pH-Responsive Morphology-Controlled Redox ... - ACS Publications

Subscriber access provided by Universitaetsbibliothek | Johann Christian Senckenberg

Article

pH-Responsive Morphology-Controlled Redox Behaviour and Cellular Uptake of Nanoceria in Fibrosarcoma RASHID MEHMOOD, Pramod Koshy, Nicholas Ariotti, Jia Lin Yang, and Charles Christopher Sorrell ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00806 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 27, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

pH-Responsive Morphology-Controlled Redox Behavior and Cellular Uptake of Nanoceria in Fibrosarcoma Rashid Mehmood1*, Nicholas Ariotti2, Jia Lin Yang3, Pramod Koshy1, Charles C. Sorrell1 1

School of Materials Science and Engineering, Faculty of Science, UNSW Sydney, Sydney, NSW 2052, Australia 2 Electron Microscope Unit, Mark Wainwright Analytical Centre, UNSW Sydney, Sydney, NSW 2052, Australia 3 Sarcoma and Nanooncology Group, Adult Cancer Program, Lowy Cancer Research Centre, Prince of Wales Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW 2052, Australia *Corresponding Author: [email protected] Keywords:

Ceria, nanoceria, oxygen vacancies, reactive oxygen species, fibrosarcoma,

cancer therapy

Abstract The present work reports structural/microstructural correlations with biological performance for three nanoceria morphologies, aiming to elucidate the major factors in their interactions with fibrosarcoma. These include the pH of the in vitro medium and the crystallinities, stoichiometries, surface areas and chemistries, and maximal oxygen vacancy concentrations ([V•• ]Max). While the [V••]Max is dominant in the redox behavior, the role of the morphology was manifested in the order of effectiveness of the redox regulation, which was nanocubes (NC) < nanorods (NR) < nanooctahedra (NO). The proposed mechanism illustrates the role of V•• in explaining antioxidant behavior at physiological pH 7.4 and prooxidant behavior in the tumour microenvironment pH 6.4. Cellular uptake at pH 7.4 was dominated by the morphology of the nanoparticle, demonstrating the order NO < NC < NR. Control of the [V••]Max, morphology, and dependent structural and microstructural parameters can be used to optimise the uptake and redox performance of nanoceria.

Introduction Biomedical research has expanded significantly in order to develop effective cancer treatments owing to the multifarious nature and rapid proliferation of cancer cells, an

ACS Paragon Plus (1) Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

important one of which is fibrosarcoma. In common with other cancers, it shows complex and rapid modes of angiogenesis, cell adhesion, and cell migration.1,2 These enable it to develop resistance to chemotherapeutics, leading to increasing recurrence in the body even after treatment. Therefore, there is a need for the development of a range of therapeutic approaches to inhibit of tumour cell growth by selectively inducing apoptosis without harming the surrounding normal cells. Cerium oxide nanoparticles (nanoceria, CeO2-x) recently have received considerable attention as a potential cancer therapeutic agent owing to their ability to mimic the biological reactions to regulate reactive oxygen species (ROS) at the enzymatic level through redox reactions on their surfaces.3-8

Owing to this redox behaviour nanoceria biomedical properties have been explored for different diseases including cancers where it can inhibit angiogenesis, cell proliferation, and inflammation in tissues and cells.9-11 Once in the tumor microenvironment, nanoceria can induce cytotoxic behavior via ROS production by, inter alia, redox reaction12-15 while providing cytoprotection to healthy normal cells.16 This redox behaviour of nanoceria is associated with maximal oxygen vacancy concentrations ([V••]Max) present at the surface, where continuous reversible switching of the Ce3+/Ce4+ pair occurs due to low reduction potential values of the pair.17,18 Consequently, the Ce3+/Ce4+ couple has the capacity to demonstrate antioxidant properties for the scavenging of ROS at basic physiological pH while, at acidic tumour pH, the couple acts as a prooxidant for the production of ROS.19 This unusual redox capacity of nanoceria can be utilised to provide cytoprotective effects for healthy cells and cytotoxic effects for cancer cells.20-22

The potential functionality and stability of nanoceria in biological systems depend on a range of physicochemical and related factors, which are dependent largely on the surface rather than the bulk of particles. Consequently, the morphology, exposed surface facets, surface area, and grain size are critical and these depend principally on the fabrication method.23-25 Despite the biomedical potential of nanoceria, only limited work on the structure-activity relationship (SAR) of CeO2 in cancer diagnosis and therapy has been undertaken,23 although nearly all studies have used what are considered to be spherical nanoparticles.3-20

Other common morphologies of nanoceria, including hexagonal and

square rods, octahedra, and cubes, have not been investigated extensively for cancer


Page 2 of 24

Page 3 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


prevention.

Further, there are few biomedical data on relevant materials properties,

including crystallinity, morphology, surface redox chemistry, and associated [V••]Max.23 Also, potential issues concerning the blockage of active V••26 from sources such as agglomeration and/or the use of capping agents as deflocculants during particle development have not been addressed properly. Consequently, the present work reports the structure-activity relationship and cellular uptake of nanoceria in contact with fibrosarcoma cells at different biological pH values.

