Cerium oxide nanoparticles as antioxidant agents: study of interaction with muscle cells under simulated microgravity

Oxidative stress is a well-known condition underlying several pathological conditions, such as heart failure and neurodegenerative diseases, but also skeletal muscle atrophy that occurs after prolonged periods of muscle disuse, such as bed immobilization and space travel. The commonly used strategy for oxidative stress treatment/prevention is the administration of antioxidant agents. Among the latter, cerium oxide nanoparticles, also called nanoceria, show self-regenerative antioxidant action mimicking that one of the main human antioxidant enzymes, and are excellent candidates for a persistent alleviation of oxidative stress. Nanoceria would ensure overcoming of limitations related to the usage of low molecular weight antioxidant agents, normally requiring repeated administrations. Purpose of this Master's Thesis is to investigate the interaction between nanoceria and skeletal muscle cells in mechanical unloading conditions, i.e. under simulated microgravity, for a possible application of this kind of nanomaterials against muscle waste induced by spaceflight. The experimental work started with nanomaterial synthesis by wet chemistry approach. In-depth characterization of the nanoparticles was then performed to assess their chemical composition, size, stability, and antioxidant capabilities. To the purpose, transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering, and photometry were used. Biocompatibility investigations were conducted with two different cell models for skeletal muscle tissue, namely C2C12 mouse myoblasts and primary human skeletal myoblasts. Afterwards, identification of the rotational conditions for microgravity simulation and for induction of mild oxidative stress in the skeletal muscle cultures was pursued with a random positioning machine. Interaction of nanomaterials and cell cultures was studied under different regimes of rotation: it was documented by phase contrast optical microscopy and by confocal microscopy upon application of cytochemistry techniques. Quantitative analyses concerning cell-nanomaterial interaction under simulated microgravity were conducted by flow cytometry and inductively-coupled plasma-optical emission spectroscopy. Cell viability was assessed upon administration of increasing concentrations of nanoceria in both static and dynamic conditions, denoting a high biocompatibility. Simulated microgravity was moreover found to be a pro-proliferative stimulus for mouse muscle cells, with potential applications in tissue engineering and regenerative medicine. On the other hand, simulated microgravity induced a mild oxidative stress on differentiating human cell cultures that deserves further investigations. The study of the interaction of nanoceria and muscle cells under microgravity conditions showed that, under the conditions tested only a small fraction of nanoceria is internalized, suggesting a low antioxidant effect of nanoceria under these conditions. The poor interaction could be due to the continuous mixing of fluids induced by the movement of the random positioning machine. Based on these results, this Thesis work leaves open several issues such as the promotion of the nanomaterial-cell interaction in simulated microgravity, the evaluation of the antioxidant effect of nanoceria and the validation of the results achieved during myoblast proliferation and differentiation for the stimuli the random positioning machine appears likely to impose.