Cerium oxide nanoparticles: applications and ... - Future Medicine

Review For reprint orders, please contact: [email protected]

Cerium oxide nanoparticles: applications and prospects in nanomedicine Promising results have been obtained using cerium (Ce) oxide nanoparticles (CNPs) as antioxidants in biological systems. CNPs have unique regenerative properties owing to their low reduction potential and the coexistence of both Ce3+ /Ce4+ on their surfaces. Defects in the crystal lattice due to the presence of Ce3+ play an important role in tuning the redox activity of CNPs. The surface Ce3+:Ce4+ ratio is influenced by the microenvironment. Therefore, the microenvironment and synthesis method adopted also plays an important role in determining the biological activity and toxicity of CNPs. The presence of a mixed valance state plays an important role in scavenging reactive oxygen and nitrogen species. CNPs are found to be effective against pathologies associated with chronic oxidative stress and inflammation. CNPs are well tolerated in both in vitro and in vivo biological models, which makes CNPs well suited for applications in nanobiology and regenerative medicine. KEYWORDS: antioxidant therapy n catalytic antioxidant nanoparticle n cerium oxide nanoparticle n neurodegeneration n oxidative stress n pH-sensitive nanoparticle n regenerative medicine

The ‘Web of Knowledge’ search engine retrieves approximately 131,706 scientific articles when ‘reactive oxygen species’ (ROS) are used as key words, with the oldest article being ‘active oxygen’, and published on 17th May 1947 by Kaplan in Nature [1]. These studies explored both the beneficial and detrimental role of ROS. Low levels of ROS play a critical role in the signal transduction process, acting as a second messenger in physiological environments; however, excessive amounts of ROS are associated with different pathological disorders, such as cancer, neurodegenerative diseases, infertility, diabetes, cardiovascular diseases, arthritis and aging [2]. Other than ROS, reactive nitrogen species (RNS) are also known to be associated with the pathogenesis of a variety of diseases [3]. In the past decade, antioxidant compounds have gained a lot of interest among the world’s leading scientists to determine if these compounds could remove ROS and decrease/mitigate pathological conditions. Antioxidants became more popular in the 1990s when a large human study (87,000 participants) suggested that there was a beneficial effect of vitamin E on heart health [4]. In a recent study by Frankel and Finley, questions were raised about the reliability of testing and predicting the effect of antioxidant delivery [4,5]. This depends on the final quantity of antioxidant absorbed by the body, which depends on the balance of sugar, fat and ethanol accompanying them to the small intestine.

In addition to naturally occurring antioxidants, there are two types of synthetic antioxidant particles; particles conjugated or loaded with naturally occurring antioxidants or enzymes (nanoparticles used as carriers), and synthetic nanoparticles with intrinsic antioxidant activity. A few examples of antioxidant-molecule-loaded or -conjugated nanoparticles are superoxide dismutase (SOD)-loaded poly(lactic-co-glycolic acid) particles, glutathione- or trolox-conjugated gold nanoparticles, quercetin-encapsulated polylactide polymeric network or poly(vinyl alcohol), and chitosan–ascorbic acid derivative nanoparticles [6]. On the other hand, metal (gold and platinum) and metal oxide (cerium [Ce] oxide, yttrium oxide and nickel oxide) nanoparticles have been shown to exhibit antioxidant properties [6]. The main limitation of naturally occurring small molecule antioxidants is their absorption in the body [4]. Therefore, to achieve targeted or systematic release of antioxidants/small molecules, nanoparticles have been investigated as delivery vehicles for antioxidant molecules [6]. In recent years, Ce oxide nanoparticles (CNPs) have gained a lot of interest owing to their regenerative antioxidant property. Ce oxide is a very important material for industrial use, with applications in glass polishing, catalytic convertors for removing toxic gases, solid oxide fuel cells, electrochromic thin-film applications, sensors and catalysts [7]. Ce can exist in two oxidation states; Ce3+ (electronic

10.2217/NNM.13.133 © 2013 Future Medicine Ltd

Nanomedicine (2013) 8(9), 1483–1508

Soumen Das‡1, Janet M Dowding2, Kathryn E Klump3, James F McGinnis3,4,5, William Self2 & Sudipta Seal*‡1,6,7 Advanced Materials Processing Analysis Center, Nanoscience Technology Center, University of Central Florida, Orlando, FL, USA 2 Burnett School of Biomedical Science, University of Central Florida, Orlando, FL, USA 3 Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA 4 Department of Ophthalmology, Dean A McGee Eye Institute, University of Oklahoma Health Sciences Center, 608 Stanton L Young Blvd, Oklahoma City, OK 73104, USA 5 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA 6 Material Science & Engineering, University of Central Florida, FL, USA 7 College of Medicine, University of Central Florida, FL, USA *Author for correspondence: Tel.: +1 407 823 5277 Fax: +1 407 882 1156 [email protected] ‡ Authors contributed equally 1

part of

ISSN 1743-5889

1483

Review

Das, Dowding, Klump, McGinnis, Self & Seal

configuration: [Xe]4f1) and Ce4+ (electronic configuration: [Xe]). Therefore, Ce oxide can have two different oxide forms, CeO2 (Ce4+) or Ce2O3 (Ce3+), in bulk material. At the nanoscale, the Ce oxide lattice has a cubic fluorite structure, and both Ce3+ and Ce4+ can coexist on the surface. Charge deficiency due to the presence of Ce3+ was compensated for by oxygen vacancy in the lattice; therefore, at nanoscale, Ce oxide contains intrinsic oxygen defects. These oxygen defects are actually ‘hot spots’ of catalytic reaction. The concentration of oxygen defects increases with a reduction in particle size [8]. Therefore, CNPs have improved redox properties with respect to the bulk materials. In 2007, we first reported that CNPs can also scavenge superoxide radicals efficiently [9]. Owing to the lower reduction potential (~1.52 V) of the Ce3+/Ce4+ couple, they can easily switch back and forth. This interchangeable property between Ce3+ and Ce4+ makes them regenerative [10]. CNPs have been tested for this unique regenerative antioxidant property in different areas of biotechnology and medicine where pathologies are associated with excessive oxidative stress. In the first part of the review we discuss the literature on different procedures for synthesis of CNPs, and correlation of catalytic activity and biological response related to the CNPs’ physiochemical properties. Next, we discuss the radical scavenging properties of CNPs as a function of various physiochemical parameters. Finally, a brief review on the potential application of CNPs in nanomedicine is presented.

Synthesis methodologies of CNPs & correlation with biological applications Numerous synthesis methods for producing Ce oxide nanomaterials for different applications have been reported. These methods include hydrolysis, wet chemical, microemulsion, solgel, sonochemical synthesis, electrochemical synthesis, hydrothermal (HT), solvothermal, gas condensation and spray pyrolysis [7]. For biological applications, the synthesis method of nanoparticles is very important, as the physiochemical properties of nanoparticles mostly depend on the synthesis procedure. The physical properties (size, agglomeration status in liquid, surface charge, and coating or residual contamination of the surfactant on the surface) of nanoparticles mainly influence interactions at the nano–bio interface [11]. Other than physical properties, the surface Ce3+:Ce4+ ratio (chemical 1484

Nanomedicine (2013) 8(9)

property) also influences the biocatalysis (discussed in detail in the ‘ROS and RNS in biology’ section), and the manipulation of the surface Ce3+:Ce4+ ratio, which can alter the biological interactions [12]. On the other hand, coating the nanoparticles with biocompatible/organic polymers increases dispersion/stability, decreases nonspecific interactions with cells and proteins, increases blood circulation time and reduces the toxicity of the nanoparticles. Therefore, a few studies also synthesized/coated CNPs with biocompatible polymers, such as PEG, dextran (Dex), poly(acrylic acid) (PNC), citric acid and oleic acid, for different biological applications. Table 1 summarizes various synthesis methods, and their physiochemical properties, used in different biological studies. Size, morphology & agglomeration state Table 1 shows that the size, morphology and dispersion of the particles are different, and depends on the synthesis parameter. Using a surfactant or polymer during the preparation or coating after synthesis improves dispersion, and decreases the extent of agglomeration in water or biologically relevant media. The dispersion level of the nanoparticles largely influences protein corona formation in cell culture media or in vivo systems. Formation of the protein corona determines the fate of nanoparticle cell uptake and clearance from the body [13]. The presence of a biomolecule/polymer on the surface also affects protein absorption/interaction on the surface of the nanoparticles, and alters the formation of the protein corona. Therefore, the surface coating thickness, or presence of unwanted molecules used in the synthesis procedures, needs to be characterized/analyzed extensively for any biological application [11]. It is interesting to mention that spray pyrolysis, or high temperature sintering of the nanoparticles after synthesis, could remove surface contaminations. However, all high temperature synthesis methods increase the particle size and extent of agglomeration. High temperature synthesis is not able to produce a homogeneous particle size, and higher agglomeration is usually observed. By contrast, room temperature wet chemical or microemulsion methods give control over nanoparticle sizes. However, residual surfactant on the surface of the nanoparticles from the surfactant used in the microemulsion method could influence the nano–bio interface, as mentioned previously. Wet chemical processes produce future science group

Cerium oxide nanoparticles: applications & prospects in nanomedicine

homogeneous smaller size particles without any contamination (e.g., hexamethylenetetramine), which are suitable for bioapplications. Moreover, by adjusting the pH of the nanoparticle suspension to an acidic pH, it is possible to have a stable unagglomerated (3–5 nm) or loosely agglomerated (10–12 nm) nanoparticle suspension [14]. Coating them with a biocompatible polymer, such as glucose, PEG or Dex, or directly synthesizing particles in a biocompatible polymer, will help increase the stability of the dispersion [10,15]. Morphology is another physical property that also needs to be considered for biological applications. For example, nanoparticles in polygonal, cube or rod shapes have sharp edges, and could cause mechanical damage to cells. Therefore, the effect of nanoparticle (NP) shape cannot be ignored for biological applications. In a recent paper, it has been reported that high-aspect ratio nanorods can destroy the liposomal vesicle and damage cells [16]. Papers dealing with other nanoparticles or nanosheets have shown that the effect of NP shape can have adverse effects in biological applications [17,18]. It is important to note that by using room temperature wet chemical methods, it is possible to manufacture spherical/near spherical shape particles without any sharp edges. Surface area, charge, defects & surface oxidation state CNPs have shown unique regenerative antioxidant activity. The regenerative antioxidant property of the nanoparticles comes from their ability to switch between Ce3+ and Ce4+ present on the surface [10]. A schematic representation of CNP surface regeneration is shown in Figure 1. The Ce3+:Ce4+ ratio on the surface of the nanoparticles is very important for the redox activity of CNPs. We have also shown that surface defects are correlated with the Ce3+:Ce4+ ratio [8]. The surface Ce3+:Ce4+ ratio is one of the parameters that also determines the antioxidant and biological activity of CNPs. Size is also known to influence the surface Ce3+:Ce4+ ratio. Smaller crystalline size nanoparticles stabilize higher levels of Ce3+ on their surface [8]. The correlation of antioxidant activity of CNPs with physiochemical properties has been discussed in detail in the ‘ROS and RNS in biology’. Therefore, the synthesis method of redox active material is very important. Recent papers have also shown a surface charge dependence effect of CNPs on biological systems [19]. Localization and uptake of the particles largely depends on the surface charge of the nanomaterials [20,21]. future science group

Review

Room temperature synthesis methods produced CNPs with higher Ce3+:Ce4+ ratios [11]. The Ce3+:Ce 4+ ratios vastly differ in room temperature synthesis methods depending on what chemicals are used for oxidizing or reducing the selected Ce precursors. CNPs prepared using hexamethylenetetramine or base (Sodium hydroxide or ammonium hydroxide) possess high Ce4+ on the surface (Ce3+/Ce4+: 21–30%). On the other hand, methods standardized to prepare CNPs using hydrogen peroxide (H2O2) result in higher Ce3+ on the surface of the CNPs (Ce3+/Ce4+: 55–65%) [14]. Nevertheless, surface Ce3+:Ce 4+ ratios can be manipulated by acid treatment or doping with other rare earth materials. Ascorbic acid treatment reduces Ce4+ to Ce3+ on the surface [22]. Europium doping can increase the Ce3+:Ce4+ ratio, whereas samarium doping decreases the Ce3+:Ce4+ ratio [19,23].

