Nanoscale
c0nr00875c
FEATURE ARTICLE 1
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Pharmacological potential of cerium oxide nanoparticles Ivana Celardo, Jens Z. Pedersen, Enrico Traversa and Lina Ghibelli* 5
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Reactions involving redox cycles between the Ce3+ and Ce4+ oxidation states allow nanoceria to react with all noxious intracellular reactive oxygen species (ROS). This review outlines the biological effects of nanoceria as they emerge from in vitro and in vivo studies, considering biocompatibility and the peculiar antioxidant mechanisms.
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FEA C0NR00875C_GRABS
Nanoscale
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Dynamic Article Links <
Cite this: DOI: 10.1039/c0nr00875c
FEATURE ARTICLE
www.rsc.org/nanoscale 1
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Pharmacological potential of cerium oxide nanoparticles a
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Ivana Celardo, Jens Z. Pedersen, Enrico Traversa and Lina Ghibelli 5
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Received 17th November 2010, Accepted 28th December 2010 DOI: 10.1039/c0nr00875c
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Nanotechnology promises a revolution in pharmacology to improve or create ex novo therapies. Cerium oxide nanoparticles (nanoceria), well-known as catalysts, possess an astonishing pharmacological potential due to their antioxidant properties, deriving from a fraction of Ce3+ ions present in CeO2. These defects, compensated by oxygen vacancies, are enriched at the surface and therefore in nanosized particles. Reactions involving redox cycles between the Ce3+ and Ce4+ oxidation states allow nanoceria to react catalytically with superoxide and hydrogen peroxide, mimicking the behavior of two key antioxidant enzymes, superoxide dismutase and catalase, potentially abating all noxious intracellular reactive oxygen species (ROS) via a self-regenerating mechanism. Hence nanoceria, apparently well tolerated by the organism, might fight chronic inflammation and the pathologies associated with oxidative stress, which include cancer and neurodegeneration. Here we review the biological effects of nanoceria as they emerge from in vitro and in vivo studies, considering biocompatibility and the peculiar antioxidant mechanisms.
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1. Introduction
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Nanotechnology allows the fabrication and manipulation of materials at the nanometre scale, promising novel developments for a huge variety of applications, including pharmacology.1–3 Ad a
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Dipartimento di Biologia, Universit a di Roma Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy. E-mail:
[email protected]; Fax: +39-06-2023500; Tel: +39-320-4317094 b International Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan
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Ivana Celardo
Ivana Celardo in July 2007 graduated in Pharmaceutical Biotechnology at the University of Naples Federico II, summa cum laude. In December 2010, she obtained from the University of Rome Tor Vergata her PhD in Molecular and Cellular Biology, under the supervision of Dr L. Ghibelli. Her research interests concern biological and toxicological effects of nanoparticles, with particular emphasis on the study of rare earth oxide nanoparticles.
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hoc nanoparticles may overcome intrinsic problems of drug efficacy, allowing targeted delivery and passage through biological barriers. Nanoparticles with therapeutic potential include tailored nanostructures where the core is a vehicle to carry the active molecules, and may include additional determinants to perform specific functions like docking. A novel category considers nanoparticles where the material itself may act directly as the therapeutic agents, primarily focusing at materials performing reversible antioxidant redox reactions. The antioxidant effect is paramount, since practically any disease, including
Jens Z. Pedersen received his Cand. Scient. degree in Biochemistry at the University of Southern Denmark in 1985. After various fellowships, and a staff position at the National Research Council (CNR) Institute for Experimental Medicine in Rome, Italy, he returned to the University of Southern Denmark in 1996 to build up an EPR Laboratory at the Dept. of Chemistry. Since 2000 he is an Jens Z: Pedersen Associate Professor at the Dept. of Biology, University of Rome Tor Vergata. He is interested in all possible, and several impossible, aspects of radicals in biological systems, and has published more than 80 papers on this topic.
