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Redox-sensitive cerium oxide nanoparticles protect human keratinocytes from oxidative stress induced by glutathione depletion Ragini Singh, Ajay Singh Karakoti, William T Self, Sudipta Seal, and Sanjay Singh Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03022 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 30, 2016
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Redox-sensitive cerium oxide nanoparticles protect human keratinocytes from oxidative stress induced by glutathione depletion Ragini Singh1, Ajay Karakoti1,2, William Self3, Sudipta Seal4 and Sanjay Singh1*
1
Division of Biological and Life Sciences, School of Arts and Science, Ahmedabad University, Navrangpura, Ahmedabad-380009, Gujarat, India. 2
School of Engineering and Applied Ahmedabad-380009, Gujarat, India.
Sciences, Ahmedabad University,
Navrangpura,
3
Department of Molecular Biology and Microbiology, Burnett School of Biomedical Science, University of Central Florida, Orlando-32816,Florida, USA. 4
Advanced Materials Processing and Analysis Centre, Nanoscience Technology Centre (NSTC), University of Central Florida, Orlando-32816, Florida, USA.
Corresponding author address Division of Biological and Life Sciences, School of Arts and Science, Ahmedabad University, Navrangpura, Ahmedabad-380009, Gujarat, India Phone: +91-79-26302414, Fax: +91-79-26302419 *Corresponding author Email Id:
[email protected] 1 ACS Paragon Plus Environment
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Abstract Cerium oxide nanoparticles (CeNPs) have gathered much attention in the biomedical field due to its unique antioxidant property. It can protect cells and tissues from oxidative stress induced damage due to its auto-regenerative redox cycle. Our study explores the antioxidant and antigenotoxic behaviour of PEGylated CeNPs towards oxidative insult produced by BSO (Buthionine sulfoximine)
in
human
keratinocytes
(HaCaT
cells).
BSO
inhibits
the
γ-
glutamylcysteinesynthetase (γ-GCS) enzyme and thus acts as a glutathione (GSH) depleting agent to modulate the cellular redox potential.GSH is a natural ROS scavenger present in the mammalian cells, and its depletion causes generation of reactive oxygen species (ROS). In this study, we challenged HaCaT cells (keratinocytes) with BSO to alter the redox potential within the cell and monitored toxicity, ROS generation and nuclear fragmentation. We also followed changes in expressions of related proteins and genes. We found that PEGylated CeNPs can protect HaCaT cells from BSO induced oxidative damage. BSO exposed cells, pre-incubated with PEGylated CeNPs, showed better cell survival and significant decrease in the intracellular levels of ROS. We also observed decrease in LDH release and nuclear fragmentation in CeNPs treated cells that were challenged with BSO as compared to treatment with BSO alone. Exposure of HaCaT cells with BSO leads to altered expression of antioxidant genes and proteins, i.e. Thioredoxin reductase (TrxR) and Peroxiredoxin 6 (Prx6) whereas, in our study, pre-treatment of PEGylated CeNPs reduces the need for induction of genes that produce enzymes involved in the defence against oxidative stress. Since, growing evidence argued the involvement of ROS in mediating death of mammalian cells in several ailments, our finding reinforces the use of PEGylated CeNPs as a potent pharmacological agent under the lower cellular GSH/GSSG ratios for the treatment of diseases mediated by free radicals. Keywords: Cerium oxide nanoparticles, Buthionine sulfoximine (BSO), Antioxidant nanoparticles, Oxidative stress, Thioredoxin reductase, Peroxiredoxin 6.
