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Synthesis of Biocompatible Cellulose-Coated Nanoceria with pH-Dependent Antioxidant Property MubarakAli Davoodbasha, Kandasamy Saravanakumar, Akbarsha Mohammad Abdulkader, Sang-Yul Lee, and Jung-Wan Kim ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00647 • Publication Date (Web): 19 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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ACS Applied Bio Materials

Graphics for Manuscript Fig. 1

Fig. 1 Synthesis and physical characterization of C/nanoceria by SPP: (a) UV-Vis spectroscopy of C/nanoceria showed a peak at 304 nm due to plasmon resonance of CeO2 ; (b) FTIR spectra of C1Ce0 and C1Ce5; (c) EDS and (d) XPS analysis

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Fig.2

Fig. 2 Structure of the 3D scaffold-type biocomposites: FESEM analysis showed that the micro-porous structure of C1Ce0 (a) and C1Ce5 (b) had oval-shaped pores with spongy appearance; C1Ce5showed pores with micro-fibrils at the edges; (c) EDS mapping showing distribution of ceria in the composite (d) . Scale bar: 10 µm.


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Fig. 3 TEM analysis of nanoceria in the biocomposites. The size and shape of the nanoceria in the biocomposites were observed. a) The C1Ce5 biocomposite had slightly agglomerated particles with the size ranging 3.2-32.4 nm and had an average cubical structures of 14.10±8.8nm; b) Schematic representation of fig 3a. (c) HRTEM image of cubical structure of nanoceria with distinct lattice fringes; (d) Particle size distribution of C1Ce5 (d).



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Fig. 4 pH-depended antioxidant activity of C1Ce5: (a) DPPH radical scavenging activity; (b) Hydroxyl radical scavenging activity; (c) Superoxide radical scavenging activity, (d) Hydrogen peroxide radical scavenging activity (values are mean ± SE of measurements in triplicate).


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Fig. 5 Cell viability assay (WST) of HeLa cell exposed to C/nanoceria at different concentrations (0-1600 μg.mL-1) for 24 hr showed no toxicity (a) and 48 hr exposure produced slight anti proliferative effect at non toxic level (b)



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Fig. 6 In vitro antioxidant property of C/nanoceria on HeLa cells: (a, d) Normal contral ; (b) cells treated with STS (1 μM) showing ROS production; and (e) damaged nucleus ; (c, f) cells treated with C/nanoceria (1000 μg.mL-1) showing no reduction of cells and no nuclear damage


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Synthesis of Biocompatible Cellulose-Coated Nanoceria with pH-Dependent Antioxidant Property MubarakAli Davoodbasha1,2,3*,Kandasamy Saravanakumar4, Akbarsha Mohammad Abdulkader5, Sang-Yul Lee,3*, Jung-Wan Kim2,3 * 1School

of Life Sciences, BSA Crescent Institute of Science and Technology, Chennai - 600048,

Tamil Nadu, India 2Division

of Bioengineering, College of Life Sciences and Bioengineering, Incheon National

University, Incheon - 22012, Republic of Korea 3Centre

for Surface Technology and Applications, Department of Materials Engineering, Korea

Aerospace University, Goyang- 10540, Republic of Korea 4Department

of Medical Biotechnology, Kangwon National University, Chuncheon, Republic of

Korea 5MGDC,

Bharathidasan University, Tiruchirappalli 620024 &Research Division, National College

(Autonomous), Tiruchirappalli – 620001, Tamil Nadu, India

*Corresponding authors: MubarakAli, Davoodbasha. Ph.D. ([email protected]) 1School

of Life Sciences, BSA Crescent Institute of Science and Technology, Chennai – 600048,

Tamil Nadu, India Jung-Wan, Kim. Ph.D. ([email protected]) Division of Bioengineering, Incheon National University 110, Yeonsu-gu, Republic of Korea Sang-Yul Lee, Ph.D. ([email protected]) Centre for Surface Technology and Applications, Department of Materials Engineering, Korea Aerospace University, Goyang 10540, Republic of Korea