Experimental Materials Cerium (III) nitrate hexahydrate (Ce(NO3)3·6H2O, 99.999 wt% trace metal basis), ammonia solution (NH3, 25wt%), 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), , redCCK-8 (cell counting kit 8) APTES (3-amino-propyl)-triethoxysilane), Fluorescein-5-isothiocyanate, ethanol (C2H5OH, 99.5%), glutaraldehyde (C5H8O2, 25% aqueous solution, Electron Microscopy Sciences, USA), osmium tetroxide (OsO4, 4% aqueous solution, Electron Microscopy Sciences, USA), LX112 resin embedding kit (Ladd Research, Williston, USA), sodium cacodylate (C2H7AsO2, Proscitech, Australia), and cerium standard (100 mg/mL Ce in HNO3) were purchased from Sigma-Aldrich, Australia unless otherwise noted. MitoSox, dulbecco’s phosphate buffered saline (DPBS, Gibco Life Technologies, USA), Trypsin-EDTA (ethylenediaminetetra acetic acid, Gibco Life Technologies, USA), and hydrogen peroxide (H2O2, 30 vol% in H2O, Ajax Finechem, Australia) were purchased from Thermo Fisher Scientific, Australia. Sodium hydroxide (NaOH, 98 wt%) was purchased from Chem-Supply, Australia. Hydrochloric acid (HCL, 32 vol%) and nitric acid (HNO3, 76 vol%) were purchased from RCI Labscan Limited, Australia. Fibrosarcoma (HT1080) cell lines were purchased from American Type of Cell Culture, USA. RPMI 1640 medium (Roswell Park Memorial Institute1640, USA) was purchased from Invitrogen, USA.

Synthesis of nanoparticles Wet-chemical and surfactant-free methodologies were used to synthesise nanoparticles of varying morphologies and sizes. To synthesise ceria nanocubes (NC) hydrothermally, equal volumes of 0.45 M Ce(NO3)3·6H2O and 20 M NaOH solutions were mixed in a 100 mL Pyrex beaker and magnetically stirred for 30 min at room temperature. The cloudy suspension



then was transferred to a 50 mL Teflon-lined autoclave reactor for hydrothermal treatment, which involved heating at 180oC for 24 h. The ceria nanorods (NR) were synthesised in a similar fashion, with the only change being an autoclaving temperature of 120oC. Ceria nanooctahedra (NO) were synthesised by stirring 0.1 M Ce(NO3)3·6H2O solution at room temperature, followed by dropwise addition of H2O2 to attain pH ~3.0 and dropwise addition of NH3(aq) to increase the pH to ~9.0. In order to bring the pH to a neutral value, nanoparticles were washed with 100 mL deionised water ten times and dried at 80oC for 24 h.

Characterisation of nanoparticles The mineralogical properties of the nanoparticles were analysed by X-ray diffraction (Philips X’Pert Multipurpose X-Ray Diffractometer [MPD], Netherlands), with CuKα radiation (0.15405 nm), at 20°-80° 2θ, with step size 0.02o 2θ and scanning speed 5.5o 2θ/min). Transmission electron microscopy (TEM) images of nanoparticles were taken using a Philips CM200, Netherlands.

The TEM samples were prepared by dispersion in ethanol and

dropping on a copper grid. Image J software was used for image analysis. The specific surface areas were determined by the Brunauer-Emmett-Teller method (BET, Micromeritics Tristar-3000, USA) and the surface chemistries were determined by X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, ESCALAB 250Xi spectrometer (UK), with a power of 13.8 kV, 8.7 mA, beam diameter 500 μm with monochromated AlKα X-rays at 1487 eV).

Stock suspension preparation Stock suspensions of nanoceria for biological tests were prepared at pH values 7.4 and 6.4, which correspond to the normal physiological environment and cancer microenvironment, respectively.27 The nanoparticles were suspended in a cell culture medium (RPMI-1640) at a concentration of 200 μg/mL and the pH values were adjusted using 1 M HCl and 1 M NaOH, monitored with an FG2 Mettler-Toledo pH meter, USA.

Cell culturing The human soft tissue fibrosarcoma HT1080 cells were cultured in the RPMI-1640 medium, which also contained 2.0 mM L-glutamine, 1% v/v penicillin/streptomycin, and 10 vol% fetal


Page 4 of 24

Page 5 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


bovine serum; this work was done in a PC2 biosafety hood. Following culturing, the cells in the medium then were transferred to a Forma 4140TS Thermo Fisher Scientific incubator, USA at 37°C with atmosphere of 5 vol% CO2 in air to assist with growth. The cells were monitored at the 48 h time point in order to ensure proper growth, after which they were extracted at 1-2 weeks for analyses.

Cell viability Cell cytotoxicity assays were performed on all three morphologies of nanoceria in order to test their redox activities at different pH values.

Cells were seeded in three 96-well

polystyrene tissue culture plates, with each well at a density of 2 × 103 cells/well with 100 μL of RPMI medium. For each plate, three wells were used for each pH value. After seeding, the cells were allowed to adhere at 37°C with 5% CO2 in air for 24 h in the incubator, after which the supernatant medium was removed from each well and replaced with 100 μL of 200 μg/mL of nanoceria suspension. Under the same incubation conditions, each of the three plates then was subjected to a different final testing interval: 24 h, 48 h, or 72 h. Following this, 10 μL of CCK-8 reagent were added as an indicator of cell proliferation and the cells were incubated under the same conditions for 3 h. Similar cell concentrations also were seeded in the remaining wells in triplicate without nanoceria to use as positive controls for comparison.

The optical absorbance at 450 nm was measured using a

Molecular Devices SpectraMax M3 (MT05412) plate reader, USA.

ROS measurements The preparation procedure was identical to that used for cell cytoxicity up to the final testing interval. The cells were washed with 1 mL DPBS solution, after which the solution was removed. The cells were dispersed with the addition of 10 μL of trypsin to each well, followed immediately by incubation for 5 min as described above. The cells were prepared for ROS detection by adding 90 μL of 10 μM solution of DCFH-DA or mitosox to each well, after which the cells were incubated for 30 min as described above. The fluorescence intensity of the dye was measured at 485/535 or 510/580 nm using a Thermo Fisher Scientific Fluoroskan Ascent Microplate Fluorometer, Finland.

