Cerium oxide nanoparticles: A promising tool for the treatment of fibrosarcoma in-vivo

Journal Pre-proof Cerium oxide nanoparticles: A promising tool for the treatment of fibrosarcoma in-vivo

Esmail Nourmohammadi, Hoda Khoshdel-sarkarizi, Nedaeinia, Majid Darroudi, Reza Kazemi Oskuee

Reza

PII:

S0928-4931(19)30433-3

DOI:

https://doi.org/10.1016/j.msec.2019.110533

Reference:

MSC 110533

To appear in:

Materials Science & Engineering C

Received date:

2 February 2019

Revised date:

3 December 2019

Accepted date:

5 December 2019

Please cite this article as: E. Nourmohammadi, H. Khoshdel-sarkarizi, R. Nedaeinia, et al., Cerium oxide nanoparticles: A promising tool for the treatment of fibrosarcoma in-vivo, Materials Science & Engineering C (2019), https://doi.org/10.1016/j.msec.2019.110533

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© 2019 Published by Elsevier.

Journal Pre-proof Cerium oxide nanoparticles: A promising tool for the treatment of fibrosarcoma in-vivo Esmail Nourmohammadia, Hoda Khoshdel-sarkarizib, Reza Nedaeiniac, Majid Darroudid*, Reza Kazemi Oskueee* a

Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad

University of Medical Sciences, Mashhad, Iran. b

Department of Anatomical Sciences and Cell Biology, Faculty of Medicine, Mashhad

Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan

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University of Medical Sciences, Mashhad, Iran.

University of Medical Sciences, Isfahan, Iran.

Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad,

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d

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Iran. e

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Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad

University of Medical Sciences, Mashhad, Iran.

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*Correspondence to: Reza Kazemi Oskuee, Pharm.D., Ph.D.

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Department of Medical Biotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, P.O. Box: 91775-1159, Mashhad, Iran. Tel: +98513800291, Fax: +985138002287, E-mail: [email protected] Majid Darroudi, PhD.

Nuclear Medicine Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, Tel.: +98 5118002286; fax: +98 5118002287. E-mail addresses [email protected], [email protected].

Running title: Cerium oxide nanoparticles for treatment of fibrosarcoma

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Journal Pre-proof ABSTRACT In this study, we used cerium oxide nanoparticles and evaluated their anti-cancer effects in a mouse model of fibrosarcoma. For evaluation of anti-cancer effects of nanoceria, tumor volume measurement, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, quantitative real-time PCR (qPCR) for Bax and Bcl2 genes, a panel of liver and kidney function tests and hematoxylin-eosin staining were done. Nanoceria dominantly accumulated in the tumor and it could significantly decrease tumor growth and volume in tumor-bearing mice that received nanoceria for four weeks. Cerium oxide nanoparticle

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showed potential anti-cancer properties against fibrosarcoma.

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Keywords: Cerium oxide nanoparticles, Fibrosarcoma, TUNEL, Bax, Bcl2

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Journal Pre-proof 1. Introduction In 2015, more than 90 million people had cancer, 14.1 million were diagnosed as new cases and about 8.8 million died due to cancer [1, 2]. It was estimated that in 2018, 1,735,350 new cancer cases and 609,640 deaths due to cancer occur only in the United States [3]. Fibrosarcoma is a rare, but highly malignant tumor that is derived from mesenchymal cells; this tumor shows low sensitivity towards chemo- and radiotherapy and high rates of recurrence [4]. Ineffectiveness of traditional approaches against cancer including surgery and radiation therapy, forced the scientists to seek out new treatments. Nanotechnology is considered as a new approach for cancer treatment and is rapidly expanding in medical

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research [5, 6]. Nanomaterials which exert unique properties, are being vastly used in medical

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and biological research [7]. Rapid advances and emerging technologies in nanoscale systems, especially nanoparticles, have significant influenced cancer diagnosis, treatment, and

