HYPOTHESIS AND THEORY published: 17 March 2022 doi: 10.3389/fimmu.2022.750175 How Does Immunomodulatory Nanoceria Work? ROS and Immunometabolism Lena M. Ernst 1 and Victor Puntes 1,2,3,4* 1 Vall d’Hebron Research Instiute (VHIR), Barcelona, Spain, 2 Instiut Català de Nanociència I Nanotecnologia (ICN2), CSIC, The Barcelona Institute of Science and Technology (BIST), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 3 Institució Catalana de Recerca I Estudis Avançats (ICREA), Barcelona, Spain, 4 Networking Research Centre for Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Instituto de Salud Carlos III, Madrid, Spain Dysregulation of the immune system is associated with an overproduction of metabolic reactive oxygen species (ROS) and consequent oxidative stress. By buffering excess ROS, cerium oxide (CeO2) nanoparticles (NPs) (nanoceria) not only protect from oxidative stress consequence of inflammation but also modulate the immune response towards inflammation resolution. Immunomodulation is the modulation (regulatory adjustment) of the immune system. It has natural and human-induced forms, and it is part of immunotherapy, in which immune responses are induced, amplified, attenuated, or prevented according to therapeutic goals. For decades, it has been observed that immune cells transform from Edited by: David Pozo, relative metabolic quiescence to a highly active metabolic state during activation(1). These University of Seville, Spain changes in metabolism affect fate and function over a broad range of timescales and cell Reviewed by: types, always correlated to metabolic changes closely associated with mitochondria number Dr. Sanjay Singh, and morphology. The question is how to control the immunochemical potential, thereby U.P. Pandit Deen Dayal Upadhyaya Veterinary University, India regulating the immune response, by administering cellular power supply. In this regard, Sanjay Singh, immune cells show different general catabolic modes relative to their activation status, linked National Institute of Animal Biotechnology (NIAB), India to their specific functions (maintenance, scavenging, defense, resolution, and repair) that can *Correspondence: be correlated to different ROS requirements and production. Properly formulated, nanoceria Victor Puntes is highly soluble, safe, and potentially biodegradable, and it may overcome current victor.puntes@vhir.org antioxidant substances limitations and thus open a new era for human health management. Specialty section: Keywords: nanoparticles, nanoceria, inflammation, macrophages, immunemetabolism, metabolism, ROS - reactive This article was submitted to oxygen species, entropy Molecular Innate Immunity, a section of the journal Frontiers in Immunology Received: 30 July 2021 INTRODUCTION Accepted: 18 February 2022 Inflammation and oxidative stress (OS), mediated by reactive oxidant species (ROS) Published: 17 March 2022 overproduction, are strictly interconnected (1). ROS refers to various biogenic free radical Citation: molecules resulting from natural metabolism characterized by being highly oxidant. These free Ernst LM and Puntes V (2022) How Does Immunomodulatory Nanoceria radicals are involved in different critical physiological processes, such as gene expression, signal Work? ROS and Immunometabolism. transduction, growth regulation, and, significantly, inflammation, where high ROS concentrations Front. Immunol. 13:750175. are not only needed for the activation of inflammatory pathways but also to sustain the energetic doi: 10.3389/fimmu.2022.750175 demands of an inflammatory response (2). Therefore, from a theoretical point of view, antioxidant Frontiers in Immunology | www.frontiersin.org 1 March 2022 | Volume 13 | Article 750175 Ernst and Puntes Nanoceria, Imunometabolism and ROS substances can both protect from oxidative stress (OS) and anti-inflammatory and immunomodulatory substance, it is facilitate the resolution of pathogenic inflammation by essential to know how immune cells employ different inhibiting ROS-dependent inflammatory reactions and metabolic pathways to sustain their energetic needs. For that, returning to homeostatic balance (3, 4). we focus on ROS production as a cause and consequence of The role of antioxidant substances became popular in the inflammation and nanoceria ROS scavenging capacities. To second half of the 20th century when Linus Pauling (1954 and propose a mechanism of action that results in effective and 1962 Nobel laureate) developed the so-called orthomolecular beneficial ROS scavenging and buffering, we need first to medicine based on nutritional supplementation and high doses review the basics of metabolism and ROS production and of ascorbic acid (5, 6). It resurfaced again in the 90s due to a large immunometabolism, from a chemist’s point of view. Then we human study suggesting that vitamin E supplements could be describe the nanoceria ROS reactivity towards unpaired electrons associated with a reduced risk of heart diseases (7). During this and free radicals. Finally, we consider pharmacological and period, other pre-clinical and epidemiological works also production aspects for the proper development of medical reported beneficial effects of antioxidant substances against formulations based on nanoceria. chronic inflammation, neurodegeneration, and cancer (8). Subsequently, antioxidant therapies were evaluated in placebo- controlled trials involving tens of thousands of patients. Despite A BRIEF OVERVIEW ON METABOLISM, the pathophysiologic, epidemiologic, and mechanistic MITOCHONDRIA, AND ROS compelling evidence, these clinical trials have been, to date, mostly negative. This has been attributed to the non-drug- In our body, nutrients and oxygen are transformed into energy, likeness of available antioxidant substances (9). These water, carbon dioxide, and other by-products as ROS. The substances have high unspecific uncontrolled reactivity, poor amounts of O2 consumed, and CO2 produced reflect the solubility, and hence limited absorption profiles, low body’s metabolism and metabolized nutrients (14). In bioavailability, and low concentrations at the target site (10, biological systems, energy is mainly provided by the controlled 11). This has given rise to a pessimistic view of antioxidant oxidation of carbohydrates and fatty acids, where the oxidizer is therapies. Today, only a few antioxidant substances have reached the limiting reagent. From a chemical point of view, oxidation of clinical use. These include N-acetylcysteine for acetaminophen nutrients corresponds to the irreversible exothermic reaction of overdose, Edaravone for ischemic stroke, alpha-lipoic acid for materials called fuels, consisting mainly of carbon and hydrogen, diabetic neuropathy, some flavonoids (polyphenolic compounds with an oxydizer. These oxidation reactions are more complex present in dietary plants) for chronic venous insufficiency, as well than they may seem. Initially, carbohydrates and fatty acids as baicalein and catechins for osteoarthritis. Unfortunately, these decompose to react with oxygen, forming unstable highly treatments have not been fully satisfactory, and as a result, new oxidant compounds called free radicals (ROS in our case). approaches are being explored. Then, these free radicals take C and H electrons, and most of In these circumstances, new antioxidant mineral substances the heat is released. Oxidation is completed when stable products like nanoceria, displaying minimal toxicity to normal tissues while are formed. It is interesting to note that incomplete oxidation providing cellular protection from ROS-dependent oxidative leads to the release of highly reactive intermediates in the form of damage, have attracted considerable attention as a potential free radicals. therapeutic tool in preventing and treating oxidative stress- These nutrient oxidation chemical reactions are a related diseases. With its mild but permanent ROS scavenging fundamental part of metabolism. Metabolism can be divided capacities and good pharmacology, nanoceria may overcome into catabolism, the energy sourcing, extracting it from chemical previous limitations and finally enable full antioxidant therapies bonds, breaking large molecules into smaller ones, and in human health. Nanoceria has already demonstrated its ability to anabolism, the synthesis of complex molecules from simpler restrict inflammation in a large number of pathologies, based on ones, using part of the produced energy. Regarding catabolism, their ability to reduce ROS levels and, consequently, most three different basic catabolic pathways exist depending on the inflammatory mediators (12). Over the last decade, the employed fuel (glucose or fatty acids derived from carbohydrates beneficial effects of nanoceria treatment have been reported in and lipids); oxidizer (O2/ROS), and combustion mode (aerobic various pre-clinical models, including cardiac diseases, diabetes, or anaerobic). The produced energy is stored in the form of ATP retinal diseases, gastrointestinal inflammation, liver inflammation, (indeed, in the form of ATP/ADP gradients (15)) and heat. These and cancer. In neurology, beneficial effects have been reported in pathways can be referred to as aerobic glycolysis, anaerobic pre-clinical models of Alzheimer’s disease, Parkinson’s disease, glycolysis, and fatty acid oxidation. multiple sclerosis, traumatic brain injury, and brain ischemia, all Glycolysis is relatively efficient in aerobic conditions (high ATP conditions associated with high ROS production and production and low ROS production) and very inefficient in neuroinflammation, reviewed in (13). anaerobic conditions, where a lot of glucose is consumed, and a The underlying hypothesis is that by scavenging excess ROS, lot of ROS produced. In fatty acid oxidation (FAO), processing tissue is protected, metabolism is controlled, immune activation one palmitic molecule efficiently produces 129 ATP molecules, suffocated, and resolution of inflammation allowed. Thus, to compared with 2 ATP molecules produced per molecule of understand how nanoceria works inside a biological system as an glucose during anaerobic glycolysis, or 36 ATP molecules per Frontiers in Immunology | www.frontiersin.org 2 March 2022 | Volume 13 | Article 750175 Ernst and Puntes Nanoceria, Imunometabolism and ROS molecule of glucose during aerobic glycolysis. FAO is peroxidases, or in a less specific way, with small molecules accompanied by a slight non-pathological ROS overproduction such as ascorbate, glutathione, alkaloids, or carotenoids (1). when compared to aerobic glycolysis. Regarding power (energy per From a chemical engineering perspective, mitochondria could unit time), anaerobic glycolysis can produce ATP 100 times faster be described as an internal combustion engine (17), transforming (16) than aerobic, thus providing the highest power to cells. FAO oxygen and organic fuels into energy and oxidized products such delivers energy at intermediate rates. These different catabolic as CO2, H2O, and H2O2 (ROS). H2O2 exists in equilibrium with pathways allow adjustments for the different cellular energetic hydroxyl species (a model ROS), and it is a common source of free requirements and needs, and as a consequence, biological radicals. Therefore, for intervening in the operating mode of an responses can be controlled by targeting the energy supply. The internal combustion engine, one can either address the fuel supply following is important for our hypothesis: biological oxidation rate (e.g., ketogenic diets are known to both be anti-inflammatory and is adjusted by ROS concentration rather than oxygen to restrict anaerobic glycolysis (18)), or the excess oxidizer (mainly concentration, which is more stable (constant) inside the body ROS), i.e., the target of antioxidant substances. than ROS, and therefore the relevance of antioxidant substances. Changes in mitochondria membrane potential parallel These different cellular metabolic pathways can be observed mitochondrial morphology (oval, spherical, or elongated/ by the lactate production, the expression of glucose transporters branched), which determines oxygen supply rate and, at the cell membrane, or mitochondria number and morphology consequently, activity (19). A high mitochondria surface-to- (See Figure 1 and Table 1). Mitochondria have been described as volume ratio (elongated/branched) allows the oxidation of high cell power-houses, converting nutrients in the form of glucose or dense fuels as FA. A low surface-to-volume ratio (spherical) will fatty acids into energy in the form of ATP, and ROS. result in the lack of oxygen and the production of highly reactive Mitochondria can rapidly adjust to the cell metabolic needs, oxidation intermediates. Consequently, considering the and play a central role in bioenergetic and biosynthetic pathways. mitochondria as an internal combustion engine where the Increased energy demand is met by mitochondrial reproduction oxygen is supplied through the membrane to the fuel inside it, and fusion. In contrast, a decrease in energy demand results in the surface-to-volume ratio is directly proportional to the oxygen the removal of superfluous mitochondria through fission provision rate. Thus, higher mitochondrial membrane and mitophagy. polarization implies a higher surface-to-volume ratio and It has been described that ROS generate from the leakage of higher oxygen provision to the fuel for oxidation. electrons in the mitochondria transport chain. Under normal