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Introduction Nanoparticles have been extensively used in therapeutic interventions and medical diagnostic applications such as in nucleic acid and drug-delivery systems, theranostic implications, photothermal therapy and imaging1-5. It is well documented that redox active elements undergo redox cycling and produce free radicals at physiological conditions in mammalian cells. In case of ionic iron, it has been reported that toxicity is controlled by redox state of iron. It was shown that ferrous (Fe2+) ions has significantly higher toxicity compared to the ferric (Fe3+) ions in neuronal cells 6, 7. Recently, CeNPs have emerged as a potent therapeutic agent due to their redox-active nature, specifically the ability to switch between 3+ or 4+oxidation states, which depend upon the surrounding physiological conditions 8, 9. Antioxidant activities possessed by CeNPs are ascribed due to the oxygen vacancy/defect in their crystal structure that leads to switching of its oxidation states 10, 11. This easy switching of oxidation states allows it to either capture or release an electron to scavenge or neutralize a variety of ROS. An excess of either 3+ and 4+ oxidation states of “Ce” atoms on the surface of CeNPs are suggested to be responsible for mimicking the naturallyoccurring antioxidant enzymes such as superoxide dismutase (SOD) and catalase, respectively1215
. Due to their protective action, analogous to natural enzymes, CeNPs are shown to be useful
in the treatment of neurodegenerative disorders and other diseases in which oxidative stress acts as a keynote11, 16-18. CeNPs have shown free radical scavenging properties in several cell culture models such as, gastrointestinal, endothelial, breast, and neuronal cells 19, 20. In this context, free radical scavenging property of CeNPs has shown excellent cyto-compatibility with mammalian cells. The surface coating of nanoparticles has also been shown to decrease the toxicity; however, a thicker coating leads to loss of intrinsic properties of nanomaterials 21.The advantage with CeNPs is that their enzyme like activity is not compromised with surface coating. Polyethylene glycol (PEG), a long chain hydrophilic and conformationally flexible polymer, is one of the most popular
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surfactants being used to coat the nanoparticle surfaces and makes them less apt to be removed through the action of the reticuloendothelial system (RES) in mammals 22. PEG coating can reduce the macrophage uptake of nanoparticles and also reduce protein adsorption at the particle’s surface, thus increase the biocompatibility and half-life of nanoparticles. In a report from Karakoti et al, it has been shown that a thick coating of PEG on the CeNPs (PEGylated CeNPs) surface does not affect the performance of superoxide dismutase (SOD) like activity of CeNPs when compared to its non-PEGylated counterparts 23. GSH, a primary cellular thiol and major contributor to redox balance in the cytosol, serves as an important factor in maintaining cellular redox balance that is critical to proliferation, differentiation and even apoptosis. Depletion of cellular GSH levels (or alteration of the GSH/GSSG ratio) causes oxidative stress and is associated with several diseases such as, asthma, acne vulgaris, cystic fibrosis, aging, AIDS/HIV and neurodegenerative diseases 24
25
. Glutathione detoxifies the
excessive amount of superoxide anions and peroxides produced in the cells and thus, protect the cells from damage caused due to ROS 24, 25. Strategies leading to the decrease in the levels of ROS could, in principle, elevate the antioxidant mediated protection of rapidly growing cancerous cells
26, 27
. Although, CeNPs are shown to scavenge ROS and RNS (reactive nitrogen species),
their cyto-protective ability has not been explored under depleted cellular GSH levels. Neurodegenerative diseases are well known to exhibit low levels of cellular GSH; thus, it is expected that the use of PEGylated CeNPs could alleviate the degenerative effects of ROS. Skin is the largest organ, and a primary site of oxidative stress induced damage in humans. Due to its anatomic location, skin cells are exposed to UV radiation as well as occupational and environmental toxic compounds. In the long-term environmental exposure to this damage may lead to immunosuppression, photo ageing and malignancy28, 29. Due to the antioxidant behaviour, PEGylated CeNPs might play a major protective role in the ROS induced skin disorders. Several studies have shown the antioxidant effect of CeNPs under in vitro and in vivo experimental 4 ACS Paragon Plus Environment