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serious ones such as tumors, neurodegenerative diseases, immunodeficiencies, etc., involves oxidative stress (Fig. 1). In the oxide form of cerium, a lanthanide or rare-earth element, cerium(IV) and cerium(III) oxidation states coexist with the former being the stable form,4 producing a redox couple that is responsible for catalytic activity.5,6 The reduction in the positive charge by Ce3+ is compensated by a corresponding number of oxygen vacancies. The concentration of defects, both Ce3+ ions and oxygen vacancies, is larger at the surface of ceria than in the
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2. Oxidative processes and oxidative stress in biological systems
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bulk;7,8 therefore nanoceria have higher Ce3+ concentration and enhanced redox activity with respect to larger particles, given the increase in the surface-to-bulk ratio.9 For these reasons, nanoceria is used in a wide range of industrial applications, including catalysis,10 solid oxide fuel cells,11,12 solar cells,13 gas sensors,14 and UV screens.15 Nanoceria is receiving much attention as a potential antioxidant agent in vivo, because of very attractive features such as high biocompatibility and the possibility to regenerate the initial oxidation state through redox cycling reactions.16 A recent firstprinciples study showed that the defect concentration at the surface increases when ceria is exposed to water, which is relevant in biological media.17 Initial biological studies have highlighted cell protective,18 neuro-protective19,20 and cardio-protective effects,21 improvement of stem cell adhesion22 suggesting potential use in tissue engineering,23 and anti-inflammatory properties,24 possibly due to the antioxidant properties of nanoceria.
Fig. 1 Oxidative stress and human pathology. Oxidative stress is involved in many human diseases, which are caused or favored by deregulated and aberrant free radical production or insufficient radical scavenging. For this reason, great emphasis is given to the development of antioxidant therapies.
All human cells produce free radicals as inevitable byproducts of a metabolism based on reduction–oxidation reactions, and this occasionally causes the formation of partially reduced oxygen forms, commonly known as reactive oxygen species (ROS). These can be highly reactive and potentially very dangerous to cells, and must be scavenged by exogenous or endogenous antioxidant systems to keep their level below a critical threshold.25 When ROS production is excessive, or the antioxidants are not sufficient to prevent the increase in ROS levels, the result is a condition termed oxidative stress, which may cause or contribute to many pathologies and disturbances.26–28 Accordingly, supplementation of exogenous antioxidants is used to
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Enrico Traversa is a Principal Investigator at the International Research Center for Materials Nanoarchitectonics (MANA) at the National Institute for Materials Science (NIMS), Tsukuba, Japan, leading a group on Nanomaterials for Fuel Cells since January 2009. He joined the University of Rome Tor Vergata in 1988 where he is a Professor of Materials Science and Technology. Since 2010 he Enrico Traversa is an Adjunct Professor at Waseda University. His research interests are in nanostructured materials for environment, energy, and healthcare, with special attention to solid oxide fuel cells. He is an author of more than 420 scientific papers (more than 270 of them published in refereed international journals). Elected in 2007 in the World Academy of Ceramics, he is currently an Associate Editor for the Journal of Nanoparticle Research and Science and Technology of Advanced Materials.
Lina Ghibelli joined the University of Rome Tor Vergata after obtaining a doctoral degree at the University of Rome La Sapienza and holding several Research Associate positions at the University of Chicago and EMBL, Heidelberg. Cell biologist and a leading scientist in the field of apoptosis for more than two decades, she coordinates a research group focused on the molecular mechanisms of Lina Ghibelli apoptosis after cell damage (intrinsic apoptotic pathway), as well as environmental modulators of apoptosis including electromagnetic fields and recently also nanostructured materials. She is the principal author of many high impact, highly cited peer reviewed scientific papers.