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monitoring [8]. One of these appealing nanomaterials is cerium oxide nanoparticles (nanoceria). Nanoceria is being vastly used in industry as a catalyst [9], gas sensor [10], in

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optical devices [11], as an ultraviolet absorber [12], in fuel cells [13] and in polishing

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materials [14]. Because of some unique properties, scientists were attracted to apply nanoceria in medical sciences. The two-valence state of Ce3+/Ce4+ on wide surface area in the nano-size confers a fascinating biological activity [15, 16]. Nanoceria, by converting these

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valence state, act like a metalloenzyme which apply transition metal ions, such as Fe3+, Cu2+, or Mn3+, to reduce reactive oxygen species (ROS) and reactive nitric oxide in cells [17]. In

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this way, it could act like superoxide dismutase (SOD) by converting Ce3+ to Ce4+ and reducing superoxide into hydrogen peroxide [18, 19]. Moreover, nanoceria by converting their oxidative state, could diminish levels of free radicals such as nitric oxide [20], hydroxyl [21], peroxynitrite [22] and hydrogen peroxide [23]. In addition, nanoceria could regenerate the valence state, which makes it an interesting material that could confer its biological activity for a long time in vivo, thereby fewer repeated dosage is required [24]. Several studies showed that the antioxidant activity of nanoceria could be used for reducing the symptoms of ROS-mediated diseases such as diabetes [25], chronic inflammation [26], neurodegeneration [27, 28], retinitis [29] and cancer [30-32]. In addition, nanoceria could act as an oxidant at acidic pH. Previous studies showed that the unique metabolism of tumor cells caused the acidity of their microenvironment that induces oxidant activity of nanoceria [33]. In the present study, we investigated anti-tumor effects of nanoceria on mouse fibrosarcoma and evaluated the potential of nanoceria for treatment of fibrosarcoma (Fig. 1.). 3

Journal Pre-proof 2. Materials and methods 2.1. Cerium oxide nanoparticles preparation The cerium oxide nanoparticles were synthesized by co-precipitation method as described in our previous work[34]. Briefly, to prepare 2.5 g of cerium oxide nanoparticle, 6.3 g of cerium nitrate hexahydrate (Ce (NO3)3.6H2O, Merck, Germany) was dissolved in 30 ml of deionized water and the mixture was stirred vigorously for 30 min. Then, cerium nitrate solution precursor was added to 30 ml of deionized water (pH 10). During the reaction, the pH was sustained at 10 by adding nitric acid and ammonium hydroxide if needed. Next, the solution

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was left for eight hours and washed twice with water and once with ethanol. Then, the solution was dried at 70 ºC for about 12 hours and calcinated at 500 ºC for two hours. Next

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the power X‐ray diffraction (PXRD, Philips, X'pert, CuKα) ,field emission scanning electron

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microscopy (FESEM, Mira 3,TESCAN) and Zeta potential measurments were used for characterization of nanoparticles. Finally, the particle size analysis was calculated by using

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the ImageJ software ver. 1.42q (National Institute of Health, Bethesda, MD, USA).

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2.2.Catalase-mimetic activity of cerium oxide nanoparticles

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MTT assay was used to perform cytotoxicity assessment. For this assay, in the first step, L929 cells (density=104 cells/well) were implemented in the well of a 96 Wells plate. These

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cells were incubated at 37°C with 5% CO2 for 24 h. In the next step, nanoparticles of cerium oxide (CeO2-NPs) at various concentrations (62.5-500 mg/ml) were added to these cells for 6 h after adding of 200 µM H2O2 per well. Cell viability was determined after 24 h using the spectrophotometric assessment of the formation of formazan. MTT solution was then added (10 µl) in each well (5 mg/ml). The incubation was repeated again for another 1 h (at 37°C). Afterwards, the aspirated materials were removed from medium. DMSO (100 µL) was added for dissolving the Formazan crystals. Microplate spectrophotometer (STAT FAX 2100 Microplate Reader, Awareness Technology in Palm City, Florida, United States) was used to measure absorbance with a wavelength at 590 nm and reference wavelength at 630 nm. Calculation of the percentages of cell viability was as follows. OD590 sample and OD590

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Journal Pre-proof control exhibit well measurements after treatment with CeO2- NPs when H2O2, is available, and untreated wells measurements.