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conditions 29, 30; however, limited information exists about unraveling the molecular mechanism behind their ROS scavenging and DNA protection effects under low cellular glutathione conditions. BSO is an irreversible inhibitor of γ-glutamylcysteinesynthetase (γ-GCS), a rate limiting enzyme in glutathione synthesis, which leads to GSH depletion and modulates the cellular redox status 31, 32. BSO administration results in GSH depletion in all the cells and tissues and has been associated with ROS production and thus induces oxidative stress 32, 33. Here, we have tested the efficacy of CeNPs to protect against the oxidative stress generated by BSO in a human keratinocytes cell culture model system (HaCaT cells). In this study, we have found that PEGylated CeNPs protect HaCaT cells against the BSO induced cytotoxicity and genotoxicity. It was also observed that PEGylated CeNPs neither induced the increase in GSH levels nor the levels of antioxidant proteins and genes, strongly suggest that HaCaT cell protection against oxidative stress is due to the in vitro catalytic effect demonstrated by CeNPs. Materials and Experimental Methods Materials: Cerium nitrate hexahydrate, 2,7-dichlorofluorescein diacetate (DCFDA), buthionine sulfoximine (BSO), ferri-cytochrome C, and xanthine oxidase were purchased from Sigma Aldrich (St. Louis, MO USA). Hydrogen peroxide (H2O2) was obtained from S D Fine Chemical Limited (Mumbai, India). Hypoxanthine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye, Propidium iodide, Phosphate Buffer Saline (PBS), 4',6-diamidino-2-phenylindole (DAPI), Cytochalasin B and Dulbecco’s minimum essential media (DMEM: F12) were purchased from Hi-Media Pvt. Ltd. (Mumbai, India). PEGylated CeNPs synthesis: PEGylated CeNPs were synthesized by the method described by Karakoti et al23. In brief, 0.1 gm of cerium nitrate hexahydrate was dissolved in 10% PEG and 1 mL of 30% hydrogen peroxide was added, which immediately turns the colourless solution into yellow color, indicating the formation of CeNPs with high 4+/3+ oxidation state. Gradually in 10-15 days, the yellow color solution turns colorless indicating the formation of PEGylated CeNPs with 5 ACS Paragon Plus Environment
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high 3+/4+ state. This transition was also monitored with UV-visible spectroscopy. Nanoparticle concentration was estimated by dry weight method. A fixed volume of nanoparticles were dried followed by weight measurement. The obtained weight was expressed in mg/mL or µg/ml). SOD-like activity measurement: SOD mimetic activity was measured, as described by Korsvik et al14. In brief, a competition for reduction of ferri-cytochrome C was utilized to assess the superoxide scavenging activity of PEGylated CeNPs. Here, superoxide is generated by hypoxanthine/xanthine oxidase system, and an excess of catalase was added to remove hydrogen peroxide that is also produced as a side reaction. Reduction of ferri-cytochrome C by superoxide was followed spectrophotometrically at 550 nm for 15 min. Reactions were carried out in triplicate in a 96 well plate with total volume of 100µL and buffered with 10 mM Tris pH 7.5. Decrease in absorbance (with time) corresponds to increase in SOD activity and vice-versa. The normalized absorbance was calculated by subtracting the absorbance values with very first value in the spectra. Fourier Transform Infra Red Spectroscopy (FTIR): The coating of PEG over CeNPs was confirmed by FTIR analysis. The spectra from pure PEG was obtained directly, whereas, for PEGylated CeNPs were prepared in KBr powder and analyzed using the ATR mode from in 4004000 cm -1 range. Transmission Electron microscopy (TEM): Sample was prepared by adding drop of PEGylated CeNPs on a carbon coated copper grid. After drying images were acquired by transmission electron microscope (TEM), JEOL JEM1400 operating at a voltage of 120 kV. Cell culture and exposure of HaCaT cells to PEGylated CeNPs: Human keratinocyte cell line (HaCaT), purchased from the national cell culture repository situated in National Centre for Cell Sciences (NCCS), Pune, India, was cultured in DMEM: F12 supplemented with 10% FBS and 1% of antibiotic and antimycotic solution at 37 oC under a humidified atmosphere of 5% CO 2. Stock suspensions of PEGylated CeNPs (200 µg/mL) were prepared in DMEM: F12 (supplemented with 10% FBS) was serially diluted to concentrations ranging from 5 to 100 µg/mL for cellular uptake, 6 ACS Paragon Plus Environment