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ameliorate acute or chronic oxidative conditions, as in neurodegenerative diseases and chronic inflammation processes, and to prevent oxidation-induced damage that may lead to cell mutation and cancer.29 However, ROS are not only noxious molecules; they are also essential endogenous participants in cell signal transduction pathways, and a basal ROS level is necessary for cell homeostasis.25,30 Therefore, antioxidants have the role of maintaining the delicate intracellular equilibrium between excessive and insufficient ROS levels (Fig. 2). The prevalent reactive oxygen species are superoxide (O2), hydrogen peroxide (H2O2) and hydroxyl radical (cOH). The cellular defense against ROS comprises antioxidant enzymes, including superoxide dismutase (SOD), catalase and glutathione peroxidase, and low molecular weight antioxidants, such as ascorbate (vitamin C), a-tocopherol (vitamin E), and glutathione (GSH).27,31 Most damage due to ROS is believed to be caused by hydroxyl or alkoxyl radicals generated through oxidation of peroxides by transition metal ions (Scheme 1, eqn (1) and (2)); eqn (1) is known as the Fenton reaction, and in human cells such Fenton-type reactions are mainly performed by Fe(II) and Cu(I), i.e., metals in partial oxidation state. However, the single unpaired electron of a radical can also be eliminated through the reaction with a suitable transition metal; this principle is exploited by SOD, which transforms superoxide into H2O2 in a dismutation reaction (Scheme 1, eqn (3a) and (3b)). Finally, H2O2 is eliminated by catalase in a second dismutation reaction (Scheme 1, eqn (4a) and (4b)). Catalase and SOD are two of the most efficient enzymes known, in part because of the relatively high reactivity and rapid diffusion times of their substrates. But the cyclic dismutation mechanisms of SOD and catalase provide other important advantages that are not immediately obvious. The reactions do not require energy or reduction equivalents. The enzymes immediately react with the first substrate and can wait for the second to arrive; this ensures the efficient scavenging also at low ROS levels. Furthermore, the transition metals in the active sites are bound in a way to prevent undesired side reactions, such as in Scheme 1, eqn (1) and (2).
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Scheme 1 Fundamental physiological reactions of ROS. Single electron oxidation of peroxides by transition metals can generate hydroxyl or alkoxyl radicals (eqn (1) and (2)); eqn (1) is known as the Fenton reaction, and in human cells such Fenton-type reactions are mainly performed by Fe(II) and Cu(I). The hydroxyl radical is extremely unstable and in practice will react with the first biomolecule it encounters, whether protein, lipid, carbohydrate or nucleic acid; it may bind covalently to a target or abstract a hydrogen atom, but always form another radical species. We have no antioxidant defense against hydroxyl radicals, with the possible exception of glutathione, present in millimolar levels in cells and therefore statistically is a likely target. Superoxide dismutases use either Cu or Mn in the active site; they react with two superoxide ions in two consecutive steps (eqn (3a) and (3b)). The sum of the two halfreactions is the disproportionation (or dismutation) of two superoxides, one is reduced to hydrogen peroxide and the other oxidized to molecular oxygen. H2O2 is actually considered potentially more dangerous than superoxide due to the Fenton reaction; it is not a radical species, but is eliminated by catalase in a dismutation process similar to the one of SOD, except that the reduced product here is water (eqn (4a) and (4b)). The mechanism details are more complicated because two electrons need to be transferred, but again a transition metal (Fe) is involved. In other organisms different metals may be found in SODs (Fe, Ni) and catalase (Mn), but the overall dismutation reactions, eqn (3) and (4), remain the same.
Many attempts have been made to construct low molecular weight analogs of SOD and catalase based on different transition ion complexes. In general the development of such models needs to solve three major problems: (a) it is difficult to design the ligand sphere of the metal in a way to allow both the reductive and the oxidative half reactions to proceed with equal efficiency; (b) it is difficult to design a complex that will not be Fentonactive or produce other unwanted products; and (c) it is difficult to construct a biocompatible complex that can survive in a biological environment. Although many metal complexes have been shown to have activity in vitro, so far there are no examples of SOD and catalase model complexes approved for clinical application.32
3. Enzyme mimetic activities of nanoceria
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Fig. 2 Fate of free radicals in the cell. Free radicals are normally produced in the living cells; several enzymes, such as SOD and catalase, are implicated in maintenance of the equilibrium of basal ROS levels. When the production of free radicals prevails on the ability of antioxidant enzymes, ROS overproduction induces oxidative stress.