2.3. Animals Female adult BALB/c mice (6-8 weeks) were purchased from Pasteur Institute, Tehran, Iran. Mice were kept under controlled conditions (at 21–24 °C with 12 hours of light/darkness cycle with natural light) and received the standard pellet with tap water ad libitum. Procedures involving animals were done in accordance with Mashhad University of Medical

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Sciences guidelines for the use and care of laboratory animal [35]. First, 2 × 106 WEHI164

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cells (mouse fibrosarcoma cells) were suspended in 100 µl of phosphate buffered saline (PBS) and subcutaneously injected into the flank of the mice. Ten days after cells inoculation,

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the tumor-bearing mice with tumor volume greater than 50 mm3, were divided into two groups, nanoceria-treated (nanoceria) and untreated (control) (n= 5 mice/group). The

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nanoceria group intraperitoneally received 0.5 mg/kg of nanoceria that were dissolved in 100

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µl PBS, twice a week for four weeks. Control group received 100 µl of PBS, concurrently.

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2.4. Tumor volume and weight measurement

Tumor volumes were estimated by external caliper measurement using the following

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formula: V = 0.52 × Length × Width × Height. Length means the greatest longitudinal diameter; width is the greatest transverse diameter, parallel to the mouse body, and height is defined as a diameter of tumor perpendicular to the length and width [36]. Tumor volumes were measured every five days. The mice were sacrificed at the end of the fourth week. Then, the tumors were removed, cleaned, and weighed using a weighing scale. 2.5. Assessment of bio-distribution of nanoceria Various tissues from the tumor, brain, kidney, lung, spleen, and liver were weighed and digested using 68% nitric oxide overnight at 50 ºC. Then, the digested tissues were reconstituted with water to 10 ml. Cerium level of samples was analyzed using inductively coupled plasma optical emission spectroscopy (ICP-OES, Specro-Arcos, Germany). Finally, the data were recorded as part per billion (ppb) per mg of tissues weight. 2.6. Cytotoxicity assay 5

Journal Pre-proof At the end of the study, before sacrificing the animals, blood samples were collected in heparin-coated tubes and kidney (urea and albumin levels) and liver (alanine aminotransferase, aspartate aminotransferase, and albumin levels) function tests were done by using the kits from Bioassay Systems (CA, USA) following the manufacturer’s instruction. 2.7. RNA extraction and cDNA synthesis Total RNA was extracted from fibrosarcoma tumor cells after the fourth week using a total RNA extraction kit (Parstous, Iran) according to the manufacturer’s instructions and RNA quality was assessed using gel electrophoresis. Then, 1 µg of RNA from each sample was

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treated with DNase 1 (Parstous, Iran). DNase1-treated RNAs were used for the synthesis of

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the first-strand cDNA using an easy cDNA synthesis kit (Parstous, Iran). We used the oligo

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(dt) as a primer for the synthesis of complementary DNA.

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2.8. Quantitative real-time reverse transcriptase-PCR

qRT-PCR was done to assess Bax and Bcl2 gene expression levels. The sequences of primers

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were obtained from the NCBI database and checked for specificity using the NCBI BLAST

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tool (Table 1).