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cytotoxicity assays, micronucleus formation assay, ROS generation and oxidative stress parameters. The experiments were performed between the passage no. 10 – 15 for HaCaT cells. For experiments involving BSO treatment, cells were pre incubated with PEGylated CeNPs for 6 hrs followed by 18 hrs of BSO treatment. Cellular uptake: Internalization of PEGylated CeNPs was analysed by flow cytometry as described by Suzuki et al34. 1.0 X 105 cells were seeded in 6-well cell culture plates for 24 hrs. Cells were treated with different concentrations (5, 20, 40 and 80 µg/mL) of PEGylated CeNPs for 6 hrs. After exposure, cells were harvested by trypsin and washed with PBS. Finally, the pelleted cells were suspended in PBS and the uptake of particles was analysed by flow cytometer (FACS Calibur, BD Biosciences, CA) with side scatter intensity (SSC). For “fold uptake” calculation, SSC intensity values of treated cells were divided by SSC values of control (untreated) cells. Mitochondrial activity/Cytotoxicity assessment: MTT assay was done to evaluate the mitochondrial activity as protocol described by Mosmann35. 1X104 cells were seeded in 96 well cell culture plates and incubated for 24 hrs. Cells were incubated with many concentrations of PEGylated CeNPs (5, 10, 20, 40, 60, 80 and 100 µg/mL) for different time periods (6, 24, 48 hrs), followed by MTT dye (5mg/mL) addition. The medium from each well was discarded and resulting formazan crystals were solubilized by adding 200µL of dimethyl sulphoxide (DMSO) and quantified by measuring absorbance at 590 nm in a multiwell plate reader (Biotek, Synergy HT spectrophotometer). Neutral red uptake (NRU) assay: Viability of cells was also assessed by neutral red uptake assay, which accumulate in the lysosomes of viable cells 36. 1X104 cells were seeded in 96 well cell culture plates and incubated at 37oC under CO 2 atmosphere for 24 hrs. Cells were incubated with many concentrations of PEGylated CeNPs (5, 10, 20, 40, 60, 80 and 100 µg/mL) for different time periods (6, 24, 48 hrs). 100µL of neutral red dye (50µg/mL) was prepared in the serum-free medium and was added to each well after discarding the media. Plate was incubated for 3 hrs at 7 ACS Paragon Plus Environment
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37 oC and the dye accumulated was dissolved in solution of 50% ethanol and 1% acetic acid. Absorbance was taken at 540 nm a multiwell plate reader
(Biotek, Synergy HT
spectrophotometer). LDH release estimation: LDH release, representative of membrane damage and cell death, was determined by LDH estimation kit (Cayman). In brief, 1X104cells were seeded in 96 well cell culture plates and pre incubated with PEGylated CeNPs for 6 hrs followed by 18 hrs of BSO exposure (10 and 20 mM). After completion of exposure, supernatant media (50 µL) was transferred into new 96 wells and incubated with reaction buffer for 30 min at room temperature to develop color based on the presence of LDH that reacts with LDH specific reagents provided in the kit. Absorbance was measured at 490 nm using multiwell plate reader. Glutathione estimation: Free sulphydryl content was measured with Glutathione estimation kit (Cayman) and expressed as µmole/mg of protein. In brief, 1X105cells were seeded in 6 well cell culture plates and incubated for 24 hrs. Cells were pre incubated with PEGylated CeNPs (5 and 10 µg/mL) for 6 hrs followed by BSO exposure (10 and 20 mM) for 18 hrs. Cells were harvested by scrapper and sonicated in a cold buffer containing EDTA, centrifuged and supernatant was collected for further quantification. Supernatant was added into 96 well plate followed by the addition of reagent mixture provided in the kit. Plate was incubated in the dark for 15-20 min at room temperature. Absorbance of standard and sample was taken at 405 nm in multiwell plate reader. ROS level measurement : The level of intracellular ROS generation (generally regarded as a measure of peroxides) was determined by using 2, 7-dichlorofluorescein diacetate dye37. Here, higher levels of peroxides were induced by BSO treatment (10 and 20 mM) in the cells with and without PEGylated CeNPs pre-treatment for further analysis. 1X104 cells were seeded in a 96well black bottom cell culture plates. After 24 hrs, the cells were pre incubated with PEGylated CeNPs for 6 hrs followed by exposure to 10 and 20 mM BSO exposure for 18 hrs. The nanoparticle containing medium was aspirated, and cells were washed twice with 1X PBS. 8 ACS Paragon Plus Environment