1 ACS Paragon Plus Environment


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Abstract: Recent developments in nanomedicine have validated nanoceria as an antioxidant of therapeutic potential. However, its clinical application is far too limited in view of its poor stability in vivo and the use of hazardous solvents during its synthesis. There is pertinent need for development of improved strategies so as for nanoceria to work better, especially by complexation with a matrix so as to improve upon its antioxidant property without toxicity. In the present study cellulose has been used as a matrix of nanobiocomposite in which nanoceria are embedded, adopting solution plasma process (SPP). This resulted in cellulose-nanoceria(C/nanoceria) biocomposite by plasma reactions for 15 min using cellulose powder and Ce(NO3)2. Three-dimensional scaffold of the C/nanoceria biocomposite was prepared by lyophilization. The biocomposite was characterized adopting UV-Vis spectroscopy, FTIR, FESEM equipped with EDS, and HRTEM analysis. The cubical nanoceria, in the size range 3.2-32 nm, were successfully internalized in the cellulose nanomatrix without agglomeration and exhibited excellent antioxidant property in pH-dependent manner. The nanobiocomposite is not cytotoxic to HeLa cell at a concentration as high as >1 mg.mL-1as revealed in the cytotoxicity assay. Thus, we describe for the first time synthesis of C/nanoceria, in a manner green and sustainable, which has potential in external clinical application as an effective anti-oxidative green material for scavenging reactive oxygen species. Keywords: Solution plasma process; cellulose; nanoceria; nanobiocomposite; antioxidant property


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1. Introduction: Advanced nano-engineering techniques of enabled synthesis of nanomaterials and their utilization for human welfare are subjects of great interest. In recent times nanomedicine is becoming a crucial area of biomedical research, particularly as a tool for preventing neuro-degenerative diseases1,2. In this context, cerium (Ce) has attracted attention in view of its inter-changeable electronic structure. The energy at the inner 4f level of CeO2 is same as that in the outer electrons. Further, little energy is required to change the electronic level. It has been reported that insertion of O2 brings about reduction in size and increase of surface area, that renders the particle a highly effective antioxidant3,4. The unique chemical and physical characteristics make nanoceria useful in industrial applications such as optics5, catalysis6, sensors7, sunscreens and coatings8. Among the several properties, nanoceria are an outstanding biomaterial that combats oxidative stress (OS) in the cell. The reactive oxygen species generated in excess of what could be dealt with by the scavengers of ROS trigger the OS9. Prolonged oxidative stress leads to cellular damage and eventually neurodegenerative diseases. Nanoceria is an alternative free radical scavenger in view of its size tunability, biocompatibility, and membrane permeability10,11. Especially, nanoceria with Ce3+and Ce4+take to catalase-mimetic and superoxide dismutase activities in the cell10-12. Currently, the biomedical applications of nanoceria are being investigated because of its unique properties such as scavenging activity13, neuro14 and cancer therapy15, and imaging16. Till date, only the highly focused scavenging activity of nanoceria has been investigated without relevance to the complexity of the cell systems. However, cytotoxicity and genotoxicity of nanoceria have been investigated using different cell lines in which toxicity of nanoceria was shown to be different between cells (HepG2, A549 and CaCo2) and exposure times (1- 10 days); especially, it has been shown that nanoceria is capable of protecting cells against H2O2 insult17. Nanoceria is synthesized adopting either physical methods such as hydrothermal18 and microwave13 or chemical methods such as surface functionalization, stabilization19 and microemulsion20. These methods often require the use of toxic chemicals, costly equipment and multiple steps. Very often the additives, detergents, and/or chemicals used in the synthesis may not be completely removed21. Very recently, polymer (chemical and bio-) coated nanoceria have been reported for their industrial and medical applications as summarized in Table 122-36. To overcome these limitations, SPP is employed to generate nanomaterials in one-step and without additives. The methodology of SPP has been described37,38,39,40. 3 ACS Paragon Plus Environment