Cellular uptake of nanoceria particles



•

Confocal microscopy imaging

HT1080 cells were seeded in a single 24-well glass cell culture plate, with each well at a density of 2 × 103 cells/well with 500 μL RPMI-1640 medium adjusted to pH 7.4. For the plate, three wells were used to correspond to each of the three morphologies of nanoparticles. After seeding, the cells were allowed to adhere at 37°C under 5% CO2 in air for 24 h initially in the incubator. To each well was added 500 μL of 100 μg/mL suspension of nanoparticles, which had been tagged with fluorescein-5-isothiocyanate on the surfaces using APTES ([3-aminopropyl] triethoxy silane).21 The plate then was incubated for 5 h as described above. The residual nanoparticles were removed by washing with 500 μL of DPBS three times, each time being followed by removal of the solution. Moisture was retained by a further addition of 500 μL of DPBS. The cells then were imaged by confocal laser scanning microscopy using a Leica SP8 DLS microscope (Wetzlar, Germany). The extent of cellular uptake was determined using Image J image analysis software from the National Institutes of Health, USA. •

Electron microscopy imaging

Cells were seeded in a single-well glass cell culture plate, with each well at a density of 2 × 103 cells/well with 500 μL RPMI-1640 medium adjusted to pH 7.4. After seeding, the cells were allowed to adhere at 37°C with 5% CO2 in air for 24 h initially in the incubator. 500 μL of 100 μg/mL suspension of nanoparticles was added to each well. The plate then was incubated for 5 h as described above. The residual nanoparticles were removed by washing with 500 μL of DPBS three times, each time being followed by removal of the solution. Cells then were fixed in 2.5 % glutaraldehyde for 1 h at room temperature in 0.1 M sodium cacodylate buffer (pH 7.4).

Cells were washed three times in a Pelco BioWave, USA

microwave processor in 1 mL of 0.1 M sodium cacodylate buffer and subsequently postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer in the microwave processor. Cells were washed in 0.1 M cacodylate buffer and twice in deionised H2O prior to serial dehydration with increasing percentages of ethanol (at 30, 50, 70, 80, 90, and twice at 100 vol%) in the microwave processor.

Cells then were infiltrated with increasing

concentrations (at 33, 67, and twice at 100 vol%) of LX112 resin, placed in 30 mm diameter tissue culture dishes, and polymerised at 60oC for 24 h. Flat embedded cells were removed, and ultrathin 60 nm horizontal sections were cut using an ultramicrotome (Leica EM UC6, Leica Microsystems, Germany), placed on Formvar-coated 200 mesh copper grids (Ted Pella


Page 6 of 24

Page 7 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Inc., USA), and imaged using a JEOL TEM-1400 transmission electron microscope (JEOL, Japan) at an accelerating voltage of 120 kV.

Quantification of nanoceria particles within cells The preparation procedure was identical to that of cellular uptake of nanoceria particles except that the nanoparticles were not tagged and nanoparticles incubation timeframes. The cells were dispersed with the addition of 10 μL of trypsin to each well, followed immediately by incubation for 5 min as described above. The cells were digested for 12 h following the addition of 50 μL of concentrated nitric acid to each well at room temperature. The samples then were heated at 90°C for 20 min by placing in an oven in order to ensure complete digestion. Since the volume of each sample had been reduced by evaporation to ~50 μL, 1 mL of aqueous solution of 1 vol% nitric acid was added to each well. Cerium standard solution was used for calibration and quantification of Ce content using a PerkinElmer quadrupole NexION inductively coupled plasma mass spectrometer (ICP-MS, PerkinElmer, USA).

Statistical analysis Graphpad Prism from Graphpad software, USA and Origin Pro 9.0 from OriginLab Cooperation, USA were used for statistical analyses (means and standard deviations) of the triplicate data.

Results and discussion Synthesis and characterisation of nanoceria The three most commonly observed morphologies for ceria nanoparticles can be obtained through appropriate experimental conditions of [NaOH]-temperature,28 and pressuretemperature.

These formalisms have been applied to fabricate surfactant-free ceria

nanocubes (NC), nanorods (NR), and nanooctahedra (NO) of varying sizes as essentially single-morphology assemblages, as shown in Figure 1 (a,d,g).



Figure 1: TEM images, SAED patterns, and XRD spectra of (a-c) nanocubes, (d-f) nanorods, and (g-i) nanooctahedra The morphologies are associated with different degrees of crystallinity, which are demonstrated by the selected area electron diffraction (SAED) patterns of the three morphologies, where the degree of crystallinity was in the order NC (diffraction spots) > NR (sharp rings) > NO (diffuse rings) as presented in Figure 1(b,e,h). These data are confirmed also by the peak widths and line broadening29 shown in the X-ray diffraction (XRD) spectra in Figure 1(c,f,i).

Image J30 processing of TEM images of fifty nanograins for each morphology were used to determine the dimensions of the nanoparticles. These results showed that the NC and NO were of relatively uniform dimensions (NC: ~60 ± 1 nm, NO: ~5 ± 1 nm) while the NR were


Page 8 of 24

Page 9 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of variable aspect ratios (NR: W ~21 ± 8 nm, L ~276 ± 112 nm). Variations in the sizes and aspect ratios can be expected to generate different surface areas and surface chemistries of all three morphologies, which resulted in specific surface areas (SSA) being in the reverse order as that of the degree of crystallinity, NC < NR < NO. The particle sizes may be contrasted with the surface areas, which were determined by measurement (BET)

X-ray photoelectron spectroscopy (XPS) surface analysis produced the same order NC < NR < NO for the surface Ce3+ concentrations ([Ce3+]), which provide a direct measure of the maximal oxygen vacancy concentrations ([V••]Max) of the three morphologies. These data are summarised in Table 1 and the XPS Ce 3d and O 1s spectra are presented in Figure 2(a-f). Table 1 also shows that the same order NC < NR < NO applies to the d spacing and the lattice strains (tensile) of the {100} plane, which is common to all three morphologies, which were calculated using the Scherrer equation31 and high-resolution TEM (images not shown here). As NR and NO also contain other index facets {110} and {111}, then the lattice strains32 in these planes can potentially contribute to the [V••]Max, where NO having higher {111} lattice strain values contained increased oxygen vacancy concentrations.