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Thereafter, DCFDA dye (20 µM) in 1X PBS was added to each well. The plate was incubated for 30 min at 37oC and the DCFDA dye was discarded. 200 µL of PBS was then added to each well and fluorescence intensity was measured in a multiwell plate reader at excitation and emission wavelengths of 485 and 528 nm, respectively. For qualitative analysis of level of ROS generated in cells after BSO exposure, imaging of cells under confocal microscope was used. Prior to imaging, cells were fixed with chilled (-20 °C) methanol for 8 min followed by incubation with a nucleus staining dye (DAPI) for 8 min. Cells were washed and mounted in anti-fade mounting media (Calbiochem),and slides were prepared. These slides were stored at 4°C until confocal imaging by Leica TCS SP5 laser scanning confocal microscope with 40X oil objective lens. In addition, the qualitative analysis of ROS generation at similar BSO and PEGylated CeNPs concentrations was also done by using a fluorescent microscope (DM 2500, Leica, Wetzlar, Germany). Nuclear fragmentation: Fragmentation of the nucleus in BSO treated HaCaT cells were analysed by DAPI staining, which was further imaged by the fluorescence microscope38. Cells were seeded on the cover slip in 12 well plate and incubated for 24 hrs. Cells were pre incubated with 5µg/mL PEGylated CeNPs followed by 10 and 20mM BSO exposure for 18 hrs. After completion of exposure time, cells were washed with 1X PBS and stained with DAPI for 5-10 min. To remove any extra background stain, cover slips were gently washed with 1X PBS then mounted on the slide. Further analysis was done by using a fluorescent microscope. To get the quantitative data ~300 cells were counted for each treatment and control. Micronucleus assay: Cytokinesis-block micronucleus (CBMN) assay: CBMN assay was done by method described by Fenech39.Cells were exposed with BSO and after completion of exposure time, the media was aspirated, and cells were allowed to grow further for 18 hrs in 1 mL of fresh complete DMEM containing Cytochalasin-B (3 µg/mL) in an incubator at 5% CO 2. 300µL of cells were loaded in cyto-funnel and harvested on slides by using Cytospin (Thermo Shandon, Hampshire, UK) at 9 ACS Paragon Plus Environment
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250g for 5min. Slides were fixed in chilled methanol, air dried and stained with 10% Giemsa in Sorenson’s buffer. Further, cells were analysed in a bright field microscope to scan the micronuclei in binucleate cells. Flow cytometer: Micronucleus formation estimation through flow cytometer was measured by the protocol described by Nusse and Kramer40. After exposure with BSO cells were harvested, washed with PBS and then dissolved in Solution-I containing sodium chloride (584 mg/L), sodium citrate (1000 mg/L), RNase (10 mg/L), EtBr (25 mg/L), Igepal (0.3 mL/L) and incubated at room temperature. After 1h, 1mL of solution II containing citric acid (15 mg/L), sucrose (0.25 M), EtBr (40 mg/L) was added into it. Further, cells were analysed by flow cytometry to access the micronuclei formation (FACS Calibur, BD Biosciences, CA). Western blot analysis: Protein was isolated from the cells seeded in 6 well plate (1 X105 cells/well). Cells were pre-treated with 5µg/mL of PEGylated CeNPs for 6hrs and then incubated with 500µM BSO for 18 hrs. Control and treated cells were harvested and lysed in a lysis buffer through sonication. Isolated protein was quantified by Bradford’s method using BSA as a standard. A 30 µg of protein was resolved on 12% SDS polyacrylamide gel electrophoresis (PAGE) and transferred on PVDF membrane for 3 hrs at the voltage of 300mV. Membrane was blocked with dry milk (5%) in Tris-buffered saline containing 0.2% Tween-20 for 2 hrs and then incubated with specific primary antibodies (Abcam, Cambridge, UK) for TrxR, Prx6 and GAPDH overnight at 4oC. After appropriate secondary antibody incubation and three washes, the blot was developed by using chemiluminescence (Super Signal West Femto chemiluminescent reagent, Pierce, Rockford, IL) and analysed through Image Quant LAS 500 software (GE Healthcare BioSciences AB, Sweden). Densitometry analysis of protein bands was performed by Image-J Software. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR): Cells were seeded in 6 well plate and pre incubated with 5 µg/mL of PEGylated CeNPs followed by500µM BSO exposure. Cells were harvested by trypsinization and washed twice with PBS. Total RNA was isolated using 10 ACS Paragon Plus Environment
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Trizol method and quantified at 260 nm using a UV-Visible spectrophotometer. A 0.5 µg of purified RNA was used as a template for the generation of cDNA using the cDNA synthesis kit (Thermofisher). The cDNA was diluted 1:100 before adding to the reaction mixture. The oligonucleotides used for this analysis were TrxR (forward, 5´-AGCTCAGTCCACCAATAGTGA3´;
reverse,
5´-GGTATTTTTCCAGTCTTTTCAT-3´)
and
Prx6
(forward,
5’-