ROS & RNS in biology The role of ROS has received considerable attention since Harman first proposed the ‘free radical theory of aging.’ More recently, RNS have been shown to have a direct role in cell signaling, vasodilatation and the immune response [3]. Nitrosative stress, defined by the excessive production of RNS, causes damage to macromolecules and can lead to degenerative diseases, contribute to metabolic diseases and, if in great excess, can lead to cell death through a variety of molecular mechanisms. The role of RNS in many age-related diseases is just starting to be appreciated. CeO2 nanoparticles & their ability to reduce ROS/RNS In a biological environment, the measurement of ROS and oxidative damage can be challenging, and the methods for detecting ROS have been controversial [24]. Therefore, to assess CNPs effect on ROS, the results need to be carefully assessed and multiple approaches should be utilized to validate changes in the level of ROS. Understanding the nature of the specific ROS/RNS being measured aids in the interpretation of biological catalytic properties of CNPs. However, quite often ROS and RNS are grouped together, and as new catalytic abilities for CNPs are being discovered [9,25–29], studying nanoparticles in biological systems is becoming more challenging. CNPs are consistently shown to exhibit SOD mimetic activity [9,25], catalase mimetic activity [27] or ·NO scavenging abilities [28], whereas high temperature synthesis methods result in CNPs without comparable www.futuremedicine.com

1485

1486


1,2-distearoyl-sn-glycero-3phosphoethanolamine-N(methoxy[PEG]-2000)

Poly(acrylic acid)

Aminated poly(acrylic acid)

Dextran

Ethylene glycol

–

–

Cationic surfactant

–

–

–

–

–

Citric acid

Reverse micelle

Precipitation

Precipitation

Precipitation

Precipitation

Precipitation

Precipitation

Precipitation

Precipitation

Hydrolysis

Hydrolysis

Hydrothermal

Hydrothermal

Hydrothermal

future science group

SD: Standard deviation.

–

Sodium bis(2-ethylhexyl) sulphosuccinate

Microemulsion

Hydrothermal

Hexamethylenetetramine

Solvothermal

Citric acid

PEG

Wet chemical

Hydrothermal

N,N,N’,N’-tetramethylethylenediamine 6

Wet chemical

Citric acid


Solvothermal

Hydrothermal


Solvothermal

Citric acid

5–10


Solvothermal

Hydrothermal

3–5

Wet chemical (using reducing agent) Dextran

5

55.0

31.2

12

4.6

45

22

–

20–25

8

5

15

6

16–22

3–4

3–4

3–4

3–4

3–5

8.5

3–5

6.9

15–20

5–8

Wet chemical (using reducing agent) –

Crystalline size (nm) 3–5

Coating/surfactant used

Wet chemical (using oxidizing agent) –

Method of preparation

–

–

41

25

7

126.8

65.7

100

–

–

7.6

38

–

–

14

5

5

17–18

8–15

38.3

–

5–16

40.2

–

–

5–7

20–30

10–15

Hydrodynamic size (nm) ± SD

–

-32

-56

-57

-53

–

–

-15

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

21

–

–

–

21

27

57

Ce3+ on the surface (%)

Truncated, octahedral –

Polyhedral

Cubic

Polyhedral

Polyhedral

Cubic

Cubic

Irregular

–

Spherical

Spherical

-53 39.6

Spherical

Spherical

Irregular shape

Spherical

Spherical

Spherical

Spherical

Spherical

Polyhedral

Spherical

Polyhedral/spherical

Octahedral

Polyhedral

Polyhedral

Spherical

Spherical

Spherical

Morphology

–

–

-46.2

-2

27

-45

–

–

45.6

–

-22.6

32.9

–

–

-2.09

34

22

Surface charge (mV)

Table 1. Physiochemical properties of cerium oxide nanoparticles synthesized using different methods.

[94]

[43]

[43]

[43]

[43,93]

[92]

[92]

[56]

[84]

[21]

[83]

[92]

[92]

[91]

[20]

[20]

[20]

[63]

[78]

[82]

[90]

[19]

[89]

[39]

[39]

[15]

[14]

[14,42]

Ref.

Review Das, Dowding, Klump, McGinnis, Self & Seal

[86]

– Cubic -10.8 241 17.3 – Flame spray pyrolysis

Review

Catalase mimetic activity Generally, H2O2 was primarily considered to be a toxic byproduct of respiration, coming from the spontaneous or catalytic breakdown of superoxide radicals. Eukaryotic cells have multiple endogenous enzymes (catalase, glutathione peroxidase and peroxiredoxins) for which the localization and expression is highly regulated for the detoxification of H2O2. Exposure to CNPs (containing 6% Ce3+/94% Ce4+) has been shown to protect HT22 cells (rat hippocampal nerve cell line) from H2O2-induced cell death in an early publication [34]. CNPs lead to a decrease in the steady state level of H2O2 [12,27]. We have recently shown that CNPs with increased Ce4+ exhibit catalase mimetic activity [27], yet CNP nanoparticles with a higher concentration of reduced Ce sites are not effective catalase mimetics.

SD: Standard deviation.

[97]

–

–

–

– –

8.9

–

–

2-ethylhexanoic acid Flame spray

Flame synthesis

–

–

–

[96]

[56]

– – – Hydrothermal

–

115

Spherical

[95]

– Spherical – 7 Hydrothermal

Uncoated/poly(acrylic acid)/citric acid

9–13

[16]

– Nanorod Diameter: 6.7–9.5; 153 ± 18 to length: 33.2–495.7 735 ± 24


Hydrothermal

–

–

[16]

– – 7 Hydrothermal

–

55 ± 4

Cubic

[85]

– Spherical – – 6.5 Hydrothermal

Citric acid

Surface charge (mV)

SOD mimetic activity SOD belongs to a diverse group of enzymes that have metal ions that are usually coordinated by nitrogen, oxygen or sulfur centers, to assist in the process of electron transfers, called metalloenzymes [30]. Since its discovery, many chemists and biochemists have developed synthetically produced metal-based small molecule catalysts to try and mimic the active site of SOD. These so called ‘SOD mimics’ have been shown to increase the lifespan of nematodes [31], and their ability to scavenge superoxide radicals and/or H2O2, utilizing various metalloprophyrin motifs [32,33]. When reviewing the literature, we have attempted to categorize the size as well as the surface Ce3+:Ce4+ ratio (if reported) with specific ROS/RNS species tested. Synthesis methods were also considered when determining the surface chemistry of CNPs, and some specific examples are described in Table 2 .

Crystalline size (nm)

Hydrodynamic size (nm) ± SD

antioxidant properties [11]. In an effort to catalog the protective effects of CNPs, we have reviewed studies that suggested that CNPs were interacting with changing ROS in various environments in biological settings.

Coating/surfactant used Method of preparation

Table 1. Physiochemical properties of cerium oxide nanoparticles synthesized using different methods (cont.).

Morphology

Ce3+ on the surface (%)

Ref.


Nitric oxide scavenging property Nitric oxide (NO) has many physiological functions (including neurotransmission and blood vessel dilation), but NO can also be converted into a highly reactive and toxic molecule that readily reacts with proteins, DNA and lipids to alter their function. NO and its reactive intermediates can trigger nitrosative damage to biomolecules, which in turn may lead to www.futuremedicine.com

1487

Review


Scavenging superoxide radical Ce3+ + O2-• + 2H+ → Ce4+ + H2O2

ROS H2O2, O2-•

Ce4+

Ce4+ + H2O2 → Ce3+ + H+ + HO2

Ce3+

Scavenging ROS while regenerating surface

Figure 1. Reactive oxygen species scavenging and surface regeneration properties of cerium oxide nanoparticles. H2O2: Hydrogen peroxide; ROS: Reactive oxygen species.

age-related diseases due to structural alteration of proteins, inhibition of enzymatic activity and interferences in regulatory function [3]. Studies suggest that CNPs may alter the level of RNS in vivo, although it is certainly possible that changes in ROS would also affect RNS [35,36]. A better understanding of which ROS/RNS are targeted in vivo is needed; however, each of these studies suggests that CNPs may indeed act as regenerating catalytic antioxidants. Hydroxyl scavenging property The hydroxyl radical (·OH) is a potent, highly reactive, one-electron oxidant. Production of ·OH occurs in a variety of reactions, however, the most relevant for biological systems is the Fenton reaction. ·OH radicals are also produced during the UV-light dissociation of H2O2 [37] .

Various redox-active nanomaterials have been shown to scavenge ·OH, including fullerene derivatives [38] and CNPs. One comprehensive mechanistic study using CNPs ability to scavenge ·OH shows size dependence as well as a positive correlation with Ce3+ at the surface of the nanoparticle [39]. Antioxidant activity & correlation with physiochemical properties Reduction of Ce4+ to Ce3+ causes oxygen vacancies or defects in the surface of the crystalline lattice structure of the particles, generating a cage for redox reactions to occur. The specificity of the catalytic activities depends on the ratio of Ce3+:Ce4+ [12,28,40]. Figure 2 shows how higher surface Ce3+ can only act as a SOD mimetic where as higher Ce4+ can act as a catalase mimetic [41].

Table 2. Catalytic activity and correlation with physiochemical properties. Catalytic activity CNP high Ce3+/Ce4+

CNP low Ce3+/Ce4+

Size (nm) ± SD

Ref.

SOD mimetic

Yes

No

3–5

[9,25]

Catalase mimetic

No

Yes

16 ± 2.4

[48]

8–10

[27]

NO scavenger

OH scavenger

No

Yes

Yes

No

10

[35]

7

[36]

3–5

[51]

5–8

[28]

5–10

[39,98]

CNP: Cerium oxide nanoparticle; NO: Nitric oxide; OH: Hydroxyl; SD: Standard deviation; SOD: Superoxide dismutase.

1488




Potential applications for CNPs in different fields of medicine In vitro and in vivo experiments studied CNP uptake by cells, and CNPs were found to be present inside the cells [12,42–46]. An in vitro cell culture model study has explored the mechanism of CNP uptake and subcellular localization [44]. To study the uptake mechanism and track the nanoparticles inside the cells, CNPs were conjugated with carboxyfluorescein molecules and these fluorescence-conjugated CNPs (F-CNPs) were used in the experiment. In the presence of an ATP-depleting agent, or at low temperatures (4°C), negligible or no F-CNPs were found inside the cell, which revealed an energy-dependent internalization of CNPs. Moreover, clathrin- and caveolae-dependent endocytosis was observed for the cellular uptake of F-CNPs. A colocalization study revealed the presence of CNPs in subcellular organelles, such as lysosomes, the endoplasmic reticulum and mitochondria, in addition to the cytoplasm and nucleus. It is important to note that the presence of carboxyfluorescein on the surface of the nanoparticles may alter cellular uptake as well as localization. Therefore, uptake and localization of F-CNPs should not be generalized to future science group

0.8

Normalized absorbance

A

0.6

0.4

0.2

0 0

5

10

15

20

Time (min)

B

Normalized absorbance

Therefore, the design of nanoparticles is very important for biological application. Table 2 highlights our catalytic studies of CNPs. We have compared the reactivity of two major preparations of CNPs over the past 5 years. We have found that CNPs that have higher levels of reduced Ce sites (Ce3+) at the surface are more effective SOD mimetics. By contrast, CNPs that have fewer reduced Ce sites exhibit better catalase mimetic and ·NO scavenging capabilities (Table 2). Analyzing reports in the literature that have used other preparations of CeO2 NPs are more difficult since in each case the catalytic effectiveness of the materials is rarely described. However, when surface oxidation states have been reported, we have attempted to correlate these data to catalytic activity observed in vitro to better understand how these nanomaterials can prove to be beneficial in vivo. To further complicate matters, some of the catalytic activities that we have observed can also be affected by the presence or absence of oxidants [25] or anions (i.e., phosphate), which alter catalytic activities [40]. Future directions for researchers must include further characterization of CNPs Ce3+:Ce4+ ratios that coordinate CNPs ability to affect signaling, stress responses and specific ROS/RNS.

