Cerium oxide nanoparticles: antioxidative potential and effects on the metabolism of glial cells

Abstract

Cerium oxide nanoparticles (CeONPs) are considered a solid candidate for the new generation of antioxidants due to their protective potential against oxidative stress demonstrated in vitro and in vivo and their self-regeneration potential. Cellular or systemic CeONPs protection against radical-induced toxicity is based on their redox cycling capacity coupled with oxygen buffering. However, their antioxidant capacity as well as the side-effects of their application strongly depend on their synthesis and the cells targeted. In this line, previous studies have demonstrated protective effects of CeONPs against neuro-pathologies with a strong background of oxidative stress, such as Alzheimer´s disease, Parkinson´s disease or ischemia. However, despite this growing scientific evidences, the non-acute effects of CeONPs in glial cells remain a vast unexplored area of research. Therefore, it is of utmost importance to explore the novel formulations and the particular cellular interactions of CeONPs.

In the presented thesis, the coating with dimercaptosuccinate (DMSA) of commercial CeONPs was tested to obtain a colloidally stable DMSA-CeONPs dispersion. These nanoparticles (NPs) were thoroughly analyzed for their physicochemical properties such as size, shape and zeta potential. In addition, DMSA-CeONPs were fluorescence functionalized with the Oregon Green dye (OG), allowing the microscopical observation of their time- and temperature-dependent cellular uptake by rat C6 glioma cells and astrocytes. Cell viability and the effects on glucose metabolism was also investigated in astrocytes after incubation with DMSA-CeONPs. Different experimental conditions revealed that astrocytes remained viable over time and accumulated cerium in a concentration-dependent manner. However, the glycolytic flux was stimulated and thus, the extracellular concentration of lactate was significantly increased in astrocytes treated with DMSA-CeONPs. Comparison to astrocytes exposed to ionic cerium showed that the stimulation of the glycolytic flux depends on the presence of cerium, independently of the form in which it was presented. However, the intracellular cerium accumulation was significantly enhanced after exposure to DMSA-CeONPs compared to cells treated with ionic cerium. Incubations with hypoxia inducible 1 α (Hif-1α) stabilizers revealed that such stimulation of the glycolytic flux showed similarities to the stimulation observed when astrocytes were incubated with DMSA-CeONPs and therefore, it is concluded that cerium may interfere with the intracellular oxygen availability.

Two different approaches were taken to assess the potential antioxidative capacity of DMSA-CeONPs against reactive oxygen species (ROS): 1) the removal of exogenous H2O2 and the scavenging of cellularly formed superoxide was tested extracellularly and 2) intracellularly, by pre-incubating glial cells with these DMSA-CeONPs and then incubating them in the presence of exogenous H2O2 or under the cellular formation of superoxide. The results demonstrate that DMSA-CeONPs have only a limited extracellular ROS scavenging capacity. Internalized DMSA-CeONPs did not act as antioxidants in glial cells, nor did they show catalyst activities under the conditions tested.

In conclusion, the coating and fluorescence functionalization of CeONPs presented in this thesis are suitable tools to study the uptake, intracellular fate and accumulation in glial cells. However, these NPs cannot be applied as antioxidants due to their limited ROS scavenging capacities. DMSA-CeONPs did not affect the viability of glial cells but altered glucose metabolism, most likely via Hif-1α stabilization. In this line, this thesis highlights the importance of the investigation of the cellular effects beyond cell viability and under non-pathological conditions of CeONPs treatments.