Cerium Oxide Nanoparticles: Structure, Applications, Reactivity, and Eco-Toxicology

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Book Title Nanomaterials: A Danger or a Promise?Series Title

Chapter Title Cerium Oxide Nanoparticles: Structure, Applications, Reactivity, and Eco-Toxicology

Copyright Year 2012

Copyright HolderName Springer-Verlag London

Corresponding Author Family Name JobbágyParticle

Given Name MatíasSuffix

Division Laboratorio de Superficies y Materiales Funcionales INQUIMAE-DQIAQF,Facultad de Ciencias Exactas y Naturales

Organization Universidad de Buenos Aires Ciudad Universitaria

Address Pab. II, C1428EHA, Buenos Aires, Argentina

Email [email protected]

Author Family Name PerulliniParticle

Given Name MercedesSuffix




Email

Author Family Name Aldabe BilmesParticle

Given Name Sara A.Suffix




Email

Abstract In this chapter, the physical, chemical, and ecotoxicological features of nanometric cerium oxide will bediscussed on the basis of the recent research. In contrast with other oxides such as SiO2, ZnO, ZrO2, orTiO2 with relevant industrial applications, ceria presents a unique redox chemistry that expanded itsapplication to fields that take advantage of its chemical reactivity, as heterogeneous catalysis anddetoxification of gaseous exhausts. In the past, several studies were strictly focused on the exploration of itseventual damage to environment and human health. CeO2, as other rare earths oxides, is basically a lowtoxicity substance[1] and nowadays there is vast and increasing evidence pointing to its potential role asprotective compound in terms of human health. The aim of this chapter is to offer a wide scope of descriptionof the intrinsic physicochemical behavior of this unique compound, with deep emphasis in the inherentchallenge that represents a definitive understanding of its surface chemistry. The apparent contradiction

between toxicity and health benefits will be discussed according to the present evidence and the intrinsiclimitations of these complex studies.

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1 Chapter 122 Cerium Oxide Nanoparticles:3 Structure, Applications, Reactivity,4 and Eco-Toxicology

5 Mercedes Perullini, Sara A. Aldabe Bilmes and Matías Jobbágy

6 Abstract In this chapter, the physical, chemical, and ecotoxicological features of7 nanometric cerium oxide will be discussed on the basis of the recent research. In8 contrast with other oxides such as SiO2, ZnO, ZrO2, or TiO2 with relevant9 industrial applications, ceria presents a unique redox chemistry that expanded its

10 application to fields that take advantage of its chemical reactivity, as heteroge-11 neous catalysis and detoxification of gaseous exhausts. In the past, several studies12 were strictly focused on the exploration of its eventual damage to environment and13 human health. CeO2, as other rare earths oxides, is basically a low toxicity sub-14 stance[1] and nowadays there is vast and increasing evidence pointing to its15 potential role as protective compound in terms of human health. The aim of this16 chapter is to offer a wide scope of description of the intrinsic physicochemical17 behavior of this unique compound, with deep emphasis in the inherent challenge18 that represents a definitive understanding of its surface chemistry. The apparent19 contradiction between toxicity and health benefits will be discussed according to20 the present evidence and the intrinsic limitations of these complex studies.

21 12.1 Introduction: Physicochemical Properties of CeO2

22 and its Relevant Applications

23 Cerium that holds a 4f25d06s2 electronic configuration has a chemistry mostly24 governed by the trivalent and tetravalent oxidation states, in contrast with most25 of the lanthanides stabilized at the trivalent state exclusively. In natural

M. Perullini � S. A. Aldabe Bilmes � M. Jobbágy (&)Laboratorio de Superficies y Materiales Funcionales INQUIMAE-DQIAQF,Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires CiudadUniversitaria, Pab. II, C1428EHA, Buenos Aires, Argentinae-mail: [email protected]

Layout: T1 Standard SC Book ID: 211395_1_En Book ISBN: 978-1-4471-4212-6Chapter No.: 12 Date: 24-5-2012 Page: 1/27

R. Brayner et al. (eds.), Nanomaterials: A Danger or a Promise?,DOI: 10.1007/978-1-4471-4213-3_12, � Springer-Verlag London 2012

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26 environments, this element typically lies in several minerals as alanite, basta-27 nite, monazite, cerite, and samarskite, partially substituted by other trivalent28 rare earths. However, only bastnaesite, a hexagonal Ce(OH)CO3 also containing29 fluoride anions and monazite, CePO4, represents the most commercial sources.30 The relevant phase of this element from a technological point of view is its31 pure oxide, CeO2, also known as Cerianite. This phase crystallizes in the32 Fluorite structure, which is named after the mineral form of CaF2. It has a33 face-centered cubic unit cell (f.c.c.) with space group Fm3 m, having a char-34 acteristic lattice parameter a = 0.541134(12) nm [2]. In this structure, each35 cerium cation is coordinated by eight equivalent nearest neighbor oxygen36 anions at the corner of a cube, each anion being tetrahedrally coordinated by37 four cations. Figure 12.138 The pure oxides of cerium constitute a vast family of mixed valence compounds39 ranging from the fully oxidized CeO2 form to the totally reduced Ce2O3, the40 C-type sesquioxide, regarded as a fluorite type in which 25% of the anion sites are41 vacant and ordered (a = 1.116 nm) [3], depending on the temperature and oxygen42 partial pressure [4]. Reduced ceria results from the removal of O2- ions from the43 CeO2 lattice, which generates an anion-vacant site according to the following44 scheme:45

4 Ce4þ þ O2� ! 4 Ce4þ þ 2e� =hþ 0:5 O2 ! 2 Ce4þ þ 2Ce3þ þhþ 0:5 O2

ð12:1Þ

474748 where h represents an empty position (anion-vacant site) originated from the49 removal of O2- anions from the lattice, here represented as an oxygen tetrahedral50 site (Ce4O). Electrostatic balance is persevered by the reduction of two cerium

Fig. 12.1 Scheme of thecubic unit cell of CeO2; grayspheres represent O2- anionswhile yellow ones Ce4+

cations. Dotted lines indicatedistance between the nearest8 oxygen neighbors of eachcerium atom. Colored planesindicate the base of the foursubcells occupied by Ce4+

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51 cations from the tetravalent to the trivalent state. Historically, the oxides of cerium52 in the range Ce2O3–CeO2 were treated using the classical point-defect model of53 nonstoichiometry, in which oxygen-vacant sites were considered to be present in54 the lattice in a randomized fashion. However, bulk stoichiometric phases with55 ordered vacancies were described in terms of geometric models for defect ordering56 [5–10]. Beyond this, a lattice expansion results from the reduction of Ce4+ ions to57 Ce3+, the radius of the Ce3+ ion being larger than that of Ce4+ (1.14 Å vs. 0.97 Å),58 according to the data of Shannon and Prewitt and in good agreement with the59 observations made over several doped forms of CeO2 [11–13]. It was observed by60 several authors a lattice expansion of ceria when the crystal size drops to a few61 nanometers [14]; there is a general agreement in assigning that expansion to the62 stabilization of Ce3+ ion [14–19]. Early computer simulations [20] indicated that63 for substances with predominantly ionic type of bond (in particular, for oxides) the64 change in the unit cell parameter on passing to the nanostate is related to the65 change in the formal oxidation state of atoms. For the case of ceria nanoparticles66 (CNP), the extrapolation of lattice parameter values suggested that the C-type67 sesquioxide exists in the pure form once their diameter drops to less than68 1.5–1.1 nm and most of Ce3+ ions are located near the surface. This observation is69 consistent with the enhanced stability of oxygen vacancies in the surface of ceria70 in comparison with oxygen vacancies in the bulk [21], and with the enhanced71 reducibility of small ceria clusters compared to bulk ceria [22]. Detailed inspec-72 tions based on electron energy loss spectroscopy revealed that the oxygen73 nonstoichiometry of CNP can be envisaged as core–shell nanostructures; in rela-74 tively large particles, the core has a composition close to stoichiometric cerium75 dioxide and the surface is close to Ce2O3 [17, 23]. Very recently, a systematic76 structural exploration supported by Rietveld refinement of the nanostructures was77 reported, confirming that this common trend describes a vast population of par-78 ticles obtained under diverse preparation methods [24] Fig. 12.2.