Review

0.025

0

-0.025

-0.050

-0.075

0

5

10

15

20

25

30

Time (min)

Control

CNPs 3+ pH7

CNPs 4+ pH7

Figure 2. Effect of surface Ce3+:Ce4+ ratio on cerium oxide nanoparticles’ antioxidant activity. (A) CNPs with higher Ce3+ on the surface have high superoxide dismutase (SOD) mimetic activity where as CNPs with higher Ce4+ on the surface show no SOD mimetic activity. (B) Higher catalase mimetic activity was observed with CNPs with higher surface Ce4+, where as no catalase mimetic activity was observed in CNPs with less Ce4+. The superoxide radical and hydrogen peroxide scavenging activities of CNPs were analyzed by using WST1 (2-[4-Iodophenyl]3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt) [41] and Amplex® (Aarhus, Denmark) red dye [41] , respectively. Change in absorbance in the presence and absence of CNPs was estimated, and the inhibition of absorbance as compared with the control refers to the SOD and catalase activity of CNPs in respective assay systems. Experiments were carried out in triplicate and results were plotted as average ± standard deviation (error bars). CNP: Cerium oxide nanoparticle. Reproduced with permission from [41] .

bare and/or coated CNPs. However, this study provides an overall theory for CNP localization and uptake. In vivo real-time imaging with F-CNPs showed CNPs mainly deposited in the liver, www.futuremedicine.com

1489

1490 Higher


–

–

–

–

–

Nanoparticles from NanoScale Corp. (KS, USA)

Microemulsion and surfactant stripped off



Spray pyrolysis technique

–

–

–

–

–

Higher Ce3+

0.00001 mg/kg of bodyweight

Female athymic nude mice (NCI-nu) Gliosarcoma cells

15 nM and 15 µM 50 µg/ml

24 h preincubation

Biweekly ip. injections

Protection against 10 MV photon beam

Protection against 30 Gy radiation and faster recovery

Improvement in survival rate as a result of protection against 30 Gy of total radiation

Mice model of colon cancer


Biweekly ip. injections

Protection against 10 Gy radiation

CRL8798 and MCF-7

Protection against 200 µM H2O2

Protection against oxidative stress-induced apoptosis by H2O2, PMC and VP-16

24 h pretreatment 10 nM

U937 and Jurkat cells

Protection against oxidative stress induced by cigarette smoke extract

HT-1080 cells

200 µg/ml

H9c2 cell line

24 h pretreatment 0.17 ng/ml

–

24 h pretreatment 10 nM

Both pre- and post-treatment

Male Sprague–Dawley Protection against liver toxicity rats induced by plant-derived pyrrolizidine alkaloid

Protection against stimulated oxidative-induced damage by carbon tetrachloride


Female CD-1 mice

2 week pretreatment

Protection against stimulated oxidative-induced damage by lipopolysaccharide and IFN-g

J774A.1 cell line

Protection against oxidative damage induced by DEP

Application

24 h pretreatment 0–10 µM

Cell line/model

RAW 264.7 and BEAS-2B cell line

Concentration/dose

24 h pretreatment 25 µg/ml

Treatment

[53]

[52]

[59]

[68]

[48]

[19]

[51]

[50]

[42]

[49]

[47]

Ref.

ANC: Aminated poly(acrylic acid); CAM: Cell adhesion molecule; CNP: Cerium oxide nanoparticle; CPS: Cardiac progenitor cell; CSC: Cancer stem cell; DEP: Diesel exhaust particle; Dex: Dextran; EAE: Experimental autoimmune encephalomyelitis; H2O2: Hydrogen peroxide; HMSC: Human mesenchymal stem cell; ip.: Intraperitoneal; MS: Multiple sclerosis; PLGA: Poly(lactic-co-glycolic acid); PMC: Puromycin; PNC: Poly(acrylic acid); VP-16: Etoposide.

–

Wet chemical

10–15

Higher Ce3+

30

Wet chemical

Higher

Wet chemical

30

Wet chemical

–

–

2610

Flame spray pyrolysis

Surface Ce3+:Ce4+ ratio

Nanoparticles from – Sigma-Aldrich (MO, USA)

Hydrodynamic size (nm)

Synthesis method/ source/coating

Table 3. Synthesis method, physiochemical properties and concentrations tested for different uses of cerium oxide nanoparticles in biomedical applications.




www.futuremedicine.com

10

30

200

–

–

Wet chemical and Dex coated

Wet chemical

Nanoparticles from NanoScale Corp.

Wet chemical, transferrin


–

Lower

Lower

Higher

Lower

Lower

CRL-5803 cell line L929 cell line

100 nM 0.001–5 mg/ml

150 µM and every alternate A375 and A375 day, with the dose of xenograft model of 0.1 mg/kg bodyweight for nude mice 30 days

Human dermal fibroblasts and myofibroblasts

A549 cell line

1 mM

150 µM

L929 and VERO cell line

Cell line/model

0.32–5 nM

Concentration/dose

Inhibits malignant melanoma growth in vivo

Inhibits tumor–stroma interactions and anticancer activity

Anticancer activity

Anticancer activity

PNC and ANC showed anticancer activity

Pretreatment showed maximum protection immediately before and 30 min after radiation showed concentration-dependent protection

Application

–

–

Continuous injection for 6 weeks

Enhanced uptake in cancer cell lines

A549 and WI-38 cell line Organotypic rat brain cortical culture

100 nm to 100 µM 10 nM

[21]

[41]

[45]

[15]

[57]

[56]

[55]

[20]

[54]

Ref.

Increased cell survival and protection [58,59] against H2O2

Causes radiation sensitization and increases the efficacy of radiation therapy in pancreatic cancer

L3.6pl and L3.6pl injected nude mouse model

Twice weekly 0.01 mg/kg

A2780, SKOV3, C200, Effective at eliminating ovarian 3 days 100 µM and 0.1 mg/kg of postinoculation of bodyweight every third day CaOV3 and TOV21G cancer cell lines; A2780 cells for 30 days injected nude mouse model

Two treatment groups; treatment started 1 (group 1) and 10 days (group 2) posttumor cell injection

–

–

–

–

30 min pretreatment immediately before radiation and 30 min after radiation

Treatment


10

Wet chemical and Dex coated

120–140

Hydrolysis and hydrothermal

–

–

–

Wet chemical

–

–

–

Wet chemical


Wet chemical, PNC, ANC Dex particles: 14 and Dex coated PNC and ANC: 5



Table 3. Synthesis method, physiochemical properties and concentrations tested for different uses of cerium oxide nanoparticles in biomedical applications (cont.).


Review

1491

1492

–

–

Micoremulsion, PEG coated and anti-b-amyloid conjugated

Nanoparticles from Sigma-Aldrich


Higher

10–15

10–15

10–15

10–15

Wet chemical

Wet chemical

Wet chemical

Wet chemical

–

–

Pretreatment for 3 days

–

7 days after start of MS induction

Postinduction of ischemia

Within 4 h of postischemia

4 h posttreatment with b-amyloid

4 h posttreatment with b-amyloid

8 h postaddition of glutamate

Time of plating

Treatment

Induces HMSC differentiation

Differentiation of osteoblasts (HMSCs) Embedded in bioglass scaffold

Prolongs photoreceptor survival, and preserves retinal structure and function

Tubby mutant mice (tub/tub) in C57BL/6J

172 ng

Vldlr knockout mouse Prevents oxidative stress effects in vivo and therapeutic potential for age-related macular degeneration and diabetic retinopathy

172 ng

Protects photoreceptor cells against light-induced degeneration

Improves motor recovery, particularly the use of the animal forelimbs

EAE model

2.5 µg/kg of bodyweight

20 nM for in vitro study Primary cell cultures and 2 µl of 1 µM for in vivo of rat retina and study albino rats

Decreases brain damage and protects against apoptosis

In vivo model of rat ischemic stroke

0.5 and 0.7 mg/kg of bodyweight

In vitro model of brain Mitigate ischemic brain injury ischemia model

1 µg/ml

Protection of neuron against b-amyloid challenge or in vitro Alzheimer’s disease model

SH-SY5Y cells

[73]

[68]

[66]

[67]

[65]

[63]

[35]

[62]

[61]

Protects neurons against b-amyloid challenge in vivo or in an Alzheimer’s disease model in vitro

200 nM

[34]

[60]

Ref.

Decreases reactive oxygen species generation induced by glutamate

Increased cell survival and protection against H2O2

Application

SH-SY5Y cells

HT22 hippocampal nerve cells

Adult rat spinal cord neurons

Cell line/model

100 µg/ml

20 and 200 µg/ml

10 nM

Concentration/dose


Higher

Higher

Higher

–

–

–

Chemical method, citrate – coated

18–30

–

–

Precipitation and high temperature calcination

Reverse micelle method, phospholipid–PEGcoated CNPs

–

Wet chemical/ – hexamethylenetetramine

–

–

–










–

Nanoparticles from Navarrean Nanoproducts Technology (L’urederra, Spain)

Treatment

–

–

–

High

–

Time of diabetes induction

Single dose postinduction

Pretreated for 3 days

Immediately after wound formation

–

Two different – CNPs; one with higher Ce3+:Ce4+ and the other with lower Ce3+:Ce4+


Application

60 mg/kg of bodyweight


10, 25 and 50 µM

Mitigates endometrial lesions

Diabetic-induced male Increase antioxidant enzymes and Wistar rats decrease oxidative stress

Endometriosisinduced CD-1 strain Swiss albino female mice

CPSs

Protects CPSs from H2O2-induced toxicity

Induces wound healing

10 µM daily

C57BL/6 mice

Induces proliferation of CSCs

HUVEC cells and CAM Induces angiogenesis assay

Cell line/model

5–20% of CNPs embedded Mesenchymal stem in PLGA scaffold cells (CSCs)

1 µM

Concentration/dose

[79]

[78]

[77]

[75]

[74]

[14]

Ref.


10

20–30

Wet chemical


–

Solvothermal and high temperature calcination

–

10–15

Wet chemical

Wet chemical





Review

1493

Review


spleen and lungs. F-CNPs were still found deposited in tissue after 2 weeks of F-CNPs injection using intravenous (iv.) route. Quantification of CNPs in the organs using inductively coupled plasma mass spectroscopy revealed bare CNPs mainly deposited in the spleen and liver when injected using iv. or intraperitoneal routes. Very low amounts of CNPs were deposited in the lungs and kidneys. Negligible or no CNPs were found in the heart or brain. CNPs administered using the oral route showed minimal organ deposition, other than in the lungs, which is due to the gavage procedure or slight aspiration. Multiple weekly systemic injections of nanoceria over a period of 5 weeks had no toxic effects on the lungs, heart, spleen, liver, brain or kidneys [42]. Histopathological analysis of tissues from the major organs did not show any overt pathology. Similar biodistribution of CNPs was observed in a mouse model of ovarian cancer [45]. However, Yokel et al. have shown the presence of citrate CNPs administered via the iv. route in the brain, in addition to the spleen and liver. Moreover, blood circulation and tissue deposition depended on the size of the nanoparticles [46]. CNPs were also found in the blood circulation after 30 days following administration, reflecting a long blood circulation time. Longer blood circulation times were observed in 5-nm CNPs as compared with 55-nm CNPs. This may be due to the fact that CNPs used in these studies had different physiochemical properties. Recently, CNPs have emerged as interesting and attractive materials for biomedical research. Several research papers and book chapters have been published, and patents recorded, in the last few years on the different possible application potentials of CNPs in medicine. Here, we review the different applications that have been documented in the literature. Table 3 summarizes the concentration and physiochemical properties of CNPs used in different areas of medicine. Anti-inflammatory & antiapoptotic activity High levels of ROS or RNS can contribute to chronic inflammation, which can initiate complex immunological disorders and result in irreversible organ damage. Chronic inflammation often promotes the development and progression of several diseases. The antioxidant activity of CNPs can reduce such inflammation, and protect cells or tissue from damage. Antioxidant properties of CNPs have been tested in a few studies using in vitro cell culture models. CNP oxidative stress was induced 1494