Mitochondria display an oval shape most of the time, conditions, the potentially harmful effects of ROS are successfully providing a sufficient O2 supply for pyruvate oxidation in the restrained by protective and reparative mechanisms. Natural TCA cycle and OXPHOS. In contrast, reduced O2 supply in antioxidant defenses may remove ROS either in a highly spherical mitochondria, when the surface-to-volume ratio is specific manner, e.g., by catalase, SOD, or glutathione minimal, induces incomplete oxidation (anaerobic glycolysis) FIGURE 1 | Metabolic pathways and biocombustion modes inside the cell. Frontiers in Immunology | www.frontiersin.org 3 March 2022 | Volume 13 | Article 750175 Ernst and Puntes Nanoceria, Imunometabolism and ROS TABLE 1 | The three different mitochondria operating modes. CATABOLIC PATHWAY Fuel Oxidizer Oxydation mode Mitochondria Mitochondria Power ROS shape S-to-V ratio suppy production Aerobic Glycolysis Glucose Oxygen Aerobic Elongate Medium Low Basal Mitochondrial ROS assisted cellular Glucose Oxygen Anaerobic/ Spherical Low High High metabolism &ROS truncated aerobic glycolysis Fatty Acid b-oxidation Fatty Oxygen Aerobic Hyperfused High Medium Medium Acid and excessive ROS production, decreasing the cell redox different free radicals. Free radicals propagate in chain potential and igniting the pyruvate in the cytoplasm. Later, reactions. Thus, once a reactive free radical is generated, it can mitochondria can fuse and expand their surface, increasing react with stable molecules forming new free radicals. For polarization and creating elongated and branched structures example, the unpaired electron transfer from O to N and S with a higher surface-to-volume ratio, allowing for molecules produces reactive nitrogen species and reactive sulfur FA consumption. species, all highly oxidant. Chain termination occurs when two Accordingly, mitochondria reduce their number and surface free radical species react with each other to form a stable, non- area (depolarize) when the cell power requirement is high. In this radical adduct. This, as discussed below, can be promoted by case, glucose is fully oxidized and directly in the cytoplasm at nanoceria. In addition, ROS, in the form of H2O2, easily cross high rates. This can be clearly observed in the connection biological membranes escaping their compartment and leaking between glucose transport and mitochondrial mode of work in into the tissue, diffusing in and outside cells. conditions of OS (1), where stimulation of cellular glucose All this is especially critical regarding the immune system uptake is frequently concomitant to inflammation. Therefore, (25), where the different immune responses have clearly different during anaerobic glycolysis, a high glycolytic flux and impaired energetic needs and power requirements, especially during oxidative phosphorylation are associated with increased ROS inflammation (26). For decades, it has been observed that levels (20, 21). Interestingly, glucose uptake has also been immune cells transform from relative metabolic quiescence to described a s an anti-ROS mechanism since excess a highly active metabolic state during inflammation (1). mitochondrial ROS is consumed, burning the extra uptake of Inflammation requires high power consumption. This glucose (22). This is consistent with the fact that the oxidizer is encourages the immune system to increase the production of the limiting reagent, and then the oxidizer concentration can be cytokines and chemokines, phagocytosis, immune cell decreased by increasing the fuel supply. When ROS levels are too recruitment and activation. These changes in essential high and/or remain increased during a prolonged time, a vicious metabolic processes affect fate and function over a broad range circle of ROS-stimulated glucose uptake and glucose-stimulated of different timescales and cell types, making the expression of ROS production can be triggered. This pathological cycle can be inflammation in different organs and conditions complex. In broken by restoring mitochondrial ROS production to normal contrast, its basal metabolic pathways are very conserved. levels, a phenomenon that has stimulated interest in Therefore, it is possible to target metabolic processes by antioxidant therapies. scavenging ROS during an immune response, modulating thus Antioxidant therapies should not eliminate all ROS. Under immune activity. normal conditions, the potentially harmful effects of ROS are Accordingly, macrophages, fundamental cells of the innate successfully restrained by protective and reparative mechanisms. immune system responsible for detecting, categorizing and Compartmented controlled ROS levels act as signaling molecules eliminating pathogens or aberrant cells, tissue repairing, to mediate localized events via the oxidative modification of development, and resolution of inflammation (27) follow the redox-sensitive mediators, which are needed at low doses for same metabolic trends. Macrophage functions can be grouped many normal biological functions, such as DNA replication and into three well-described macrophage functional phenotypes repair. However, they become toxic at high concentrations when (also called cellular polarization) (28): M0 for resting, the antioxidant cell defenses are overwhelmed. High ROS quiescent, macrophages; M1 for classical pro-inflammatory concentration induces OS, damaging phospholipids and DNA, activation; and M2 for alternative activation when resolving an inducing cell alterations, provoking mutations, and cell death. inflammatory reaction and promoting tissue repair (29). It has Consistently, abnormal ROS overproduction has been involved, been independently and repeatedly observed that M0 works on directly or indirectly, in the pathogenesis and progression of aerobic glycolysis, M1 on anaerobic glycolysis, and M2 on FAO, many diseases, to the point that it is fair to ask if there is any providing different amounts of power and different ROS disease without associated abnormal ROS production. And the concentrations in each case (2) (Figure 2). Thus, changes in answer seems to be no (23, 24). the basic macrophage metabolism occur following immune ROS are highly reactive free radicals and, therefore, short- activation, shifting from reliance on aerobic glycolysis to lived molecules, and can therefore be used by the cell to produce increased anaerobic glycolysis, or FAO. The different rapid and local responses. They are also challenging to target macrophage polarization energy processing has been studied since they are highly mutable and quickly transform into under the concept of immunometabolism (30). Frontiers in Immunology | www.frontiersin.org 4 March 2022 | Volume 13 | Article 750175 Ernst and Puntes