Antioxidant Enzyme-Mimetic Activity and Neuroprotective Effects of Cerium Oxide Nanoparticles Stabilized with Various Ratios of Citric Acid and EDTA

Abstract

Cerium oxide (CeO2) nanoparticles (CeNPs) are potent antioxidants that are being explored as potential therapies for diseases in which oxidative stress plays an important pathological role. However, both beneficial and toxic effects of CeNPs have been reported, and the method of synthesis as well as physico-chemical, biological, and environmental factors can impact the ultimate biological effects of CeNPs. In the present study, we explored the effect of different ratios of citric acid (CA) and EDTA (CA/EDTA), which are used as stabilizers during synthesis of CeNPs, on the antioxidant enzyme-mimetic and biological activity of the CeNPs. We separated the CeNPs into supernatant and pellet fractions and used commercially available enzymatic assays to measure the catalase-, superoxide dismutase (SOD)-, and oxidase-mimetic activity of each fraction. We tested the effects of these CeNPs in a mouse hippocampal brain slice model of ischemia to induce oxidative stress where the fluorescence indicator SYTOX green was used to assess cell death. Our results demonstrate that CeNPs stabilized with various ratios of CA/EDTA display different enzyme-mimetic activities. CeNPs with intermediate CA/EDTA stabilization ratios demonstrated greater neuroprotection in ischemic mouse brain slices, and the neuroprotective activity resides in the pellet fraction of the CeNPs. The neuroprotective effects of CeNPs stabilized with equal proportions of CA/EDTA (50/50) were also demonstrated in two other models of ischemia/reperfusion in mice and rats. Thus, CeNPs merit further development as a neuroprotective therapy for use in diseases associated with oxidative stress in the nervous system.

Variable in Vivo and in Vitro Biological Effects of Cerium Oxide Nanoparticle Formulations

“…In particular, synthesizing all three CeNPs with the same stabilizer or equilibrating the size of all three CeNPs by making larger CNRx CeNPs or smaller TI and NP CeNPs would aid in clarifying which characteristic is most relevant for these biological outcomes. In other experiments, we have demonstrated that the stabilizer dictates biological effects of CNRx CeNPs (Estevez et al, 2019), though these different CeNPs were not tested in the in vivo EAE model.…”

“…All protocols involving humane use of animals were approved by and conducted in accordance with standards of the St. Lawrence University Institutional Animal Care and Use Committee (#SU 11-12-1, S11-11, and SU 12-10). Harvest of hippocampal brain slices from healthy CD1 mice, subsequent ischemic exposure, and quantification of oxidative stress have been previously described (Estevez et al, 2019). SYTOX Green (Molecular Probes, Invitrogen; Eugene OR) was used to stain dead cells as described (DeCoteau et al, 2016).…”

“…For these experiments, the brain slices were exposed to ischemic conditions simultaneously with exposure to 5.8 mM CNRx, TI, or NP CeNPs. Artificial cerebrospinal fluid was used as the vehicle (Estevez et al, 2019). The staining of treated samples is expressed as a percentage of staining of control samples; a percentage less than 100% indicates greater viability vs control.…”

“…Exposure of murine brain slices to hypoglycemic and ischemic conditions ex vivo induces oxidative stress that triggers cell death and mimics an ischemic stroke (Estevez et al, 2019). Brain sections subjected to ischemic conditions for 30 minutes with or without CeNP treatment were stained with SYTOX Green to quantify cell viability.…”

“…Though nanomaterials typically adsorb a high number of proteins into their corona (Monopoli et al, 2012), only a relatively small number of proteins adhere to the CNRx CeNPs (Heckman et al, 2014): a profile of molecules that would promote receptor mediated uptake (ApoE) and transcytosis (albumin). These CeNPs exhibit catalase and SOD-like activity in vitro, enabling the reduction of ROS levels in murine hippocampal brain slices exposed ex vivo to ischemic conditions (Estevez et al, 2019). Further, the CeNPs oppose peroxide or ischemia induced shifts in the oxidation-reduction potential of brain tissue (DeCoteau et al, 2016).…”