Figure 2: XPS spectra of nanoceria: (a-c) Ce 3d and (d-f) O 1s regions



Page 10 of 24

Table 1. Nanoceria structural, microstructural and redox performance correlations with [V•• ]Max Redox Effects TEM Morphology

Particle Size (nm)

Stoichiometry

TEM Lattice Strain (Tensile) (%)

Lattice Deformation

(100) 3+

[Ce ] (XPS)

[V•• ]MaxB

d-

Spacing (nm)

NC

60

NR

D

{100} =0.002

(DCF) (%) at pH

SSA (m2/g)

C

XRD Lattice

7.4

Strain {100} Anti(%)

6.4 Pro-

oxidant oxidant

13.6

17.48

8.74

0.273

+0.08

34

29

104.9

18.59

9.30

0.278

+0.24

21

20

198.7

19.67

9.84

0.289

+0.40

12

9

{100} =0.005 21

{110} =0.009 {111} =0.008

NO

5

{100} =0.019 {111} =0.023

A

Calculated on the basis of ideal geometries and planar facets using average dimensions Theoretical calculated stoichiometric value from Ce3+ XPS data on the basis of one oxygen vacancy charge compensating for two Ce4+ → Ce3+ redox reactions C The (100) plane is common to all three morphologies and its d-spacing is expected to be directly proportional to the lattice strain D Width dimension selected on the premise that its vibrational mode requires less energy than that required by the length B

Nanoceria cellular interactions The nanoparticles in vitro pH-dependent cell proliferation and redox behavior for all three morphologies with fibrosarcoma cells are shown in Figure 3.


Page 11 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3: Cell viability and redox performance of nanoceria at pH 7.4 and pH 6.4 as a function of time: (a,b) cell viability, (c,d) redox performance in terms of DCF dye intensity, and (e,f) redox performance in terms of mitosox dye intensity The cell viability assays at both pH 7.4 and pH 6.4 conditions, as shown in Figure 3(a,b), demonstrate that cells exposed to nanoceria were significantly viable under slightly basic



(pH 7.4) conditions while cytotoxic behavior was observed under slightly acidic (pH 6.4) conditions. These data are in agreement with prior work on spherical nanoceria under similar acid-base conditions.19 Further, the data in Figure 3(b) also show the pronounced effect of morphology with increasing exposure time. It can be seen that the cytotoxicity at pH 6.4 is the order NC < NR < NO, which corresponds to that of the data in Table 1: reverse crystallinity, SSA, [Ce3+], [V•• ]Max, d(100), and lattice strain.

To examine the effect of varying pH conditions in more detail, the in vitro redox behavior of nanoceria was determined by using two ROS specific dyes; where DCFH-DA is generally considered to be specific for H2O2 or •OH while mitosox is to detect the mitochondrial •O2–. These dyes generate fluorescent signal upon oxidation by ROS. Figure 3(c-d) shows the antioxidant (pH 7.4) and prooxidant (pH 6.4) performances in terms of dyes intensities where nanoceria particles at physiological pH 7.4 produced an antioxidant effect by scavenging the ROS, which is indicated by a reduction in the dyes intensities. The trends in both morphology and exposure time are consistent with those for cytotoxicity. This is as expected because the ROS produced by cells are scavenged by nanoceria (antioxidant effect), thereby increasing cell viability and associated reduction in dye signal. Conversely, at tumour pH 6.4, the acidic environment facilitates the production of ROS by the nanoceria (prooxidant effect). These data also allowed to compare the specific ROS behavior in terms of dyes intensities where pH 7.4, the scavenging of H2O2 were higher than •O2–. Similarly, at pH 6.4 conditions, the prooxidant activities to produce •OH were higher than •O2–. These effects can be attributed to the fact that lower •O2– detection by mitochondrial specific dye compared to H2O2 and •OH which are generated by different cell organelles and are present in higher amount at cellular levels.

The unique redox behavior of nanoceria, which facilitates Ce3+ ↔ Ce4+ switching, derives from the defect structure of ceria owing to the presence of intrinsic oxygen defects, which not only are responsible for charge compensation but also for initiation of the redox reaction at the surface.33 The data in Figure 3(c-f) and Table 1 show that the dyes intensities for antioxidant behavior (pH 7.4) are higher than those for prooxidant behavior (pH 6.4), which suggests that the former occurs more readily than the latter. This is in agreement with the comment that lower reduction potentials occur at higher pH values.17,18 This


Page 12 of 24

Page 13 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


greater antioxidant effect increases the extent of scavenging of ROS, which yields the two advantages of (1) protection of normal cells and (2) hindrance of proliferation of cancer cells. Since there are pH fluctuations inside cells within the range 4.7-8.034 and Ce3+ ↔ Ce4+ switching occurs readily over the pH range ~4.5-8.532, the reversibility of the switching under essentially all cellular conditions is implicit. In effect, the direction of the Ce3+ ↔ Ce4+ equilibria favour the Ce4+ → Ce3+ reaction but the activation energy for the reverse reaction must be sufficiently low that cellular pH fluctuations are sufficient to overcome it. These effects allow the nanoceria to become available for repeated antioxidant behavior. The data in Figure 3(c-f) and Table 1 again emphasise the importance of morphology, where the redox effects at both pH values are in the reverse of the order exhibited by the other structural and chemical parameters: NC < NR < NO. Since nanoceria has been involved in different signalling pathways11 and it is also reported to scavenge ROS (H2O2) in neutral pH environment where the pH is weak basic (7.4) and it can produce ROS in tumor microenvironment like pH,35 the present work can provide basis to understand the signalling mechanisms; particularly by considering regulation of specific ROS (mitochondrial ROS, •O2–) at different biological pH conditions. Consequently, the phenomenological model for the redox mechanism is given in Figure 4.