diameter / nm15 258421

a/Å

5.40

5.44

5.48

5.52

5.56

5.60Fig. 12.2 Expansion ofCNPs lattice parameter, a, asfunction of particle0sdiameter reported byBaranchikov et al.[24] Dottedline represents the latticeparameter of pure Ce2O3

sesquioxide and the emptydot the limit diameterpredicted by several authors

12 Cerium Oxide Nanoparticles: Structure, Applications 3


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79 Among reactive rare earth oxides, CeO2 plays a key role in industrial catalysis80 due to the reversibility of its redox cycle that gas–solid equilibrium implies and81 this property is regarded as oxygen storage capacity (OSC) [25]. CeO2 has82 potential uses in two of the most important commercial catalytic processes as the83 three-way catalysis (TWC) [26] and the fluid catalytic cracking (FCC). It is also84 involved in solid-state reactions, as the removal of soot from diesel engine exhaust85 [27], for the catalytic wet oxidation of organics from wastewaters [28], as an86 additive for combustion catalysts and processes [29], and the emerging field of IT-87 SOFC fuel cell technology, either as a solid electrolyte [30] or anode [31]. Then,88 much effort is still focused in studying the role of ceria and its substituted forms in89 well-established industrial processes; [32] the most relevant reactions of ceria are90 summarized on Table 12.1.91 Beyond the aforementioned reactions, more detailed exploration of CeO2

92 reactivity at an atomic level revealed that the crystal plane of ceria dramatically93 affects its catalytic properties for CO oxidation [33]. Single-crystalline CeO2

94 nanorods reveal that the predominantly exposed 001 and 110 planes are more95 reactive for CO oxidation in contrast with the 111 stable ones that prevail in96 irregular nanoparticles [33]. According to high-resolution transmission electron97 microscopy, different exposed crystal planes prevail on each kind of single crystal98 morphology: 111 and 100 for polyhedral, 110 and 100 for rods, and 100 for cubes.99 Reactivity is also affected by morphology; OSC measurements recorded at 400 �C

100 revealed that reversible reduction takes place both at the surface and the bulk in the101 case of CeO2 nanorods and nanocubes, but is restricted at the surface for the102 nanopolyhedra, just like the bulk one. This result suggests that high OSC materials103 might be designed and obtained by a shape-selective synthetic strategy [34].104 A tuned morphology can improve the OSC achieving useful activities at a tem-105 perature 250 �C less than the recorded for irregular ones [35, 36]. Nowadays, there106 is a vast number of synthesis methods, including highly shape-selective ones, for107 the preparation of CNPs [37], ranging from mechanochemical procedures [38–44],108 high temperature combustion [45], and mild thermal decomposition [46, 47], to109 microwave- or sonochemical-based ones [48].110 The basic aspect underlying this structure-dependent reactivity triggered in111 silico-based research; the formation of oxygen vacancies through depletion of112 oxygen from CNP (CenO2n with n \ 80) was found to be greatly facilitated

Table 12.1 Main gas–solid heterogeneous redox reactions driven by ceria at moderatetemperatures

CeO2-x ? x/2 O2 ? CeO2 (Eq. 12.1)CeO2 ? x CO ? CeO2-x ? x CO2 (eq.2)CeO2 ? x/3 CnH2n ? CeO2-x ? nH2O ? n CO2 (eq.3)CeO2 ? x/2 SH2 ? CeO2-x ? x/2 S ? x/2 H2O (eq.4)CeO2-x ? x/2 SO2 ? CeO2 ? x/2 S (eq.5)CeO2-x ? x/2 NO2 ? CeO2 ? x/2 N2 (eq.6)

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113 compared to extended surfaces, which explains the observed spectacular reactivity114 of nanostructured ceria [49–51].115 The redox properties of elemental cerium also played a relevant role in the116 metallurgic industry, as an anticorrosion agent. The high affinity of cerium for117 oxygen and sulfur underlies the use of cerium-containing ferro-alloys to improve118 the physical properties of high strength low-alloy (HSLA) steels [52–66] or119 aluminum-based alloys [67]. In the iron casting process, cerium is considered to120 remove free oxygen and sulfur from the melt, improving significantly this121 oxidation resistance [68]. In electrolysis, self-forming anode technology is used122 whereby cerium oxide coatings are deposited onto conducting ceramic substrates.123 Cerium oxide provides an alternative to thorium oxide, a common additive in124 welding electrodes that is now being phased out for environmental reasons. The125 addition of cerium oxide to other oxides as zirconia produces a material with126 exceptional toughness and good strength [69, 70]. Cerium oxide-doped zirconia is127 also used in thermal barrier spray coatings on metal surfaces.128 Beyond the chemical properties of cerium oxides, increasing attention has been129 paid to applications dealing with the interaction of this large-gap semiconductor130 phase with light. As might be expected, nanoconfinement affects the phase’s131 intrinsic band diagram and CNPs exhibited noticeable changes in its absorptive132 properties as a function of size; Masui et al. [71] reported that a decrease in micelle133 stabilized particles sized from 4.1 to 2.6 nm is accompanied by an increase in the134 band gap energy (Eg) from 2.73 to 2.87 eV for indirect transition and from 3.38 to135 3.44 eV for direct transition. Other researchers found a similar trend for colloidal136 CNP however the found Eg values markedly exceeded those reported earlier,137 probably due to the inherent high error involved in linearization of UV spec-138 troscopy data [72]. This tendency observed for colloidal ceria was also reproduced139 in thin films of nanocrystalline cerium dioxide [73]. Zhang et al. [74] reported an140 expression linking the dependence of the CeO2-x band gap for direct transition on141 the particle size of radius R and a relative dielectric constant of cerium dioxide142 equal to 24.5. Beyond this issue, it was stated that the observed change obeys to143 the increasing reduction of Ce4+; increasing the energy difference between the O144 2p and Ce 4f orbitals, resulting in a hypsochromic shift of the absorption band of145 this phase. In this scenario, it is not surprising to find differences among different146 colloids depending on the preparation procedure [75, 76]. The relative contribution147 of quantum confinement and partial reduction is still a matter of debate [77]. The148 ability of CNP to drive electron–hole splitting under light absorption was exploited149 for its potential application in solar cells, photocatalytic degradation of organic150 pollutants as well as photocatalysis sensitizing agents for TiO2 [78–85].151 UV-shielding property of certain nanocrystalline semiconductor materials is152 widely used in sunscreen cosmetics; most of inorganic UV-blocking filters are153 based on titanium dioxide (TiO2) and zinc oxide (ZnO). However, it was reported154 that the aforementioned oxides can eventually exert certain degree of cell damage155 on brain cells [86], blood lymphocytes [87], and lymphoblast cells damage by156 titania nanoparticles [88]. Both zinc and titanium oxides nanoparticles exhibit157 remarkable photocatalytic activity under UV-irradiation, even immersed within



