Nanoceria distribution and effects are mouse-strain dependent

Abstract

Prior studies showed nanoparticle clearance was different in C57BL/6 versus BALB/c mice, strains prone to Th1 and Th2 immune responses, respectively. Objective: Assess nanoceria (cerium oxide, CeO₂ nanoparticle) uptake time course and organ distribution, cellular and oxidative stress, and bioprocessing as a function of mouse strain. Methods: C57BL/6 and BALB/c female mice were i.p. injected with 10 mg/kg nanoceria or vehicle and terminated 0.5 to 24 h later. Organs were collected for cerium analysis; light and electron microscopy with elemental mapping; and protein carbonyl, IL-1β, and caspase-1 determination. Results: Peripheral organ cerium significantly increased, generally more in C57BL/6 mice. Caspase-1 was significantly elevated in the liver at 6 h, to a greater extent in BALB/c mice, suggesting inflammasome pathway activation. Light microscopy revealed greater liver vacuolation in C57BL/6 mice and a nanoceria-induced decrease in BALB/c but not C57BL/6 mice vacuolation. Nanoceria increased spleen lymphoid white pulp cell density in BALB/c but not C57BL/6 mice. Electron microscopy showed intracellular nanoceria particles bioprocessed to form crystalline cerium phosphate nanoneedles. Ferritin accumulation was greatly increased proximal to the nanoceria, forming core-shell-like structures in C57BL/6 but even distribution in BALB/c mice. Conclusions: BALB/c mice were more responsive to nanoceria-induced effects, e.g. liver caspase-1 activation, reduced liver vacuolation, and increased spleen cell density. Nanoceria uptake, initiation of bioprocessing, and crystalline cerium phosphate nanoneedle formation were rapid. Ferritin greatly increased with a macrophage phenotype-dependent distribution. Further study will be needed to understand the mechanisms underlying the observed differences.

Keywords: BALB/c mice; C57BL/6 mice; caspase-1; ferritin; liver; nanoceria.

Figure 1.

Physicochemical properties of the as-dosed nanoceria. The as-dosed nanoceria is shown in TEM images with increasing magnification as agglomerates on lacy carbon support (A, B, C and D). Circles in C indicate agglomerates and circles in D individual CeO₂ crystallites. E and G are STEM images at atomic resolution displaying rows of atoms (primary particles are crystalline as indicated by d-spacings in E and G and the X-ray diffractogram in F with characteristic ring structure signifying faces {100} and {111}). The surface of the primary nanoceria has an increased defect density (lower atom density) as indicated by the dotted rectangular areas in E and G. The EELS measurement in H was obtained at the nanoceria surface inside the dotted rectangular area and the M5 and M4 Ce peaks at 883 eV and 901 eV, respectively, are marked. The taller M5 peak indicates presence of predominantly Ce⁺⁺⁺. The M5 peak is missing a satellite peak (at 895 eV) which is characteristic of Ce⁺⁺⁺ dominance. Ce⁺⁺⁺ dominance is caused by the increased defect density or oxygen vacancies in the nanoceria crystal surface.

Figure 2.

Organ cerium as a percent of the injected dose. Results are mean±S.D. of cerium, determined by ICP-MS. They are expressed as a percent of the injected nanoceria dose, in the whole organ, for the mouse strain, treatment and sacrifice times shown. Results are from five control (vehicle treated) and three nanoceria-treated mice. + = Statistically different by protected LSD. * = statistically different in two-way ANOVA, at p < 0.05.

Figure 3.

Liver caspase-1 levels. Results are mean ± S.D of 10 control (vehicle treated) mice and six nanoceria-treated mice. *, **, ***, and *** indicate statistically significant differences at p < 0.05, 0.01, 0.001, and 0.0001, respectively.

Figure 4.

Nanoceria uptake in the liver of C57BL/6 and BALB/c mice at various times after nanoceria injection. All EM images are STEM images except C2 that is a TEM image. 0.5, 1, 6, and 24 h indicate the time between nanoceria injection and mouse termination. Intracellular nanoceria (Ce-NP, inside yellow circles and red squares) is shown at 0.5 h (A and B), 1 h (C and D), 6 h (E and F), and 24 h (G–J). Nanoceria, which occurs in agglomerates, looks dark in TEM and white in STEM imaging mode and is surrounded by copious ferritin (Ferritin NP) accumulations (A2, B2, C2, D2, G2, and I2). Individual ferritin nanoparticles are ~5–10 nm. Elemental maps (EDS mapping) are shown for corresponding regions imaged in STEM mode and show verification of cerium and oxygen and co-localization of cerium, oxygen, phosphorus, and iron after 6 h (Figure 4(E1; STEM) with maps E2–5 and 4F1 (STEM) with maps 4F2-5) and after 24 h (Figure 4(H1; STEM) with maps 4H2-5 and 4J1 (STEM) with maps 4J2–5) after nanoceria injection.