This model

represents the switching of Ce3+ ↔ Ce4+, where Ce3+ associated V•• take up the ROS (H2O2) at physiological pH values and converts it to neutral molecules through oxidation-reduction reactions at the surface or near-surface of the nanoparticles. On the other hand, these V•• can take up H2O2 (ROS) and other neutral molecules (H2O2, O2) and convert them to ROS. The V•• defects can be filled and unfilled temporarily under the dynamic Ce3+ → Ce4+ equilibrium conditions required for charge compensation through the oxygen in the ROS (H2O2, •OH, •O2–) as well as the O2- in H2O. Consequently, changes in the pH facilitate reversible antioxidant (scavenging ROS) and prooxidant (producing ROS) effects.



Figure 4: Redox reaction mechanisms for nanoceria at cellular pH values

The role of the [V••]Max is explored in more detail in Figure 5, which plots the relevant data in Table 1 against it. These data establish direct correlations between the [V•• ]Max and the (a) various structural and microstructural parameters and (b) the redox performance. Where the increase in redox effects with [V••]Max provided the concepts to enhance structural defects to get the improved performance. The other factors including particles size, surface area, d-spacing, and lattice strain contributed in the development of V•• defects. The variations in structural, microstructural, and V•• defects data yielded the following order of redox performance of nanoceria: NC < NR < NO. Hence, these data illustrate the critical importance of the [V•• ]Max, which is a function of structural and microstructural parameters, to the redox performance, which is supported by the work of others.36,37


Page 14 of 24

Page 15 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5: Correlations of nanoceria materials parameters and redox performance as a function of [V•• ]Max. Note: The central dotted curve having three (red, green, blue) colors is representing three data points (antioxidant performance = red, lattice strain = green, prooxidant performance = blue). This curve is drawn in such a way because of significantly close values of these three data points. Reports have been published where ‘’naked’’ nanoparticles interacted with cell membranes via membranes lipids and were up-taken in the cells which were confirmed by light and electron microscopy techniques.38

The electrically charged surfaces of nanoceria can

interact with cell outer surfaces and potentially internalized into the cells.39-41 Further, since nanoceria react via scavenging and production of ROS, its presence within the cell is not necessary for redox reaction.

It can generate or scavenge ROS in extracellular

microenvironments where it can induce cell apoptosis by lipid peroxidation or other mechanisms.42 Consequently, present work measured the zeta potential values for all three morphologies which were as follow: NC = – 30, NR = – 26, NO = – 33. These data revealed negative surface charges on nanoparticles where NR possess the least value.

The

mechanism and effects of surfaces charges on the cellular uptake of nanoceria is not clear but NR having least negative charges were high in concentration inside the cells then NC and NO. Further, Figure 6(a,d,g) show confocal optical microscopy images suggesting uptake of nanoceria by fibrosarcoma cells (the green regions are the fluorescein-tagged nanoceria). Since these images cannot confirm uptake or subcellular location, this was done by the electron microscopy images shown in Figure 6(b,c,e,f,h,i), where Figure 6(b,e,h) shows the entire cell and Figure 6(c,f,i) presents higher magnification images of the nanoceria within the endosomes (insets are at higher resolution). The uptake of nanoceria was confirmed quantitatively by the ICP-MS analyses of the Ce in the cells as a function of morphology and



time.

These data are summarised in Figure 6(j) and they reveal that the effect of

morphology on the uptake by fibrosarcoma cells is in the order NO < NC < NR, which is not the same order as observed for redox performance.

Figure 6: (a,d,g) Confocal light microscopy images (green – nanoceria, blue – mitochondria, red – plasma membrane, white/orange – lysosome), (b,c,e,f,h,i) electron microscopy images, (j) ICP-MS quantification of nanoceria within cells ACS Paragon Plus (16) Environment

Page 16 of 24

Page 17 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


It is perceived commonly43-46 that the dominant factors that enhance uptake are high surface areas relative to volume (viz., small sizes and high aspect ratios) and low wetting angles. However, there are many other potential factors that may affect uptake and hence the subsequent redox performance:

Population

Solids loading (number count of nanoparticles in suspension)

Chemistry

Dangling bonds and surface-adsorbed species, surface energies and wetting angles, surface charge

Microstructure Morphology, particle size, aspect ratio, surface area, roughness, topography, surface alteration Structure

Degree of crystallinity, lattice parameter, lattice stress, surface planes, defect types and concentrations

More importantly, although the NR, which have the maximal aspect ratio, exhibited the maximal uptake, the greatest extent of redox effect was observed for the NO, which have a minimal aspect ratio of unity. This demonstrates that the number count of nanoceria taken up by cells is not a dominant factor in the redox effect. Thus, one or more of the factors listed above are likely to provide the dominant effect on the redox behavior. Figure 5 documents that these factors include morphology, particle size, surface area, and structural defects (V••) and dislocations (from lattice strain). Other work has documented that other key parameters are surface roughness, surface energies, hydrated surface layer, degree of crystallinity, and exposed surface planes.33,47,48

Conclusions The present work demonstrates the structure-activity relationship (SAR) of nanoceria in order to identify the optimal redox behavior for potential fibrosarcoma treatment. While several features contribute to the SAR, the pH of the immediate environment was found to be the principal factor in determining nanoceria’s antioxidant (basic pH = 7.4) or prooxidant (acidic pH = 6.4) behavior, which demonstrates ceria’s potential use in physiologically distinct bioenvironments. The correlations between the (a) particle size, (b) SSA, (c) lattice strain, (d) (100) d-spacing, and (e) redox performance demonstrate the dominant role



played by the [V••]Max.