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158 sunscreen cosmetics, releasing reactive oxygen species (ROS) that can react with159 skin cells damaging their DNA [89–96]. Photocytotoxicity of titania against160 fibroblasts has also been confirmed [97].As an alternative to the aforementioned161 compounds and in accordance to its Eg, CeO2 emerge as a friendly option, due to162 the intrinsic highly defective structure of ceria lattice, in which the recombination163 of free charge carriers (electrons and holes) forming upon UV-irradiation of ceria164 proceeds very rapidly compared to TiO2. Additionally, UV-extinction coefficient165 of ceria is rather high, positioning this phase as a promising alternative UV-filter in166 sunscreen cosmetics [98–100]. CNPs exhibit protective properties against radia-167 tion-induced cellular damage, radiation-induced pneumonitis, and can prevent168 retinal degeneration by photons of harmful light [101, 102]. Very recently it was169 reported that the sun protection factor, the critical absorption wavelength, and the170 UVA/UVB-ratio of ceria nanoparticles are comparable to traditional oxide nano-171 particles holding a dramatically lower photocatalytic activity. A recent report172 describes the protective character of CNPs for mouse fibroblasts (L929) and173 fibroblast-like cells of African Green monkey (VERO) exposed to UV-irradiation174 [103].175 Concerning photoemissive properties of cerium ions, once isolated in a proper176 host are an essential luminescent component applied in several phosphors for-177 mulations. Upon excitation by energetic cathode-ray electrons, they produce useful178 light emission, finding application in numerous light sources such as compact179 fluorescent lighting and related devices [104–108]. However, there is increasing180 evidence pointing to CeO2 even in the form of CNPs, as a suitable phase to host181 and activate several photoemitting centers as Eu, Tb, or Yb, with potential182 application in biolabeling [109–114].183 Due to its intrinsic hardness, cerium oxide is the most efficient polishing184 agent for most glass compositions as well as a glass additive to diminish185 undesired Fe absorption preventing UV-driven damage or antireflective coatings186 [115–118]. Cerium oxide has a high refractive index, and is an opacifying agent187 in enamel compositions used as protective coatings on metals. Rare earth sul-188 fides, among them also cerium, are used in glass and ceramics as colorants to189 replace toxic CdS. In certain glass compositions (at low weight percentages)190 along with comparable amounts of titanium oxide, cerium oxide produces a deep191 yellow coloration.192 In the last decade, a vast amount of basic research was focused on exploring193 and elucidating the redox activity of CNPs in aqueous media, beyond all the194 well-established industrial applications of CNP in gas solid heterogeneous195 catalysis [119].This particular issue was recently reviewed in great detail and the196 following section describes the main aspects of its fascinating biomimetic197 chemistry [120]. In a straight resemblance to enzymatic redox reactions, nan-198 oceria with a high Ce3+/Ce4+ ratio on its surface is able to reduce superoxide to199 peroxide, (see Scheme 12.1) playing the role or superoxide dismutase (SOD)200 [121]. However, the mechanism for the restoration of reduced nanoceria remains201 uncertain [122]. It was also shown that H2O2 is able to oxidize ceria from Ce3+

202 to Ce4+ in a reversible fashion, in the time scale of days [101, 123, 124]. Celardo



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203 et al. [125] proposed a comprehensive mechanism of regeneration of nanoceria,204 based on the combination of both the SOD mimetic [121] and the catalase-205 mimetic activity [124] (see scheme 12.1). Interestingly, by coupling both redox206 steps (superoxide to peroxide, peroxide to O2) the paradoxical toxic effects of207 SOD enzymes observed for cell systems possessing low catalase levels could be208 skipped [126] Fig. 12.3.209 These simply coupling of redox cycles easily explain the ROS scavenging210 activity of ceria; however, it should be kept in mind that the degree of hydrox-211 ylation, the pH as well as the presence of competing anions as phosphates with212 high affinity for Ce3+ sites could severely affect the aforementioned pathway and213 the intrinsic activity of ceria [127]. CNP (unlike SOD) can inactivate also the214 hydroxyl radical OH [128]. This is in line with the recent discovery of the key role215 that Ce3+, instead of oxygen vacancies, plays in the intracellular antioxidant effect

Fig. 12.3 Scheme of the main cycle for the oxidation of hydrogen peroxide by nanoceria viareduction by superoxide and the active site regeneration, according to the catalytic pathwayproposed by Celardo et al.[125]. A surface oxygen-deficient site on the nanoceria (extreme left)offers a two Ce4+ binding site for H2O2, the release of protons is coupled with two-electrontransfer to the two cerium ions and molecular oxygen is released from the now fully reducedoxygen vacancy site (extreme right). Superoxide binds to this site, and is reduced by one Ce3+;the uptake of two protons releases H2O2. With the coordination of a second superoxide molecule,the oxygen vacancy site returns to the initial stable state, with two Ce4+, releasing a second H2O2

molecule. A plausible reaction mechanism for hydrogen peroxide’s disproportion can beenvisaged as a shortcut of the former mechanism (cycle around blue shadow): The reductive site(extreme right) binds a second H2O2 molecule to the two neighbors Ce3+ centers, with asubsequent uptake of two protons and breakage of the O–O bond with transfer of electrons to thetwo Ce3+, and release of the water molecules to regenerate the initial Ce4+ site (extreme left) in ananalogous way to the previous scheme

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216 on leukocyte cell lines [129]. In silico studies suggest that reduced ceria, CeO2-x, in217 contact with molecular oxygen leads to the formation of peroxo, O2

2-, and super-218 oxo, O2

- , species. The formation of the former can be explained by the interaction219 of O2 with two electron-donor oxygen vacancies at the ceria surface. In contrast,220 the latter form can be explained by direct interaction of O2 with low-coordinated221 Ce3+ ions on reduced ceria nanoparticles [130]. Beyond the ability to catalyze222 these ROS scavenging cycles [131], CNP (3–5 nm in size) are able to hydrolyze223 phosphate ester bonds, cleaving phosphate groups from biologically relevant224 molecules [132]. Eventually, as can be envisaged from the inherent capacity of225 CNP to decompose NOx-carrying exhausts, Nitrogen reactive species (NOS) can226 also be effectively scavenged by CNP under physiological conditions [119].227 Concerning the attempts to achieve a comprehensive exploration of the enzy-228 matic mimetic reactivity and the eventual toxicity of CNP, several critical issues229 should be taken into account. Numerous factors limit the straight comparison of230 the results reported by different authors. As was stated by Celardo et al. [125], and231 in agreement with the dramatic shape-dependant reactivity found, CNPs obtained232 under different protocols could exhibit very different intrinsic surface reactivity.233 Instead of mass based concentrations, effective available surfaces seems to be a234 more robust parameter to define CNP activities, however, the coalescence of235 nanoentities in the form of larger aggregates, an expectable phenomenon that236 inevitably occurs in biological studies. In this sense, strategies to prevent237 agglomeration include capping nanoceria with organic compounds [133] provided238 that the external layer does not alter the ceria biological effects, and that it is239 biocompatible and biodegradable [134]. Citrate capping was recently shown to240 promote nanoceria cell uptake without cytotoxic effects [135].241 Finally, another important source of uncertainty is the eventual release of Ce3+

