In vivo formation of Ce-phosphate Nanoparticles following Intratracheal Instillation of CeCl₃: Subcellular sites, Nanostructures, Precipitation Mechanisms and Nanoparticle 3D-Alignment

Abstract

We demonstrate the in vivo formation of nano-particulate Ce-containing structures in lungs instilled with 5 mg/kg CeCl3 using high resolution electron microscopy (HRTEM), energy loss spectroscopy (EELS), and elemental mapping (EDS). The observed high lung retention of Ce after instillation of CeCl3 (75-92% retained at 28 days) is unexpected since metal ions are usually readily transported across the air-blood barrier. The binding of cerium ions to lung constituents has been suggested [1], and the formation of cerium phosphate has been shown previously, but without discussions on the mechanisms involved in nanoparticle nucleation and growth [2]. Determining the form of Ce after lung exposure to CeCl3 has been challenging due to the difficulties in distinguishing ions from particulate forms when using radioactivity or ICP-MS. We have identified cerium nanoparticles at the sub-cellular level in lung macrophages after CeCl3 instillation. This observation provides insights on the cell structures and components that will help distinguish which cellular areas are the sites of in vivo nanoparticle formation.Lung tissue samples were fixed by vascular perfusion with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1M HEPES buffer. The samples were processed for electron microscopy at 2 hours, 7 days and 4 weeks post-instillation. High-resolution imaging revealed that nanoparticles had formed at all of the selected time-points and were located in lysosomal regions, along membrane surfaces and inside mitochondria. The nanoparticles formed in a narrow particle size range (~1-5 nm) and were bundled together in 3D-agglomerates up to several hundred nm (Fig. 1a). Individual nanoparticles appear self-aligned into rodshaped structures or needles that reach up to 10 nm in length and 2-5 nm in width, with some structures arranged in a spider-like form (Fig. 1b). The typical crystalline structures of individual ultra-fine nanoparticles are shown in Fig. 1c. EELS analyses demonstrate co-localization of Ce and P for individual nanocrystals (Fig. 2). The in vivo nucleation and growth of cerium phosphate nanoparticles in the observed large agglomerates are demonstrated by the EDS elemental maps obtained in the STEM mode (Fig. 2). EDS maps and corresponding spectra indicate that cerium ions formed cerium phosphate nanoparticles. Furthermore, the elemental map also reveals the presence of abundant Fe, which may correspond to the ultrafine ferritin nanoparticles (~5nm) that formed in the vicinity of the cerium phosphate nanoparticles (Fig. 2). Inflammatory responses have been previously shown to activate abundant ferritin formation in localized regions with invader nanoparticles [3]. The current study shows for the first time that the same process occurs after in vivo delivery of ions, followed by their transport, and precipitation.

Analytical High-resolution Electron Microscopy Reveals Organ-specific Nanoceria Bioprocessing

“…Although nanoceria may act as a phosphatase to release phosphate, other regions where nanoceria undergoes greater bioprocessing/solubility, such as inside the liver (Graham et al 2014), may favor ion transport over phosphate precipitation. The formation of Ce-phosphate precipitates that form from available metal ions in tissue was previously demonstrated (Berry et al 1997;Graham, Wang, et al 2017) and is independent of nanoceria's phosphatase mimetic activity, suggesting there are several pathways to form Ce-phosphate precipitates. We suggest that Ce-phosphate nanoneedle formation may counteract the toxicity caused by free Ce ions derived from dissolving (bioprocessing) nanoparticles.…”

“…The high retention of Ce (75–92% at 28 days) in the lung after Ce-ion instillation was unexpected. Follow-up HRTEM and EELS revealed that in vivo formation of Ce phosphates had occurred (Graham, Wang, et al 2017). Ce-phosphate precipitation observed in the spleen in the current study also requires a source of Ce ions, which we suggest is a result of the in vivo breakdown and dissolution of nanoparticles in the liver (Figure 6).…”

“…One approach is the utilization of advanced high-resolution transmission electron microscopy (HRTEM) coupled with in situ spectroscopic methods (energy-dispersive X-ray spectroscopy, EDS-mapping) and electron energy loss spectroscopy (EELS). The beginnings of this field have been described (Graham et. al.…”