Nanoceria, Imunometabolism and ROS The final question is how to modulate the immunochemical disorders. Indeed, it gained unaccountably rapid and widespread potential. As high ROS concentrations are needed to sustain popularity to treat sickness and coughing, and other nervous anaerobic glycolysis, the cell response can be controlled by disorders such as chorea and epilepsy. Its fall into oblivion was controlling ROS concentration. Modulating ROS should almost as quick as it rose and for equally unclear reasons. It was change the cell chemical potential, inducing metabolic said that: “Here, perhaps, is a good case of the right drug being adjustments and phenotypic changes towards inflammation used for the wrong reason” (34). Diversity of opinion regarding resolution and homeostasis restoration in the case of the therapeutic value of cerium oxalate has existed ever since. It is macrophages. For that, ROS flooding the tissue and mutating possible that the lack of a mechanistic description of its action, and have to be removed in many forms from everywhere, for a the lack of standardized materials, were at the root of the sufficiently long period of time, conditions that can only be controversy. Afterward, in the late 1950s, cerium oxide and achieved at unrealistically high doses with currently available phosphate tested in rats showed anti-inflammatory efficacy substances and traditional molecular medicine. attributed to their dual valence state of oxidation (35). At these times, cerium and other lanthanide compounds found use as bio- imaging contrast agents in light and electron microscopy for the in THE NANOCERIA IMMUNOMODULATORY situ detection of oxidase (36, 37) and phosphatase (38) activity MECHANISM since oxidized cerium precipitates in the form of highly electron- dense NPs. Later, in 1999, Telek et al. (39) used cerium chloride for Nanoceria has recently raised as an anti-inflammatory agent the in vivo histological detection of oxygen-derived free radicals in working very well in a wide variety of disease models. Indeed, inflammatory conditions, and observed anti-inflammatory effects. cerium and other rare-earth compounds have been employed in Although the precise mechanisms were not elucidated, the report medicine since the 19th century (31–33). The first reported use hinted at a possible role of cerium precipitates in the observed was that of Ce3+ oxalate as an antiemetic agent during pregnancy, decrease of ROS concentration. Besides, during the 20th century, reported by the Scottish doctor J.Y. Simpson in 1854. nanoceria was extensively developed in the petrochemical and Subsequently, it came to be prescribed for other gastrointestinal automobile industry as catalysts. Currently, nanoceria is also FIGURE 2 | Macrophage phenotypes, mitochondria morphology and corresponding catabolic pathways. Frontiers in Immunology | www.frontiersin.org 5 March 2022 | Volume 13 | Article 750175 Ernst and Puntes Nanoceria, Imunometabolism and ROS employed as a polishing agent, as a glass constituent to prevent 51). Notably, the nanoceria cubic fluorite crystal structure is solar discoloration, and in coatings to protect metallic materials preserved while Ce4+ is reduced to Ce3+ and oxygen vacancies from corrosion. Its promising past having been forgotten, the formed. Consequently, Ce3+ can be easily re-oxidized (recycled) nanoceria biomedical potential was re-discovered in Virginia Tech to Ce 4+ and the vacancy covered, completing thus its less than two decades ago, when it was observed that nanoceria of catalytic loop. less than 20 nm prolonged the lifespan of brain cell cultures for The observed competitive nanoceria advantages, such as its periods of up to 6–8 months (40). This finding was described by electron sponge effect, catalytic behavior, and potential Professor Berverly Rzigalinski, and collaborators Sudipta Seal, biodegradability, should be looked for in its electronic David Bailey, and Swanand Patil, as “somewhat serendipitous”, structure. Metallic cerium has an electronic configuration [Xe] according to her words (41) since she was studying nanoceria as a 4f15d16s2, Ce3+ has an electronic configuration [Xe] 4f15d06s0, drug carrier. Since then, many studies have been performed by and Ce 4+ has an electronic configuration [Xe] 4f 05d06s0, them and others, and the potential therapeutical effects of indicating that the 4f electron is the labile one. The main nanoceria examined in many animal models of disease. difference of rare earths from other elements is to have these 4f The first diseases subject to nanoceria treatment in pre- orbitals, whose electrons are shielded by 4d and 5p orbitals. This clinical models have been related to inflammation and diseases orbital shield makes 4f electrons weakly bound to the nucleus, where antioxidants substances were previously assayed with allowing for the Ce3+/Ce4+ tautomery. Thus, the two oxidation positive results, from sepsis to age-related degeneration. states of the cerium element in the face-centered cubic crystal Regarding sepsis, nanoceria has shown promising results lattice make possible the formation and occupation of oxygen regarding septic shock treatments where the mortality rate vacancies essential to its oxygen (electron) buffering capabilities, induced by LPS sepsis in rats decreased from 73% to 11% (34, and thus its ability to act as a catalyst for both oxidation and 42, 43). Indeed, nanoceria has also been proposed to counteract reduction reactions. the lethal effects of cytokine storms in COVID-19 patients (44). Nanoceria has been described as an antioxidant and anti- Regarding aging (45), nanoceria has shown significant protective inflammatory agent since it produces effects similar to those effects in age-related diseases such as retinal degeneration (46), substances. By measuring the nanoceria electronic structure in Alzheimer’s, and Parkinson’s (47, 48). Nanoceria has also real-time by X-ray absorption spectroscopy during the catalytic demonstrated a positive impact in metabolic disorders, rather degradation of H2O2, a rapid uptake of electrons by the NPs was orphan of treatment, such as the metabolic syndrome or non- evidenced, followed by a later and slower release of these alcoholic fatty liver disease (NAFLD) (49), where ROS contribute electrons, and corresponding pH modifications, in such a way to the initiation and progression of the disease. Another that nanoceria was described as electron sponges (52). A labile prominent example of metabolic disorder related to disease is unpaired electron from the free radical can be passivated by cancer, illustrated by the Warburg effect and the metabolic either pairing it with another electron (antioxidants provide this reprogramming of cancer and other cells