242 ions to the liquid media, since the reduced form may be soluble in water for pH243 values lower than 7.5 [136], which are found in most of the reports. This could be244 critical for the biological effect of nanoceria, since soluble Ce4+ salts might be245 toxic in vivo [137]. Additionally, from a mechanistic point of view, Ce3+ ions246 demonstrated to rapidly react in aqueous solution with H2O2, producing hydroxyl247 radicals in a Fenton-type reaction [138]. These side effects have been the main248 hindrance for the development of catalase and SOD model compounds in the past249 [139] and the true limits between heterogeneous and homogeneous reactivity of250 cerium is still a matter of debate [140]. The high reactivity of ceria nanoparticles in251 aqueous suspensions and its role on assorted redox processes [134, 141] combined252 with their inherent low toxicity [142] open the gate for its application linked with253 the inactivation of some of the most toxic ROS, such as superoxide radical [121],254 hydrogen peroxide [124], and nitroxyl radical [143]. Nowadays, CNP is consid-255 ered one of the most promising inorganic nanomaterials in the field of nano-256 medicine [120, 144–150].



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257 12.2 Interaction of CNPs with the Environment258 and Representative

259 Before the industrial era the fate of cerium compounds was governed by the260 inherent geochemical cycle of rare earths, which is affected by several complex261 and interactive processes as any weathering process, including atmospheric262 phenomena, geologic activity, physical and chemical weathering, hydrologic263 cycles, etc. [151]. Once CNPs are exposed to reducing environments the264 eventual presence of free Ce3+ is unlikely since in the soil, like the other265 lanthanides, this cation is immobile under a wide range of pH conditions, due to266 the low solubility of its typical solid phases, such as carbonates, fluorohy-267 droxides, and phosphates. Since lanthanides sorbs strongly to silicates and humic268 material, the bulk of the Ln content including cerium is associated with such269 colloidal particulates present in most natural waters [152]. The fate of CNPs in270 aquifers is controlled by the inherent complexity of colloidal physicochemistry,271 in general agreement with DLVO theory. Advanced modeling is subject of272 current research [153].273 Concerning the anthropogenic sources of CNPs, the massive emission by274 refineries and automobiles [154, 155] into the atmosphere and hence, the whole275 environment has been a matter of concern since decades. Ten years ago, the276 National Institute of Health (USA) reported a comprehensive study summarizing277 the impact of these emissions on human health [156]. Another report from the278 Health Effects Institute also analyzes this topic in great detail [157]. More recently,279 the Environmental Protection Agency (USA) published a toxicological review of280 cerium oxide and cerium compounds, in the frame of the Integrated Risk Infor-281 mation System (IRIS) [158]. In the following section, the most recent research282 concerning the interaction of CNPs with the environment and the human health283 will be summarized and discussed. As a first reductionist approach to envisage the284 eventual environmental impact of a certain nonentity, toxicity assays performed285 over common wild microorganism can offer a first glance of the potential damage286 that those entities represent. Yet, this approach is matter of debate and has intrinsic287 limitations [159]. Most of the work on ecotoxicological effects has been done with288 algae and aquatic organisms. Some work has been done with bacteria as model289 organisms and few studies with seeds or plants in order to determine the effect of290 NPs in germination and the possible translocation in leaves. Data on ecotoxico-291 logical effects of CNPs are scarce and seems to be contradictory. However, most of292 published work does not compare similar shapes and surface derivatization con-293 ditions. The concentration doses are also very dissimilar. The effect of size on294 survival or growth has been established: the smaller the NPs, the more important295 the damage.



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-cel

lco

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tce

lls

wit

hda

mag

edm

embr

ane

shor

teni

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dna

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lam

ents

(Ana

baen

a)

Lum

ines

cenc

ein

hibi

tion

ofA

naba

ena

CP

B43

37

Ref

.[1

62]

Pse

udok

irch

ne-r

iell

asu

bcap

itat

aD

aphn

iam

agna

and

Tha

mno

ceph

alus

plat

yuru

sem

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sof

Dan

iore

rio

14,

20an

d29

nmsu

ppli

edby

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stry

part

ners

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42an

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g-1

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edi

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utio

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2–32

mg/

L.

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uore

scen

ceof

extr

acte

dch

loro

phyl

l.T

EM

for

NP

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lco

ntac

tA

mm

onia

and

phos

phat

e(n

utri

ent

depl

etio

n)

EC

10fo

r2.

6–5.

4m

g/m

LD

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ased

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yno

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Por

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ith

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oxic

ity

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eto

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etio

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nutr

ient

s.R

educ

tion

ofgr

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whe

n60

%ph

osph

ate

depl

eted

(50

%re

duct

ion

ingr

owth

rate

inph

osph

ate-

free

med

ium

)N

oev

iden

ceof

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ing

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ans:

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ter

24h

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eto

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00(D

.M

agna

)an

d50

00(T

.pla

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mg/

mL

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ngor

gani

sms

afte

r72

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EM

for

NP

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bryo

sof

Dan

iore

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to20

0m

g/m

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Pad

here

nce (c

onti

nued

)



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(con

tinu

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Ref

.[1

63]

daph

nia

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nane

onat

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Ps

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ma

Ald

rich

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lm

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icro

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ount

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feed

ing

orin

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sed

met

abol

ism

and/

orex

cret

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rate

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ef.

[164

]da

phni

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agna

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rio

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eri

Syn

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idat

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4]64

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entr

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agna

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rio

fisch

eri

biol

umin

esce

nce

eIn

hibi

tion

C80

%at

0.06

4m

g/m

L

Ref

.[1

65]

daph

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mag

nane

onat

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rius

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pio

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pato

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s

CN

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ma

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rich

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dehy

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hnia

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nane

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Chi

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size

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hydr

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cal

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er;

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d30

nm(T

EM

);56

and

9m

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mg/

L

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onin

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etas

say

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ced

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wit

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NP

s)no

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ifica

ntch

ange

sin

grow

than

dre

prod

ucti

onR

ef.

[166

]ze

brafi

sh(D

anio

reri

o)S

igm

a-A

ldri

ch,

10.2

±1.

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byT

EM

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5–5

mg/

L.

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hex

pose

dto

NP

sin

wat

erco

lum

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der

sem

ista

tic

24h

and

7da

ys.

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offi

shti

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s.

Sig

nifi

cant

upta

kein

live

rsof

fish

expo

sed

to0.

5m

g/L

but

noin

fish

expo

sed

to5

mg/

L.