The morphological effect is shown through their consistent

exhibition of the order NC < NR < NO. This key variable provides the basis for the different performances of each morphology, where antioxidant and prooxidant performances are directly proportional to the [V••]Max.

Finally, this approach can be used to engineer

nanoceria of specific morphologies and surface areas in order to optimise the redox effect. The present work reveals that the uptake of nanoceria by fibrosarcoma was in the order NO < NC < NR but that the structural and microstructural correlations were in the order NC < NR < NO (or, in the case of crystallinity, NO < NR < NC). The inconsistency of the uptake data with all of the other data can be attributed largely to the effects of the morphology and its associated features.

Competing financial interests The authors declare no competing financial interests.

Acknowledgements This work is supported by the Australian Research Council (DP170104130). The electron microscopy imaging was conducted at the Electron Microscope, UNSW, Sydney. Authors contribution R.M. designed the project; undertook the syntheses, characterisation, imaging, biological testing, and data analysis; wrote the first draft of the manuscript; and worked on all subsequent drafts. N.A helped in Bio-TEM imaging. P.K., J.L.Y, and C.C.S provided the data analysis support. C.S.S worked on all subsequent drafts of the manuscript, and supervised the overall project.

References (1) Fthenou, E.; Zong, F.; Zafiropoulos, A.; Dobra, K.; Hjerpe, A.; Tzanakakis, G. N. Chondroitin sulfate A regulates fibrosarcoma cell adhesion, motility and migration through JNK and tyrosine kinase signalling pathways. In vivo. 2009, 23, 69–76. PMID: 19368127


Page 18 of 24

Page 19 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(2) Aljabab, A. S.; Nason, R. W.; Kazi, R.; Pathak, K. A. Head and neck soft tissue sarcoma. Indian J Surg Oncol. 2011, 2, 286–290. DOI: 10.1007/s13193-012-0127-5 (3) Xu, C.; Qu, X.

Cerium oxide nanoparticle: a remarkably versatile rare earth

nanomaterial for biological applications.

NPG Asia Mater.

2014, 6, e90.

DOI:

10.2147/IJN.S124855 (4) Celardo, I.; Pedersen, J. Z.; Traversa, E.; Ghibelli, L. Pharmacological potential of cerium

oxide

nanoparticles.

Nanoscale.

2011,

3,

1411–1420.

DOI:

10.1039/c0nr00875c (5) Walkey, C.; Das, S.; Seal, S.; Erlichman J.; Heckman, K.; Ghibelli, L.; Traversa, E.; McGinnis, J.F.; Self, W. T. Catalytic properties and biomedical applications of cerium oxide nanoparticles. Environ. Sci. Nano. 2015, 2, 33–53. DOI: 10.1039/C4EN00138A (6) Sahu, T.; Bisht, S. S.; Das, K. R.; Kerkar S. Nanoceria: Synthesis and biomedical Applications. Current Nanoscience. 2013, 9, 000. DOI: 10.1007/s11837-008-0029-8 (7) Alili, L.; Sack, M.; Von Montfort, C.; Giri, S.; Das, S.; Carroll, K. S.; Zanger, K.; Seal, S.; Brenneisen, P. nanoparticles.

Downregulation of tumor growth and invasion by redox-active Antioxid

Redox

Signal.

2013,

19,

765–778.

DOI:

10.1089/ars.2012.4831 (8) Das, S.; Dowding, J. M.; Klump, K. E.; McGinnis, J. F.; Self, W. T.; Seal, S. Cerium oxide nanoparticles: applications and prospects in nanomedicine. Nanomedicine. 2013, 8, 1483–1508. DOI: 10.2217/nnm.13.133 (9) Karakoti, A.; Singh, S.; Dowding, J. M.; Seal, S.; Self, W. T. Redox-active radical scavenging nanomaterials. Chem. Soc. Rev. 2010, 39, 4422–4432. DOI: 10.1039/b919677n (10) Babu, S.; Cho, J-H.; Dowding, J. M.; Heckert, E.; Komaski, C.; Das, S.; Colon, J.; Baker, C. H.; Bass, M.; Self, T. W.; Seal, S.

Multicolored redox active upconverter cerium

oxidenanoparticle for bio-imaging and therapeutics. Chem. Commun. 2010, 46, 6915– 6917. DOI: 10.1039/C0CC01832E (11) Cimini, A .; D'Angelo, B.; Das, S.; Gentile, R. Benedetti, E.; Singh, V.; Monaco, A. M. Santucci, S.; Seal, S. Antibody-conjugated PEGylated cerium oxide nanoparticles for specific targeting of Aβ aggregates modulate neuronal survival pathways.

Acta

biomaterialia. 2012, 8, 2056–2067. DOI: 10.1016/j.actbio.2012.01.035 (12) Kim, C. K.; Kim, T.; Choi, I. Y.; Soh, M.; Kim, D.; Kim, Y. J.; Jang, H.; Yang, H. S.; Kim, J. Y.; Park, H. K.; Park, S. P.; Park, S.; Yu, T.; Yoon, B. W.; Lee, S. H.; Hyeon, T. Ceria



Page 20 of 24

nanoparticles that can protect against ischemic stroke. Angew. Chem.-Int. Ed. 2012, 51, 11039–11043. DOI: 10.1002/anie.201203780 (13) Chen, J.; Patil, S.; Seal, S.; McGinnis, J. F. Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides.