The 3+/4+ valence switch of nanoceria resembles the mechanism of redox enzymes, which use metals as co-factors to catalyze reversible redox reactions in cells and tissues. The reduction of superoxide requires nanoceria with a high Ce3+/Ce4+ ratio, because only Ce3+ is able to get oxidized and produce peroxide. This reaction corresponds to the reduction of superoxide by SOD (Scheme 1, eqn (3b)) and hence was termed SOD-mimetic.33 Restoring the reduced state of the metal present in SOD requires enzymatic recycling (Scheme 1, eqn (3a)); it has been suggested
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that nanoceria may ‘‘spontaneously’’ recycle, via a still obscure mechanism.19 In other studies, it was shown that also H2O2 is able to oxidize ceria from Ce3+ to Ce4+. This is also a reversible oxidation, and reduction occurs via an unclarified mechanism after a long permanence (15 days) in aqueous solution after complete H2O2 degradation.34 However, it was recently found that nanoceria can react with H2O2 in an additional way,35 with very important implications in terms of radical scavenging in vivo. This mechanism involves H2O2 reaction with Ce4+, which is reduced to Ce3+ with the concomitant oxidation of H2O2 to molecular O2 by a reaction similar to that of catalase (Scheme 1, eqn (4a)). This process is thus favored by a low Ce3+/Ce4+ ratio. The combination of the reactions with superoxide and hydrogen peroxide may provide nanoceria with an extra, very important antioxidant function that could make it very attractive as a biological ROS scavenger. In the literature much emphasis is given to the ability of nanoceria to spontaneously switch from the oxidized (4+) to the reduced (3+) state, thus recycling its antioxidant ability. The enthusiasm is understandable since a self-regenerating antioxidant would be an extremely valuable tool as a pharmacological agent,19,36 but to our knowledge a plausible mechanism of redox regeneration has not been proposed so far. However, if we put together the SODmimetic33,34 and the catalase-mimetic35 activities, we may envisage a bio-related mechanism of regeneration of nanoceria. When nanoceria reduces superoxide, H2O2 is formed and Ce3+ is oxidized to Ce4+; then Ce4+ and H2O2 can react together to regenerate Ce3+ and oxidize H2O2 to O2. This would be a very elegant way of regenerating reduced nanoceria and eliminating, in a sequential set of reactions, both superoxide and hydrogen
peroxide. The stoichiometry requires two superoxides reduced for each H2O2 oxidized (Fig. 3). Alternatively, a second molecule of H2O2 may oxidize the reduced Ce3+, as described in the literature,19,34 leading to Ce4+ formation and reduction of H2O2 to H2O; in this case a true catalase-like dismutation cycle is created (Fig. 4). If this mechanism actually works, nanoceria could prove to be an ideal antioxidant, because it may scavenge two abundant types of ROS as a never-ending machine. The coupling of the two sequential redox steps (superoxide to peroxide, peroxide to O2) might also provide an efficient way to avoid the paradoxical toxic effects of SOD enzymes. Indeed, excessive H2O2 is believed to have more toxic potential than superoxide and should be rapidly eliminated after formation, and in many cell systems possessing low catalase levels, SOD appears to be toxic rather than protective.37 Two different enzyme-type reactivities of nanoceria were recently reported. Dextran-coated nanoceria (4 nm ceria core) catalyzed the oxidation of various dyes with an oxidase-like behaviour.38 Again the activity was believed to involve the Ce3+/ Ce4+ switch, but the regenerative process was not investigated. Another very interesting study reported that the Ce3+ ions of nanoceria (3–5 nm in size) can break the phosphate ester bonds and remove phosphate groups from biologically relevant molecules.39 Reversible phosphorylation/dephosphorylation reactions are the basis of energy and signaling metabolism in cells, and this molecular activity opens a new perspective in the evaluation of the biological effects of nanoceria. It must be considered that all these studies have been made in abiotic systems, and it remains to be demonstrated whether activities such as SOD or catalase mimesis may be observed also in biological systems, i.e. cell cultures or animal models.
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2 Fig. 3 A model of the reaction mechanism for the oxidation of hydrogen peroxide by nanoceria and the regeneration via reduction by superoxide. An 55
oxygen vacancy site on the nanoceria surface (1) presents a 2Ce4+ binding site for H2O2 (2), after the release of protons and two-electron transfer to the two cerium ions (3) oxygen is released from the now fully reduced oxygen vacancy site (4). Subsequently superoxide can bind to this site (5), and after the transfer of a single electron from one Ce3+, and uptake of two protons from the solution, H2O2 is formed (6) and can be released. After repeating this reaction with a second superoxide molecule (7) the oxygen vacancy site returns to the initial 2Ce4+ state (1). It is also possible that the third Ce3+ indicated, which gives rise to the oxygen vacancy, could participate directly in the reaction mechanism. The square Ce–O matrix is shown here only to illustrate the model and does not correspond to the actual spatial arrangement of the atoms in the crystal structure.