No

upta

kede

tect

able

inot

her

tiss

ues

Ref

.[1

67]

Cae

norh

abdi

tis

eleg

ans

incu

ltur

em

ediu

mse

eded

wit

hE

sche

rich

iaco

li

synt

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zed

byhy

drot

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alm

etho

din

supe

rcri

tica

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ater

;15

and

45nm

(TE

M).

1m

g/L

.

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rtil

ity

and

surv

ival

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i-qu

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inte

rfer

ence

feed

ing

CN

Ps

expo

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oke

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ifica

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fect

ongr

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and

surv

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posu

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CN

Ps

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%(1

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sed

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onof

cyp3

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gene

whi

chha

sne

gati

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fect

onfe

rtil

ity

(con

tinu

ed)



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tho

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f

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RR

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DPR

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(con

tinu

ed)

Ref

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8]C

aeno

rhab

diti

sel

egan

sfe

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ith

Esc

heri

chia

coli

Syn

thes

isby

oxid

atio

nw

ith

HM

T.[

74]

8.5±

1.5

nmby

TE

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BE

T10

7.8

m2/

g.1–

500

nM

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coli

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ilit

ycu

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ith

H2D

CF

DA

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mM

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emat

ode

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feed

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ico

unti

ngA

ccum

ulat

ion

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ith

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one

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tro

capt

ure

ofO

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olve

das

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III)

).U

ptak

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CN

Ps

act

asan

exog

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urce

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CN

Ps

foun

din

inte

stin

allu

men

CN

Ps

adhe

red

toE

.Col

ice

llm

embr

anes

Ref

.[1

69]

Esc

heri

chia

coli

Rho

dia

from

prec

ipit

atio

nat

low

pH.

Ell

ipso

idal

7nm

;40

0m2

g-1,

pzc

10.5

Ce(

III)

:C

e(IV

)by

XA

NE

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0.46

–73

0mg/

L.

CF

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LB

petr

idi

shes

adso

rpti

onon

cell

mem

bran

e50

%su

rviv

al5

mg/

L;

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rviv

alab

ove

230

mg/

LR

educ

tion

ofN

Ps

byba

cter

iaas

defe

nse

toox

idat

ive

stre

ssC

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oxic

ity

requ

ires

dire

ctsp

atia

lco

ntac

tce

ll-N

PR

ef.

[170

]N

itro

som

onas

euro

paea

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ioru

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size

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LS

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rex

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reto

med

iaw

ith

NP

sm

orph

olog

ical

chan

ges

byT

EM

noch

ange

ince

llsi

zeN

Ps

adhe

red

toce

llw

alls

and

dist

orsi

onof

mem

bran

eno

intr

usio

nof

part

icle

sin

the

cell

lR

ef.

[171

]3-

5ol

dm

aize

plan

tsfl

ame

spra

ypy

roly

sis

from

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um2-

ethy

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anoi

cin

xyle

ne(8

wt

%),

37nm

,11

0m

2/g

.10

lg

(Ce)

/L

expo

sure

ofpl

ants

toai

rw

ith

NP

sir

riga

tion

ofpl

ants

wit

hN

Ps

susp

ensi

ons

expo

sure

ofvi

able

leav

esto

10pp

mce

ria

susp

ensi

ons

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byIC

P-

MS

TE

Mof

leav

esqu

ench

edin

liqu

idN

2

aggl

omer

ated

CN

Ps

adso

rbed

onle

aves

expo

sed

toce

ria

aero

sol

inde

pend

ent

onil

lum

inat

ion

(ope

nor

clos

edst

omat

a)in

corp

orat

ion

ofC

NP

sin

tole

aves

notr

ansl

ocat

ion

ofce

ria

inne

wle

aves

noin

corp

orat

ion

ofC

NP

sby

irri

gati

onR

ef.

[164

]se

eds

Syn

thes

isby

oxid

atio

nw

ith

HM

T.[

74]

64–6

40m

g/L

germ

inat

ion

and

root

sle

ngth

0%

germ

inat

ion

afte

r5

days

expo

sed

to0.

64m

g/L

;20

%ex

pose

dto

0.06

4m

g/L

(con

tinu

ed)



Au

tho

r P

roo

f

UN

CO

RR

ECTE

DPR

OO

F

(con

tinu

ed)

Ref

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0]S

oybe

an(G

lyci

nem

ax)

seed

scu

bic,

NP

sM

elio

rum

Tec

hnol

ogie

s.7n

mcr

ysta

llin

edo

mai

nby

XR

Dd.

10–

4,00

0m

g/L

.

DN

Ais

olat

ion

and

yiel

daf

ter

trea

ted

wit

h2,

000

and

4,00

0m

gL

-1.

RA

PD

geno

toxi

colo

gy,U

ptak

eby

XA

NE

S.

All

the

CN

Ps

conc

entr

atio

nssi

gnifi

cant

lyin

crea

sed

root

elon

gati

on..

Ce

inti

ssue

sin

crea

sing

wit

hco

ncen

trat

ion

ofN

Ps

Roo

tsup

take

and

stor

eC

NP

sw

ith

sam

eox

idat

ion

stat

eas

inN

Ps.

At

2,00

0an

d4,

000

mg

mL

-1

CN

Ps

are

geno

toxi

ca

Par

alle

lel

ectr

onen

ergy

loss

spec

trom

etry

bIn

hibi

tory

conc

entr

atio

ngi

ving

50%

redu

ctio

nin

alga

lgr

owth

rate

cT

hiob

arbi

turi

cac

idre

acti

vesu

bsta

nces

dIn

All

enan

dA

rnon

Mod

ified

Med

ium

Dil

uted

1/10

and

Adj

uste

dto

pH6

in2m

MH

EP

ES

aggr

egat

esre

ach

2,00

0nm

eE

ffec

tive

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entr

atio

n,E

C50

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fine

das

the

conc

entr

atio

nth

atpr

oduc

esa

50%

ligh

tre

duct

ion

mea

sure

daf

ter

5an

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min

cont

act

tim

e.