The primers were synthesized by Macrogen Company (Seoul, Korea). The final mixture

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reaction volume was 20 µl, and the final primer concentration was 0.2 pmol/μl. A SYBR Green master mix kit was provided from Parstous, Iran. The ABI StepOnePlus real-time PCR System (Applied Biosystems, Foster City, CA, USA) was used for real-time PCR experiments for 40 cycles 10 min at 94ºC as a pre-incubation (15 seconds at 95ºC, 30 seconds at 62ºC and 30 seconds at 72 ºC), and melting was done at 62-95ºC. The relative Bax and Bcl2 expressions were calculated using the 2-ΔΔCT method. Gene expression data were normalized against β-actin as reference gene. 2.9. Histological Studies The tumor tissues were excised and fixed in 10% formalin. After tissue processing, tumor samples were embedded in paraffin, sectioned at 5-µm thickness, and stained with hematoxylin and eosin (H&E). The extent of necrotic area [necrotic area / total tumor area * 100] was determined using the Image J software (version 1.48). In addition, the mean number of mitotic cells was estimated as explained later. 6

Journal Pre-proof 2.10. TUNEL assay DNA fragmentation that occurs in the apoptotic cells, was detected using Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labelling (TUNEL) kit (Roche, Germany). First, sections were deparaffinized using xylene, rehydrated through decreasing ethanol concentrations, and rinsed with 0.1 M PBS for 15 min. The sections were treated with 3% H2O2 in methanol for 10 min in the dark at room temperature to inactivate endogenous peroxidase, and afterwards were treated with proteinase K (Roche, Germany) for 20 min at room temperature. Then, tissues were rinsed with PBS and incubated in the labeling reaction

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mixture of TUNEL kit (Roche, Germany) at 4 °C, overnight. After rinsing the sections in PBS, they were treated with horseradish peroxidase (POD, 1:500) for 1 hour at room

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temperature and then incubated with 0.03% diaminobenzidine solution (DAB; SigmaAldrich, USA) at room temperature for 15 min [25]. After that, sections were washed with

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running water and counterstained with hematoxylin. Finally, the sections were dehydrated by

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increasing ethanol concentrations, cleared in xylene and mounted with a coverslip. Microscopic images were captured by a light microscope (Olympus, BX51, Japan). In this

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method, apoptotic nuclei were stained dark brown [37]. The number of TUNEL-positive cells

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was estimated using a 1000 μm2 counting frame and then, by the following formula:

NA is the number of apoptotic cells per unit area, ΣQ is the sum of counted particles in sections, a/f is the area associated with each frame, and ΣP is the sum of counted frames in sections.

2.11. Statistical analysis All experiments were performed in triplicate and repeated at least twice. Data were presented as mean ± SD (Standard deviation). Statistical analyses were performed by SPSS 20 using ANOVA followed by Tukey's post hoc test to compare the groups. The level of significance was was set at *P<0.05 ,**P<0.01, ***P<0.001, and ****P<0.0001. 3. RESULTS

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Journal Pre-proof 3.1. Cerium oxide nanoparticles preparation In the present study, a co-precipitation method was used to synthesize the nanoparticles while power X-ray diffraction (PXRD) and field emission scanning electron microscopy (FE-SEM) were used to characterize the nanoparticles. Bragg peaks in a powder X-ray diffraction (PXRD) pattern of calcined nanoceria showed miller indices of (111), (200), (220), (311), (222), (400), (331), (420), and (422) (Fig. 2A). It can be indexed in the form of fluorite cubic structure. PXRD peaks broadening will show that the crystallite sizes of synthesized nanoparticles are less than 50 nm (Fig.2B). PXRD data indicated the small size of

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nanoparticles and it was confirmed by FESEM (Fig. 2C and D). Nanoceria showed good stability in water in neutral pH that could be attributed to the negative value of zeta potential

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(Fig. 2E)

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3.2.Catalase-mimetic activity of cerium oxide nanoparticles

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As shown in Fig.3 in the presence of H2O2, nanoceria could act as the antioxidant and protect cells from oxidative stress and death that could be attributed to catalase activity of nanoceria.