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20 Fig. 4 A model of the reaction mechanism for the complete dismutation of hydrogen peroxide. The oxidative half-reaction is identical to the sequence shown in Fig. 3 (1–4). The reductive half involves binding of H2O2 to the 2Ce3+ site (5), uptake of two protons and homolysis of the O–O bond with transfer of electrons to the two Ce3+ (6), and release of the water molecules to regenerate the initial Ce4+ site (1). This reaction sequence would be analogous to the one found in catalases.
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Many convincing studies demonstrate effects of nanoceria on cells in culture. This may result from nanoceria internalization: confocal and transmission electron microscopy analyses demonstrated that nanoceria in a dose and size-dependent manner enter the cytoplasm of epithelial cells,40 possibly by phagocytosis,41 but the small size of nanoceria particles (3–5 nm in many preparations) may allow other routes of entry. Indeed, in a normal human keratinocyte cell line fluorescent nanoceria was found internalized via clathrin- and caveolae-mediated endocytic pathways, whereas phagocytosis seems not to be involved; the internalized nanoceria is distributed throughout the cell.42 But also extracellular nanoceria might affect cell behavior, e.g. by scavenging ROS released from cells, by adhering to and disturbing the plasma membrane, or by mimicking specific molecular interactions (i.e., ligand–receptor) and promoting intracellular signaling cascades. Methodological issues hamper the comparison of different studies on the biological effects of nanoceria (and of nanoparticles in general) due to the intrinsic properties of nanoparticles. First, nanoceria may be prepared by different protocols, with Ce ions in different redox states, or retaining trace elements that may exert biological effects of their own. Second, oxide nanoparticles are not molecules, which makes it impossible to establish a true ‘‘molarity’’; concentrations should instead be provided as weight/volume. Since the part reacting with biomolecules is the surface, it is important to calculate the total specific surface area that, at equal mass, is determined by the size and shape of the particles. Third, a major concern comes from agglomeration, a phenomenon difficult to quantify that occurs when nanoceria is suspended in aqueous solution, as in
biological studies. In these conditions precipitates form that dramatically change the properties of nanoceria, decreasing its active surface area, hampering its mobility in body fluids, and affecting the uptake by cells. Strategies to reduce agglomeration include capping nanoceria with organic compounds,43 provided that the external layer does not alter the ceria biological effects, and that it is biocompatible and biodegradable.44 Citrate capping was recently shown to promote nanoceria cell uptake without cytotoxic effects.45 Another important issue is the loss of ions from ceria in water and biological media. The Ce4+ ions in ceria are insoluble in water at any pH value, whereas Ce3+ ions may be soluble in water for pH values lower than 7.5,46 which are found in many biological environments. This could be critical for the biological effect of nanoceria, since soluble Ce4+ salts might be toxic in vivo.47 Recently it was shown that Ce3+ ions in aqueous solution react rapidly with H2O2 and produce hydroxyl radicals in a Fenton-type reaction.48 This is precisely the kind of unwanted side effects that has been the main hindrance for the development of catalase and SOD model compounds in the past.49 So far it is not known whether this problem may exist also for nanoceria under biological conditions.
5. Effects of nanoceria on cells in vitro A number of studies have addressed the effect of nanoceria on cells in culture of different histotypes, including established cell lines and primary cultures from tumors or normal tissues. Two main issues have been addressed: the effect on basal and exogenous ROS, and an eventual cyto-protective or cytotoxic effect. Table 1 summarizes the literature results. Nanoceria was found to scavenge free radicals in murine insulinoma cells treated with hydroquinone (HQ)50 and in
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Table 1 The effect of nanoceria on ROS and cell death in tumor vs. normal cellular systems. The cells types used in the published in vitro studies are listed (h: human; m: murine; r: rat). The effect of nanoceria on cell viability and basal oxidative level is reported, as well as the effect on induced oxidative stress. The following pro-oxidant treatments have been used: diesel exhaust particles (DEP), X-rays, hydrogen peroxide (H2O2), and hydroquinone (HQ). ND ¼ not determined
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NP incubation time
Basal ROS
Basal viability
Effect on added ROS
Protection from insults
48 h
ND
Z
ND
24 h
ND
¼
ND
+ (X rays) + (X rays)
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2–5 nm
10 nM (0.0017 mg ml1) 10 nM
N
8 nm
25 mg ml1
1–16 h
¼
¼
Z (DEP)
(DEP)
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N
40 mg ml1
24, 48, 72, 96 h
\
Z
ND
ND
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N
5–10–15– 20–25– 30–40 nm 5 nm
5 mg ml1
ND
¼
ND
ND
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N
3–5 nm
1, 10, 100 nmol
24, 48, 72, 96 h 24 h
ND
ND
3–5 nm
24 h
Z
¼
24
N
?