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The present study was aimed at synthesizing C/nanoceria adopting SPP with no additive chemicals. The physico-chemical reaction conditions were optimized for enhanced preparation of the biocomposite. The synthesised C/nanoceria biocomposite was assessed by XPS, FESEM with EDS, HRTEM, FTIR, and UV-Vis spectroscopy. The C/nanoceria was investigated adopting various antioxidant assays including DPPH, HO, H2O2, and SOD. Further, the cytotoxicity of the C/nanoceria was tested in human cervical cancer cell (HeLa) by WST assay. This is the first report of utilization of cellulose as matrix for synthesis of nanoceria with definite cubical structure by SPP for biomedical applications. The structure and pH of the nanoceria thus synthesized were dependent on its biological function as antioxidant. 2. Materials and methods: 2.1. Chemicals and cell line Cellulose powder was obtained from Samsung Fine Chemicals, Korea, and Ce(NO3)2was supply from Sigma-Aldrich, USA. All other chemicals and reagents used were of the best quality. De-ionized water was used to prepare the reagents. HeLa cell was used for the cytotoxicity and ROS assays. 2.2. C/nanoceria biocomposite synthesis Briefly, 300 mL of water containing1or 5 mM Ce(NO3)2and 1% cellulose were mixed in a beaker for SPP under conditions as described previously41. A schematic representation of synthesis of C/nanoceria is presented in Supplementary file as Scheme S1. 2.3. Characterization of C/nanoceria Generation of nanoceria in the matrix was confirmed using Shimadzu (Japan), UV-Vis spectrophotometer (wavelength range, 200 to 1200 nm). The structure of the biocomposite in 3D scaffold was determined in a field emission scanning electron microscope (FE-SEMJEOL-JSM7001F, Japan). The oxidative state was studied by X-ray photospectroscopy (VersaProbe II, PHI, Japan). The biocomposite’s functional associations were deduced using an FTIR Fourier transform infrared spectroscope (Vertex 8VBruker, Germany) in the 400 to 4,000 cm-1 range. The nature and size distribution of nanoceria in the matrix were observed in a high-resolution transmission electron microscope (JSM-2010, JOEL, Japan) at 200 keV. 2.4. Antioxidant properties of C/nanoceria 2.4.1. Assay of scavenging of DPPH The antioxidant activity of C/nanoceria was assessed by DPPH assay42,43. Different concentrations of the C/nanoceria were used to react with the DPPH radical. The scavenging activity (%) was determined as described. 4 ACS Paragon Plus Environment

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2.4.2. Assay of scavenging of hydroxyl radical Deoxyribose method was adopted to assay the hydroxyl radical scavenging44,

45.

The

reaction mixture consisted of 2-deoxy-2-ribose (2.8 mM), phosphate buffer (0.1 mM; pH 7.4), ferric chloride (20 μM), EDTA (100 μM), hydrogen peroxide (500 μM), ascorbic acid (100 μM) and nanoceria at increasing concentrations (range: 0-1000 μg.mL-1).The hydroxyl radical scavenging activity (%) was calculated based on already available methods. 2.4.3. Assay of superoxide radical scavenging The superoxide radical was generated adopting the photoreductionmethod46,47. It was detected by photoreduction of riboflavin by NBT. The reaction mixture consisted of EDTA (0.1 M), sodium cyanide (0.0015%), riboflavin (0.12 mM), NBT (1.5 mM)and different concentrations of nanoceria in phosphate buffer (67 mM; pH 7.8),all in 3 mL volume. The mixture was exposed to white light for 10-20 min and the absorbance was recorded before and after exposure to light at 530 nm. All experiments were conducted at sample pH 6, 7 or 8.The percentage of superoxide radical scavenging was determined. 2.4.4. Assay of hydrogen peroxide radical scavenging The hydrogen peroxide scavenging was assayed with H2O2 (2 mM) in phosphate buffer (50 mM; pH 7.4). Briefly, 100 μL of nanoceria of different pH were made up to 400μL using phosphate buffer (50 mM; pH 7.4) and H2O2 solution (600 μL). The contents were mixed, incubated for 10-15 min and the absorbance was read at 230 nm against reagent blank. The percentage of H2O2 scavenging was calculated48. 2.5. Cytotoxic and antioxidant activities HeLa cells were used as the in vitro model. WST-1 assay was adopted to find the effect of C/nanoceria49. The cells (5 x104cells.cm2) were seeded in 96 and 24 well plates containing DMEM incorporated with 10% FBS and antibiotics. After 24 hr, the cells were exposed to C/nanoceria (01600 μg.mL-1) for 24 and 48 hr. Finally, the cell viability was determined using the EZ-Cytox reagent, as prescribed in the manufacturer’s instructions. Trypan blue dye exclusion assay was applied to count the viable cells using a haemocytometer and an inverted microscope (TMS-F, Nikon, Japan). For observation of the change in cellular morphology in the context of ROS production, the cells (5 x 104.cm2) in chamber slides were treated with STS (1 μM). The cells were washed with cold PBS, stained with Hoechst 33342 and observed in a fluorescent microscope (Carl Zeiss, Germany).



