed swCNTs increase immunotoxicity, but use of highly dispersed swCNTs (COOH-max) shows lower level of immune function.


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 serum WBC count and the spleen.Notes: Various swCNTs (1 mg/kg/day) were administered to mice by intravenous injection through the tail for 2 weeks. (A) At the end of animal treatment, the mice (n=10) were fasted overnight and euthanized using carbon dioxide. Serum was collected, and WBC count was obtained using a hemocytometer after lysis of RBCs. (B) Flow cytometry analysis of T lymphocyte (CD3+) populations. T-cell population comprised the absolute number of cells that expressed CD3+. (C) Splenocytes were isolated and cultured into 96-well plates containing Con A (10 μg/mL) for 72 hours. Cell proliferation is represented by the relative absorbance from Con A-stimulated control splenocytes. Gene expression of (D) IL-2, (E) IL-1β, and (F) TNF-α in the spleen was analyzed using real-time PCR. The results are presented as mean ± SE (n=10). *P<0.05 significantly different from control.Abbreviations: CON, control; Con A, Concanavalin A; IL, interleukin; PCR, polymerase chain reaction; RBC, red blood cell; SE, standard error; swCNT, single-walled carbon nanotube; TNF, tumor necrosis factor; WBC, white blood cell.
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Related In: Results  -  Collection

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f5-ijn-10-2697: Effects of dispersed single-walled carbon nanotubes on serum WBC count and the spleen.Notes: Various swCNTs (1 mg/kg/day) were administered to mice by intravenous injection through the tail for 2 weeks. (A) At the end of animal treatment, the mice (n=10) were fasted overnight and euthanized using carbon dioxide. Serum was collected, and WBC count was obtained using a hemocytometer after lysis of RBCs. (B) Flow cytometry analysis of T lymphocyte (CD3+) populations. T-cell population comprised the absolute number of cells that expressed CD3+. (C) Splenocytes were isolated and cultured into 96-well plates containing Con A (10 μg/mL) for 72 hours. Cell proliferation is represented by the relative absorbance from Con A-stimulated control splenocytes. Gene expression of (D) IL-2, (E) IL-1β, and (F) TNF-α in the spleen was analyzed using real-time PCR. The results are presented as mean ± SE (n=10). *P<0.05 significantly different from control.Abbreviations: CON, control; Con A, Concanavalin A; IL, interleukin; PCR, polymerase chain reaction; RBC, red blood cell; SE, standard error; swCNT, single-walled carbon nanotube; TNF, tumor necrosis factor; WBC, white blood cell.
Mentions: To confirm the cytotoxicity of differently dispersed swCNTs, an in vivo experiment was performed to analyze the immunotoxicity of dispersed swCNTs (1 mg/kg/day, administered by intravenous injection during 2 weeks). Neither mean body weight (Figure S4A) nor food and water consumption (data not shown) was altered by swCNTs treatment. No significant changes in tissue weights were detected in given administration (Figure S4B). However, the white blood cell (WBC) counts in the serum decreased significantly upon exposure to COOH-low particles, but remained at normal levels for COOH-max (Figure 5A). Because WBCs help defend the body against pathogen invasion, a change in the serum WBC count implies an alteration of the immune response. Furthermore, exposure to swCNTs altered the T lymphocyte population in the spleen (Figure 5B). The spleen, a reticuloendothelial system organ, is a part of the immune system that consists of phagocytic cells located in reticular connective tissue. The spleen is highly sensitive to damage by xenobiotics, and the lymphocyte population of the spleen is important in the immune response.36,37 Significant increases in both CD4+ and CD8+ T cells 1 day following intravenously injection of multiwall CNTs (mwCNTs) have been reported, as well as the continued elevation of CD4+ T cells through 7 days.38 As shown in Figure 5B, although the T lymphocyte population was increased by all dispersed swCNTs, the COOH-max produced a lesser effect on the T lymphocyte population than that produced by the COOH-low and COOH-mid. In addition, a splenocyte proliferation assay showed similar effects on the T lymphocyte population (Figure 5C). Other studies indicate good tolerability and no tissue accumulation for water-dispersible highly functionalized mwCNTs in mice. In this regard, a higher degree of functionalization leads to less retention of the nanotubes in tissues.39 Therefore, our results suggest that less-dispersed swCNTs increase immunotoxicity, but use of highly dispersed swCNTs (COOH-max) shows lower level of immune function.

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