ND
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ND
\
Z (H2O2) ND
ND
3–5 nm
0.5, 12, 24, 96 h 15–30 days
ND
N
1, 10, 100 nM 1, 10 mM 1, 3, 5, 10, 20 nM ?
+ (X rays) ND
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N
Z (X rays) ND
19
ND
+ (H2O2) ND
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5 Cell system
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h. lung fibroblasts (CCL 135) h. mammary epithelial (CLR9798) h. bronchial epithelial (BEAS-2B) h. bronchial epithelial (BEAS-2B) r. cardiomyocytes (H9C2) h. colon (CRL 1541) m. macrophage (J774A.1) m. primary retinal neurons r. spinal cord neurons h. glioblastoma (T98G) h. bronchoalveolar carcinoma (A549) m. macrophage (RAW 264.7) m. insulinoma (bTC-tet cells) h. mammary epithelial (MCF7)
Normal/ tumor
NP size
NP dose
N
?
N
1
5 Ref.
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T
5 nm
5 mg ml
24487296 h
ND
T
20 nm
3.5, 10.5, 23.3 mg ml1
24, 48, 72 h
Z
Z
ND
ND
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T
8 nm
25 mg ml1
1––16 h
¼
¼
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50, 100 mM
18 h
ND
ND
(DEP) ND
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T T
2–5 nm
10, 50 nM
24 h
ND
Z
\ (DEP) Z (HQ) ND
cultured retinal neurons treated with H2O2.51 In murine macrophages stimulated with lipopolysaccharide (LPS), nanoceria reduced the LPS-dependent ROS production and cell activation (measured as induction of nitric oxide synthases, iNOS), without alleviating LPS-induced macrophage death.24 Nanoceria prolonged the survival of explanted adult rat spinal cord neurons (whereas glial cells were unaffected), and increased survival to peroxide challenge.12 Normal lung fibroblasts were protected from radiation-induced cell death by nanoceria.52 A different study showed that nanoceria improved the viability after irradiation of cultured normal mammary epithelial cells, whereas the tumor counterparts (MCF7 cells) irradiated under identical conditions were not rescued.36 However, nanoceria was able to reduce cell death induced by pro-oxidant diesel exhaust particle (DEP) extracts in transformed human bronchial epithelial (Beas2B) cells.53 A study on normal human colon cells showed that nanoceria rescued cell viability and reduced ROS production after irradiation.18 In this set of studies nanoceria was non-toxic, decreased both endogenous and induced ROS, and preserved the viability of damaged cells. Other studies instead convincingly demonstrate pro-oxidant effects and cell death. In human bronchial epithelial cells (Beas2B),54 nanoceria stimulated intracellular ROS and ROSdependent signals, including phosphorylation of MAP (mitogen-
(X rays)
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activated protein) kinases and induction of antioxidant enzymes;55 concomitantly nanoceria induced cell death by apoptosis. The same study54 reported that nanoceria did not affect the viability of two additional, very different cell types (rat cardiomyocytes and human brain fibroblasts), but in this study the effects on ROS production were not examined. Other authors reported pro-radical and cytotoxic effects of nanoceria on human bronchoalveolar carcinoma cells.56 Commercially available nanoceria (25 nm) was not toxic to C3A tumor hepatocytes.57 Nanoceria particles (20 nm) rendered fluorescent by doping with Yb and Er proved cytotoxic to lung cancer CLR5883 cells, whereas the non-tumor HUVEC endothelial and WI38 macrophage cells were unaffected.58 Taken together, these studies suggest that the effects of nanoceria on cell viability/apoptosis depend on the effect on the intracellular oxidation state: when nanoceria behaves as an antioxidant, it produces a pro-survival effect, whereas when it exerts a pro-oxidant effect, apoptosis is induced. Apoptosis is a mechanism of cell death partially dependent on oxidative stress,59–61 and the effects of nanoceria are thus acceptable from a logical point of view, considering that the types of damage imposed on the cells in these studies (such as X-rays, H2O2, DEP extracts), all induce oxidative stress. The cell antioxidant effects of nanoceria may be explained by its antioxidant capability reported in abiotic experiments.