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3. Results and discussion 3.1. Synthesis of C/nanoceria Solution plasma generates reactive oxygen species, UV and visible light in the solution containing cerium salt and cellulose under continuous stirring in ambient conditions. During this process, cellulose gets coated on the surface of the nanoceria and prevents nucleation resulting in cellulose-coated nanoceria (C/nanoceria). The initial pH (7.0), temperature (28°C) and pale colour of the solution were recorded. Once the plasma was discharged, the solution turned cloudy and became pale yellow in course of time, when the pH was decreased from 6 to 3 while the temperature was increased to 90 °C (data not shown). SPP is an eco-friendly process. It can be used for synthesis of nanoparticles. These nanoparticles have unique properties that enable their application in various medical devises. The SSP-based C/nanoceria does not aggregate or settle down as pellet when subjected to centrifugation at 10000 rpm. This approach of synthesis is advantageous over one which is solvent-based50. 3.2. Spectroscopic investigation of C/nanoceria Results from UV-Vis spectroscopy confirmed the successful fabrication of nanoceria (containing Ce(NO3)2and cellulose). Various concentration of Ce(NO3)2wereused for the synthesis of the C/nanoceria. UV-spectra results indicated that 5mM of Ce(NO3)2was the optimal concentration for nanoceria synthesis which was evidenced by the high absorption peak at 300310nm (Fig 1a). The peak of absorbance for 1mMnanoceria was not prominent because of the less Ce(NO3)2 concentration due to which it embedded deeply in cellulose (data not shown).The absorbance peak increased with increase of reaction time. The high absorption peak at 304 nm revealed the successful synthesis of nanoceria. This is in accordance with previous report of nanoceria synthesis by solution-combustion method51.Although several reports support the present finding of successful synthesis of nanoceria by observing the high absorption peak at 304 nm, a few studies proved the synthesis of nanoceria by observing the absorption peak at 360 nm because of charge transfer in the Ce4+22. UV-Vis analysis of the bimetallic nanoparticles of nanoceria and gold nanoparticles showed the absorption peak at557 nm due to the localized SPR (surface Plasmon resonance) of the particles52.The size of the particles is increased with decrease of the absorption peak which indicates that the particle size has correlation with intensity of the absorption peak 53. The resultant C/nanoceria was characterized by FTIR spectroscopy (Fig 1b). The spectra of C/nanoceria were wide, and the presence or absence of peaks was dependent on the cellulose molecules interacting with Ce3+ or Ce4+. The vibrations at 3419 cm-1 (OH bending), and 2932cm-1 (CH2 bending) had enabled the formation of CeO2-cellulose composites. Furthermore, vibrations 6 ACS Paragon Plus Environment

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at1646 cm-1(C=H) and 728 cm-1 (CH3) were present in the HPMC. There were vibrations at 2361 cm-1 and 2116 cm-1 which might be corresponding to the formation of C/nanoceria matrix but not cellulose, which would representCH2stretch and CH stretch, respectively. The peaks such as O-H, C-H, C-O stretching, C-H bending, and C-H wagging seen in the range 900-3500 cm146werecorresponding

to cellulose. Another IR peak in 3400cm-1(O-H stretching) indicates the

hydroxyl group of polymers. When dextran was used in the place of cellulose, there were vibrations at 800-1300cm-1range which indicated that nanoceria were coated with dextran54,9. The EDS results of C/nanoceria showed the presence of O (K-series) and Ce (L-series) (Fig 1c).There was no impurity indicated in the EDS spectrum, which emphasizes the ultra-purity of the biocomposite, and suggests that it would be useful for therapeutic purposes. The XPS analysis (Fig 1d) revealed the prevalence of nanoceria as Ce3+ and Ce4+ in C/nanoceria matrix, which was evidenced by observation of the biding energies 880,888, 898, 905, and 918 eV. This spectrum proves that the cellulose matrix does not interfere with the bivalent state of nanoceria. It is known that the mixed valence states offer vital roles to the nanoceria in the context of anti-oxidant activity by Ce3+ and Ce4+ on the surface of the particles55. Previous studies have suggested that Ce3+at high concentration would readily react with SOD as excellent free radical scavengers which would be dependent on the valance of nanoceria56,57. The treatment of the Ar ions trigger the oxygen vaccines to archive the high concentration of the Ce3+, which convert Ce4+intoCe3+[4]. In an earlier study, the concentration of Ce3+has been determined theoretically based on size of the nanoceria, which indicated that 30.4% and 20.9% of the concentration of the Ce3+were corresponding to the size of nanoceria at 5-10nm and 15-20nm, respectively58. These results, which indicated the copious smaller sized Ce3+particles on the surface, are in accordance with the existing literature57, and in this state the nanoceria particles are enhanced in free radical scavenging activity. 3.3. Characterization of C/nanoceria Microporous scaffold of the C/nanoceria was generated through the UV radiation-based cross linking followed by adopting freeze-drying (Fig 2).The FE-SEM was utilized to visualize the microporous nature of cellulose and C/nanoceria, in which there was a slight difference between the two, i.e., oval-shaped microporous nature of cellulose and round and spongy appearance of C/nanoceria. The diameter of the pores ranged from 6 to 47 μm (Fig 2a-c). The biocomposite showed micro fibril-like structures at the edges of C/nanoceria. In our earlier studies, cellulose/AgNPs biocomposite appeared to be similar and micro-fibril structures are important features of cellulose41. The micro-fibril structures would be useful as scaffold for 3D cell culture in 7 ACS Paragon Plus Environment