CGTGTGGTGTTTGTTTTTGG-3’; reverse, 5’-TCTTCTTCAGGGATGGTTGG-3’) (Xcelris, India). Amplified products were analysed through Image Quant LAS 500 software (GE Healthcare BioSciences AB, Sweden) and the densitometry analysis of bands was performed by Image-J Software. Results Characterization of PEGylated CeNPs: Presence of PEG coating on CeNPs surface was confirmed by the FTIR analysis (Fig. ESI: 1A). As expected, PEG alone (red curve) and PEGylated CeNPs )black curve) showed transmission peak centering about ~3450, 2873, 1456, 1096 and 939 cm -1 representing the O-H stretch, CH2-stretch, CH2-bend, C-O stretch and C-C stretch, respectively from PEG, which suggest the successful capping of CeNPs with PEG molecules 23. Additionally, the presence of transmission bands at ∼833 cm -1and 439 cm −1,due to Ce-O and CeO 2stretching vibrations from cerium oxide further confirm the formation of PEG coated CeNPs 41, 42.The size and shape of PEGylated CeNPs was analyzed by TEM imaging (ESI:1B and C). It is evident from the TEM images that particles are non-agglomerated, quasispherical in shape and ~10 nm in diameter. The particles also shows the presence of an amorphous polymer layer over CeNPs, which could be ascribed due to the PEG coating23. SOD mimetic activity of PEGylated CeNPs in cell culture media. It has been previously shown that the physico-chemical properties and behaviour of engineered nanomaterials (ENMs) are altered upon suspension in biologically relevant buffers. The biological responses are predominantly due to the interplay between biologically relevant components and intrinsic properties of ENMs
43
. Bare ENMs, due to their high reactivity, undergo aggregation in the 11 ACS Paragon Plus Environment
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complex biological environment causing misrepresentation of results and impede experimental reproducibility of the cellular uptake and the toxicity profile. Although surface coating of bare ENMs has been suggested as an alternative to avoid aggregation, particles sometimes lose their inherent properties after thick coating of biomolecules or polymers 21. PEGylated CeNPs are well known for retaining their intrinsic SOD mimetic activity, even after thick coating of PEG 23. Despite the enormous potential of PEGylated CeNPs in biomedical applications, the colloidal stability and intrinsic properties in cell culture media have not been explored in detail. Therefore, we first studied the effect of cell culture media on SOD mimetic activity of PEGylated CeNPs. It was found that PEGylated CeNPs suspended in cell culture media (DMEM: F12) for 24 hrs, did not show any decrease in SOD mimetic activity (Figure 1A, solid triangles), and displayed superoxide radical scavenging property comparable to aqueous suspended PEGylated CeNPs (Figure 1A, hollow spheres). Interaction between BSO and PEGylated CeNPs were evaluated by following the absorbance pattern of CeNPs and the SOD-like activity of CeNPs (Fig. ESI: 2A and B). As expected, neither the UV-Vis spectra nor the SOD-like activity pattern of CeNPs exhibited any change, which confirms that BSO and CeNPs do not interact when mixed together. The colloidal stability of PEGylated CeNPs was estimated with hydrodynamic size and zeta potential measurements (Table 1), which revealed that there was no significant change in the hydrodynamic size and zeta potential values of PEGylated CeNPs suspended in water (~245 nm and -7.8 mV) and cell culture media (~247 nm and-10.4 mV). In case of PEGylated nanoparticles, the colloidal stability is controlled by steric repulsion induced by layer of PEG molecules and not by electrostatic repulsion between the charged nanoparticles. Therefore, despite of low zeta potential value, PEGylated CeNPs are colloidally stable in water as well as in cell culture media. This observation is also supported by TEM images showing well dispersed PEGylated CeNPs. These results demonstrate that PEGylated CeNPs have a steady layer of PEG on their surface, which imparts stability to CeNPs in cell culture medium and thus helps to retain the SOD mimetic activity in cell culture media. 12 ACS Paragon Plus Environment