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Instead the pro-oxidant effect may be due to multiple reasons. In some cell/particle experimental systems, the Fenton effect described in vitro for nanoceria48 may occur and overrule the nanoceria antioxidant effect. Alternatively, a signal transduction chain of events induced by association of nanoceria with the cell structures may activate a pro-oxidant process, or the balance of the catalase- vs. SOD-mimetic effect may change in different cell/ particle systems producing opposite effects. The discrepancy of the results (pro- vs. anti-radical; pro- vs. anti-survival) is not easy to interpret, since a meta-analysis of the published studies (see Table 1) does not allow establishing simple correlations with cell type or particle size/preparations. Thus we must assume that a complex interplay between cell features (genetic or metabolic redox asset) and particle size (i.e., different bulk/surface ratios implying different redox properties) determines the overall effects.
6. In vivo effects of nanoceria Nanoceria is considered essentially non-toxic.62,63 The published studies show that administration of nanoceria to laboratory animals is well tolerated; no histological alterations were reported up to 30 days after injection,24 and no increase in animal deaths occurred within 9 weeks.53 Nanoceria was still detected in the tissues of treated animals at 15 days post-injection;24 however, time-course experiments of tissue permanence, that might reveal an eventual trend of discharge, were not performed. The lack of acute toxicity leads to the general belief that
nanoceria is not harmful, but studies must be performed in the long run to establish possible adverse effect of chronic exposure. Nanoceria administration to animals protects organ functions after specific insults. This includes protection from radiationinduced pneumonitis52 or gastro-intestinal epithelial damage,18 and from peroxide-induced retinal degeneration.51 Irradiationinduced tissue damage mainly depends on the ability of X-rays to evoke secondary oxidative stress, hence these effects may depend on the ROS scavenging ability of nanoceria. In the same studies the authors applied similar treatments (irradiation or H2O2) to ex vivo or in vitro cell models mimicking the target organs, and showed that nanoceria-induced cell protection was accompanied by decreased ROS production. In a pioneering study, nanoceria was injected into transgenic mice expressing monocyte chemo-attractant protein-1 (MCP-1) in the cardiac tissue. These mice develop myocardial inflammation leading to fatal ischemic cardiomyopathy.64 Nanoceria injection relieved many clinical symptoms of the transgenic mice, possibly due to its radical scavenging properties that reduce inflammation.65 Moreover, nanoceria reduced ROS production and apoptosis induced on H9c2 cardiomyoblasts cultured in high glucose levels to mimic diabetes conditions.21 A very interesting study showed that Drosophila fed with nanoceria-containing food lived significantly longer than control flies.66 If the pro-ageing effect of ROS and the anti-ageing effect of antioxidants are now common knowledge, in Drosophila it has been shown that transgenic flies simultaneously over-expressing catalase and SOD have a 30% increased life-span with respect to wild-type.67 This is suggestive of a systemic SOD and catalase
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Fig. 5 Reactive oxygen species and inflammatory response. ROS activate key redox-sensitive kinases such as Jun kinase, p38, and IKK. p38 activates the arachidonic acid pathway leading to production of lipid inflammatory mediators such as prostaglandins (PG) and leukotrienes (LT) by cyclooxygenase (COX) and lipoxygenase (LOX), respectively. IKK activates the NF-kappaB pathway, which leads to de novo synthesis of a set of proinflammatory cytokines belonging to the interleukin (IL) family. JunK activates the nitric oxide synthase (iNOS) pathway, which leads to the formation of the gas inflammatory mediator NO. In addition to responding to oxidations, all of these pathways exert pro-oxidant functions, thus creating an oxidation-dependent auto-amplification of the inflammatory process.