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tho

r P

roo

f

UN

CO

RR

ECTE

DPR

OO

F

296 12.3 Interaction of CNPs with Cells and its Impact297 on Human Health

298 The mechanisms of cellular entry by NPs are a topic under intense debate [172–299 174]. On one side, given the small size CNPs (3–5 nm in many preparations),300 direct transport across the membrane has been claimed for uncovered NPs or NPs301 with hydrophobic coatings [175], whereas most of the work points to their302 incorporation by endocytosis, an active transport in which the cell encloses the303 objects in vesicles or vacuoles pinched off from its cytoplasmic membrane [176,304 177]. The uptake of NPs involves their interaction with the non-rigid and non-305 uniform cell membrane [172]. The cell surface is heterogeneous both in306 phospholipid composition and presence of embedded proteins and other structures,307 and can be thought as patches with a length scale of 10–50 nm. If a NP were to308 interact with one patch at a time, the interaction energy would vary depending on309 NPs properties such as surface charge, surface roughness, and degree of curvature,310 as well as on its location on the cellular membrane. Moreover, the interaction of a311 particle with the phospholipid bilayer leads to a new particle surface different from312 the initial one. This introduces the concept of time-dependent dynamic interface313 that allows describing complex phenomena such as endocytosis [178, 179]. On the314 other hand, the mechanisms of direct entrance of NPs inside the cells (i.e., without315 endocytic compartments) are not less complex. It has been observed that subtle316 changes (for example, by tuning the non-specific binding forces of spiked uncoated317 particles or by modifying the arrangement of the ligands on coated NPs) allow or318 impede the direct penetration of the lipid bilayer [174]. The mechanisms of cel-319 lular uptake and further fate inside the cells may vary depending on NP properties320 (size, shape and surface chemistry) as well as on the cellular type. For instance, in321 a normal human keratinocyte cell line fluorescent-labeled nanoceria was found to322 be internalized via endocytic pathways, and further distributed throughout the cell323 [180].324 It is worth mentioning that particles much larger than the membrane patch325 length, i. e., microparticles or big-size NPs aggregates, rarely enter non-phago-326 cytotic cells. The surface properties of nanoparticles can drive their agglomeration327 into larger aggregates; in turn, these properties are also determined by the phys-328 icochemical scenario of the dispersion media, in particular the ionic strength, pH,329 and the eventual presence of complexing agents and capping macromolecules330 [181]. The properties of NPs assemblies interacting with a cell membrane are far to331 be a problem with a trivial solution. Moreover, multiple particles might form rafts332 with different properties than the sum over those of the single particles. Further333 complexity is added when one considers that the surface of NPs is usually covered334 with adsorbed molecules resulting from the synthesis process in order to get stable335 and monodispersed nanocrystals or specially designed for some purpose as for336 example for drug delivery or imaging [182].337 Due to the high area to volume ratio, the shape (i.e. spherical, cubic, rod-like,338 triangular), the surface composition, the surface charge, and surface roughness



Au

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UN

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F339 play an important role in the properties of NPs that must be taken into account340 either when considering medical or technological applications, as well as their341 toxicity [173, 183]. The nanosize can also give them access to biological systems342 that are normally inaccessible to both single molecules [184] or to larger particles343 [185].344 Figure 12.4 presents a simplified schematic representation of the mechanisms345 of NPs cellular uptake and toxicity. Three major mechanisms of NPs toxicity are346 shown. One common mechanism relays on the ability of NPs to organize around347 them a protein corona and generate adverse biological outcomes through protein348 unfolding, loss of enzymatic activity, and fibrillation [186]. For example, SiO2-349 NPs have been shown to generate nucleoplasmic protein aggregates impairing350 normal nuclear function [187]. Another paradigm of NPs toxicity is the release of351 toxic ions when the thermodynamic properties favor particle dissolution in the352 biological environment [188]. It is worth mentioning that while most organisms353 live in rather neutral pH ranges, intracellular compartments (vesicles, lysosomes)354 and specialized organs (stomach) significantly extend the possibility for degra-355 dation or chemical modification. An example is ZnO that dissolves to form356 hydrated Zn2+ [189], inducing apoptosis in mammalian cells [190]. Limbach et al357 [191] demonstrated that metal-oxide NPs internalized in human lung epithelial358 cells by a so-called Trojan-horse mechanism provoked an up to eight times higher359 oxidative stress if compared to reference cultures exposed to aqueous solutions of360 the same metals. NPs can also interfere with the antioxidant defense mechanism361 [192], leading to reactive oxygen species generation, the initiation of an inflam-362 matory response, and perturbation and destruction of the mitochondria causing

Fig. 12.4 Schematic representation of the mechanisms of NPs cellular uptake and toxicity.Insets depict the complex NP-cellular membrane interactions that allow the direct penetration ofthe lipid bilayer (A) or the specific ligand (adsorbed to the nanoparticle)-receptor (at the cellmembrane) interactions that can drive the endocytosis pathway for cellular uptake (B)

B&

WIN

PR

INT



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363 apoptosis or necrosis [193]. This last toxicity mechanism is generally attributed to364 nanoceria [169, 194, 195]. On the other hand, several studies have reported that365 CNPs are not nocive [196] or even that they mitigate oxidative stress at a bio-366 logical level [197]. [198] As will be shown, the discrepancies regarding the367 antioxidant/oxidant effects of cerium oxide NPs could be attributed to the fact that368 health effects vary significantly depending on the type of cells used for the study369 and the physicochemical characteristics of the used CNPs.370 NPs have shown to produce cytotoxic, genotoxic, inflammatory, and oxidative371 stress responses in different mammalian cells [199, 200]. The nature of the372 interface between NPs and biological systems affects the in vivo biocompatibility373 and toxicity. Evaluation of NPs safety has to consider their interaction with pro-374 teins, DNA, lipids, membranes, organelles, cells, tissues, and biological fluids375 [172]. It is important to consider that extracellular nanoceria might also affect cell376 behavior, e.g., by ROS generation or scavenging, by adhering to and disturbing the377 plasma membrane, or by mimicking specific molecular interactions (i.e., ligand–378 receptor) and promoting intracellular signaling cascades.379 The release of nanoparticles into the environment can occur through many380 processes, such as spilling and washing consumer products incorporating nano-381 particles; during synthesis and production; as an accidental release during transport382 or use; from industries that exploit nanotechnology, for example wastewater383 treatment and drug delivery. The way of contact of NPs with the biological target384 is a key factor to take into account when assessing toxicity. As mentioned in the385 previous sections, CNPs has found increasing use in polishing and computer chip386 manufacturing [201], [202] but mainly as an additive to decrease diesel emissions387 [203]. Thus, the principal way of exposure to CNPs is the respiratory tract.388 Figure 12.5 summarizes the biodistribution and the mechanisms of detoxifi-389 cation of CNPs administered by the gastrointestinal (GI) tract, intratracheal (IT)390 instillation, or by intravenous (IV) injection. In the center of the figure is depicted391 the lung deposition and extrapulmonary translocation of CNPs after intratracheal392 instillation, according to Xiao He et al. [204]. After administration, well-dispersed393 NPs of 6.6 ± 0.9 nm in diameter, aggregate in contact with the intratracheal fluid.394 The deposited nanoceria was slowly cleared from the host lung tissue (male Wistar395 rats), with an elimination half-life of 103 days. It was found that most of the396 particles on the surface of airways, i.e., before reaching the alveoli, (about 23 % in397 the cited study) were removed from the lungs within 1–2 days by mucociliary398 clearance, and swallowed by the animals into the GI tract and finally eliminated399 via feces. Following the same terminal, the main clearance route of aggregated-400 NPs deposited in alveoli, at 4–7 days post exposure, is phagocytosis by alveolar401 macrophages (AM) and AM-mediated re-entrainment into the airway lumen, with402 the consequent elimination via the GI tract. Moreover, it has been demonstrated403 that over 99 % of oral administered CNPs is eliminated through feces during the404 first 3 days after administration, rendering the absorption in the GI tract barely405 discernible. These results are illustrated in Fig. 12.5 (left).406 At long term, the binding to proteins present in biological fluids would lead to407 the redispersion of the big size NPs aggregates and the protein-NPs binding affinity