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MTT assay indicated that cell death observed at various concentrations of CeO2-NP. The

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maximum decrease in cell viability, about 80%, was shown when the highest concentration of CeO2-NP (500 mg / ml) was present. Moreover, CeO2- nanoparticles effect on the viability

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of cell is dependent on its dose.

3.3. Tumor volume and Tumor weight Fig. 4 shows that nanoceria-treated group had a significant decrease in tumor volume and weight compared to the control group. 3.4. Bio-distribution of nanoceria At the end of the study, tumors along with major organs including the liver, kidney, lung, the brain, and spleen from nanoceria-treated group, were removed for examination of nanoceria accumulation. As shown in Fig. 5, nanoceria mainly accumulated in the tumor tissues, followed by liver, spleen, kidney, lung and brain tissues, respectively. 8

Journal Pre-proof 3.5. Toxicity of nanoceria A panel of liver and kidney function tests (albumin, alanine transaminase, aspartate aminotransferase, blood urea nitrogen, creatinine, and glucose levels) as well as blood glucose test were performed on plasma samples (prepared from untreated and nanoceriatreated mice) for evaluation of nanoceria toxicity. The results showed that nanoceria had no significant toxicity and all values were found in the normal range in both groups (Fig. 6). 3.6. Bax/Bcl2 expression

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Bax and Bcl2 are well‐ known apoptosis marker genes. Bcl2 gene acts as an apoptotic

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inhibitor, while Bax is an apoptotic inducer. The expressions of Bax and Bcl2 in the tumors from treated and untreated groups, were evaluated by Real-time PCR and the results showed

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that Bax expression level significantly increased, whereas Bcl2 mRNA level had a significant decrease in the treated group. Therefore, Bax/ Bcl2 ratio was increased in the nanoceria-

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3.7. Histopathological studies

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treated group (Fig. 7).

Micrographs obtained from tumors stained with hematoxylin and eosin were quantified using

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Image J software to measure the necrotic area and total tumor area. The results showed that the ratio of necrotic area to total tumor area in nanoceria group was significantly higher than that of the untreated group (Fig. 8). However, counting the number of mitotic cells showed no significant difference between nanoceria and untreated groups (Fig. 9). 3-8. Apoptotic cell density TUNEL assay results demonstrated that nanoceria significantly increased numerical density of apoptotic cells in the tumor, compared to the untreated group (Fig. 10). 4. DISCUSSION Application of nanoparticles in medicine provides novel weapons against human diseases. Various nano-metals have been designed and applied which exhibited therapeutic effects in various animal models especially in the cancer research field [8]. As chemotherapy and 9

Journal Pre-proof radiation therapy have limitations such as side effects and high mortality, the use of nanoparticles as an anti-cancer agent can potentially improve the patient's quality of life [31]. In this regard, we studied the potential anti-cancer effects of cerium oxide nanoparticles (nanoceria) in a fibrosarcoma mouse model. Nanoparticles were synthesized by copercipitation method. The charatirzation of nanoparticles showed that CeO2 NPs have narrow size distribution and mean diameter of it is about 31.84 nm. In addition synthesized nanoparticles have good zeta potential that could confer it a good stability in water and in vivo condition. Nanoceria can reduced the cytotoxicity effects of H2O2. It seems the catalase activity of nanoceria decreased the cell damage that comes from H2o2. In vivo results showed

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that in mouse fibrosarcoma tumor, nanoceria at the concentration of 0.5 mg/kg had the

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potential to attenuate tumor growth. The results revealed that nanoceria could increase apoptosis in the tumor cells whereas no significant toxicity in normal organs such as the

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kidney and liver was found. In addition, nanoceria in the tumor cells, increased a proapoptotic gene (Bax) expression and decrease the expression of an anti-apoptotic gene (Bcl2)

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(Fig. 9). These findings are in agreement with a previous study done in nude mice showing that at 135 mg/kg , nanoceria did not cause any remarkable side effect [38]. Hardas et al.