2006, 1,

Nat. Nanotechnol.

142−150. DOI: 10.1038/nnano.2006.91 (14) Tarnuzzer, R. W.; Colon, J.; Patil, S.; Seal, S. Vacancy engineered ceria nanostructures for protection from radiation-induced cellular damage.

Nano Lett.

2005, 5,

2573−2577. DOI: 10.1021/nl052024f (15) Pagliari, F.; Mandoli, C.; Forte, G.; Magnani, E.; Pagliari, S.; Nardone, G.; Licoccia, S.; Minieri, M.; Di-Nardo, P.; Traversa, E. Cerium oxide nanoparticles protect cardiac progenitor cells from oxidative stress.

ACS Nano.

2012, 6, 3767–3775.

DOI:

10.1021/nn2048069 (16) Giri, S.; Karakoti. A.; Graham, R. P.; Maguire, J. L.; M. Reilly, C. M.; Seal, S.; Rattan, R.; Shridhar, V.

Nanoceria: A rare-earth nanoparticle as a novel antiangiogenic

therapeutic agent in ovarian cancer.

PLoS One.

2013, 8, e54578.

DOI:

10.1371/journal.pone.0054578 (17) Hayes, S. A.; Yu, P.; O’Keefe, T. J.; O’Keefe, M. J.; Stoffer, J. O. The phase stability of cerium species in aqueous systems I. E-pH diagram for the Ce-HCLO4-H2O.

J.

Electrochem. Soc. 2002, 149, C623–C630. DOI: 10.1149/1.1516775 (18) Yu, P.; Hayes, S. A.; T. J.; O’Keefe, O’Keefe, M. J.; Stoffer, J. O. The phase stability of cerium species in aqueous systems II. The Ce(III/IV)-H2O-H2O2/O2 system. Equilibrium considerations and Pourbaix diagram calculations. J. Electrochem. Soc. 2006, 153, C74–C79. DOI: 10.1149/1.2130572 (19) Alpaslan, E.; Yazici, H.; Golshan, N. H.; Ziemer, K. S.; Webster, T. J. pH-Dependent activity of dextran-coated cerium oxide nanoparticles on prohibiting osteosarcoma cell proliferation.

ACS

Biomater.

Sci.

Eng.

2015,

1,

1096–1103.

DOI:

10.1021/acsbiomaterials.5b00194 (20) Alili, L.; Sack, M.; Karakoti, A. S.; Teuber, S.; Puschmann, K.; Hirst, S. M.; Reilly, C. M.; Zanger, K.; Stahl, W.; Das, S.; Seal, S.; Brenneisen, P. Combined cytotoxic and antiinvasive properties of redox-active nanoparticles in tumour-stroma interactions. Biomaterials. 2011, 32, 2918–2929. DOI: 10.1016/j.biomaterials.2010.12.056


Page 21 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(21) Lord, M. S.; Jung, M.; Teoh, W. Y.; Gunawan, C.; Vassie, J. A.; Amal, R.; Whitelock, J. M. Cellular uptake and reactive oxygen species modulation of cerium oxide nanoparticles in human monocyte cell line 937.

Biomaterials.

2012, 33, 7915–7924.

DOI:

10.1016/j.biomaterials.2012.07.024 (22) Asati, A.; Kaittanis, C.; Santra, S.; Perez, J. M. The pH-tunable oxidase-like activity of cerium oxide nanoparticles achieves sensitive fluorigenic detection of cancer biomarkers at neutral pH.

Anal Chem.

2011, 83, 2547–2553.

DOI:

10.1021/ac102826k (23) Grulke, E.; Reed, K.; Beck, M.; Huang, X.; Cormack, A.; Seal, S. Nanoceria: factors affecting its pro- and anti-oxidant properties. Environ. Sci. Nano. 2014, 1, 429–444. DOI: 10.1039/C4EN00105B (24) Kumar, A.; Das, S.; Munusamy, P.; Self, W. T.; Baer, D. R.; Sayle, D. C.; Seal, S. Behavior of nanoceria in biologically-relevant environments. Environ. Sci. Nano. 2014, 1, 516– 532. DOI: 10.1039/C4EN00052H (25) Reyes, G. P.; Palomares, I. R.; Das, S.; Sakthivel, T. S.; Leganes, F.; Rosal, R.; Seal, S.; Fernandez-Pinas, F. Untangling the biological effects of cerium oxide nanoparticles: the role of surface valence states. Sci. Rep. 2015, 5, 15613. DOI: 10.1038/srep15613 (26) Koirala, R.; Pratsinis, S. E.; Baiker, A. opportunities and challenges.

Synthesis of catalytic materials in flames:

Chem. Soc. Rev.

2016, 45, 3053–3068.

DOI:

10.1039/C5CS00011D (27) Zhang, X.; Lin, Y.; Gillies, R. J.; Koirala, R. Tumor pH and its measurements. J Nucl Med. 2010, 51, 1167–1170. DOI: 10.2967/jnumed.109.068981 (28) Sakthivel, T.; Das, S.; Kumar, A.; Reid, D. L.; Gupta, A.; Sayle, D. C.; Seal, S. Morphological phase diagram of biocatalytically active ceria nanostructures as a function of processing variables and their properties. Chem. Plus Chem. 2013, 78, 1446–1455. DOI: 10.1002/cplu.201300302 (29) Shafi, P. M.; Bose, A. C. Impact of crystalline defects and size on X-ray line broadening: a phenomenological approach for tetragonal SnO2 nanocrystals. AIP Advances. 2015, 5, 057137. DOI. 10.1063/1.4921452 (30) Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; Tinevez, J. Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.; Tomancak, P.; Cardona, A. Fiji: an open-source platform for



biological-image

analysis.

Nat.

Methods.

2012,

Page 22 of 24

9,

676–682.