in tumor electron), or removing it (by antireducers), with opposite effects microenvironments, where anaerobic glycolysis is favored (44), on pH. Thus, nanoceria is antireducer (Ce4+ to Ce3+), and suggesting that proliferation contra naturam costs extra energy. antioxidant (Ce3+ to Ce4+) during recycling, providing a high Another field where nanoceria could be beneficial is regenerative capacity to remove excess ROS from its surroundings. Normally, medicine. One of the biggest challenges in regenerative medicine substances that uptake electrons are called electron sinks or and tissue engineering is to deal with inflammation. Typically, antireducers, rather than antioxidants, even if the ROS the tissue to reconstruct is under oxidative stress due to tissue scavenging effects are similar. Such molecules, antireducers, are damage that impedes proper regeneration. In this regard, in a ubiquitous in nature, especially in the photosynthesis reaction partial hepatectomy animal model, rats treated with nanoceria chain, where the generation of ROS by-products is higher than showed a significant increase in liver regeneration compared during metabolism. with controls (50). Similarly, in an acetaminophen overdose Taking the hydroxyl molecule as model ROS, the oxygen atom experimental model, nanoceria and N-acetyl-cysteine in the OH· molecule is surrounded by 7 electrons in its valence treatments decreased early liver damage. However, only band, and it is unsatisfied, longing for 8 (the octet). This situation nanoceria was associated with a significant increment of can be overcome if the unpaired electron is removed, and Ce4+ hepatocellular proliferation (50). passes to Ce3+, and the OH· becomes ½ O2 plus the formation of The nanoceria chemical formula is commonly written as H+ (equation 1). Alternatively, the OH· molecule may get an CeO2 since its primary oxidation state is Ce4+. Nevertheless, electron from the nanoceria, and Ce3+ recycles to Ce4+, and the defects in the crystal structure are usually present at the OH· transforms into OH- (equation 2). Thus, the nanoceria nanoscale, and some Ce ions present a Ce3+ instead of a Ce4+ catalytic cycle, with corresponding pH modifications and oxygen valance state. Having Ce3+ instead of Ce4+ induces a deficiency of generation, can be described as follows: positive charge, compensated with oxygen vacancies, usually occurring at the NP surface. The Ce3+ concentration in the Ce4+ (s) + OH·(I) − > Ce3+ (s) + 1=202(g) + H+(I) (1) NP, hence oxygen vacancies and redox activity, increases as the NP size decreases, achieving its maximum capacity and thermodynamic stability at diameters of a few (2 to 5) nm (50, Ce3+ (s) + OH·(I) − > Ce4+ (s) + OH− (I) (2) Frontiers in Immunology | www.frontiersin.org 6 March 2022 | Volume 13 | Article 750175 Ernst and Puntes Nanoceria, Imunometabolism and ROS If we add the two equations: Nanoceria biodegradation can also be described with the above equations. Cerium oxide is known to be a non- 2OH·(I) − > H2 O(l) + 1=202(g) (3) biodegradable material. However, in its nanometric form, at Accordingly, when mixing nanoceria and H2O2, oxygen neutral pH and low oxygen concentration, nanoceria in water generation is clearly observed in the form of vigorous thermodynamically prefers to stay in the Ce3+ soluble valence bubbling. Note that oxygen generation by nanoceria could be state rather than in the Ce4+ insoluble valence state (see Pourbaix related to their observed pro-angiogenic properties (53). diagram in SI (55)). Thus, during the catalytic cycle, an However, we have to take two other equations into account. “activated” state, Ce3+, can leak from the NP and swim away. At low OH· concentration, reaction (2) can be outcompeted by Consequently, in vivo, nanoceria can degrade into innocuous Ce3 + (4): ions, expulsed from the body through the urine. The degradation of nanoceria during its biological action was Ce4+ (s) + 1=2 O2(g) + H+ (I) − > Ce4+ (s) + OH·(I) (4) reported for the first time in 2014, after observing how intracellular antioxidants dissolve man-made antioxidant and the ROS scavenging activity stopped, or even its production nanoparticles and proposing to use the redox vulnerability of promoted. Additionally, at low ROS and O2 concentrations, the nanoceria to develop a responsive drug delivery system (56). Ce3+ ion, which is soluble at pH below 8 (see Pourbaix diagram Recent data show how nanoceria distributes, accumulates, and is in SI), may dissolve away from the NP (indeed, it is the Ce4+ expulsed from healthy mice after intravenous injection (54). At crystal structure that holds the Ce3+ soluble ions in the solid few hours after injection, the NPs are accumulated in the liver phase), and nanoceria slowly disintegrates, (equation 5). and spleen. From this point, the cerium concentration progressively decreases following an exponential decay where Ce3+ (s) − > Ce3+ (I) (5) half of the dose has been expulsed in 6 weeks. During this In other words, nanoceria can uptake a limited number of experiment, cerium was found in urine and feces (57). Probably electrons maintaining their fluorite crystal structure and NP ions in the first case and NPs in the second, expulsed through the integrity. Due to the water solubility of Ce3+ ions and an hepatobiliary route. This degradation of nanoceria can be increasing concentration of oxygen vacancies, if Ce3+ ions are promoted by increasing the reducing environment, decreasing not recycled to Ce4+, at some point, the fluorite crystal structure pH (as in endolysosomes), and complexing molecules that cannot be maintained, and the NP disintegrates. absorb Ce3+ ions in solution and remove them from the The combination of these equations makes nanoceria act as a equilibrium. As a final consideration, the smaller the NP, the redox buffer. Free radicals have to be continuously supplied to the higher their dissolution rate. Regarding nanoceria excretion, it is NP surface to allow their combination into more stable species. worthy to mention the observed excretion of nanoceria coated When the ROS concentration is low, and this condition cannot be with dextran when administered orally as a computed fulfilled anymore, the NP loses its catalytic activity, and reactions tomography contrast agent for imaging the gastrointestinal 1 and 4 combine instead of 1 and 2. This ROS concentration tract in a mice model of Inflammatory Bowel Disease (58). threshold needed to trigger nanoceria activity is apparently found to be between M1 and M2 phenotypes. In such a way that during M1 polarization, ROS is efficiently scavenged by nanoceria, but no PHARMACOLOGICAL DEVELOPMENT nanoceria activity or biological effect has been observed when OF NANOCERIA cells are expressing an M0 or M2 