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tissue engineering applications59 and for development of artificial tissues and skin60. EDS mapping study revealed distribution of Ce (blue), O (green) and carbon (red) in the selected areas of nanoceria (Fig 2d).The C signal also appeared as was expected due to the carbon tape used61. The morphological characteristics of nanoceria in the cellulose matrix were examined by TEM and the data is shown in Figure 3. Cubical structures ranging in size from 3.2-32.4 nm, without agglomeration, were observed. The average size of individually countable particles was determined as 14.1±8.8 nm (Fig 3a). The nanoceria were embedded in the matrix to overcome particle nucleation (Oswald ripening) and became stabilized. The nanoceria maintained the cubical structure throughout the matrix, which was decorated schematically (Fig 3b).It is known that the nanomaterials can be stabilized by polymer capping40. In addition, the HR-TEM micrographs exhibited distinct lattice fringes (1nm gap) of nanoceria (Fig 3c).The surface area of the rhombohedral-shaped nanoceria is larger than the spherical-shaped nanoceria, and the shape, design, distribution and size play an incredible role in the catalysis process of nanoparticles54,62. Generally the smaller nanoparticles are endowed with higher density of oxygen vacancies and solubility than particles of larger size58. The size distribution of the nanoceria in the cellulose matrix is shown (Fig. 3d). Furthermore, the TEM analysis also enabled measurement of size distribution of the nanoceria, which ranged from 3 to 32nm, and its shape remained unchanged. Nanoceria of similar size (25 nm) has been synthesised by precipitation method

61which

had a

cluster appearance with average size of 17 to 22 nm17; microwave method resulted in agglomeration in the size range 15 to 20 nm13; and polyhedral-shaped nanoceria with 8-14 nm particles was generated by hydrothermal method16. 3.4. Antioxidant potential of C/nanoceria Data in Figure 4a show the scavenging effects at various concentrations and different pH of C/nanoceria on DPPH radical. The nanoceria-induced DPPH scavenging was observed from the colour changes after the 6 hr of incubation. All other protocols for incubation for 1- 2 hr are described. In this experiment, 6 hr incubation was found optimum to the end point (data not shown). The scavenging of the DPPH was significantly increased with the increase of the nanoceria concentrations; it was 54.12, 40.38 and 37.8% at pH 8, 7 and 6, respectively, at the maximum nanoceria concentration of 500 μg.mL-1. This was found to be slower than the scavenging activity of the natural antioxidant ascorbic acid which was 87.15%. However, it is important to note that metallic composites are not comparable with natural antioxidants. Principally, the antioxidants donate the hydrogen to the free radicals (DPPH, SOD, H2O2) resulting the non-oxidant species 63. Nanoceria showed dose-dependent hydroxyl radical scavenging (Fig 4b).The results 8 ACS Paragon Plus Environment