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reported toxic effects of nanoceria in rodents that could be attributed to the high dose of nanoceria used which was at least 200 times higher than what we used (100–250 mg/kg body

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weight) [39]. One of the fascinating properties of nanoceria is its capacity to serve as a free radical scavenger. Nanoceria has two valence states (+4 and +3) that can convert to each

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other depending on environmental factors especially pH [40]. Because of its size, nanoceria offers a wide surface area of mixed valences (Ce+4 and Ce+3) that could effectively scaveng free radicals in the biological system. In addition, it has the capacity to regenerate its valence state (+4 ↔ +3) and confer its beneficial effects for a long period of time and decrease the number of frequent dosages required [31]. Various studies demonstrated that nanoceria could protect normal cells by attenuating ROS levels [26, 41-43]. In this regard, the potential of biomedical application of nanoceria was investigated in a variety of disease states such as gastrointestinal [44], ophthalmologic [45, 46], and neurological conditions [28, 47], as well as “multiple sclerosis“ [28], “gastric ulcer“ [44] endometriosis [43] and diabetes [25]. In addition, several studies showed that pretreatment with nanoceria prior to radiation therapy (RT), reduced the RT-induced cell death in normal tissues of the breast [48], gastrointestinal tract [49], head and neck [50] and also prevented radiation-induced pneumonitis in an animal model [38]. In contrast, nanoceria were found to be toxic in the tumors and could sensitize cancer cells to RT. Some studies revealed that pretreatment with nanoceria prior to RT both 10

Journal Pre-proof in culture and tumors, significantly increased apoptosis rate in cancer cells without damaging the normal tissues [48, 49, 51]. Considering nanoceria as a novel RT sensitizer for many types of cancers, it has been proposed that nanoceria could protect normal cells by their antioxidant activity (at neutral pH) while at acidic pH (such as tumor microenvironment), the catalase activity of nanoceria could be inhibited. Therefore, nanoceria in cancer cells is only capable of conversion of unstable superoxide to the more stable H2O2, and in this way could exert anti-cancer effects [51]. 5. Conclusion

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In this study, we showed that nanoceria dominantly accumulates in cancer cells and since the

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size of our nanoparticles was <50 nm, it seems that enhanced permeability and retention (EPR) plays a crucial role in delivering nanoceria to tumor cells and their

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microenvironment [52, 53]. The liver, spleen, and kidney were the next destinations of nanoparticles. The blood-brain barrier (BBB) effectively prevented nanoparticles entry to the

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brain. Overall, our data revealed that nanoceria could show anti-tumor properties and could

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Table Caption

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be potentially employed in clinical practice for treatment of fibrosarcoma.

FIG. Legends

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Table 1 Sequence of the oligonucleotide primers used in real‐ time PCR.

Fig. 1. Cerium oxide nanoparticles exert double properties regarding environmental pH. At acidic pH (tumor microenvironment) it could generate and stabilize reactive oxygen species (ROS), while at neutral pH (normal cells), could act as an antioxidant enzyme and attenuate free radicals. In this study, we used cerium oxide nanoparticles and evaluated its anti-cancer effects in a mouse model of fibrosarcoma. Fig. 2. Cerium oxide nanoparticles preparation (A) Powder X-Ray Diffraction (PXRD) patterns of synthesized nanoceria (B) Size distribution of nanoceria (C) and (D) FESEM images of synthesized nanoparticles (SEM magnification: 50.kX, Worked distance: 5.43) (E) Nanoceria zeta potential measurement.