in mouse leukemic monocyte macrophage (RAW 264.7) and human bronchial epithelial (BEAS‑2B) cells by administering pro-oxidative organic diesel exhaust particle extract. Interestingly, cells pretreated with CNPs showed a significant decrease in intracellular ROS levels as compared with stimulated cells [47]. A similar study with a human breast fibrosarcoma cell line also showed that CNPs significantly reduced apoptosis caused by H2O2 challenge in a concentration-dependent manner [48]. ROS generation decreased by half in CNP-treated murine macrophage cells (J774A.1) in a concentrationdependent manner [49]. Interestingly, reduced expression of iNOS mRNA and protein was observed in the presence of CNPs in both stimulated and control cells. In a continuation of this study [42], the same group further estimated ·NO production in both stimulated and control cells using the Griess assay (measuring nitrate levels). A decrease in nitrate concentration was observed in CNP-treated cells, which further supports the anti-inflammatory effect of CNPs. Next, CNPs were tested in an in vivo oxidative stress-induced mouse model. CNP injection showed a significant decrease in lipid peroxidation at 2 and 3 weeks. In summary, this report supports the hypothesis that CNPs could act as antioxidant/anti-inflammatory agents not only in in vitro, but also in vivo. Monocrotaline (MCT) is a plant-derived pyrrolizidine alkaloid, and exposure to MCT could cause oxidative veno-occlusive disease of the liver [50]. Potential applications of CNPs to reduce MCT-mediated toxicity have been explored. CNPs restore the reduced glutathione, glutathione reductase, glutathione S-transferase and GPX levels. Moreover, increase in catalase and SOD levels were found to be increased in MCT-stimulated rats, whereas CNPs reverse SOD and catalase levels to normal levels. This study also reflects that CNPs could decrease oxidative stress-induced cellular or tissue damage. Another interesting study tested CNPs effect on oxidative stress induced by cigarette smoke extract on H9c2 rat heart-derived embryonic myocytes [51]. CNP treatment showed significantly decreased expression levels of proinflammatory cytokines, which indicates an anti-inflammatory property of CNPs. Moreover, in this study both gene and protein expression levels of iNOS were also significantly decreased by CNPs in cigarette smoke extract simulated cells. In summary, anti-inflammatory CNP treatment can minimize this cellular damage by inhibiting NF-kB activation and increasing future science group

Review


the antioxidant defense mechanisms of the cell. In another in vitro study CNPs antioxidant and anti-inf lammatory behavior has been explored with a human tumor monocyte cell line (U937) and Jurkat cells, as a model of well-studied inflammatory cells [19]. Reduction of intracellular ROS was observed in a concentrationdependent manner in both cell lines, even at longer time periods (48 and 72 h). Apoptosis was induced in cells using three different agents H2O2, the protein synthesis inhibitor puromycin and the topoisomerase II inhibitor etoposide. CNP treatment showed decreased apoptosis

and increased cell survival against all three by interfering with intracellular signaling pathways. Further cotreatment with cystathionine (a known antioxidant agent that partially prevents apoptosis against etoposide challenge) with CNPs did not show any further improvement, suggesting that CNPs inhibit the same target as cystathionine. In this study, intracellular antioxidant and antiapoptotic activity was correlated with oxygen vacancy and the Ce3+:Ce4+ ratio of CNPs. Interestingly, Ce3+/Ce4+ concentration, not oxygen vacancy, was found to be key for CNPs antioxidant and antiapoptotic activity.

A Normal breast epithelial cells

B Normal breast epithelial cells 3.0

8000

Cells (n)

6000

4000

2000

0

Control

0 Gy 48 h TUNEL-positive cells (%)

1 Gy 10 Gy

2.5

10 Gy 48 h

2.0 1.5 1.0 0.5 0

Treated CNPs

Control

Treated CNPs

C Survival curves 100

Control Survival (%)

Radiation CNPs 50

Amifostine Radiation + CNPs Radiation + amifostine

0 0

100 Days after radiation exposure

200

Figure 3. In vitro and in vivo radiation protection of cerium oxide nanoparticles. Pretreatment with CNPs protects normal breast epithelial cells (CRL8789) against (A) 10 Gy of radiation and (B) oxidative stress-induced DNA damage. (C) In vivo experiment with male athymic nude mice also showed improved survival with CNP treatment. CNP: Cerium oxide nanoparticle; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling. (A & B) Reproduced with permission from [99] . (C) Reproduced with permission from [37] .



1495

Review


Stromal cell (e.g., [myo]fibroblast)

Tumor cell

TGF-β1 ROS

CNP

Invasion

CNP

Proinvasive signals

αSMA

Tumor weight 9

Weight (g)

8 7 6 5 4 3 Control

CNPs

Control

CNPs

Figure 4. Anticancer activity of cerium oxide nanoparticles. (A) CNPs inhibit myofibroblasts and the ROS-dependent cytotoxic and anti-invasive effect on squamous tumor cells. (B) CNP treatment causes a significant decrease in the tumor weight in an in vivo model of ovarian cancer. CNP: Cerium oxide nanoparticle; ROS: Reactive oxygen species. (A) Reproduced with permission from [57] . (B) Reproduced with permission from [45] .

Radiation protection properties Radiation exposure to living tissue generates free radicals through ionizing reactions (UV, laser, g-ray or particle radiation) such as the photoelectric, Compton and Auger effects. These free radicals damage tissue, cells, proteins, lipids and even genetic material (DNA and RNA). On the other hand, radiation therapy alone, or in combination with chemotherapy, is still the primary method for treatment of a wide range of human malignancies. However, radiation exposure does not differentiate between normal and cancer cells, thus, it delays the recovery and reduces quality of life. Therefore, new classes of agents are being used in an effort to improve outcomes for cancer patients and to reduce the potential toxicity of radiation to normal tissue surrounding the tumor. 1496


CNPs are potential candidates to reduce the radiation toxicity to normal cells, owing to their unique regenerative antioxidant activity. In 2005, our group showed that CNPs could protect (>90% cell survival) immortalized normal breast epithelial cells (CRL8789) against 10 Gy radiation in the presence of CNPs (Figure 3A). Along with cell survival, CNPs were also found to decrease DNA damage in normal breast epithelial cells (Figure 3B). Interestingly, CNPs did not protect breast cancer cells (MCF7) against the same radiation exposure. In another study, treatment with CNPs showed significant protection against normal fibroblast cells (CCL 135) against a single dose of 20 Gy radiation [37]. In vivo experiments with athymic nude mice, in which mice were exposed to fractionated doses of 30 Gy radiation (weekly future science group


administration of 5 Gy) in the presence of twice weekly CNPs (iv. injections), showed 80% survival until week 33 (Figure 3C). Tissue analysis showed a 50% decrease in caspase-3 and cells with DNA damage as compared with those without nanoparticles. A similar study, dealing with a neck radiation mouse model, also showed a decrease in apoptotic acinar cells and an increase in salivary production in CNP-treated mice [52]. This suggests CNPs have the potential to be a radiation protector for radiation therapy. A recent study has shown that the radiation protection effect of CNPs also depends on modification of the radiation electron spectra in the local area by CNPs [53]. Radiation protection of CNPs was observed against a 10 MV photon beam in gliosarcoma cells (9L); however, no

Live and dead cells/cover slip (n)

900

Review

significant effect was observed against 150 kVp x-ray exposure. This observation was explained in terms of the photoelectric effect at kilo voltage x-ray energies, which influence low-energy electron production with high liner energy transfer and may cause cellular damage. This study will help to choose a photon field use with CNP-based material for radiation protection. Finally, CNPs have also been shown to protect cells against UV radiation-induced oxidative damage. According to this study, a citrate stabilized CNP solution did not show any photocatalytic activity [54]. Moreover, CNPs showed a decrease in the degradation of methyl orange under UV exposure, which indicates partial blocking of UV radiation. However, HT preparation of CNPs showed some extent of photo

Number of cover slips in both the control and CNP-treated groups (n = 6) 50 µm

*

600

*

CNP-treated adult rat nerve cell culture

300

** 0

Live cells

Dead cells 15-day culture β-amyloid

Live cells

Dead cells

30-day culture β-amyloid + CNPs

NF-H200

Control

*

Figure 5. Cerium oxide nanoparticles sustained survival and protection of neuron and photoreceptor cells against oxidative stress. (A) CNPs increase the lifespan of neuron cells isolated from an adult rat’s spinal cord. (B) CNPs targeted to b-amyloid plaques protect the neuronal network of cultured neuron cells against b-amyloid challenge (scale bars: 17 µm). (C) CNP treatment prevents light-induced degradation of photoreceptor cells in albino rats (scale bars: 25 µm). *p < 0.05; **p < 0.01. CNP: Cerium oxide nanoparticle; NF-H200: Heavy neurofilament. (A) Reproduced with permission from [60] . (B) Reproduced with permission from [62] . (C) Reproduced with permission from [66] .



1497

Review


Nanoceria (ng)

400

300

200

100

0 0

30

60

90

120

Duration (days)

Figure 6. Retention of cerium oxide nanoparticles in the eye after intravitreal injection. A total of 91% of the injected cerium oxide nanoparticles (344 ng) are retained in the eye for at least 4 months. Supplementary data indicate retention of nanoceria in the eye for 1 year with a calculated half-life of 525 days [92] . Reproduced with permission from [72] .

degradation with increasing size (4.6–14 nm), while 4.6-nm particles still showed partial blocking of UV. Citrate stabilized ceria solutions led to an activation/increase in cell proliferation of both mouse fibroblasts (L929) and fibroblast-like cells of the African green monkey (VERO). Pretreatment of cells with a CNP solution 30 min prior to UV radiation gave cells full protection. However, dose-dependent protection was observed in the case of CNP treatment immediately before and after 30 min of UV irradiation. Cancer therapy Cancer is the leading cause of death worldwide. Excessive ROS may play a role in the pathogenesis of many diseases, including cancer. CNPs have been shown to have selective anticancer activity in both in vitro and in vivo models of different cancer types [15]. An in vitro basic study with an emphasis on mechanisms revealed that PNC and aminated PNC-coated CNPs showed toxicity towards a lung cancer cell line (A549) [20]. No toxicity towards A549 was observed in the case of DexCNP. Surface charge, higher endocytosis/uptake and lysosomal localization of PNC-CNPs and aminated PNC-CNPs were found to be toxic toward lung cancer cells. Oxidase-like activity of polymer-coated CNPs in the acidic microenvironment of cancer cells may be utilized in the further development of anticancer agents. An 1498


antiproliferative activity and the induction of apoptotic (caspase-3/7) upconvertor nanoparticles (doped CNPs: CeO2: 20% Ytterbium, 2% Europium) have been reported for a lung cancer cell line (CRL-5803) [55]. An antiproliferative effect of HT- and hydrolysis-prepared CNPs towards prostate cancer cell lines (PC-3) was also reported [56]. It is important to mention that at similar concentrations upconvertor nanoparticles (HUVEC), HT-CNPs (L929 cell line) and LH-CNPs (L929 cell line), were found to be nontoxic towards normal cell lines. In cancer pathogenesis, the main concern is the interaction between cancer cells with stroma environments, because stroma is involved in tumor progression and invasion. Dex-CNPs almost completely eliminate the TGF-b1-triggered a-smooth-muscle actin induction after 24-h treatment, and decreased the number of myofibroblasts [57]. Moreover, Dex-CNPs were found to be selectively toxic towards squamous tumor cell lines, whereas no effect was observed in normal dermal fibroblasts. Dex-CNPs also decreased the invasion property of squamous tumor cells by 2.3-fold. A possible schematic diagram has been shown in F igur e 4A . Interestingly, a concentrationdependent increase in ROS production was observed in squamous tumor cells, whereas no alteration of ROS production in normal dermal fibroblasts was observed when treated with Dex-CNPs. This phenomenon was described in terms of a pH-dependent antioxidant effect of Dex-CNPs. Dex-CNPs also showed a decrease in human melanoma cell (A375) proliferation, whereas cell viability was not altered in normal dermal fibroblast and endothelial cells [15]. Similar to the squamous tumor cells, a decrease in the invasion property of A375 cells was observed. An in vivo study also revealed the anticancer activity of Dex-CNPs against malignant melanoma [15]. Both CNP treatments started at day 1 and day 10 post-tumor cell injection, and showed a significant decrease in tumor volume (day 1: 75% and day 10: 85%) and weight (70%). Expression of the CD-31 antigen was decreased by 60% in tumor tissue in both DexCNP treatment groups, which confirms in vivo antiangiogenic activity of Dex-CNPs. The anticancer activity of bare CNPs was tested in both in vitro and in vivo ovarian cancer models. In ovarian cancer cell lines (A2780), bare CNPs decreased the amount of ROS generation compared with the control [45]. Similar to a previous study, CNPs were found to inhibit migration and invasion of ovarian cancer cells in vitro [45]. future science group


