High dispersity of carbon nanotubes diminishes immunotoxicity in spleen

Soyoung Lee1 CMRI, Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea

Dongwoo Khang2 Department of Molecular Medicine, Graduate School of Medicine, Gachon University, Incheon, Republic of KoreaCorrespondence[email protected]

Sang-Hyun Kim1 CMRI, Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Republic of KoreaCorrespondence[email protected]

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.

Keywords:

Supplementary materials

Figure S1 Carboxyl functionalization (FTIR) and amount of functionalization on swCNTs (thermal gravimetric analysis).

Notes: (A) FTIR signals for functionalized swCNTs showed increased carboxylate bonds (C=O) at 1,720 (cm⁻¹), whereas unmodified swCNTs did not exhibit any notable peaks around the C=O energy. Signals from crystalline carbon lattice structures (C=C) diminished after acidic treatment (for functionalization) as identified around 1,600 (cm⁻¹). (B) The carboxyl weight percentages of COOH-low, COOH-mid, and COOH-max were 12%, 21%, and 22% (at 600°C), respectively.

Abbreviations: FTIR, Fourier transform infrared spectroscopy; swCNT, single-walled carbon nanotube.

Figure S1 Carboxyl functionalization (FTIR) and amount of functionalization on swCNTs (thermal gravimetric analysis).Notes: (A) FTIR signals for functionalized swCNTs showed increased carboxylate bonds (C=O) at 1,720 (cm−1), whereas unmodified swCNTs did not exhibit any notable peaks around the C=O energy. Signals from crystalline carbon lattice structures (C=C) diminished after acidic treatment (for functionalization) as identified around 1,600 (cm−1). (B) The carboxyl weight percentages of COOH-low, COOH-mid, and COOH-max were 12%, 21%, and 22% (at 600°C), respectively.Abbreviations: FTIR, Fourier transform infrared spectroscopy; swCNT, single-walled carbon nanotube.

Figure S2 Macrophage uptake of differently dispersed single-walled carbon nanotubes.

Notes: 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). Most of swCNTs were sufficiently uptaken by macrophages, regardless of carboxylation order. The fluorescence was visualized using confocal microscopy (×200).

Abbreviations: CON, control; DAPI, 4′,6-diamidino-2-phenylindole; swCNT, single-walled carbon nanotube.

Figure S2 Macrophage uptake of differently dispersed single-walled carbon nanotubes.Notes: 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). Most of swCNTs were sufficiently uptaken by macrophages, regardless of carboxylation order. The fluorescence was visualized using confocal microscopy (×200).Abbreviations: CON, control; DAPI, 4′,6-diamidino-2-phenylindole; swCNT, single-walled carbon nanotube.

Figure S3 Effects of dispersed single-wall carbon nanotubes on Jurkat and THP-1 cytotoxicity.

Notes: (A, B) Jurkat cells. (C, D) THP-1 cells. For cell viability, 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. For ROS production, cell viability was determined by the relative absorbance compared to control. Cells (2×10⁴ cells/well in 96-well plates) were treated with 1 μg/mL swCNTs for 24 hours. After treatment, the cells were stained with 10 μM DHR 123 for 30 minutes. Production of ROS was determined using DHR 123 staining. The fluorescent intensity of DHR was recorded using a fluorescent plate reader. FeSO₄ (100 μM) was used as a positive control. The results are presented as mean ± SE of three independent experiments. *P<0.05 significantly different from control.

Abbreviations: CON, control; DHR, dihydrorhodamine; MTT, 3(4,5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide; ROS, reactive oxygen species; SE, standard error; SOD, superoxide dismutase; swCNT, single-walled carbon nanotube.

Figure S3 Effects of dispersed single-wall carbon nanotubes on Jurkat and THP-1 cytotoxicity.Notes: (A, B) Jurkat cells. (C, D) THP-1 cells. For cell viability, 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. For ROS production, cell viability was determined by the relative absorbance compared to control. Cells (2×104 cells/well in 96-well plates) were treated with 1 μg/mL swCNTs for 24 hours. After treatment, the cells were stained with 10 μM DHR 123 for 30 minutes. Production of ROS was determined using DHR 123 staining. The fluorescent intensity of DHR was recorded using a fluorescent plate reader. FeSO4 (100 μM) was used as a positive control. The results are presented as mean ± SE of three independent experiments. *P<0.05 significantly different from control.Abbreviations: CON, control; DHR, dihydrorhodamine; MTT, 3(4,5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide; ROS, reactive oxygen species; SE, standard error; SOD, superoxide dismutase; swCNT, single-walled carbon nanotube.

Figure S4 Body and tissue weight changes by dispersed swCNTs.

Notes: (A) Body weight. (B) Tissue weight. Various swCNTs (1 mg/kg/day) were administered to mice by intravenous injection through the tail for 2 weeks. At the end of the treatment period, the mice were fasted overnight and euthanized using carbon dioxide. Various organs were aseptically excised and weighed. The results are presented as mean ± SE (n=10).

Abbreviations: CON, control; SE, standard error; swCNT, single-walled carbon nanotube.

Figure S4 Body and tissue weight changes by dispersed swCNTs.Notes: (A) Body weight. (B) Tissue weight. Various swCNTs (1 mg/kg/day) were administered to mice by intravenous injection through the tail for 2 weeks. At the end of the treatment period, the mice were fasted overnight and euthanized using carbon dioxide. Various organs were aseptically excised and weighed. The results are presented as mean ± SE (n=10).Abbreviations: CON, control; SE, standard error; swCNT, single-walled carbon nanotube.

Acknowledgments

This research was supported by grants from the Ministry of Food and Drug Safety of Korea (13182MFDS607), the National Research Foundation of Korea (2014R1A5A2009242, 2013R1A1A3009525, 2012M3A9B6055416, 2012R1A1A2041157, and 2014R1A2A1A11052615), and the Korea Health Technology R&D Project through the KHIDI, funded by the Ministry of Health and Welfare (HI14C1802).

Disclosure

The authors report no conflicts of interest in this work.