Cerium oxide nanoparticles as novel radio-protective and radio-sensitizing agents

Nanomedicine is the novel frontier of the prevention and treatment of an impressive series of severe human diseases. The search for therapeutic nanomaterials is fervent. In particular, finding a reliable and effective nano-antioxidant (as opposed to molecular anti-oxidants) is a main focus of current pharmacological research, since many serious diseases, including tumors, chronic inflammation, diabetes, neurodegenerative diseases, etc., imply oxidative stress. Among intrinsically active nano anti-oxidants, special attention has been given to cerium oxide nanoparticles (CNPs), showing promising anti-inflammatory and anti-degenerative properties. CNP biological properties may be associated to the presence of two different surface defects on their surface: the co-existence of Ce3+ and Ce4+ oxidation states and the consequent formation of charge-compensating oxygen vacancies. The pharmacological potential of CNPs as nanoantioxidant has been always attributed to the redox changes in the Ce oxidation state (Ce4+/Ce3+ redox switch), which trigger the abatement of the noxious intracellular reactive oxygen species (ROS), thereby protecting from the cytotoxic effects induced by oxidative stress. Very recently CNPs have been shown to possess a peculiar anti-cancer activity. CNPs seem able to selectively sensitize cancer cells to apoptosis, while protecting healthy tissues from exogenous carcinogenic sources. Therefore a promising use of CNPs in cancer therapy is emerging. However the mechanisms behind their selective anti-cancer activity are still unclear. Here we present a mechanistic analysis of the effects of CNPs against two different carcinogenic radiation sources, UV-rays and X-rays, on in vitro cell models. First, we demonstrate that CNPs are able to strongly counteract the pro-oxidant and pro-mutagenic effects of UV-rays and X-rays, although with different mechanisms. Second, we show that CNPs are able to radio-sensitize resistant cells to X-ray-induced apoptosis by enhancing the stringency of their defective DNA damage response. By Sm doping, which decreases the number of Ce3+ sites while keeping the same content of oxygen vacancies, is possible to perform a mechanistic analysis of the role of CNPs surface defects (Ce3+/Ce4+ redox couple vs. oxygen vacancies) in their anti-cancer activity. First, we correlate the protective effects of CNPs against UV-rays cytotoxicity to the activity of their Ce3+/Ce4+ redox couple. Second, we show that in cells irradiated with X-rays CNPs act as direct anti-oxidants, thanks to the activity of the Ce3+/Ce4+ redox couple, as well as by a peculiar nonredox mechanism, probably involving oxygen vacancies. Investigating this peculiar non-redox activity of CNPs, we show that CNPs are able to inhibit the activity of lipoxygenases (LOX) and cyclooxygenases (COX), strongly reducing their pro-oxidant, pro-survival and pro-mutagenic effects. A specific cause-effect relationship is found between the inhibition of 5-LOX enzymes and the protection against X-ray-induced damage to cellular membranes and to DNA. Interestingly, the inhibition of 5-LOX by CNPs increases the X-ray-induced apoptosis by increasing selectively the sensitivity of radio-resistant, e.g. transformed, cells to radiation treatments. The finding that CNP inhibit LOX and COX metabolism will help to better focus their pharmacological potential in radiation therapy as well as in pathologies involving the hyperactivation of these enzymes, including cancer, neurodegenerations and chronic inflammation. CNPs seem promising nanomaterials for the treatment of many diseases, including cancer. However the synthesis of CNPs suitable for biomedical applications is still a challenging task. One of the main challenges for a safe and efficient use of CNPs as pharmacological agents is their tendency to agglomerate in biological media. The formation of precipitates is expected to decrease the active surface area of CNPs, thereby reducing their biological activity. Importantly, nanoparticle agglomeration could also cause toxicity and deleterious side effects by accumulating on target organs (e.g. spleen and kidney) and by depressing the inflammatory response. Therefore, to reduce CNP agglomeration, we develop a base-catalyzed precipitation approach exploiting ethylene glycol as synthesis co-factor, followed by a post-synthesis surface functionalization with a poly (ethylene glycol)-terminated silane. We demonstrate that in situ silanization improves CNP colloidal stability with respect to the non-functionalized nanoparticles, at the same time allowing maintaining their pristine biological activity, yielding thus functionalized CNPs suitable for in vivo applications.