Figure 4.

Nanoceria uptake in the liver of C57BL/6 and BALB/c mice at various times after nanoceria injection. All EM images are STEM images except C2 that is a TEM image. 0.5, 1, 6, and 24 h indicate the time between nanoceria injection and mouse termination. Intracellular nanoceria (Ce-NP, inside yellow circles and red squares) is shown at 0.5 h (A and B), 1 h (C and D), 6 h (E and F), and 24 h (G–J). Nanoceria, which occurs in agglomerates, looks dark in TEM and white in STEM imaging mode and is surrounded by copious ferritin (Ferritin NP) accumulations (A2, B2, C2, D2, G2, and I2). Individual ferritin nanoparticles are ~5–10 nm. Elemental maps (EDS mapping) are shown for corresponding regions imaged in STEM mode and show verification of cerium and oxygen and co-localization of cerium, oxygen, phosphorus, and iron after 6 h (Figure 4(E1; STEM) with maps E2–5 and 4F1 (STEM) with maps 4F2-5) and after 24 h (Figure 4(H1; STEM) with maps 4H2-5 and 4J1 (STEM) with maps 4J2–5) after nanoceria injection.

Figure 5.

Nanoceria was bioprocessed in the liver. Examples of bioprocessed nanoceria 0.5, 6, and 24 h after its injection are shown as STEM (A, C, G, and I) and TEM (E and K) images and associated EDS elemental maps (B1-3, D1-3, F1-3, H1-3, J1-2, and L1-2). The STEM images indicate the presence of needles around the nanoceria. The nanoceria and needles in the STEM images are marked by arrows and shown in the corresponding EDS maps as Ce, O, and P rich regions. Only the needles are P-rich and are identified with X-ray diffraction pattern (B5 and L4) as Ce-phosphate. The diffraction patterns also confirm the crystalline nature of the needles. The nanoceria are replaced by Ce-phosphate during bioprocessing. The nanoceria and Ce-phosphate nanoneedles are surrounded by copious ferritin nanoparticles (5A, C, G, I, and K) which are ~5–10nm and are identified in the corresponding EDS Fe maps (B4, D4, F4, H4, J3, and L3). Cellular regions that do not include nanoceria/Ce-phosphate have a very low density of ferritin while the ferritin density around nanoceria/Ce-phosphate grains is significantly higher.

Figure 6.

Biomineralized iron nanoparticle distribution. Biomineralized iron nanoparticles (ferritin) accumulated in mouse liver after nanoceria injection and occur in locally enriched zones around nanoceria. Biomineralized iron nanoparticles are shown in the STEM images (A, B, and C) as white ~5–10 nm spots while the nanoceria agglomerates are larger and denser (indicated in B and C with yellow squares). Tissue regions that are not invaded by nanoceria have a low concentration or “normal” distribution of ferritin (A). B and C are at 24 h post-nanoceria dosing. Both ferritin and nanoceria associate with lysosomal regions or granules (B and C). Some granules are completely filled with ferritin and nanoceria, while others appear to have a core-shell type occupancy in the case of the C57BL/6 mice. Image D1 is an EDS spectrum identifying both Ce and Fe and D2-4 are EDS maps obtained from the region defined by the red box in C. The BALB/c mice liver uptake of nanoceria is shown in STEM images E1 to E2 which show EELS mapping for Ce and P and a corresponding EDS map of Fe in E3 which indicates that iron accumulation as ferritin occurs in the vicinity of Ce-phosphate after bioprocessing of nanoceria in the liver.

Figure 7.

EELS oxygen and iron analyses of Fe⁺⁺- and Fe⁺⁺⁺-containing standard minerals Amosite (Fe⁺⁺) and Hematite (Fe⁺⁺⁺) and the biomineralized iron nanoparticles (ferritin) that accumulate around nanoceria in liver as shown in Figure 6(B,C). A shows the oxygen edge of amosite and hematite indicating a large edge in the Fe⁺⁺⁺ (hematite) but not Fe⁺⁺- (amosite) containing standard. B shows the edge for iron with the standard materials amosite and hematite indicating a satellite peak for the Fe⁺⁺- but not Fe⁺⁺⁺-containing standard mineral. There is also a slight energy shift of ~ 1 eV (B) to distinguish Fe⁺⁺ versus Fe⁺⁺⁺ rich phases. C shows the oxygen edge of ferritin that formed in the liver and hematite standard. The EELS comparison in C shows an ~ 1/3 reduction in the oxygen edge of ferritin compared to the standard hematite. D illustrates the iron edge of ferritin and hematite standard showing the presence of pre-edge and satellite peaks in ferritin only and the alignment of the core edges of ferritin and hematite standard.