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mimetic role played by nanoceria eaten by the flies, as expected from the abiotic experiments described above. Intriguingly, ceric sulfate in the molecular form exerts opposite effects on Drosophila, decreasing the activity of SOD and catalase and life expectancy, proving that metals in nanoparticles and in free ionic forms may exert very different results.49 Some of the in vivo studies mention that higher nanoceria levels were less successful in relieving pathological conditions,20 suggesting either that excessive ROS scavenging may exert negative effects on animal health, or that higher doses of nanoceria may promote radical formation, possibly via a Fenton effect. Nanoceria is supplemented to laboratory animals by intraperitoneal or intravenous injection or as a food additive, and significant effects are reported in spite of the distance between the injection point and the organ specifically examined, i.e., heart, eye, lungs, etc.21,24,51 This may imply that nanoceria is delivered to many different target organs after injection, where it then exerts local effects. In one transmission electron microscopy nanoceria was detected 24 h after intravenous injection within cells of distal organs such as kidney or liver,24 showing that nanoceria travels within the body. However, pharmacokinetic analyses are required to study accumulation and secretions routes, to evaluate if nanoceria may reach local levels sufficient for the biological effects. Alternatively, nanoceria may exert systemic effects, ameliorating the body’s generic responsiveness to a broad range of insults. Such a systemic effect might involve the control of the inflammatory response. This is conceivable, since inflammatory cells (leukocytes) reside in the blood stream, thus being easy targets of the injection routes utilized for nanoceria. Moreover, leukocytes exert pro-oxidant effects that are essential for the inflammatory response, and which may be easily intercepted by nanoceria. The oxidative environment created by activated leukocytes is by itself a mediator of inflammation, helping recruiting more leukocytes and producing more proinflammatory molecules (Fig. 5), thus producing more oxidations, in an auto-implementing cycle. The scavenging of free radicals is a way of breaking this cycle and attenuating the inflammatory response. Interestingly, many of the above mentioned effects of nanoceria include alteration of inflammatory parameters. However, high nanoceria doses promote the expression of inflammatory cytokines in cultured human endothelial cells.68 This issue remains a challenge for future studies.
7. Conclusions and perspectives The potential pharmacological effects of nanoceria rely on the hypothesis, so far partially supported by abiotic studies, that nanoceria acts as a regenerative redox machine, cycling its redox state while transforming superoxide to hydrogen peroxide, and then peroxide to water. This antioxidant activity combined with an apparent lack of toxicity makes it an extremely promising therapeutic tool; however, both of these aspects still require thorough analyses and clarifications. The regenerative redox cycle needs to be demonstrated in biological fluids, cells, tissues and animals. The toxicity must be tested in model animals for longer periods, especially considering that the nanoceria cellrescue ability also might favor survival of tumor cells, increasing the tumor incidence. However, as shown in Table 1, the studies report that after cell damage nanoceria promotes rescue of
normal cells (four out of five studies), but not of tumor cells (two out of two studies). If confirmed, this would suggest the use of nanoceria as adjuvant in anti-tumor therapies, because nanoceria might protect healthy cells from aggressive anti-tumor therapies without impairing the efficacy of tumor cell elimination. Protocols for in vivo administration must be optimized to avoid adverse effects of agglomerates in critical areas such as microcirculation or renal glomerular passage. Passage through different biological barriers requires attention to establish administration protocols. Special care should be taken with nanoceria dosage, standardizing the parameters. Pharmacokinetics studies need to be carried out to assess movements, body permanence and excretion routes, and to detect eventual accumulation in specific organs and/or dissolution of the nanoparticles. As to the possible therapeutic applications, much work needs to be done. Animal models mimicking human pathologies implying oxidative stress should be examined, investigating whether nanoceria might ameliorate symptoms in animals developing oxidation-related pathologies, such as neurodegenerations, or early ageing. Very important will be the analysis of nanoceria effects on inflammation, both in animal and in vitro cell models. Novel applications of nanoceria in the growing field of nanomedicine are appearing every day, including tissue engineering and specific drug targeting. It was recently shown that nanoceria improves the culture of mesenchymal stem cells and cardiac progenitors grown in a biodegradable polymer matrix, by promoting their adhesion and proliferation, possibly via its antioxidant mechanism.22 Moreover, nanoceria was successfully conjugated to the tumor marker transferrin69 as a means to improve selective antitumor therapy. In conclusion, the premises are so promising for the pharmaceutical use of nanoceria to foresee the passage to clinical trials in the next future.
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Acknowledgements This work was supported in part by the 2008 PRIN project ‘‘miRNA in diagnosis and experimental therapy via nano-vectors of pleural malignant mesothelioma’’ of the Italian Ministry of Education, University and Research (MIUR), and by the World Premier International Research Center Initiative of MEXT, Japan.
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