Moreover, in an in vivo model of mouse ovarian cancer, CNP treatment significantly decreased abdominal circumference and tumor weight (Figure 4B). This study showed antiangiogenic and anticancer activity of CNPs in both in vitro and preclinical in vivo models of ovarian cancer [45]. In another study, CNPs were explored as radiation sensitizers for pancreatic cancer radiation therapy [41]. This experiment revealed a pHdependent pro-oxidant effect of CNPs in a radiation environment. A twofold increase in ROS was observed in a CNP-treated pancreatic cancer cell culture model (L3.6pl) after 30 min and up to 24 h of postradiation exposure, whereas a 50% decrease in ROS was observed in CNPtreated normal pancreatic cells. An in vivo pancreatic cancer model showed that the presence of CNPs significantly decreased tumor volume and weight compared with radiation alone [41]. This study further showed that CNPs can be used as potential radiation sensitizers for cancer therapy, while protecting normal tissue surrounding the tumor from radiation damage. In previous papers, passive targeting of CNPs has been investigated for cancer therapy [15,21,45]. In this study, transferrin-coated CNPs showed enhanced cellular uptake in human lung adenocarcinoma epithelial cells (A549) as compared with bare CNPs [21]. However, minimal cellular uptake was observed in human embryo lung fibroblast cells (WI-38) for transferrin-coated CNPs. This study revealed that the anticancer efficacy of CNPs could be enhanced by conjugation or coating with cancer cell targeting molecules, which will enhance cellular uptake by concentrating CNPs inside the tumor cells, while decreasing side effects by reducing nonspecific interactions to normal cells.

Review

observed when cultured in the presence of CNPs [60]. Significantly higher amounts of live neuron cells were observed after day 15 and day 30 after culturing compared with the control (Figure 5A). No differences in the number of glial cells were observed, with or without CNPs; however, significantly higher neural cell populations were observed in CNP-treated cultures at both of the estimated time points, and they were observed to be functioning similarly to the other adult rat CNS cultures. Moreover, the CNP pretreated neuronal culture showed a higher amount of H2O2 detoxification, compared with the control, when exposed to H 2O2 , which further confirms the neuron protective activity of CNPs against ROS. The pathogenesis of Alzheimer’s disease is associated with the agglomeration of b-amyloid and the formation of plaques. Precipitation- and high temperature-calcined CNPs were found to reverse the effect of neuron survival, in in vitro culture, against b-amyloid challenge [61]. CNPs also significantly decreased the number of apoptotic cells, compared with b-amyloid challenge, and restored them to a similar level as the control. Restoration of neuronal morphology was observed in CNPtreated cells. PPARb and BDNF are both known to be involved in neuron survival and proliferation. These results prove the involvement of CNPs in the modulation of BDNF signaling pathways to minimize the damage by b-amyloid-induced ROS. This in vitro mechanistic study showed

400

Neurodegenerative diseases In 2003, the neuroprotective effect of engineered CNPs was reported for the first time, and this study has shown that CNPs enhanced the average lifespan of neuron cells [58]. Moreover, neuron protection of CNPs after in vitro trauma has also been reported [59]. The protective effect of different sized CNPs (6 nm, 12 nm and micron size particles) was tested in neurons, and no significant correlation was observed between the size of the nanoparticles and the extent of protection against the stress condition. The neuron protective property was only observed in nanoparticles, while the precursor (Ce nitrate) did not show any antioxidant activity [34]. Similar increases in the lifespan of neuron cells, isolated from an adult rat spinal cord, were future science group

Nanoceria (ng)

300

200

100

0 0

30

60

90

120

Duration (days)

Figure 7. The retina retains almost 70% of injected cerium oxide nanoparticles for at least 4 months. Supplementary data indicate retention of cerium oxide nanoparticles in the retina for 1 year with a calculated half-life of 415 days [92] . Reproduced with permission from [72] .


1499

1500


369–1634 depending on concentration

55–735

22; shape unknown

33–495; rod

–

THP-1 cell line

-14.1 ± 0.04 Green alga and cyanobacterium

-53 ± 7, Male Sprague–Dawley -57 ± 5, rats -56 ± 8 and -32 ± 2

Male Sprague–Dawley rats

HUVEC cell line

50 µg/ml for 24 h

0.01–100 mg/l

100 mg CNPs/kg for the 5-, 15- and 30-nm CNPs, and 50 mg/kg for the 55-nm CNPs

85 mg/kg in water using a volume of 2 ml/kg over 1 h, iv. route

0–17 µM

20 µg/ml

1–100 nM

Caenorhabditis elegans HT22 murine neuronal cells

Intratracheal instillation of 0.1 ml 2 mg/ml CNPs

100–200 µg/ml for 24 h

10, 20 and 100 µg/ml for 48 and 72 h

[104]

[16]

≥200 nm CNP nanorods induced proinflammation

[43,46]

[83]

[12]

[103]

[82]

[102]

[101]

[71]

[100]

Ref.

Oxidative stress and cell death

Oxidative stress seen in the spleen and liver

Induction of oxidative stress in liver

ATP depletion and higher cell internalization

Altered genes related to nemological disease, cell cycle control and growth

Induction of oxidative damage

–

Increase in the intracellular levels of ROS

After 72 h, concentrations >10 µg/ml showed DNA damage

641 mg/m3 aerosolized CNPs Inhalation of CNPs caused for 4 h oxidative stress and inflammation in the lung

Wistar rats

HaCaT and A549

Human lens epithelial cells

Wistar rats of either sex

Toxicological effect

CNP: Cerium oxide nanoparticle; iv.: Intravenous; ROS: Reactive oxygen species; SD: Standard deviation; SEM: Scanning electron microscopy; TEM: Transmission electron microscopy.

–

–

Citric acid

-53 ± 7

7, 25, 41


5, 10, 55 (polyhedral) and 30 (cubic)

–

6 and 100; polygonal

Hexamethylenetetramine 30.1

–

12.8 ± 2.2

8.5; polygonal

Hexamethylenetetramine 30.1

–

7.6

12.8 ± 2.2

6.6 ± 0.9; polygonal

–

–

5; spherical

–

14 and 400; shape unknown

3-phosphonopropionic acid

–

Hexamethylenetetramine 34–38

–

5–6; irregular

–

Surface Cell line/plant/animal Concentration/dose charge model (mV) ± SD

10–15; polygonal/round 80–150

–

15–30; irregular

TEM/SEM analysis and morphology (nm) radii (nm) ± SD

Coating of Hydrodynamic nanoparticles

Size of CNPs

Table 4. Toxicological effect of cerium oxide nanoparticles.




CNP: Cerium oxide nanoparticle; iv.: Intravenous; ROS: Reactive oxygen species; SD: Standard deviation; SEM: Scanning electron microscopy; TEM: Transmission electron microscopy.

[106]

CeO2 nanoparticle-induced oxidative injury 400 or 800 mg/kg Corn plants -22.8 2124 10; shape unknown

–

Induced apoptosis and decreased cell viability 0.5–10 µg/ml for 20 and 40 h Human peripheral blood monocytes -15 – 231

TEM/SEM analysis and morphology (nm) radii (nm) ± SD

10–30; shape unknown

Surface Cell line/plant/animal Concentration/dose charge model (mV) ± SD Coating of Hydrodynamic nanoparticles Size of CNPs

Table 4. Toxicological effect of cerium oxide nanoparticles (cont.).

Toxicological effect

Ref.

[105]


Review

CNPs could be a promising nanomedicine for Alzheimer’s disease treatment. In another study, a targeted regenerative antioxidant to the plaque appeared to enhance the antioxidant therapeutic efficacy of CNPs in Alzheimer’s disease. Well-dispersed CNPs prepared using a microemulsion method were coated with PEG, acting as a spacer, and conjugated with anti-b-amyloid antibodies, and were tested in an amyloid-b challenged or in vitro Alzheimer’s disease model [62]. Similar to the previous study, targeted CNPs also inhibited neuronal cell agglomeration, and neurite losses were analyzed using immunostaining (NF-H200 staining is shown in Figur e 5C) at significant low concentrations as compared with bare CNPs [61]. This study clearly suggests that by improving the stability of the nanoparticle suspension (physical properties), decreasing the nonspecific interaction and making nanoparticles target specific, it is possible to decrease the effective concentration and side effects associated with nanoparticle therapeutic applications. Cerebral ischemia or hypoxia caused by stroke induces oxidant stress, inflammation and subsequent tissue damage due to the reduction of glucose and oxygen supplies to the brain. A recent study using an in vitro model of brain ischemia showed that the presence of CNPs at the onset of ischemia could significantly decrease cell death [35]. CNP treatment within 4 h of postischemia showed significant neuroprotection. A significant decrease in both ROS (superoxide radical and H 2O2) and RNS (NO) was observed in the presence of CNPs. The neuroprotective effect of CNPs was also established in an in vivo rat ischemic stroke model [63]. In this study, well-dispersed phospholipid– PEG-coated CNPs (PPEG-CNPs) were used. Low concentrations of PPEG-CNPs did not show any protection, however, concentrations between 0.5 and 0.7 mg/kg of bodyweight showed a significant decrease in brain infarct volumes by 50% compared with the control. In addition, high concentrations (>1 mg/kg of bodyweight) failed to show any neuroprotective efficacy. Most important to note is that PPEG-CNPs did not significantly penetrate the normal blood–brain barrier; however, PPEGCNPs crossed the blood–brain barrier after a stroke, which minimizes the possible toxicity concern. Interestingly, 3D reconstruction of the brain showed that PPEG-CNPs were mainly localized in the peri-infarct area in the ischemic hemisphere. This study demonstrated www.futuremedicine.com

1501

Review


A

B 5

Wound size (nm)

Day 1

4

Control

CNPs

*

**

**

**

**

1

3

5 Days

8

13

3 2 1

Day 8 0

Figure 8. Application of cerium oxice nanoparticles in regenerative medicine. (A) 3D scaffold made with bioglass material; CNPs incorporation into the scaffold showed an increase in osteoblastic differentiation and collagen production. (B) Topical application of CNPs in full-thickness dermal wounds (arrows) causes them to close significantly faster compared with the control (scale bars: 4 mm). *p < 0.05; **p < 0.01. CNP: Cerium oxide nanoparticle. (A) Reproduced with permission from [73]. (B) Reproduced with permission from [75] .

