Elucidation mechanism of different biological responses to multi-walled carbon nanotubes using four cell lines

Figures & data

Table 1 Basic properties of VGCF in PBS containing 0.1% gelatin

	VGCF in gelatin	Testing method
Diameter (nm)	80–130	FE-SEM
Length (μm)	7–10	FE-SEM
d002 (Å)	3.385	X-ray diffraction
R value (Id/I)a	0.088	Raman spectroscopy (514 nm)
Specific surface area (m^2/g)	15	N2 adsorption
Real density (g/cm^3)	2.1	Pycnometer
Zeta potential (mV)	1.18 ± 1.66	Particle sizing and zeta potential analyzerb
Agglomerate diameter (nm)	1442	Particle sizing and zeta potential analyzerc

a Notes: R refers to the intensity of D band over the intensity of G band;

b phase-shift analysis for electrophoretic light scattering;

c dynamic light scattering technique.

Abbreviations: VGCF, vapor-grown carbon fibers; PBS, phosphate buffered saline; FE-SEM, field emission scanning electron microscope.

Figure 1 Light microscopy image of cells exposed to VGCF. Images from the bright-field mode, in which VGCF or SB can be easily seen, are merged with images from the phase-contrast mode, in which the cells can be clearly seen. (A) Cells were exposed to varying concentrations of VGCF for 24 hours. (a, d, g, j and m) DM only. (b, h, k and m) 10 μg/mL SB. (c, i, l and n) 10 μg/mL VGCF. (e) 1 μg/mL SB. (f) 1 μg/mL VGCF. Arrow indicates accumulated VGCF around the nuclei. (B) Cells pretreated with CytoD were exposed to varying concentration of VGCF for 24 hours. (a, d and g) DM only. (b and h) 10 μg/mL SB. (c and i) 10 μg/mL VGCF. (e) 1 μg/mL SB. (f) 1 μg/mL VGCF.

Note: Scale bar indicates 50 μm.

Abbreviations: VGCF, vapor-grown carbon fiber; SB, Sumi black; DM, dispersion medium.

Figure 2 TEM image of cells exposed to VGCF. Cells were exposed to varying concentrations of VGCF for 24 hours. (A, D and G) DM only. (B and H) 10 μg/mL SB. (C and I) 10 μg/mL VGCF. (E) 1 μg/mL SB. (F) 1 μg/mL VGCF.

Abbreviations: TEM, transmission electron microscope; VGCF, vapor-grown carbon fiber; SB, Sumi black; DM, dispersion medium.

Figure 3 Cytotoxicity of VGCF in each cell line. The cells were exposed to varying concentrations of VGCF for 24 hours. (A–E) Cell viability by Alamar blue assay (n = 8). The data are compared to DM. (F–J) Plasma membrane permeability by LDH assay (n = 3). PC; 0.01% Triton X-100.

Notes: LDH activity = (Experimental value – DM value)/(PC value – DM value) × 10 × 100%. Mean ± SE, *P < 0.05, **P < 0.01, ***P < 0.001.

Abbreviations: VGCF, vapor-grown carbon fiber; SB, Sumi black; LDH, lactate dehydrogenase; DM, dispersion medium.

Figure 4 Cytokine production. Differentiated or undifferentiated THP-1 cells were exposed to VGCF for 24 hours. The supernatant of the culture medium was assayed for cytokine production. (A and B) Differentiated THP-1 cells exposed to 50 μg/mL of VGCF or CB secreted TNFα and IL-1β. (C) Undifferentiated THP-1 cells exposed to 100 μg/mL of VGCF or SB secreted IL-8.

Note: n = 3, mean ± SE, *P < 0.05, **P < 0.01, ***P < 0.001.

Abbreviations: VGCF, vapor-grown carbon fiber; SB, Sumi black; DM, dispersion medium.

Figure 5 Cellular ROS production. Cells were exposed to varying VGCF concentrations for 24 hours.

Notes: PC; 100 μM H₂O₂ (n = 3, mean ± SE, *P < 0.05, **P < 0.01, ***P < 0.001).

Abbreviations: VGCF, vapor-grown carbon fiber; ROS, reactive oxygen species.

Movie S1 Time-lapse confocal imaging of MESO-1 cells exposed to 10 μg/mL of VGCF. The cells were monitored every 10 minutes for 24 hours by using laser-scanning confocal microscopy with a ×20 PlanFluor objective. The images were converted to a movie file at 10 frames/second.

Abbreviation: VGCF, vapor-grown carbon fiber.

Movie S2 Time-lapse confocal imaging of the BEAS-1 cells exposed to 1 μg/mL of VGCF. The cells were monitored every 10 minutes for 24 hours by using laser-scanning confocal microscopy with a ×20 PlanFluor objective. The images were converted to a movie file at 10 frames/second.

Abbreviation: VGCF, vapor-grown carbon fiber.

Movie S3 Time-lapse confocal imaging of the differentiated THP-1 cells exposed to 10 μg/mL of VGCF. The cells were monitored every 10 minutes for 24 hours by using laser-scanning confocal microscopy with a ×20 PlanFluor objective. The images were converted to a movie file at 10 frames/second.

Abbreviation: VGCF, vapor-grown carbon fiber.