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408 would also induce strong adhesion of NPs to the membrane or intracellular sub-409 stances of cells in alveolar walls. This would aggravate the retention and inter-410 stitialization of NPs in the lung tissue, which has been shown to cause411 inflammatory responses such as granuloma and/or fibrosis through persistent412 damage to the lung [205].413 Another important consequence of the disaggregation of CNPs is that small size414 particles are now able to penetrate through the alveolar-capillary barrier into the415 systemic circulation. It was proposed that NPs in blood are taken up by the416 phagocytic cells in tissues, so they would be accordingly accumulated in the417 phagocytic cell-rich tissues. Yokel et al. [206] studied the biodistribution and418 oxidative stress effects of a systemically introduced commercial nanoceria in mice.419 The used NPs were mostly platelets, highly crystalline, and had a bimodal size420 distribution (TEM average particle sizes: 8 nm and 24 nm). Zeta potential mea-421 surements showed that the system would be stable at physiological pH (-35 mV at422 pH 7.4). Different doses of CNPs up to 0.75 % animal weight were intravenously423 (IV) infused. The initial t� of ceria clearance from blood after termination of the424 infusion was 7.5 min. Tissue Ce concentration is dose dependent, being highest in425 the spleen while the factor organ weight X Ce concentration was found to follow426 the trend: liver [ spleen [ blood [ brain. Ceria agglomerations were seen in the427 spleen (cytoplasmic localization), although no obvious histopathology was428 detected in this organ. By the contrary, some was observed in the liver and kidney.429 In contrast to the significant accumulation of ceria agglomerates in reticuloendo-430 thelial organs, much less ceria was seen in the brain (and almost no evidence of431 toxicity, except for some lipidic peroxidation in hippocampus), and no micro-432 scopic evidence of disruption of the blood–brain barrier (BBB) was observed.433 These results are illustrated in Fig. 12.5 (right).

Fig. 12.5 Biodistribution and mechanisms of detoxification of CNPs

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434 The reticuloendothelial system (RES) is the first line of defense against xeno-435 biotic intrusion. Thus, the spleen, liver, and kidney constitute a specific subpop-436 ulation of organs with phagocytic potential to take up ceria, but the aggregation437 state of the NPs is determinant, because only external bodies [100 nm in size are438 recognized by RES. Small size NPs exert a higher toxicity, as they can injure the439 target tissue by entering to non-phagocytotic cells in one hand, and in the other440 hand by penetrating into the systemic circulation and reach secondary organs. As a441 counterpoint, they may be (at least partially) eliminated by renal (NPs\ 8 nm) or442 hepatobiliary clearance (NPs\ 20 nm) [172].443 While admittedly it is impossible to perform risk assessment and management444 without in vivo toxicological data, the strategy of using animal studies as the445 primary means of analysis method when confronted with the great diversity of446 commercial NPs and exposure conditions is unsustainable. Efforts have been done447 to develop predictive in vitro toxicological screening to rank NPs for priority in448 vivo testing: target-specific and predictive in vitro science that utilizes mecha-449 nisms of injury and toxicological pathways to guide the judicious use of in vivo450 studies [173]. Toward this end, quick screening approaches can be used to speed451 up the safety analysis on a scale that commensurate with the rate of expansion of452 nanotechnology development [207]. Moreover, in vitro analysis can be based on453 detection methods that are more difficult to utilize in vivo, such as tracking of454 radioactive marks, and may provide complementary insight at the molecular and455 subcellular level [208]. Due to the redox couple Ce3+/Ce4+, the main toxicity456 exerted by CNPs is via oxidative stress and their intracellular toxicity can be457 assessed according to the hierarchical oxidative stress paradigm (HOSP) [173], in458 which the different levels of oxidant stress have been classified as antioxidant459 defense (Tier 1), pro- inflammatory (Tier 2), and cytotoxic (Tier 3) cellular460 responses.461 Since the respiratory tract is the first target attack by CNPs aerosols, many of462 the in vitro studies are being performed in lung epithelial cells. Studying cytotoxic463 effects in a human bronchoalveolar carcinoma-derived cell line, significant dose-464 and time-dependent ROS generation, lipid peroxidation, and cellular membrane465 breakage (revealed by LDH levels in culture medium) were reported [194], thus466 confirming that cellular damage caused by CNPs results from elevated oxidative467 stress. Park et al. [195, 209] showed that different sizes of CNPs (15, 25, 30,468 45 nm) cause dose-dependent ROS increase, glutation (GSH) decrease, and469 induced antioxidant defense genes such as heme oxygenase-1, catalase, glutathi-470 one-S-transferase, and thioredoxin reductase (HOSP-Tier 1). It was also reported471 that the increased ROS induced by these NPs triggered the induction of pro-472 inflammatory pathways, as revealed by nuclear factor kappa-B (NFjB) augmented473 expression (HOSP-Tier 2). Moreover, morphological changes to these cells such as474 chromosome condensation and apoptosis were observed (HOSP-Tier 3). Surpris-475 ingly, the authors did not find significant differences in toxicity among NPs with476 different sizes (and hence, with different surface areas). They assumed that it may477 be due to the aggregation state of NPs inside the cells, determining similar surface478 areas regardless of the NP size.



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479 By means of X-ray absorption spectroscopy, Auffan M. et al [210] defined the480 causal mechanisms linking the physico-chemical properties of CNPs (ellipsoidal481 crystallites; hydrodynamic diameter in solution of 15 nm) with their biological482 effects. They examined the potential in vitro cytotoxicity and genotoxicity toward483 human dermal fibroblasts and the interactions occurring at the NP/biological media484 interface. They found that even though NPs stability at physiological pH (7.4)485 would be expected (isoelectric point pH 7.9–10.5), the suspension was destabilized486 after 24 h in the culture medium, as a consequence of interactions with proteins487 and salts that neutralized the NPs charge, decreasing electrostatic repulsion.488 Concerning the oxidation state of CNPs, 8 ± 2 % of cerium was reduced to tri-489 valent state in the abiotic culture medium, but no increase in Ce3+/Ce4+ ratio was490 detected after 24 h of incubation with fibroblasts. TEM images showed that during491 the incubation with fibroblasts, CNPs aggregates were adsorbed onto the cell492 membrane and further internalized into the cytoplasm inside vesicles. No mito-493 chondria or nuclear presence of NPs was detected. The cytotoxicity (found to be494 similar to that of the positive control, TiO2-NPs) was evaluated in terms of cell495 viability, which decreased 20–40 % for concentrations larger than 1.5 g/L.496 Genotoxicity was studied by monitoring DNA single strand breaks (SSB) for-497 mation and micronuclei (MN) induction, and was found to be even greater than498 that observed for TiO2-NPs. They found a strong dose-dependent effect in chro-499 mosome damage caused by CNPs, generated by oxidative stress. Additionally, the500 genotoxicity was compared to that caused by micro-CeO2 (particle501 size = 320 nm), finding that CNPs are much more genotoxic per unit of mass, but502 that the toxicity effects become similar when nano- and micro-CeO2 doses are503 normalized by surface area. Another important conclusion is that genotoxic effects504 appeared at concentrations 2–3 orders of magnitude lower than the concentration505 at which cytotoxic effects occurred, highlighting the importance of taking into506 account DNA damage effects in risk assessment studies.507 To study particle-cell interactions in cell culture systems representing the air-508 way epithelial barrier, it is important to mimic the in vivo interactions of particles509 with cells as closely as possible. As stated before, the physical and chemical510 properties of NPs rapidly change when suspended in biological fluids or artificial511 culture mediums. To simulate accidental exposure to NPs in a relevant state of512 agglomeration and surface coating, Rothen et al. [211] directly combined the513 synthesis of NPs to the exposure of alveolar epithelial cell cultures at the air–liquid514 interface in a glove box. The deposition of the particles was monitored by TEM. In515 contrast to other studies performed with particles in suspension, in the present516 study a homogeneous distribution of CNPs with only a few aggregates was found517 on culture cells. No cytotoxic reaction and no remarkable change in the cyto-518 skeleton or cellular ultrastructure of the epithelial cells due to particle exposure519 (highest dose 0.024 mg/cm2) were observed. However, they report short-term520 (30 min) dose-dependent decrease in epithelial tightness and increase in perme-521 ability, possibly due to disorganization of the tight junctions and long-term522 decrease of lamellar body volumes. This last effect may be due to surfactant523 release triggered by NPs.