CNP’s neuroprotective effect for the first time in an ischemic stroke model of a living animal. CNPs were also tested in a mouse model of multiple sclerosis (MS). MS is an autoimmune disease where oxidative stress and inflammation play a key role in the pathology, and result in focal demyelinated plaques and nerve loss throughout the brain and spinal cord [64]. Preliminary studies with citric acid-stabilized CNPs were carried out on an experimental autoimmune encephalomyelitis model of MS [65]. After 7 days of MS induction, the CNP injected mice showed improvements in motor performance (estimated by daily testing, including rotorod, hanging wire and balance beam tests). These preliminary results also indicate that CNPs could be further explored for MS treatment. 1502


Vision loss & eye disease Among the leading causes of vision loss in the developed world is blinding disease resulting from pathologic neovascularization or from selective degeneration of retinal neurons. A common node in the etiology of diseases such as age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, glaucoma and retinopathy of prematurity is the production of ROS in excess of endogenous redox-active defenses, a condition termed oxidative stress. Irrespective of whether the observed increase in ROS in pathologic neovascular and degenerative eye disease is causative or occurs as a result of mechanisms leading to cell death, it is clear that targeting ROS with catalytic antioxidant nanoparticles reverses pathology in both induced future science group


and heritable models of retinal degeneration. The seminal in vivo experiments of Chen et al. demonstrated protection of mammalian retinal neurons with the injection of less than 0.5 ng of CNPs into the eye (Figure 5C) [66]. A higher dose of 344 ng provided almost complete preservation of retinal morphology and function under conditions of light damage, which caused blindness in uninjected or saline-injected rodents. Nanoceria dramatically decrease the expression of VEGF, and result in a greater than fourfold reduction in pathological intraretinal and subretinal neovascular lesions in the VLDLR knockout mouse, a model for retinal angiomatous proliferation and the ‘wet form’ of age-related macular degeneration [67]. Hereditary retinopathies affect approximately one in 2000 individuals in the USA, and produce progressive photoreceptor degeneration, leading to complete blindness. In the tubby (tub/tub) mouse, a model of the human Usher syndrome, intravitreal dosing of nanoceria reduced the level of ROS in the retina, and resulted in prolonged protection of photoreceptor cells by upregulation of survival pathways and inhibition of apoptosis [68,69]. In addition to successful application for the treatment of retinal degenerative and proliferative vascular diseases of the eye in animal models, CNPs demonstrate a potential avenue for vision-sparing therapy in pediatric eye cancer [70]. Advantages of CNPs for the treatment of ocular cancer include the capability for local delivery and lack of toxicity to surrounding normal tissues [71,72]. In experiments using p53TKO mice that exhibit a heritable model of retinoblastoma, a single intravitreal injection of CNPs was shown to inhibit tumor growth within 1 week and decreased proliferation of tumor-forming cells in vitro, suggesting a novel use for CNPs in the treatment of ocular malignancy [70]. Collectively, these studies demonstrate that ROS scavenging by CNPs is effective in reducing pathologic neovascularization and retinal damage, and strongly supports therapeutic applications for nanoceria in the treatment of vision loss and eye disease. Moreover, multiple intracardial injections had neither detectable toxicity nor negative effects on retinal morphology or function [69], and there were no negative effects 1 month after a single intravitreal injection of nanoceria in the tubby mouse [68]. In rats, a single intravitreal injection had no toxic effects on retinal morphology or function when examined up to 1 year after the injection [72]. Owing to the panretinal protection provided by a single intravitreal injection, we think that nanoceria become evenly distributed future science group

Review

across the entire retina [66]. Most surprising to us was our discovery that more than 90% of the intravitreally injected nanoceria are retained in the eye for at least 4 months [72]. In addition, the retina rapidly takes up the injected nanoceria and also retains most of them (69%) for 4 months (Figure 6 & 7). Additional longitudinal studies carried out for 1 year showed that the half-life of nanoceria in the eye was 525 days and its half-life in the retina was 415 days. The molecular basis for this long retention time is unknown but currently under investigation. Tissue regeneration or regenerative medicine CNPs were shown to increase the lifespan of photoreceptors and neuron cells, which gives the rationale to explore CNPs for tissue engineering and regeneration fields. CNPs incorporated in the scaffold, either for soft or hard tissue regeneration, or the direct application of CNPs in wound healing procedures, have been reported [14,73–75]. Free radical formation during cellular growth hinders cell differentiation, and CNPs could promote cell growth/tissue engineering by acting as antioxidants. Scaffolds are 3D templates that stimulate tissue growth. Scaffolds could be used for hard or soft tissue growth, where CNPs could be used as a stimulus and antioxidant for faster growth or healing. Bioactive glass materials (Novabone Products LLC, FL, USA) have been used clinically for dental applications and have been shown to enhance bone formation by inducing differentiation of osteoblasts [73]. Two different CNPs, bare CNPs and Dex-CNPs, were incorporated in the bioactive glass for comparison purposes. CNPs were incorporated in the scaffold so that it was entrapped within the silica network and pore wall of the foam (Figure 8A). A large adhesion area and well-spread morphology were observed for both of the CNP-incorporated scaffolds, however, no osteoblast-like structures were observed. Increases in osteoblastic differentiation of human mesenchymal stem cells (HMSCs) in the bare CNP-incorporated scaffold was observed, however, no change was observed in Dex-CNP-incorporated CNPs in the absence and presence of osteogenic supplements. Moreover, Dex-CNP-incorporated scaffolds showed significantly lower alkaline phosphatase activity, compared with ceria-free scaffolds. Collagen production was also increased (11.2%) in culture for bare CNP-incorporated scaffolds, compared with CNP-free and Dex-CNP-incorporated scaffolds. This study showed that the www.futuremedicine.com

1503

Review


presence of CNPs could influence and help in the differentiation of HMSCs. It is important to note that the influence of HMSC differentiation depends on the CNP physiochemical properties and culture conditions. In another study, CNP-incorporated biocompatible scaffolds were explored for culture and differentiation of cardiac and mesenchymal cancer stem cells [74]. In particular, CNP-embedded biodegradable poly(lactic-co-glycolic acid) matrices were investigated for in vitro stem cell cultures. Up to 10% of CNP incorporation increased the number of cardiac and mesenchymal stem cells observed at all of the time points (24 h, 3 day and 6 day), compared with CNP-free scaffolds. This study further revealed that for the best result in therapy or tissue regeneration, an optimum concentration of CNPs is needed. Recently, proangiogenesis properties of CNPs were also reported [14]. Endothelial tube formation assays with HUVEC cells and cell adhesion molecule assays showed an increase in tube or blood vessel formation for both CNPs. However, CNPs with higher Ce3+/Ce4+ showed a higher induction (40% increase) of tube or blood vessel formation, as compared with CNPs having less Ce3+/Ce4+ (11% increase). CNPs with different sizes and shapes were also tested. CNPs larger than 15 nm did not show any increase in tube formation as compared with the control. This study also clearly suggests that CNPs decrease molecular oxygen transiently, and, therefore, induce HIF1a stabilization and nuclear translocation, which regulates several genes along with genes regulating angiogenesis, including VEGF. This study revealed that the oxygen buffering capacity of CNPs not only depends on the physical properties of the nanoparticles, but that the surface Ce3+:Ce4+ ratio plays a key role in governing catalytic activity in the cellular microenvironment. Induction of angiogenesis and antioxidant activity of CNPs provides a motivation to explore CNPs with a high Ce3+:Ce4+ ratio in wound healing applications [75]. To verify that the CNPs could work in vivo, a skin wound healing mouse model was selected. The CNP-treated wounds closed significantly faster compared with the control, with the wounds being completely closed on days 8 and 13, respectively (Figure 8B). Similar to this study, 160 nm of sol-gel-prepared CNPs (2%) were also shown to possess wound healing activity. CNPs increased the OH-proline content, wound tensile strength and wound closure time [76]. The potential applications of progenitor cells in regenerative medicine are enormous and wide open; however, the challenge will be successfully 1504


culturing progenitor cells in vitro. The efficacy of CNPs to support the growth of cardiac progenitor cells (CPSs) by reducing oxidative stress has been investigated [77]. A time-dependent response has been noted when counting CPSs in the presence and absence of CNPs, and CNP-treated CPSs maintain their pluri-/multi-potency. Moreover, CNP-treated CPSs cocultured with murine neonatal cardiomyocytes proceeded to cardiac commitment. For CPS pretreated with CNPs, a significant decrease in intracellular ROS was observed at all time points (after day 1, 3 and 7) when challenged with H2O2. This study opens a new frontier for CNP applications in regenerative medicine. Other applications Endometriosis is a chronic gynecological disorder, associated with pelvic pain, dysmenorrhea and infertility. Like cancer, the pathology of this disease is also strongly associated with oxidative stress and angiogenesis, therefore, the therapeutic efficacy of a novel antioxidant and antiangiogenic oxide nanoparticle have been explored in endometriosis-associated symptoms [78]. This study showed the potential of CNPs to mitigate endometrial lesions induced in a mouse model, by decreasing oxidative stress and inhibiting angiogenesis. This finding opens a window to further explore CNPs in oxidative stress-related pathology in the female reproductive system. Treatment with CNPs along, or in combination, with sodium selenite showed a significant decrease in oxidative stress in a diabetes-induced mice model [79]. Increase in antioxidant enzymes and high-density lipoproteins, and a decrease in oxidative stress, adenosine diphosphate/adenosine triphosphate levels, cholesterol, triglyceride and low-density lipoproteins were observed in diabetic-induced rats that received CNPs or combination therapy. In the near future CNPs will be used as a fuel additive, as addition of CNPs improves the burning efficiency of fuel. A recent paper showed that CNPs as a fuel additive could limit the proatherosclerosis by decreasing the inflammatory response associated with regular diesel fuel by reducing the number of particles in the exhaust [80].

Toxicological aspects of CNPs The unique physiochemical properties of nanoparticles raise concerns about toxicity towards the environment. Toxicity aspects of CNPs have been studied by different research groups. In vitro and in vivo studies reported toxicological effects of CNPs are summarized in Table 4. future science group


Recently, a study using the wild-type Caenorhabditis elegans model showed the toxicity of CNPs mediated by nonspecific inhibition of feeding. The C. elegans strains with known sensitivity to metal did not show any additional oxidative stress as expected, owing to the presence of the metal-containing CNPs [81]. Therefore, toxicity is probably due to the properties of the CNP, including surface coating, size and agglomeration status, surface charge and morphology of the synthesized nanoparticles, and not the property of bulk material. Supporting studies comparing different CNPs in both in vitro and in vivo model systems are needed to understand the mechanism of toxicity caused by differences in coating, surface charge, shape, size/agglomeration status and chemical properties. These studies will help to identify CNPs with minimum or no toxicity.

Conclusion Many diseases progress through oxidative stress, and because ROS are a common connection between the primary cause of disease and their downstream consequences, ROS represent an Achilles’ heel that CNPs can successfully target because of their broad spectrum catalytic antioxidant activity, small size and prolonged retention in tissues. However, the health effects of CNPs are not as yet well understood, and there are reports that claim that CNPs are both protective and toxic [11, 43,82–88]. Moreover, material characteristics, protocols for biological/toxicological processing, surface composition, and specific biological systems and end points being tested are also important variables in these studies. In a recent review, researchers have discussed how preparation procedure, handling and storage of nanoparticles could alter their catalytic properties as well as their biological response [11]. In considering CNPs as potential therapeutic agents, it is important to pay attention to their synthesis method, concentration and surface

Review

chemistry. The preparation/synthesis methods of CNPs result in particles with a wide variety of surface functionalities and modifications, and these properties will dictate whether a nanoparticle will be protective or deleterious. From the literature it can be concluded that CNPs are regenerative antioxidant agents. The property that makes CNPs different from other antioxidants is their ability to self-regenerate their surface. Therefore, one small dose can work for a long time before being cleared from the body. We anticipate that over the next decade, CNPs will become ‘the global equivalent of an aspirin for oxidative stress-associated diseases’.

Future perspective Oxidative stress is associated with most chronic diseases such as cancer, cardiac disease, neurodegeneration and autoimmune disease. Different outcomes reported for antioxidant therapy are mainly due to differences in delivery methods and antioxidant absorption levels in the body. CNPs were recently shown to have regenerative antioxidant activity. Thus, a small amount of CNPs can work for extended time periods. However, due to their nanoscale size, it also raises toxicological concerns. The toxicity of CNPs depends on their surface coating and charge, agglomeration and concentration of the nanoparticles in the biological media/fluids and surface charge of the nanoparticles. Therefore, by manipulating the synthesis method it is possible to tailor CNPs with minimum or no toxicity to living systems. Biocompatible polymer coatings and/or targeting CNPs to the disease site could enhance the therapeutic activity by minimizing side effects and improving pharmacokinetics. It is essential to investigate different CNPs to establish an array of nontoxic formulations with enhanced catalytic activity for clinical application. Finally, systematic studies to explore the long-term effects of CNPs using large animal models are needed.

Executive summary Pharmaceutical potential of cerium oxide nanoparticles Cerium oxide nanoparticles (CNPs) are regenerative antioxidant agents. This regenerative antioxidant property of CNPs is facilitated by the ability of CNPs to mediate between 3+ and 4+ oxidation states with equilibrated oxygen defect sites. The synthesis method, and the handling and storage of nanoparticles could alter their catalytic properties as well as their antioxidant or pro-oxidant biological response. Different studies have shown that CNPs can protect biological tissues, including neurons, against oxidative stress induced by different agents or radiation, help prolong the survival of neurons or photoreceptor cells, act as a proangiogenesis modulator and enhance wound healing, reduce chronic inflammation, control or reduce the growth and proliferation of tumors, and reduce brain damage in ischemic stroke. The health effects of CNPs are not yet well understood, and there are reports that claim that CNPs are both protective and toxic. Moreover, material characteristics, the protocols for biological/toxicological processing, surface composition, and the specific biological system and end point being tested are also important variables in these studies.