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indicated the highest pH-dependent hydroxyl radical scavenging activity at 60μg.mL-1 concentration of nanoceria while other concentrations did not show any difference in scavenging activity with variation of pH (6-8). It is evidenced that 60μg.mL-1 of nanoceria scavenged hydroxyl radicals to the extent of 76.2 % at pH 6 and 78.5% at pH 7. An earlier work reported the highest hydroxyl radical scavenging of 60% at 1 μM concentration of nanoceria, when compared with the control with which the inhibition was only 51%57. The hydroxyl radicals trigger oxidative damage in nucleus, DNA, proteins and lipids 64. The superoxide radical scavenging effect of nanoceria ranged from 25 to 500 μg.mL-1 (Fig 4c). At 200μg.mL-1concentration, the scavenging activity was 90% at pH 7 and the highest activity of 96%was found at pH 8. However, the activity decreased with increase of concentration in the range 300-500 μg.mL-1. The superoxide radicals are potentially the most deleterious among the harmful sources of ROS65. Although superoxide is a weak oxidant compared to singlet oxygen and hydroxyl radicals which trigger oxidative stress through conversion of the harmful ROS including hydroxyl radicals and H2O2, other oxidants that damage bio-molecules would result in chronic diseases66,67. Thus, the potential superoxide scavenging properties of nanoceria fulfils the requirement for therapeutic application towards treatment of oxidative stress-related diseases. Our study is significant since nanoceria prepared by the microwave techniques did not show the scavenging of superoxide which might be due to the occurrence of Ce4+in the preparation13. The H2O2 scavenging effect of nanoceria at different pH is shown (Fig 4d). The 100μg.mL-1 of nanoceria showed the highest H2O2 scavenging activity (94%) at pH 6 and 7, whereas at pH 8 the same concentration of nanoceria produced only 88% H2O2 scavenging activity. The scavenging activity of nanoceria with respect to H2O2was much powerful compared to the other free radicals. To-date there has been no report of H2O2 scavenging activity of nanoceria. It is worth mentioning here that nanoceria prepared by SPP showed excellent activity at the lowest concentration tested (25 μg.mL-1). On the whole, nanoceria prepared by SPP showed highly potent antioxidant activity against DPPH, hydroxyl, superoxide and H2O2 radicals compared to those prepared by other process so far reported13, 52, 57,58. 3.5. Cytotoxic and antioxidant potentials of C/nanoceria The cytotoxicity of the nanoceria was assessed by using the WST assay and the results are shown in Fig 5. Exposure of HeLa cells to nanoceria at concentrations from 200 to 1600 μg.mL-1 for 24 and 48 hr did not affect to any significant level the viability of the cells. The cells showed normal growth (Fig 5a). With doubling of the exposure time to 48hr, a slight anti-proliferative 9 ACS Paragon Plus Environment


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activity was indicated but the values were not significant (Fig 5b). Inability of the nanoceria to inhibit growth of tumour cells has been previously reported68. Thus, our study has proved that the C/nanoceria that we produced is non-toxic even at a very high concentration. On the other hand there are also reports of cytotoxic property of nanoceria. Nanoceria are significantly cytotoxic to human prostate cancer cell (PC2), lung cancer cell (A549), and HepG2 but not cytotoxic to normal cell (L-929); Caco2 was suffered little nanoceria toxicity17, 69. These contradictory results indicate that the toxic effect or safety may depend on the methods of synthesis of the nanoceria. Essentially, the nanoceria synthesized adopting SPP did not affect viability of the cells, i.e., is not cytotoxic. The intracellular antioxidant activity of the nanoceria was evaluated by treating HeLa cells with1000 µg.mL of nanoceria for 24-48 hr. DCFH-DA staining enabled to measure the level of ROS generation by adopting fluorescent microscopy. It was revealed that the treatment of the nanoceria to HeLa cells for 48 hr was not toxic, and increased the growth through reducing the oxidative stress particularly ROS generation (Fig 6c-f). Whereas, the treatment of STS to HeLa cells for 24 hr produced significant cytotoxicity through triggering the oxidative stress including ROS (Fig 6b, e).The untreated cells showed normal growth, without any cellular damage (Fig 6a, d). Furthermore, the antioxidant potential of C/nanoceria has garnered significant applications such as in treatment of neurodegenerative diseases. The nanoceria prepared by the precipitation method produced high cytotoxicity and cellular damage in HepG2 and A549 cells at a very lowconcentration15. But in the human lens epithelial cells treated with nanoceria (5 or 10µg.mL) did not suffer any DNA damage70. Thus, the cytotoxicity of the nanoceria is conflicting with respect to the method of its preparation and the cell types. In a previous work, the impact of nanoceria of various sizes (7, 14 and 94 nm) on U937 cells was investigated at concentrations of 5 and 200 mg.mL-1 for 144 hr when it was found that cell proliferation was reduced within 24 h, compared to cells exposed to normal growth conditions54. The protective effect was dose-dependent, and there was 1.5-fold improvement of metabolic activity over superoxide-treated controlcells9. Dextran-coated nanoceria did not show cytotoxic effect at 1mM concentration but showed pH-dependent activity of inhibiting bone cancer cells and not normal osteoblast cells33,56. It has been proposed that the effect of valence state of nanoceria on cell proliferation was due to the wettability and the surface charges4.The C/nanoceria was synthesized by SPP method (cubical morphology with average of

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