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Journal Pre-proof Fig. 3. Cell viability was measured by MTT assay. L929 cells were exposed to different concentrations of cerium oxide nanoparticles (CeO2- NPs) for 6h following H2O2 was added to each well. After 24 h cell viability was determined based on spectrometric measurement of formazan formation. The values mentioned as a percentage of viability compared to control group. The level of significance from CeO2- NPs concentrations represented mean ± S.D. * P<0.05 compared to the control-H2o2 group. Fig. 4. Tumor volume changes in the nanoceria (2 A)-treated and untreated groups on days after cells inoculation. Differences in tumor weight between nanoceria-treated and untreated

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groups at the end of the experiment. (2 B) Differences in tumor size between nanoceriatreated and untreated mice. (2 C) Values represent mean ± S.D. *P<0.05, **P<0.01,

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***P<0.001, and ****P<0.0001 compared to the untreated group.

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Fig. 5. Bio-distribution of nanoceria in the tumor, liver, spleen, kidney, lung, and brain.

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Values represent mean ± S.D. ** P<0.01 compared to the tumor tissue. Fig.6. Effect of the nanoceria on liver and kidney parameters: (4 A) Effect of nanoceria

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on levels of albumin, ALT (alanine transaminase) (4 B), AST (aspartate aminotransferase)

represent mean ± S.D.

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(4C), BUN (blood urea nitrogen) (4 D), creatinine (4 E) and glucose (FIG. 4F). Values

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Fig. 7. The ratio of Bax and Bcl2 expression in the nanoceria-treated and untreated groups. Values represent mean ± S.D. **** P<0.0001 compared to the untreated group. Fig. 8. Photomicrographs show the necrotic area in total tumor area in nanoceriatreated and untreated groups. (6 A) Necrotic areas were outlined by yellow color line (H & E staining, Scale bar=1000 µm). (6 B) Ratio of the necrotic area to total tumor area. (6 C) Values represent mean ± S.D. *** P<0.001 compared to the untreated group. Fig. 9. Photomicrographs show the distribution of mitotic cells in the tumor in nanoceria-treated and untreated groups. (7 A) Yellow arrows point to mitotic cells (H & E staining, Scale bar=100 µm). (7 B) the mean number of mitotic cells per unit area in nanoceria-treated and untreated groups. (7 C) Values represent mean ± S.D. *** P<0.001 compared to the untreated group.

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Journal Pre-proof Fig. 10. Photomicrographs demonstrating the distribution of apoptotic cells in the tumor in nanoceria and untreated groups. (8 A) Yellow arrows point to apoptotic cells (visualization done using DAB, Counterstaining with Harris hematoxylin, Scale bars=100 and 20 µm). (8 B) the mean number of TUNEL-positive cells per unit area in nanoceriatreated group compared to the untreated. Values represent mean ± S.D. *** P<0.001 compared to the untreated group. Conflict of interest

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The authors declare that there are no conflicts of interest.

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Acknowledgments

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This work was financially supported by a research grant from the Vice Chancellor for

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Research of Mashhad University of Medical Sciences, Mashhad, Iran (Grant No. 921955).

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Clinical oncology 7(11) (2010) 653-64.

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Journal Pre-proof

Table 1 Sequence of the oligonucleotide primers used for real‐ time PCR Primers

Genes Bax

Oligonucleotides primers 5′- GCC GCC CCA GGA TGC -3′

Amplicons (bp) 178

5′- GCC CCA GTT GAA GTT GCC AT -3′

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5′- GTC CAT CTG ACC CTC CGC C -3′

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5′- CTC CCC CAG TTC ACC CCA TC -3′

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β-Actin 5′- GCG GGC GAC GAT GCT C -3′

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5′- GGA TAC CTC TCT TGC TCT GGG -3′

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Mice

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130

Journal Pre-proof highlights 

We successfully designed CeO2 NPs for the treatment of fibrosarcoma in-vivo model.

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Our findings demonstrated the novel therapeutic potential of CeO2 NPs in

fibrosarcoma.

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Our data revealed that nanoceria could show anti-tumor properties and could be

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potentially employed in clinical practice for treatment of fibrosarcoma.

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Figure 1

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Figure 3

Figure 4

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