DOI:

10.1038/nmeth.2019 (31) Patterson, A. L. The Scherrer formula for X-Ray particle size determination. Phys. Rev. 1939, 56, 978–982. DOI. 10.1103/PhysRev.56.978 (32) Desppande, S.; Patil, S. S.; Kuchibhatla, V. N. T. S.; Seal, S. Size dependency variation in lattice parameter and valency states in nanocrystalline cerium oxide. Appl. Phys. Lett. 2005, 87, 133113. DOI. 10.1063/1.2061873 (33) Channei, D.; Phanichphant, S.; Nakaruk, A.; Mofarah, S. S.; Koshy P.; Sorrell, C. C. Aqueous and surface chemistries of photocatalytic Fe-doped CeO2 nanoparticles. Catalysts. 2017, 7, 45. DOI: 10.3390/catal7020045 (34) Casey, J. R.; Grinstein, S.; Orlowski, J. Sensors and regulators of intracellular pH. Nat Rev Mol Cell Biol. 2010, 11, 50–61. DOI: 10.1038/nrm2820 (35) Wason, M. S.; Colon, J.; Das, S.; Seal, S.; Turkson, J.; Zhao, J.; Baker, C. H. Sensitization of pancreatic cancer cells to radiation by cerium oxide nanoparticle-induced ROS production. Nanomedicine. 2013, 9, 558–569. DOI: 10.1016/j.nano.2012.10.010 (36) Yang, Y.; Mao, Z.; Huang, W.; Liu, L.; Li, J.; Li, J.; Wu, Q. Redox enzyme-mimicking activities of CeO2 nanostructures: Intrinsic influence of exposed facets. Sci. Rep. 2016, 6, 35344. DOI: 10.1038/srep35344 (37) Deml, A. M.; Holder, A. M.; O’Hayre, R. P.; Musgrave, C. B.; Stevanovic, V. Intrinsic material properties dictating oxygen vacancy formation energetics in metal oxides. J. Phys. Chem. Lett. 2015, 6, 1948–1953. DOI: 10.1021/acs.jpclett.5b00710 (38) Bhattacharya, R.; Mukherjee, P. Biological properties of “naked” metal nanoparticles Adv Drug Deliv Rev. 2008, 60, 1289–306. DOI: 10.1016/j.addr.2008.03.013 (39) Singh, S.; Kumar, A.; Karakoti, A.; Seal, S.; Self, W. T. Unveiling the mechanism of uptake and sub-cellular distribution of cerium oxide nanoparticles. Mol. BioSystems. 2010, 6, 1813–1820. DOI: 10.1039/c0mb00014k (40) Vincent, A.; Babu, S.; Heckert, E.; Dowding, J.; Hirst, S. M.; Inerbaev, T. M.; Self, W. T.; Reilly, C. M.; Masunov, A. E.; Rehman, T. S.; Seal, S. Protonated nanoparticle surface governing ligand tethering and cellular targeting. ACS Nano. 2009, 3, 1203−1211. DOI: 10.1021/nn9000148


Page 23 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(41) Gupta, A.; Das, S.; Neal, C. J.; Seal, S. Controlling the surface chemistry of cerium oxide nanoparticles for biological applications. J. Mater. Chem. B. 2016, 4, 3195−3202. DOI:10.1039/C6TB00396F (42) Lin, W.; Huang, Y. W.; Zhou, X. D.; Ma, Y. Toxicity of cerium oxide nanoparticles in human lung cancer cells.

2006, 25, 451-457.

Int J Toxicol.,

DOI:

10.1080/10915810600959543 (43) Stylianopoulos, T.; Jain, R. K. Design considerations for nanotherapeutics in oncology. Nanomedicine. 2015, 11, 1893–1907. DOI: 10.1016/j.nano.2015.07.015 (44) Toy, R.; Peiris, P. M., Ghaghada, K. B., Karathanasis, E. Shaping cancer nanomedicine: The effect of particle shape on the in vivo journey of nanoparticles. Nanomedicine. 2014, 9, 121–134. DOI: 10.2217/nnm.13.191 (45) Geng, Y.; Dalhaimer, P.; Cai, S.; Tsai, R.; Tewari, M.; Minko, T.; Discher, D. E. Shape effects of filaments versus spherical particles in flow and drug delivery. Nat Nano. 2007, 2, 249–255. DOI: 10.1038/nnano.2007.70 (46) Moore, M. C.; Guimard, N.; Shi, L.; Roy, K. Designer nanoparticles: Incorporating size, shape, and triggered release into nanoscale drug carriers. Expert Opin Drug Deliv. 2010, 7, 479–495. DOI: 10.1517/17425240903579971 (47) Liu, Z.; Li, X.; Mayyas, M.; Koshy, P.; Hart, J.N.; Sorrell, C.C. Growth mechanism of ceria nanorods by precipitation at room temperature and morphology-dependent photocatalytic performance.

CrystEngComm.

2017, 19, 4766–4776.

DOI:

10.1039/C7CE00922D (48) Chen, W. F.; Koshy, P.; Adler, L.; Sorrell, C.C. Photocatalytic activity of V-doped TiO2 thin films for the degradation of methylene blue and rhodamine B dye solutions. J Aust. Ceram. Soc. 2017, 53, 569–576. DOI. 10.1007/s41779-017-0068-0



“For Table of Contents Use Only pH-Responsive Morphology-Controlled Redox Behavior and Cellular Uptake of Nanoceria in Fibrosarcoma Rashid Mehmood, Pramod Koshy, Nicholas Ariotti, Jia Lin Yang, Charles C. Sorrell

ACS Paragon Plus Environment

Page 24 of 24

pH-Responsive Morphology-Controlled Redox ... - ACS Publications

Recommend Documents