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524 Paradoxically, other studies with ceria nanostructures showed high biocom-525 patibility [212]. CNPs have been shown to serve as free radical scavengers [213,526 214] providing protection against chemical, biological, and radiological insults527 that promote the production of free radicals. The intracellular CNPs promote cell528 longevity and decrease toxic insults by virtue of their antioxidant effects [215],529 preventing the accumulation of ROS, reducing the activation of the apoptotic530 response and death of the cells [216], and avoiding retinal degeneration induced by531 intracellular peroxidases [213]. Additionally, on previous studies, CNPs showed532 no toxic effect on normal breast epithelial cells (CRL 8798) and only a slight effect533 on breast cancer (MCF-7) cells at concentrations [50 nM [101]. Furthermore,534 CNPs selectively conferred radioprotection to the normal cells (CRL 8798) as535 compared with the tumor cells (MCF-7), representing a novel approach to the536 protection of normal cells from radiation-induced cell damage in vitro on normal537 lung fibroblast cells (CCL 135) and in vivo on athymic nude mice [102].538 Thus, CNPs may offer a novel therapeutic alternative for scavenging environ-539 mentally elevated ROS. Recently, the use of nanoparticle-based antioxidants as a540 potential treatment for hepatotoxicity, which is a life-threatening problem, was541 explored. One obvious use of the nanoparticles would be for enhancing the per-542 formance of antioxidants, such as those normally present in the body or those543 administered as medicines for this kind of injury. It was shown that CNPs provided544 protection against Monocrotaline (MCT), a plant-derived pyrrolizidine alkaloid545 that causes oxidative veno-occlusive disease of the liver [217]. Electron micro-546 scopic examinations of liver samples from rats receiving CNPs alone demonstrated547 a homogeneous intrahepatocellular distribution of nanoparticles without pheno-548 typic alteration of hepatocellular architecture. Liver samples obtained from the549 CeO2 ? MCT group also demonstrated regular intracellular distribution of550 nanoparticles and, importantly, did not exhibit alterations in cellular morphology,551 which is likely to be due to CeO2 protection against MCT-elevated oxidative552 damage to the liver. This puts in evidence the protective effects of cerium oxide553 nanoparticles against hepatic oxidative damage.554 This apparent discrepancy may be due to the surface oxidation state of nan-555 oceria to scavenge superoxide or act in a catalytic manner, to the aggregation state556 of particles (that will depend not only on the their isoelectric point, but also on the557 interaction with particular biomolecules present at the biological fluid) and, even558 more important, to the pH micro conditions of the biological matrix (for instance559 subcellular organelle) that hosts the NPs. A. Asati et al. [212] synthesized poly-560 mer-coated nanoceria with enhanced aqueous stability and unique pH-dependent561 antioxidant activity, demonstrating optimal antioxidant properties at physiological562 pH, and behaving as an oxidase at acidic pH [131]. As shown in Figure X, the563 ability of NPs to permeate the cellular membrane depends on the hydrophobic/564 hydrophilic properties of their surface. Thus, the polymer coating and function-565 alization of the NP’s surface play a major role in cellular uptake and subcellular566 localization. In turn, as mentioned before, the pH in the medium surrounding the567 NPs (highly acidic in lysosomes or neutral in cytoplasm) will play a critical role in568 NP’s beneficial (antioxidant) vs. harmful (oxidant) properties. The same authors



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569 showed that CNPs coated with either poly-acrylic acid (negatively charged),570 aminated poly-acrylic acid (positively charged), or dextran (with no charge),571 present a completely different uptake pattern in several cell lines, including cardiac572 myocytes (H9c2) and human embryonic kidney (HEK293) normal cells and lung573 (A549) and breast carcinoma cell lines (MCF-7) [218]. Positively charged NPs574 internalized in all cells except for breast carcinoma, and localized preferentially in575 the lysosomes, resulting toxic to these cells. Negatively charged NPs were inter-576 nalized only by lung carcinoma, localizing in lysosomes and consequently577 exhibiting toxicity selectively to this cell line. NPs with neutral charge resulted578 nontoxic to normal or cancer cells, as these NPs primarily localized in the cyto-579 plasm of these cells.

580 12.4 Concluding Remarks

581 In the context of this book it should be remarked that CNPs seem to be more a582 promise than a danger for life, both from a chemical and a biological perspective.583 From the evidence presented in previous reports as well as this chapter it can be584 concluded that the toxicity of CNPs to human health is mainly related with air-585 borne particles that straightly affect the respiratory track, lungs, and subsequently586 other organs. However, at the same time, the same CNPs are offering increasing587 evidence pointing toward their unique ability to catalytically decompose ROS588 under physiological conditions. This apparent contradiction is an expectable result589 from an inherently complex system that requires deep and systematic investigation590 under very controlled experimental boundary conditions. Regarding this, some591 critical issues should be summarized in order to give guidance for future research592 on reactivity and/or toxicity of CNPs.593 Since the cerium centers located on CNP’s surface can be easily reduced to the594 trivalent state, much attention should be paid to the redox conditions. In the595 reduced state, the surface is more susceptible to irreversible chemical transfor-596 mations as partial dissolution or other transformations as inner sphere coordination597 of phosphate or carboxylic groups. This will result in the modification of the598 inherent redox reactivity as well as the surface charge, affecting the intrinsic ability599 of CNP’s to diffuse through tissues and cell membranes, due to electrostatic600 repulsion, steric hindrance due to the binding of bulky macromolecules, or,601 eventually, massive agglomeration of CNPs into large micrometric clusters.602 A common observation in nanotextured systems is that not only particle size but603 also shape governs several relevant properties of nanoparticles. This is particularly604 valid for the CNPs, beyond the particle size, the morphology should be known and605 controlled since it dramatically affects the surface reactivity and eventually, the606 rate of dissolution.

607 Acknowledgments MP, SAB and MJ are members of CONICET.



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608 References

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Author Queries

Chapter No.: 12

Query Refs. Details Required Author’s Response

AQ1 Please provide correct font in place of ‘h’ in Eq. 12.1.

AQ2 Please check Eqs. 2, 3, 4, 5, 6 is given in ‘Table 12.1’ butnot provided in the chapter.

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Cerium Oxide Nanoparticles: Structure, Applications, Reactivity, and Eco-Toxicology

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Transcript of Cerium Oxide Nanoparticles: Structure, Applications, Reactivity, and Eco-Toxicology