Intracellular analysis of the antioxidant mechanism of ceria nanoparticles

Ceria nanoparticles (CNPs) display remarkable biological antioxidant properties. Their enzyme-like behaviour arises from the Ce³⁺/Ce⁴⁺ equilibrium, enabling reduction of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide and hydroxyl radicals. Unlike most rare-earth elements, which predominantly occur in a stable 3+ oxidation state, cerium can adopt two stable oxidation states: Ce³⁺ and Ce⁴⁺. Traditionally, CNP activity has been explained by abiotic models in which the Ce³⁺/Ce⁴⁺ redox cycle operates at the nanoparticle surface, which is often rich in oxygen vacancies [1]. 

However, maintaining long-term antioxidant activity in this model requires regeneration of Ce³⁺ sites – a process that is not well defined – and it overlooks the complexity of the cellular environment. In cells, CNPs enter via endocytosis, progressing from early endosomes to lysosomes [2]. ROS and reactive nitrogen species (RNS) are generally cytosolic, not lysosomal, so this compartmentalisation raises questions about where and how activity occurs.

This study tracked changes in Ce³⁺/Ce⁴⁺ ratio during CNP uptake and trafficking using micro-X-ray fluorescence (μXRF) and micro-X-ray absorption near-edge spectroscopy (μXANES) at the Ce L_III-edge on beamline ID21. Together, these techniques enabled visualisation of CNP distribution within cells and quantification of cerium oxidation states at subcellular resolution. 

Polyacrylic acid-stabilized 14-nm CNPs were used in ‘pulse-chase’ experiments, in which cells were incubated with CNPs for 1 hour (‘pulse’) and then cultured without them for 2 or 6 hours (‘chase’). This protocol synchronised their intracellular distribution, concentrating most particles in lysosomes; by contrast, under continuous exposure, CNPs disperse throughout the endocytic system.

After the 1 h pulse, lysosomal cerium was predominantly Ce⁴⁺. Following a 2 h chase, Ce³⁺ (the redox state linked to antioxidant activity) increased slightly; after 6 h, the increase was substantial (Figure 1). The correlation between lysosomal residence time and Ce³⁺ proportion indicates a chemical transformation within CNPs, likely driven by the acidic lyosomal pH. In vitro solubility tests confirmed a >10-fold increase in dissolution as pH decreased from 7 to 4. 

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Fig. 1: a) Backscattered scanning electron microscopy (BS-SEM) image showing a cluster of four cells incubated with CNPs. b) Confocal microscopy images showing CNPs (red) and endolysosomes (green); yellow regions indicate colocalization, as confirmed by the signal overlap histograms (inset, bottom right). Nuclei are shown in blue. c) Representative μXRF maps of phosphorus (P, grey) and cerium (Ce, red) in cells after a 1 h pulse, followed by a 2 h chase in CNP-free medium (P1–C2). d) μXANES spectra from pulse-chase experiments: pulse 1 h (P1), pulse 1 h + chase 2 h (P1+C2), and pulse 1 h + chase 6 h (P1+C6). The inset shows reference spectra for Ce³⁺ (Ce(NO₃)₃) and Ce⁴⁺ (bulk CeO₂). The spectra were normalized at the unit absorption coefficient at 400 eV above the edge, where the extended X-ray absorption fine structure (EXAFS) oscillations were negligible. e) Quantification of Ce³⁺ content for each experimental condition. The Ce³⁺ fraction was estimated by fitting the patterns reported in (d) with a linear combination of the reference patterns. XANES spectra were quantified by linear-combination fitting (LCF) against appropriate reference compounds; for μXANES, LCF was applied pixel-wise to generate speciation maps.  

Ce³⁺ ions generated in intracellular vesicles can diffuse into the cytosol (pH »7) along a concentration gradient, possibly via Ca²⁺ channels. In the cytosol, Ce³⁺ can neutralize ROS. The pH gradient between lysosomes and cytosol maintains the solubility of CNPs, supporting sustained ion release. 

The pH dependence of antioxidant activity was tested by inhibiting the vacuolar H⁺-ATPase (V-ATPase) proton pump – responsible for establishing and maintaining endolysosomal pH – with bafilomicyn A1, which raised lysosomal pH to ~7. Under these conditions, CNPs no longer attenuated ROS (Figure 2), confirming that acidic dissolution is essential for their activity.

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Fig. 2: a) Confocal microscopy images of control cells (endolysosomal pH 4.5–5) and cells treated with bafilomycin A1 (BA1) to block V-ATPase, raising endolysosomal pH to 7–7.5. Acridine Orange staining marks acidic lumina as red puncta and neutral lumina as green puncta. b) CNP antioxidant activity under different endolysosomal pH conditions (± BA1) and with/without induced oxidative stress (+tBHP). Fluorescent intensity reflects intracellular ROS levels. c) Schematic model: Ce³⁺ ions (green) are released via partial reductive dissolution of CNPs (yellow) in acidic endolysosomes and diffuse into the cytosol, where they reduce ROS.

The findings indicate that CNPs in endolysosomes act as dynamic reservoirs of Ce³⁺, producing a controlled, prolonged release into the cytoplasm. This dissolution-driven mechanism engages the particle bulk rather than only its surface, enabling durable antioxidant action without continual generation of redox sites. Furthermore, the Ce³⁺ concentration remains low and is buffered by stable solubility equilibria between cellular compartments, thereby avoiding cytotoxicity.

In contrast to conventional antioxidant molecules, which degrade quickly and require frequent replenishment, CNPs persist intracellularly, steadily supplying active ions over extended periods. This mechanism  does not require direct ROS-nanoparticle surface contact; instead, antioxidant activity is mediated by Ce³⁺ ions released via partial dissolution in acidic compartments.

Taken together, these results clarify the mechanism and explain the unusual persistence of CNP antioxidant activity, and suggest that the same principle may extend to other pH-responsive nanozymes with biomedical potential.
 

Principal publication and authors
Unveiling the Role of Intracellular Dissolution Equilibria in the Antioxidant Mechanism of Ceria Nanoparticles, P. Sommi et al., ACS Appl. Mater. Interfaces 17(15), 22474-22486 (2025); https://doi.org/10.1021/acsami.5c02505

References
[1] C. Korsvik et al., Chem. Commun. Camb. Engl. 10, 1056 (2007).
[2] P. Sommi et al., ACS Nano 15, 15803 (2021).