1505

Review


Acknowledgements The authors express their sincere thanks to R Draper and A Kumar for their help in formatting the manuscript and preparing artwork.

Financial & competing interests disclosure S Seal and W Self acknowledge the NIH grant R01AG03152901, and National Science Foundation grants CBET 0930170

impacts of ceria nanoparticles. Surf. Interface Anal. 44(8), 882–889 (2012).

References Papers of special note have been highlighted as: n of interest nn of considerable interest

and CBET 0708172. JF McGinnis acknowledges NIH grants R01EY018724 and R01EY022111. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

nn

Nanoparticle synthesis parameters, storage and handling are very important for use in biological science/medicine. Summarizes the possible challenges for potential CNP use.

1

Kaplan J. Active oxygen. Nature 159(4046), 673 (1947).

2

Uttara B, Singh AV, Zamboni P, Mahajan RT. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr. Neuropharmacol. 7(1), 65–74 (2009).

12 Dowding JM, Das S, Kumar A et al. Cellular

Drew B, Leeuwenburgh C. Aging and the role of reactive nitrogen species. Ann. NY Acad. Sci. 959, 66–81 (2002).

interactions. Nano Today 3(1), 40–47 (2008).

3

4

5

6

7

8

9

n

Hutson S. Experts urge a more measured look at antioxidants. Nat. Med. 14(8), 795–795 (2008).

24 Kalyanaraman B, Darley-Usmar V, Davies KJ

10 Karakoti A, Singh S, Dowding JM, Seal S, Self

WT. Redox-active radical scavenging nanomaterials. Chem. Soc. Rev. 39(11), 4422–4432 (2010). 11 Karakoti AS, Munusamy P, Hostetler K et al.

Preparation and characterization challenges to understanding environmental and biological

1506

et al. Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic. Biol. Med. 52(1), 1–6 (2012).

14 Das S, Singh S, Dowding JM et al.

The induction of angiogenesis by cerium oxide nanoparticles through the modulation of oxygen in intracellular environments. Biomaterials 33(31), 7746–7755 (2012).

16 Ji Z, Wang X, Zhang H et al. Designed

First paper to show that cerium oxide nanoparticles (CNPs) have superoxide dismutase mimetic activity.

23 Kumar A, Babu S, Karakoti AS, Schulte A,

13 Lynch I, Dawson KA. Protein–nanoparticle

Erica S, Daniel A, Silvana A. Artificial nanoparticle antioxidants. In: Oxidative Stress: Diagnostics, Prevention, and Therapy. American Chemical Society, DC, USA, 235–253 (2011).

Korsvik C, Patil S, Seal S, Self WT. Superoxide dismutase mimetic properties exhibited by vacancy engineered ceria nanoparticles. Chem. Commun. 10, 1056–1058 (2007).

enhanced CeO2 host matrix for energy transfer from Ce3+ to Tb3+. RSC Adv. 3, 3623–3630 (2013). Seal S. Luminescence properties of europiumdoped cerium oxide nanoparticles: role of vacancy and oxidation states. Langmuir 25(18), 10998–11007 (2009).

15 Alili L, Sack M, Von Montfort C et al.

Deshpande S, Patil S, Kuchibhatla SV, Seal S. Size dependency variation in lattice parameter and valency states in nanocrystalline cerium oxide. Appl. Phys. Lett. 87(13), 133113 (2005).

22 Wang X, Zhang D, Li Y et al. Self-doped Ce3+

interaction and toxicity depend on physicochemical properties and surface modification of redox-active nanomaterials. ACS Nano 7(6), 4855–4868 (2013).

Frankel EN, Finley JW. How to standardize the multiplicity of methods to evaluate natural antioxidants. J. Agric. Food Chem. 56(13), 4901–4908 (2008).

Sun C, Li H, Chen L. Nanostructured ceria-based materials: synthesis, properties, and applications. Energy Environ. Sci. 5(9), 8475–8505 (2012).

ligand tethering and cellular targeting. ACS Nano 3(5), 1203–1211 (2009).

Downregulation of tumor growth and invasion by redox-active nanoparticles. Antioxid. Redox Signal. doi:10.1089/ ars.2012.4831 (2012) (Epub ahead of print). synthesis of CeO2 nanorods and nanowires for studying toxicological effects of high aspect ratio nanomaterials. ACS Nano 6(6), 5366–5380 (2012). 17 Tarantola M, Pietuch A, Schneider D et al.

Toxicity of gold-nanoparticles: synergistic effects of shape and surface functionalization on micromotility of epithelial cells. Nanotoxicology 5(2), 254–268 (2011). 18 Das S, Singh S, Singh V et al. Oxygenated

functional group density on graphene oxide: its effect on cell toxicity. Part. Part. Syst. Char. 30(2), 148–157 (2013). 19 Celardo I, De Nicola M, Mandoli C, Pedersen

JZ, Traversa E, Ghibelli L. Ce3+ ions determine redox-dependent anti-apoptotic effect of cerium oxide nanoparticles. ACS Nano 5(6), 4537–4549 (2011). 20 Asati A, Santra S, Kaittanis C, Perez JM.

Surface-charge-dependent cell localization and cytotoxicity of cerium oxide nanoparticles. ACS Nano 4(9), 5321–5331 (2010). 21 Vincent A, Babu S, Heckert E et al.

Protonated nanoparticle surface governing


25 Heckert EG, Karakoti AS, Seal S, Self WT.

The role of cerium redox state in the SOD mimetic activity of nanoceria. Biomaterials 29(18), 2705–2709 (2008). 26 Kuchma MH, Komanski CB, Colon J et al.

Phosphate ester hydrolysis of biologically relevant molecules by cerium oxide nanoparticles. Nanomedicine 6(6), 738–744 (2010). 27 Pirmohamed T, Dowding JM, Singh S et al.

Nanoceria exhibit redox state-dependent catalase mimetic activity. Chem. Commun. 46(16), 2736–2738 (2010). 28 Dowding JM, Dosani T, Kumar A, Seal S,

Self WT. Cerium oxide nanoparticles scavenge nitric oxide radical (·NO). Chem. Commun. 48(40), 4896–4898 (2012). n

Reveals that CNPs can also scavenge reactive nitrogen species.

29 Dowding JM, Seal S, Self WT. Cerium oxide

nanoparticles accelerate the decay of peroxynitrite (ONOO -). Drug. Deliv. Transl. Res. doi:10.1007/s13346-013-0136-0 (2013) (Epub ahead of print). 30 Flora SJ. Structural, chemical and

biological aspects of antioxidants for strategies against metal and metalloid exposure. Oxid. Med. Cell. Longev. 2(4), 191–206 (2009). 31 Melov S, Ravenscroft J, Malik S et al.

Extension of life-span with superoxide dismutase/catalase mimetics. Science 289(5484), 1567–1569 (2000).



32 Salvemini D, Mazzon E, Dugo L et al.

Pharmacological manipulation of the inflammatory cascade by the superoxide dismutase mimetic, M40403. Br. J. Pharmacol. 132(4), 815–827 (2001). 33 Rong Y, Doctrow SR, Tocco G, Baudry M.

EUK-134, a synthetic superoxide dismutase and catalase mimetic, prevents oxidative stress and attenuates kainate-induced neuropathology. Proc. Natl Acad. Sci. USA 96(17), 9897–9902 (1999). 34 Schubert D, Dargusch R, Raitano J, Chan SW.

Cerium and yttrium oxide nanoparticles are neuroprotective. Biochem. Biophys. Res. Commun. 342(1), 86–91 (2006). 35 Estevez A, Pritchard S, Harper K et al.

Neuroprotective mechanisms of cerium oxide nanoparticles in a mouse hippocampal brain slice model of ischemia. Free Radic. Biol. Med. 51(6), 1155–1163 (2011). 36 Niu J, Azfer A, Rogers LM, Wang X,

Kolattukudy PE. Cardioprotective effects of cerium oxide nanoparticles in a transgenic murine model of cardiomyopathy. Cardiovasc. Res. 73(3), 549–559 (2007). 37 Colon J, Herrera L, Smith J et al. Protection

from radiation-induced pneumonitis using cerium oxide nanoparticles. Nanomedicine 5(2), 225–231 (2009). 38 Wang IC, Tai LA, Lee DD et al. C(60) and

water-soluble fullerene derivatives as antioxidants against radical-initiated lipid peroxidation. J. Med. Chem. 42(22), 4614–4620 (1999). 39 Xue Y, Luan Q, Yang D, Yao X, Zhou K.

Direct evidence for hydroxyl radical scavenging activity of cerium oxide nanoparticles. J. Phys. Chem. C 115(11), 4433–4438 (2011). 40 Singh S, Dosani T, Karakoti AS, Kumar A,

Seal S, Self WT. A phosphate-dependent shift in redox state of cerium oxide nanoparticles and its effects on catalytic properties. Biomaterials 32(28), 6745–6753 (2011). 41 Wason MS, Colon J, Das S et al. Sensitization

of pancreatic cancer cells to radiation by cerium oxide nanoparticle-induced ROS production. Nanomedicine 9(4), 558–569 (2012). 42 Wang X, Zhang D, Li Y et al. Self-doped Ce3+

enhanced CeO2 host matrix for energy transfer from Ce3+ to Tb3+. RSC Adv. 3, 3623–3630 (2013). 43 Yokel RA, Tseng MT, Dan M et al.

Biodistribution and biopersistence of ceria engineered nanomaterials: size dependence. Nanomedicine 9(3), 398–407 (2012). 44 Singh S, Kumar A, Karakoti A, Seal S, Self

WT. Unveiling the mechanism of uptake and sub-cellular distribution of cerium oxide nanoparticles. Mol. BioSystems 6(10), 1813–1820 (2010).


45 Giri S, Karakoti A, Graham RP et al.

Nanoceria: a rare-earth nanoparticle as a novel anti-angiogenic therapeutic agent in ovarian cancer. PLoS One 8(1), e54578 (2013).

interactions. Biomaterials 32(11), 2918–2929 (2011). 58 Ellison A, Fry R, Merchant S et al. Engineered

oxide nanoparticles protect against neuronal damage associated with in vitro trauma. J. Neurotrauma 10(20), 1105 (2003).

46 Yokel RA, Au TC, Macphail R et al.

Distribution, elimination, and biopersistence to 90 days of a systemically introduced 30 nm ceria-engineered nanomaterial in rats. Toxicol. Sci. 127(1), 256–268 (2012).

59 Callahan P, Colon J, Merchant S et al.

Deleterious effects of microglia activated by in vi-tro trauma are blocked by engineered oxide nanoparticles. J. Neurotrauma 20(10), 1057 (2003).

47 Xia T, Kovochich M, Liong M et al.

Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2(10), 2121–2134 (2008).

60 Das M, Patil S, Bhargava N et al. Auto-catalytic

ceria nanoparticles offer neuroprotection to adult rat spinal cord neurons. Biomaterials 28(10), 1918–1925 (2007).

48 Clark A, Zhu A, Sun K, Petty HR. Cerium

oxide and platinum nanoparticles protect cells from oxidant-mediated apoptosis. J. Nanopart. Res. 13(10), 5547–5555 (2011).

61 Dangelo B, Santucci S, Benedetti E et al.

Cerium oxide nanoparticles trigger neuronal survival in a human Alzheimer disease model by modulating BDNF pathway. Curr. Neurosci. 5(2), 167–176 (2009).

49 Hirst SM, Karakoti AS, Tyler RD,

Sriranganathan N, Seal S, Reilly CM. Anti-inflammatory properties of cerium oxide nanoparticles. Small 5(24), 2848–2856 (2009).

62 Cimini A, D’angelo B, Das S et al.

Antibody-conjugated PEGylated cerium oxide nanoparticles for specific targeting of Ab aggregates modulate neuronal survival pathways. Acta Biomater. 8(6), 2056–2067 (2012).

