Biocompatibility testing and antioxidant properties of cerium dioxide nanoparticles in human nervous system cells

Abstract

Cerium dioxide nanoparticles (CeO₂ NP), or nanoceria, are versatile materials with interesting properties for industry and medicine fields, particularly redox properties and catalytic activity. Because of their distinctive features, they have gained high attention in biomedical and pharmacological research to be employed in drug delivery, tissue regeneration, radioprotection, or diagnostic imaging. However, previous works reported that nanoceria may also induce reactive oxygen species (ROS) under certain conditions, leading to cellular stress, cellular damage, or cell death. In this study, the effects of CeO₂ NP on cell viability and morphology as well as their influence on oxidative stress (both oxidant and ROS scavenging capacities) were investigated in nervous system cells (SH-SY5Y neuronal and A172 glial cells) treated with a wide range of CeO₂ NP concentrations (1-100 µg/mL) for several treatment times. Results obtained showed that, despite being stable in time and effectively internalized by both cell types, CeO₂ NP did not produce significant decrease in viability, evaluated by MTT assay, morphological alterations, or intrinsic cell-free ROS, but they generated cellular ROS limited to longer exposure periods. Furthermore, CeO₂ NP demonstrated a certain intrinsic ability to scavenge ROS generated by H₂O₂ in both tested cell types, more pronounced in neuronal cells. These results confirm the good biocompatibility of nanoceria on human nervous system cells and support further exploring their potential use in biomedicine field, particularly for those therapeutic and diagnostic applications related to the nervous system.

Keywords: Antioxidant capacity; Cerium dioxide nanoparticles; Cytotoxicity; Glial cells; Neuronal cells; Oxidative stress.

Fig. 1

Role of oxidative stress in neurodegenerative disorders. Production of reactive oxygen species (ROS) is a natural cellular process that occurs mainly as a byproduct of cellular metabolism, particularly within peroxisomes or the mitochondrial electron transport chain, but can also arise from external factors like pollution, radiation, cigarette smoke, and pesticides. Once the production of free radicals, such as superoxide (^•O₂⁻), hydrogen peroxide (H₂O₂), or hydroxyl radicals (^•OH), overwhelm the cellular antioxidant mechanisms, their excessive accumulation can lead to detrimental effects compromising cellular function. Potential consequences include lipid peroxidation, causing cell membrane disruption, protein oxidation/aggregation and the buildup of misfolded proteins causing endoplasmic reticulum stress, mitochondrial dysfunction increasing ROS production, and oxidative DNA damage (primarily 8-oxo-7,8-dihydroguanine (8-oxoG)). Neurons in the central nervous system are especially susceptible to oxidative stress due to their high energy demands, the abundance of polyunsaturated lipids in their membranes, and their limited antioxidant capacity. Therefore, oxidative stress can lead to mitochondrial impairment, altered protein structures and functions, DNA mutations and genetic instability, disruptions in cellular signaling and in synaptic transmission, and chronic neuroinflammation, that trigger programmed cell death mechanisms and accelerates neurodegeneration. Created in Biorender by Fernández-Bertólez N. in 2024 https://BioRender.com/h89v100

Fig. 2

Flow cytometry analysis of cellular uptake in SH-SY5Y neurons a and A172 glial cells b exposed to CeO₂ NP for 3, 24 and 48 h. * p < 0.05, ** p < 0.01, significant differences regarding the corresponding control. PC, positive control (200 µg/mL TiO₂ NP)

Fig. 3

Cellular viability assessment of SH-SY5Y neuronal cells a, and A172 glial cells b exposed to CeO₂ NP for 3, 24 and 48 h. **p < 0.01, significant differences regarding the corresponding control. PC, positive control (1% Triton X-100)

Fig. 4

Cytomorphological analysis of undifferentiated SH-SY5Y cells (left panels) and A172 cells (right panels), untreated and exposed to different concentrations of CeO₂ NP for 3 and 24 h. In SH-SY5Y cells, white arrows point at neuronal cells with spindle-like morphology with few dendritic cytoplasmic protrusions (processes), and yellow arrows indicate cells with epithelial-like morphology. In A172 cells, white arrows point at the body of polygonal-shaped to fibroblast-like cells, and yellow arrows indicate the characteristic and numerous processes of the astrocytes, a subtype of glial cells

Fig. 5

Reactive oxygen species (ROS) generation by CeO₂ NP. Autofluorescence of CeO₂ NP at 485/530 nm (excitation/emission) a acellular ROS production b intracellular ROS generation in SH-SY5Y neuronal cells c and in A172 glial cells d. *p < 0.05, **p < 0.01, significant differences regarding the corresponding control. a.u., absorbance units; PBS, phosphate-buffered saline; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; PC, positive control (1 mM H₂O₂ + 0.04 U/mL horseradish peroxidase for acellular assay, and 1 mM H₂O₂ for intracellular assay)

Fig. 6

Intracellular scavenging capacity of CeO₂ NP in SH-SY5Y cells a, and A172 cells b. *p < 0.05, **p < 0.01, significant differences regarding H₂O₂-treated cells at time = 0 h (striped red bar). ^##p < 0.01, ^#p < 0.05, significant differences regarding the basal control

Fig. 7

Results of fpg-modified comet assay. Interference of CeO₂ NP (100 µg/mL) with fpg enzyme in SH-SY5Y neuronal cells a, and A172 glial cells b, and oxidative DNA damage induced by CeO₂ NP in SH-SY5Y cells c and A172 cells d *p < 0.05, **p < 0.01, significant difference regarding the corresponding control. PC: positive control (1.5 mM KBrO₃)