phenotype. Indeed, it has been recurrently observed that the use of nanoceria enables the These last two decades have started building a pharmacokinetic expression of M2 polarization and increased production of model for nanoceria, its behavior in physiological media, SOD, Arginase, or NOS synthetases (54), well known M2 administration, biodistribution, degradation, and excretion. enzymes and cytokines, employed to protect the cell from OS. Still, today, the pharmacological knowledge on the subject is This is probably not because nanoceria promotes M2 premature, mainly due to material uncertainty. First, NPs for polarization. This is simply because when M1 polarization is medicine should be monodisperse, biocompatible, small, and stopped, M2 is allowed to take control of the produced damage highly dispersible in physiological media, with engineered and repair the tissue. In the opposite direction, nanoceria ROS surfaces to escape from phagocytosis (59). The traditional buffering capacity increases with ROS concentration up to NP industrial basic precipitation employed for the preparation of surface saturation. Taking into account that ROS have to arrive, nanoceria is normally continued by a calcination step to fully absorb, react and desorb from the NP surface, surface saturation oxidize Ce3+ to Ce4+ and fully dehydrate Ce(OH)4 into CeO2. will determine the highest ROS concentration it can be managed Nanoceria prepared in these conditions sinters and grows, losing at once. Consequently, the nanoceria scavenging reaction rate will its therapeutical properties, and needs high temperature to be be constant while ROS is in excess (according to the NP surface). activated, which is common in its industrial applications. This reaction rate will diminish as ROS concentration decreases Interestingly, current procedures for preparing nanoceria for until it stops, corresponding to homeostatic concentrations of medical applications by basic precipitation yield small colloidal ROS. This buffering capacity is at the origin of their Ce+3/+4 hydroxide/oxide NPs that can be employed in biology. immunomodulatory properties. The Ce3+/Ce4+ ratio is not always specified in the scientific Frontiers in Immunology | www.frontiersin.org 7 March 2022 | Volume 13 | Article 750175 Ernst and Puntes Nanoceria, Imunometabolism and ROS literature despite being highly dependent on the NP size, tumors, described by Maeda et al. in 2002 (69), where due to preparation technique, NP history, and surface state. It can be abnormal angiogenesis, blood vessels supplying nutrients to solid determined by some characterization techniques, including X- tumors have defects in their tiling, and large pores (hundreds of ray photoelectron spectroscopy (XPS) (60), X-ray absorption nm) are formed, making the tumor accessible to nanocarriers, spectroscopy (52), and UV-visible absorption spectroscopy (UV/ which together with poor lymphatic drainage, facilitate their VIS) (61). accumulation (indeed albumin act as a nanometric carrier for We recommend the use of CeCl3 as cerium precursor instead cisplatin favoring its accumulation in tumors) (70). Second is the of Ce(NO3)3 when using TMAOH as the base (chosen because of increase of barrier permeability during inflammation, as in the the stabilizing effect of TMA+ counterions) since the presence of case of neuroinflammation, granting access to the brain to NPs nitrate in the synthesis process may derivate in nitrosamine after i.v. or i.p. injection (68, 71). All in all means that nanoceria contaminants which are of serious concerns for the regulatory can be designed to passively reach and stay in different organs for agencies. In this respect, the “FDA guidance for industry on drug an extended period of time, depending on NP features, medical products, including biological products, that contain state, and administration route. nanomaterials” (62) can be of great help in developing medical At the cellular level, NPs distribution has also been well nanoceria. Related to that, a paper indicating how to carry pre- described (72). For many different materials such as gold or iron clinical studies on nanoceria harmonized with FDA regulations oxide, NPs are found to be densely aggregated in endosomes has recently been published (63). It is expected that nanoceria persistent during the experimental times. The same is observed will have to follow the path other metal oxide inorganic NPs have with nanoceria (54). In these cases, one would say that the followed, such as Fe3O4 NPs, approved as a contrast agent for nanoceria will not be functional because it is kept away from the MRI (Resovist®), as iron supply in the case of iron-deficiency cytoplasm, and this is true; however, as ROS can cross the anemia (Feromuxytol®) or as hyperthermia agent to treat phospholipid bilayers in the form of H2O2, the aggregate neuroblastoma (Nanotherm®). nanoceria can perform its task scavenging cytoplasmatic ROS Special attention must be given to size, parental (as- that enters the endosome. Hypothetically, if long-lasting and synthesized), and eventually, aggregated (when dispersed). functional, these structures could be pictorially called ceriasomes, Aggregation, especially in physiological media, corresponds to a nanoceria highly loaded (hundreds of NPs) endosome which the NP natural tendency to reduce surface area and consequently scavenges free radicals as soon as they enter, as artificial surface energy. Size is critical for both the catalytic activity of intracellular OS protective organelles. Besides, nanoceria nanoceria and their pharmacological properties. This is because permeation out of the endosome can increase during endosomal the number of oxygen vacancies increases with reducing the size acidification if it loses its surface charge and become non-charged and surface accesses increases for non-aggregate NPs, and (depending on lysosome pH, nanoceria concentration, and because size is a major parameter of the administration, nanoceria isoelectric point). In addition, proton-sponge-like biodistribution, metabolization, and excretion profile of effects due to its basic oxide surface may contribute to NPs (57). endosomal disruption. Nevertheless, this appears to happen only The biodistribution of NPs is different from traditional small eventually. Therefore, most nanoceria will remain in endosomal drugs designed to cross biological barriers and membranes, and vesicles inside the cytoplasm, acting as a ROS scavenging organelle. distribute across the body. Nanoceria follows the main principles These studies are, in part, possible thanks to the easy of NP biodistribution which depends on size, hydrophilicity, and traceability of cerium. As a xenobiotic element, its background surface charge (64). The initial observation is how reducing NP presence in the body is negligible, making it possible to trace its size extends blood circulating times and reaches good levels of presence to attomolar concentrations by inductively coupled homogeneous distribution in tissues (65). For neutral and plasma mass spectroscopy (ICPMS). Additionally, thanks to its negatively charged small inorganic NPs, depending on the high Z number, it gives strong contrast, not only in optical and portal of entry, different organs can be targeted. Normally, electron microscopies but also to X-ray (58). These aspects are after i.v. administration, NPs accumulate in the liver (90%) not shared by conventional drugs that “disappear” as soon as and spleen (9%) after a few hours of blood circulation (66). they enter the body, and complex chemical and biochemical However, NPs smaller than 6 nm can be rapidly cleared through resolution-limited techniques must be employed. the urinary tract (67). To avoid this, small nanoceria can be Regarding dosing, nanoceria has been administered conjugated to different biomolecules, such as albumin (54), to formulated with BSA (54), PEG (71), sodium citrate and prevent aggregation and avoid renal clearance. The reported EDTA (68), or a series of drugs (73). In most cases, aggregates most prolonged half-life in blood for nanoceria has been about 4 of few tens nm have been employed, however, works with non hours after injection (68). It is also feasible to target the lymph aggregated NPs showed better biodistribution and increased nodes after intramuscular injection, or the eye, the skin, and the biocompatibility. In any case, despite formulation and gastrointestinal tract, by oral and topical administration. If aggregation state, successful nanoceria applications work at injected into a tumor or the brain, NPs tend to remain inside concentrations of a few micrograms (50 to 250 µg) per gram of the organ. It is also essential to consider that body barriers tissue, administered in single injections at concentrations of controlling NP dispersion are altered during the course of about 1 to 10 mg/ml, which in the case of 3 nm NPs disease. Two significant cases are worthy of mention. First is corresponds to 9.25x1015 NPs/ml to 9.25x1016 NPs/ml. It is the enhanced penetration and retention effect of NPs into solid normally administered up to 1 mg nanoceria per Kg of animal, in Frontiers in Immunology | www.frontiersin.org 8 March 2022 | Volume 13 | Article 750175 Ernst and Puntes Nanoceria, Imunometabolism and ROS 200 (mice) or 300 (rat) microliter volumes (Table 2, see an NP toxicity (83). An example is the work of Dowding et al. where extended version in the SI, which includes formulation and a similar nanoceria synthesis process was done, but using administration route, among others). different bases [NH4OH or hexamethylenetetramine (HMTA)] (86). Results showed that HMTA-nanoceria NPs were readily Nanosafety taken into endothelial cells and reduced cell viability at a 10-fold Since the seminal work of Vicky Colvin in 2003 (56), nanosafety lower concentration than the other NPs, which showed no has been one of the most significant issues when discussing NP toxicity. Finally, a paper was published recently on a woman medical, industrial, and customer applications. Since then, a who drank a large amount of nanoceria-based polishing powder great effort has been dedicated to studying the detrimental by mistake (87). The product was not described, but this aspects of NPs. Initial results were sometimes puzzling and industrial nanoceria is a mix of NP aggregates at a relatively confusing. In this regard, Prof. Harald Krug, in a 2014 review high pH. The observed transient toxic effects could be related to article (83), analyzing about 10.000 nanotoxicology papers, the basic pH of the preparation and the presence of Ce3+ soluble revealed that most of the nanotoxicity studies “do not offer a species in the formulation. Besides, observed coagulation clear statement on the safety of nanomaterials and, on the disorders had been previously described with Ce3+ at high contrary, most of them are either self-contradictory or arrive at doses because of their interference with Ca2+ homeostasis (88). completely erroneous conclusions.” Indeed, it has been observed Therefore, well-described, pure, monodisperse, and highly that at realistic doses in a controlled manner, NPs show no dispersible in physiological media nanoceria is mandatory for significant increased toxicity compared to their molecular or this promising material meaningful and controlled use for therapy. bulk counterparts. The reported toxicity often has to be One strategy to avoid nanoceria toxicity due to aggregation when attributed to NPs aggregation and NPs association with toxic dispersed in physiological media is pre-albuminization before moieties (endotoxin, surfactants, or allergens) rather than the injection (54). The albumin has not to be firmly bound to the NPs themselves, leading to apparently contradictory data. NP. Its mere presence prevents NP aggregation by interacting with Nanoceria is an illustrative case (84). While it is reported its surface. Once injected, NPs dilute in the bloodstream, or tissue, many times to be beneficial in protecting against oxidative putting away aggregation risk. stress and irradiation damage, other studies, mainly related to Finally, cerium is affordable, abundant as silver (not in veins the toxicity of nanoceria in the industrial dry form (nanometric though, which makes its mining complicated), and nanoceria is aggregate powders), show in vitro and in vivo toxicity (81). easy to produce following green chemistry principles -at RT with Similarly, while some studies show anti-inflammatory effects of simple reagents having recyclable basic waters as by-product. It is nanoceria taken up by hepatocytes (54), others report liver stable in simple storage conditions, and of universal use (the same macrophage (Kupffer cells) uptake and pro-inflammatory NPs perform well in different disease models). Additionally, it is effects (85). This often results from the challenging xenobiotic, which makes it easily traceable by imaging and dispersibility of inorganic NPs in physiological media that too spectroscopic techniques such as X-ray or mass spectroscopy, often leads to NP aggregation and sedimentation, losing their facilitating its preclinical studies. Thus, nanoceria and other beneficial properties. In vitro studies showed how the nanoceria nanozymes may represent a new era for medicine, where the ROS scavenging capacity increased with nanoceria concentration ability to buffer excess ROS allows for better general population until it was lost when NPs aggregated at higher concentrations