50 Amin KA, Hassan MS, Awad el-ST, Hashem

KS. The protective effects of cerium oxide nanoparticles against hepatic oxidative damage induced by monocrotaline. Int. J. Nano. 6, 143– 149 (2011).

n

51 Niu J, Wang K, Kolattukudy PE. Cerium oxide

nanoparticles inhibits oxidative stress and nuclear factor-kB activation in H9c2 cardiomyocytes exposed to cigarette smoke extract. J. Pharmacol. Exp. Ther. 338(1), 53–61 (2011). 52 Madero-Visbal RA, Alvarado BE, Colon JF

et al. Harnessing nanoparticles to improve toxicity after head and neck radiation. Nanomedicine 8(7), 1223–1231 (2012).

Nanoparticles that can protect against ischemic stroke. Angew. Chem. Int. Ed. Engl. 124(44), 11201–11205 (2012). 64 Estevez A, Erlichman J. Cerium oxide

nanoparticles for the treatment of neurological oxidative stress diseases. Brain 28, 30 (2011). 65 Decoteau W, Estevez A, Leo-Nyquist S,

Heckman K, Reed K, Erlichman J. Ceria nanoparticles reduce disease severity in a mouse model of multiple sclerosis. Presented at: TechConnect World Conference and Expo 2011. Boston, MA, USA, 13–16 June 2011. 66 Chen J, Patil S, Seal S, McGinnis JF. Rare earth

nanoparticles prevent retinal degeneration induced by intracellular peroxides. Nat. Nanotechnol. 1(2), 142–150 (2006).

54 Zholobak NM, Ivanov VK, Shcherbakov AB

et al. UV-shielding property, photocatalytic activity and photocytotoxicity of ceria colloid solutions. J. Photochem. Photobiol. B Biol. 102(1), 32–38 (2011).

nn

55 Babu S, Cho J-H, Dowding JM et al.

Multicolored redox active upconverter cerium oxide nanoparticle for bio-imaging and therapeutics. Chem. Commun. 46(37), 6915–6917 (2010).

57 Alili L, Sack M, Karakoti AS et al. Combined

cytotoxic and anti-invasive properties of redoxactive nanoparticles in tumor–stroma


Demonstrates that CNPs, in a concentrationdependent manner, protect normal retinal neurons in vitro and in vivo against intracellularly generated reactive oxygen species.

67 Zhou X, Wong LL, Karakoti AS, Seal S,

McGinnis JF. Nanoceria inhibit the development and promote the regression of pathologic retinal neovascularization in the VLDLR knockout mouse. PLoS One 6(2), e16733 (2011).

56 Renu G, Rani V, Nair S, Subramanian K,

Lakshmanan VK. Development of cerium oxide nanoparticles and its cytotoxicity in prostate cancer cells. Adv. Sci. Lett. 6(1), 17–25 (2012).

Targeted nanoparticles are more efficient in mitigating oxidative stress-induced damage.

63 Kim CK, Kim T, Choi IY et al. Ceria

53 Briggs A, Corde S, Oktaria S et al. Cerium

oxide nanoparticles: influence of the high-Z component revealed on radioresistant 9L cell survival under x-ray irradiation. Nanomedicine doi:10.1016/j.nano.2013.02.008 (2013) (Epub ahead of print).

Review

nn

Demonstrates that a single 172-ng injection of CNPs can regress inherited pathological vascular lesions in vivo in a mammalian retina.

1507

Review


68 Cai X, Sezate SA, Seal S, McGinnis JF.

Sustained protection against photoreceptor degeneration in tubby mice by intravitreal injection of nanoceria. Biomaterials 33(34), 8771–8781 (2012). 69 Kong L, Cai X, Zhou X et al. Nanoceria extend

photoreceptor cell lifespan in tubby mice by modulation of apoptosis/survival signaling pathways. Neurobiol. Dis. 42(3), 514–523 (2011). 70 Klump K, Seal S, Dyer M, McGinnis J. In vivo

targeting and inhibition of retinoblastoma with catalytic antioxidants. Presented at: International Society for Eye Research. Berlin, Germany, 21–25 July 2012. 71 Pierscionek BK, Li Y, Schachar RA, Chen W.

The effect of high concentration and exposure duration of nanoceria on human lens epithelial cells. Nanomedicine 8(3), 383–390 (2012). 72 Wong LL, Hirst SM, Pye QN, Reilly CM, Seal

S, McGinnis JF. Catalytic nanoceria are preferentially retained in the rat retina and are not cytotoxic after intravitreal injection. PLoS One 8(3), e58431 (2013). 73 Karakoti AS, Tsigkou O, Yue S et al. Rare earth

oxides as nanoadditives in 3-D nanocomposite scaffolds for bone regeneration. J. Mater. Chem. 20(40), 8912–8919 (2010). 74 Mandoli C, Pagliari F, Pagliari S et al. Stem cell

aligned growth induced by CeO2 nanoparticles in PLGA scaffolds with improved bioactivity for regenerative medicine. Adv. Function. Mater. 20(10), 1617–1624 (2010). 75 Chigurupati S, Mughal MR, Okun E et al.

Effects of cerium oxide nanoparticles on the growth of keratinocytes, fibroblasts and vascular endothelial cells in cutaneous wound healing. Biomaterials 34(9), 2194–2201 (2013). 76 Davan R, Prasad R, Jakka VS et al. Cerium

oxide nanoparticles promotes wound healing activity in in-vivo animal model. J. Bionanosci. 6(2), 78–83 (2012). 77 Pagliari F, Mandoli C, Forte G et al. Cerium

oxide nanoparticles protect cardiac progenitor cells from oxidative stress. ACS Nano 6(5), 3767–3775 (2012). 78 Chaudhury K, Babu NK, Das S, Kumar A,

Seal S. Mitigation of endometriosis using regenerative cerium oxide nanoparticles. Nanomedicine 9(3), 439–448 (2013). 79 Pourkhalili N, Hosseini A, Nili-Ahmadabadi A

et al. Biochemical and cellular evidence of the benefit of a combination of cerium oxide nanoparticles and selenium to diabetic rats. World J. Diabetes 2(11), 204–210 (2011). 80 Cassee FR, Campbell A, Boere AJF et al.

The biological effects of subacute inhalation of diesel exhaust following addition of cerium oxide nanoparticles in atherosclerosis-prone mice. Environ. Res. 115, 1–10 (2012).

1508

81 Arnold M, Badireddy A, Wiesner M, Di Giulio

95 Safi M, Sarrouj H, Sandre O, Mignet N, Berret

R, Meyer J. Cerium oxide nanoparticles are more toxic than equimolar bulk cerium oxide in Caenorhabditis elegans. Arch. Environ. Contamin. Toxicol. 65(2), 224–233 (2013). 82 Zhang H, He X, Zhang Z et al. Nano-CeO2

exhibits adverse effects at environmental relevant concentrations. Environ. Sci. Technol. 45(8), 3725–3730 (2011).

JF. Interactions between sub-10‑nm iron and cerium oxide nanoparticles and 3T3 fibroblasts: the role of the coating and aggregation state. Nanotechnology 21(14), 145103 (2010). 96 Gojova A, Lee JT, Jung HS, Guo B, Barakat

AI, Kennedy IM. Effect of cerium oxide nanoparticles on inflammation in vascular endothelial cells. Inhal. Toxicol. 21(S1), 123–130 (2009).

83 Tseng MT, Lu X, Duan X et al. Alteration of

hepatic structure and oxidative stress induced by intravenous nanoceria. Toxicol. Appl. Pharmacol. 260(2), 173–182 (2012).

97 Raemy DO, Limbach LK, Rothen-Rutishauser

B et al. Cerium oxide nanoparticle uptake kinetics from the gas-phase into lung cells in vitro is transport limited. Eur. J. Pharm. Biopharm. 77(3), 368–375 (2011).

84 Lin W, Huang Y-W, Zhou X-D, Ma Y. Toxicity

of cerium oxide nanoparticles in human lung cancer cells. Int. J. Toxicol. 25(6), 451–457 (2006).

98 Xue Y, Zhai Y, Zhou K et al. The vital role of

buffer anions in the antioxidant activity of CeO2 nanoparticles. Chemistry 18(35), 11115–11122 (2012).

85 Hardas SS, Butterfield DA, Sultana R et al.

Brain distribution and toxicological evaluation of a systemically delivered engineered nanoscale ceria. Toxicol. Sci. 116(2), 562–576 (2010).

99 Tarnuzzer RW, Colon J, Patil S, Seal S.

Vacancy engineered ceria nanostructures for protection from radiation-induced cellular damage. Nano Lett. 5(12), 2573–2577 (2005).

86 Demokritou P, Gass S, Pyrgiotakis G et al.

An in vivo and in vitro toxicological characterisation of realistic nanoscale CeO2 inhalation exposures. Nanotoxicology doi:10.310 9/17435390.2012.739665 (2012) (Epub ahead of print). 87 Hardas SS, Butterfield DA, Sultana R et al.

Brain distribution and toxicological evaluation of a systemically delivered engineered nanoscale ceria. Toxicol. Sci. 116(2), 562–576 (2010). 88 Ma Y, Kuang L, He X et al. Effects of rare earth

oxide nanoparticles on root elongation of plants. Chemosphere 78(3), 273–279 (2010). 89 Zhang P, Ma Y, Zhang Z et al.

Biotransformation of ceria nanoparticles in cucumber plants. ACS Nano 6(11), 9943–9950 (2012). 90 Karakoti AS, Singh S, Kumar A et al. PEGylated

nanoceria as radical scavenger with tunable redox chemistry. J. Am. Chem. Soc. 131(40), 14144–14145 (2009). 91 De Marzi L, Monaco A, De Lapuente J et al.

Cytotoxicity and genotoxicity of ceria nanoparticles on different cell lines in vitro. Int. J. Mol. Sci. 14(2), 3065–3077 (2013). 92 Pelletier DA, Suresh AK, Holton GA et al.

Effects of engineered cerium oxide nanoparticles on bacterial growth and viability. Appl. Environ. Microbiol. 76(24), 7981–7989 (2010). 93 Dan M, Tseng MT, Wu P, Unrine JM, Grulke

EA, Yokel RA. Brain microvascular endothelial cell association and distribution of a 5 nm ceria engineered nanomaterial. Int. J. Nanomedicine 7, 4023–4036 (2012). 94 Rojas S, Gispert JD, Abad S et al. In vivo

biodistribution of amino-functionalized ceria nanoparticles in rats using positron emission tomography. Mol. Pharm. 9(12), 3543–3550 (2012).


n

Demonstrates that CNPs significantly decrease radiation damage by scavenging reactive oxygen species in normal cell lines while inducing cancer death.

100 Srinivas A, Rao PJ, Selvam G, Murthy PB,

Reddy PN. Acute inhalation toxicity of cerium oxide nanoparticles in rats. Toxicol. Lett. 205(2), 105–115 (2011). 101 Horie M, Nishio K, Kato H et al. Cellular

responses induced by cerium oxide nanoparticles: induction of intracellular calcium level and oxidative stress on culture cells. J. Biochem. 150(4), 461–471 (2011). 102 He X, Zhang H, Ma Y et al. Lung deposition

and extrapulmonary translocation of nano-ceria after intratracheal instillation. Nanotechnology 21(28), 285103 (2010). 103 Lee T-L, Raitano JM, Rennert OM, Chan SW,

Chan WY. Accessing the genomic effects of naked nanoceria in murine neuronal cells. Nanomedicine 8(5), 599–608 (2012). 104 Rodea-Palomares I, Gonzalo S,

Santiago-Morales J et al. An insight into the mechanisms of nanoceria toxicity in aquatic photosynthetic organisms. Aquat. Toxicol. 122–123, 133–143 (2012). 105 Hussain S, Al-Nsour F, Rice AB et al. Cerium

dioxide nanoparticles induce apoptosis and autophagy in human peripheral blood monocytes. ACS Nano 6(7), 5820–5829 (2012). 106 Zhao L, Peng B, Hernandez-Viezcas JA et al.

Stress response and tolerance of zea mays to CeO2 nanoparticles: cross talk among H2O2, heat shock protein, and lipid peroxidation. ACS Nano 6(11), 9615–9622 (2012).
