Safe Administration of Carbon Nanotubes by Intravenous Pathway in BALB/c Mice

Abstract

Carbon nanotubes (CNTs) are nanomaterials with multiple possible uses as drug carriers or in nanovaccine development. However, the toxicity of CNTs administered intravenously in in vivo models has not been fully described to date. This work aimed to evaluate the toxic effect of pristine multi-walled CNTs (UP-CNTs), purified (P-CNTs), or CNTs functionalized with fluorescein isothiocyanate (FITC-CNTs) administered by intravenous injection in BALB/c mice. Biochemical and histopathological parameters were analyzed at 1, 14, 29, and 60 days post-exposure. Pristine CNTs were the most toxic nanoparticles in comparison with P-CNTs or FITC-CNTs, increasing serum AST (≈ 180%), ALT (≈ 300%), and LDH (≈ 200%) levels at one day post-exposure. The urea/creatinine ratio suggested pre-renal injury at the 14^th day accompanied of extensive lesions in kidneys, lungs, and liver. Biochemical and histological findings in mice exposed to P-CNTs had not significant differences compared to the controls. A lower toxic effect was detected in animals exposed to FITC-CNTs which was attributable to FITC toxicity. These results demonstrate that the purification process of CNTs reduces in vivo toxicity, and that toxicity in functionalized CNTs is dependent on the functionalized compound. Therefore, P-CNTs are postulated as potential candidates for safe biomedical applications using an intravenous pathway.

Keywords: Keywords. carbon nanotubes; lung damage; mice; nanotoxicology; renal damage; toxicity.

Figure 1

Timeline of exposure protocol for toxicological evaluation in BALB/c mice. The UP-CNTs and P-CNTs were characterized by SEM, and Raman spectroscopy; FITC functionalization was demonstrated by epi-fluorescence microscopy. Serum samples were obtained at 0, and 24 h, and later at 14, 29, and 60 days post inoculation of CNTs to determine the biochemical parameters. At the end of each time post inoculation, the animals were sacrificed, and the organs were dissected for histological observation.

Figure 2

Organ weights (g) from mice exposed to different CNTs at different exposure times. Each graph shows the data of the indicated organ, the bars represent the mean ± SD obtained for each group (n = 4). CTL, vehicle control (0.2% Pluronic F127 in sterile phosphate buffer). * indicates significant differences vs the control group (α= 0.05). Data were analyzed by Dunnett’s test.

Figure 3

Macroscopic and microscopic lesions observed in lungs of mice exposed to UP-CNTs 60 days after exposure. A, Macroscopic aspect of lungs from a mouse exposed to UP-CNTs; the black arrows indicate the accumulation of nanoparticles mainly in the peripheral areas. The dotted line on the left lung indicates a tumor growth lesion. Microphotographs show: B, UP-CNTs trapped under endothelium (arrow) found in the right lung (magnification 60×); C, general aspect of lesions detected in the left lung, the arrow indicates the accumulation of UP-CNTs (arrow, magnification 4X); D, malignant lymphoproliferative lesion of peribronchial and perivascular localization, artery (a) is surrounded by several layers of large lymphocytes, bronchiole (b) looks obstructed by neutrophils (magnification 10×); E, the bronchiole epithelium (epi) in contact with neutrophils inside luminal space (magnification 60×), and; F, an area with large lymphocytes with abnormal nuclei (plasmocyte, p) (magnification 100×).

Figure 4

Histopathological findings in kidney of mice exposed to different types of CNTs. Microphotographs show kidney sections of mice exposed to: A, control group non exposed, the normal morphology of renal cortex (g, glomerulus); B, P-CNTs exposed group. C, UP-CNTs exposed group, hemorrhagic areas (arrows) and small spherical bodies trapped in glomeruli and damaged areas (arrowheads) that corresponds to UP-CNTs. D, FITC-CNTs exposed group. Microphotographs are representative of one experiment. Magnifications 10×. Insets show magnification at 40×. H&E staining.

Figure 5

Histopathological findings in lungs of mice exposed to different types of CNTs. Microphotographs show lung sections of mice exposed to: A, control group non exposed, the normal morphology of bronchiole (b) and alveoli (a) is shown; B, P-CNTs exposed group. C, UP-CNTs exposed group, accumulation of CNTs in the artery, and inflammatory infiltrate in alveolar walls (arrow). D, FITC-CNTs exposed group, inflammatory infiltrate (arrow), of note is the decrease of the alveolar sacs in the lung. Microphotographs are representative of one experiment. Magnification 10X. Insets show magnification at 40×. H&E staining.

Figure 6

Effect of exposure with different CNTs on the relative activity of AST and ALT. Relative activity of serum aminotransferases in mice exposed to different CNTs at 1, 14, 29, and 60 days post exposure. (A) AST and (B) ALT activities. Each bar represents the mean ± SE of each group (n = 4). a: difference with respect to the control (p < 0.05). b: difference among treatments (p < 0.05), and c: difference among times post exposure (p < 0.05).

Figure 7

Effect of exposure with different CNTs on the relative activity of LDH. Relative activities of serum LDH in mice exposed to different CNTs at 1, 14, 29, and 60 days post exposure. Each bar represents the mean ± SE of each group (n = 4). a: difference with respect to the control without stimulus (p < 0.05). b: difference between treatments (p < 0.05), and c: difference between times post exposure (p < 0.05).

Figure 8

Effect of exposure to different CNTs on levels of urea and creatinine. Graphics show relative concentration of A, urea and B, creatinine, at 1, 14, 29, and 60 days post exposure. Each bar represents the mean ± SE of each group (n = 4). a: difference with respect to the control without stimulus (p < 0.05). b: difference among treatments (p < 0.05), and c: difference among times post exposure (p < 0.05).