health (anti-aging, anti-tumoral and anti-inflammatory). and started being pro-inflammatory (54). Interestingly, the Summarizing, the relationship between metabolism and disease therapeutic doses are far from these toxic doses (10 to 100 has been extensively explored during the past decade. times). Large aggregates are easily detected by the immune Understanding how cells use energy to perform their functions system, and often a pro-inflammatory response is triggered, has attracted attention concerning diseases such as obesity, diabetes, making the medical use of NPs complicated because of cancer, and neurodegeneration. Indeed, pathological inflammation material uncertainty. Ji et al. demonstrated the importance of is at the origin and progression of many diseases, from chronic, controlling NPs size, shape, and aggregation state. Inflammatory inducing accelerated aging and oncogenesis, to acute, such as immune response and toxicity were only reported when using ischemia, cytokine storms, and anaphylaxis. Antioxidant high aspect ratio nanoceria nanowires at high doses and substances have shown promising immunomodulation in pre- aggregation state. Besides, it is important to note that some clinical and epidemiological studies, and their mechanism has toxic ingredients coming from the NP formulation or derived been observed in detail. However, they are still poorly translated from chemicals employed during NP preparation can misreport to the clinic. The inconsistencies between the mechanistic and TABLE 2 | Dosing of nanoceria in different in vivo studies. Study # 1 (74) 2 (75) 3 (76) 4 (77) 5 (78) 6 (79) 7 (68) 8 (80) 9 (81) 10 (82) 11 (71) Size* 3 1, 3 10 No data 3 1-2.5 2.4 3 10 No data 3.3 (nm) Dose (µg/g) No Data No Data 0.05, 0.5, 5, 50 0.1 30, 100 10, 6 10, 20, 30 20 0.05, 0.5 0.05, 0.5, 5 0.1, 0.3, 0.5, 0.7, 1, 1.5 *parental size, independently of aggregation state. Frontiers in Immunology | www.frontiersin.org 9 March 2022 | Volume 13 | Article 750175 Ernst and Puntes Nanoceria, Imunometabolism and ROS epidemiological studies, and the clinical trials, indicate the poor by case since tissue environment, and the metabolic and immune pharmacological properties of currently available substances and the status depends on the studied tissue and medical condition. For need for new approaches and strategies. Today, nanoceria, catalytic example, the brain, despite its high consumption of glucose and mild antioxidant NPs, may provide the required pharmacokinetics tendency to suffer from oxidative stress, it is short in endogenous and overcome previous limitations, unleashing the power of antioxidant defenses. Or like pregnancy, which starts with the antioxidant prevention and therapy. immune system activating an M1 polarization to follow up with Cerium is a rare earth element that accumulates oxygen an M2 polarization from placentation up to delivery. vacancies in its nanometric oxide form capable of catalytically removing excess ROS in metabolic imbalance situations. Indeed, nanoceria act as a redox buffer, promoting immunomodulation without immune suppression. Nanoceria displays a good safety DATA AVAILABILITY STATEMENT profile to normal tissues while providing cellular protection from The original contributions presented in the study are included in various forms of ROS and irradiation. Thanks to its catalytic the article/Supplementary Material. Further inquiries can be nature, nanoceria can be used at low doses for a prolonged time directed to the corresponding author. (before NPs are degraded, dissolved, and excreted). Small nanoceria in the neutral pH and low oxidant conditions inside the body slowly dissolves in few months as the insoluble Ce4+ is progressively reduced to soluble Ce3+ ions, excreted through the AUTHOR CONTRIBUTIONS urine. Nanoceria is redox selective (only degrades ROS at high concentrations) but not ROS selective (degrades any form of LM and VP developed the hypothesis, search the literature, wrote ROS). Indeed, it is selective to a high concentration of unpaired the manuscript and approved it for publication. electrons regardless of the atomic orbital carrying them. When adequately formulated (endotoxin-free, stable, soluble), no harmful effects have been observed in in vitro and in vivo SUPPLEMENTARY MATERIAL models at applicable doses. Of note, cerium compounds (cerium oxalate and cerium nitrate) were used in the past, The Supplementary Material for this article can be found online among others, as antiemetic agents during pregnancy. at: https://www.frontiersin.org/articles/10.3389/fimmu.2022. Nanoceria formulation and dose will have to be developed case 750175/full#supplementary-material 11. Benfeito S, Oliveira C, Soares P, Fernandes C, Silva T, Teixeira J, et al. REFERENCES Antioxidant Therapy: Still in Search of the “Magic Bullet.” Mitochondrion 1. Liemburg-Apers DC, Willems PHGM, Koopman WJH, Grefte S. Interactions (2013) 13:427–35. doi: 10.1016/j.mito.2012.12.002 Between Mitochondrial Reactive Oxygen Species and Cellular Glucose 12. Xu C, Qu X. Cerium Oxide Nanoparticle: A Remarkably Versatile Rare Earth Metabolism. Arch Toxicol (2015) 89:1209–26. doi: 10.1007/s00204-015-1520-y Nanomaterial for Biological Applications. NPG Asia Mater (2014) 6:e90–0. 2. 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Cerium Oxide and do not necessarily represent those of their affiliated organizations, or those of N a n o p a r t i c l e s P r o t e c t A g a i n st MP TP - In d u ce d D opa m i n erg i c the publisher, the editors and the reviewers. Any product that may be evaluated in Neurodegeneration In A Mouse Model For Parkinson’s Disease. NSTI- this article, or claim that may be made by its manufacturer, is not guaranteed or Nanotech (2011) 3:451–4. endorsed by the publisher. 77. Hegazy MAE, Maklad HM, Abd Elmonsif DA, Elnozhy FY, Alqubiea MA, Alenezi FA, et al. The Possible Role of Cerium Oxide (CeO2) Nanoparticles in Copyright © 2022 Ernst and Puntes. This is an open-access article distributed under Prevention of Neurobehavioral and Neurochemical Changes in 6- the terms of the Creative Commons Attribution License (CC BY). The use, distribution Hydroxydopamine-Induced Parkinsonian Disease. Alexandria J Med (2017) or reproduction in other forums is permitted, provided the original author(s) and the 53:351–60. doi: 10.1016/J.AJME.2016.12.006 copyright owner(s) are credited and that the original publication in this journal is 78. Kwon HJ, Kim D, Seo K, Kim YG, Han SI, Kang T, et al. Ceria Nanoparticle cited, in accordance with accepted academic practice. No use, distribution or Systems for Selective Scavenging of Mitochondrial, Intracellular, and reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 12 March 2022 | Volume 13 | Article 750175