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PEGylated CeNPs are efficiently internalized in HaCaT cells. For any potential delivery application of ENMs, it is imperative that they are efficiently internalized in mammalian cells efficiently. Therefore, we studied the internalization of PEGylated CeNPs in a HaCaT cells by following the increase in side scattering (SSC) intensity through flow cytometer (Figure 1Band ESI: 3). It is expected that the nanoparticle internalization would lead to the increase in the granularity of the cytoplasm, which could be measured by flow cytometer as the increase in SSC intensity. Flow cytometry is an extremely powerful technique which can differentiate between the nanoparticles physically adsorbed on the cell surface (forward scattering) and particles present in the cell cytoplasm (side scattering)44. Different concentrations of PEGylated CeNPs (5, 20, 40 and 80 µg/mL) were exposed to HaCaT cells for 6 hrs. This incubation resulted in a concentration dependent increase in SSC intensity (2.1, 2.5, 3.0 and 4.0 fold, respectively) than untreated control cells. Further, the cytotoxicity analysis of PEGylated CeNPs showed that these nanoparticles lack toxicity up to 100 μg/mL concentrations, as evident by MTT and neutral red uptake (NRU) assays (ESI: 4). PEGylated CeNPs protect cells from free radicals. Buthionine sulfoximine (BSO) is a wellknown inhibitor of gamma-glutamylcysteinesynthetase that lowers cell/tissue GSH concentrations and in doing so modulates the cellular redox status 45. Loss of GSH expression in tissues is associated with the increase in ROS production, which is also linked with several diseases such as asthma, acne vulgaris and AIDS 24, 25.Therefore, in order to establish the utility of PEGylated CeNPs in the protection of GSH deprived cells/tissues, we used BSO as an artificial agent to reduce the GSH production and increase the reactive oxygen species levels in HaCaT cell cytoplasm. It is clearly evident by MTT assay (Figure 2A) that cells exposed to 10 and 20 mM BSO show decrease in cell viability; however, the cells pre incubated with PEGylated CeNPs (5 µg/mL) did not show any significant decrease in viability, which indicates that PEGylated CeNPs imparts protective effect under low GSH condition of cells after. As expected, exposure of only PEGylated CeNPs did not show any reduction in cell viability, which further confirms the 13 ACS Paragon Plus Environment
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biocompatibility of PEGylated CeNPs. The protection of cells against BSO mediated ROS by PEGylated CeNPs was further established by lactate dehydrogenase (LDH) release assay, which is a very sensitive method to measure the cell membrane permeability and thus, the cytotoxicity and cytolysis. BSO treated cells exhibited significant loss of membrane integrity, as evident by increase (~1.8 fold) in LDH release (Figure 2B), whereas, cells pre incubated with PEGylated CeNPs did not show any increase in LDH release. Taken together, these results suggest that PEGylated CeNPs could protect the cells from damage caused by the ROS generated as a result of loss of cellular GSH production. It can also be concluded that PEGylated CeNPs retain their radical scavenging activity even in the complex composition of cytoplasm. PEGylated CeNPs exposure does not induce GSH production in HaCaT cells. In order to explore the level of GSH present in PEGylated CeNPs and BSO treated cells, we performed the quantification of GSH present in treated HaCaT cells. As evident from Figure 2C, 100 – 500 µM BSO treatment can lead to ~40 % decrease in cellular GSH production, compared to untreated control HaCaT cells. Interestingly, PEGylated CeNPs (5 µg/mL) did not induce any alteration in cellular GSH production and its concentration remains same as untreated control cells. Interestingly, HaCaT cells, pre incubated with PEGylated CeNPs followed by BSO exposure also did not exhibit any increase in GSH level, which suggest that ROS scavenging activity is primarily performed by PEGylated CeNPs. This observation indicates that although pre-treatment of PEGylated CeNPs did not increase the GSH synthesis in BSO treated HaCaT cells, but helps to sustain the optimum level of reactive oxygen species through its intrinsic ROS scavenging activity, even in the absence of GSH. Cellular reactive oxygen species are scavenged by PEGylated CeNPs: It is well established that decrease in GSH can lead to increases in cellular ROS levels. In our experiments, generation of ROS was determined using 2′,7′-dichlorodihydrofluorescein diacetate, which diffuses across the cell membrane and is subsequently oxidized in to membrane impermeable, highly fluorescent DCF by intracellular ROS. We exposed HaCaT cells with BSO and imaged the elevated levels of 14 ACS Paragon Plus Environment
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ROS in the cell cytoplasm by confocal microscope (Figure 3A – H). Cells exposed to BSO and stained with H2DCF-DA dye exhibited green fluorescence signal from their cytoplasm (Figure 3B) that suggests extensive formation of ROS than untreated control. In comparison, PEGylated CeNPs pre incubated cells did not show any green fluorescence signal (Figure 3F) suggesting that the ROS generated by BSO treatment were scavenged efficiently by PEGylated CeNPs. Further, the quantitative information about generated ROS in HaCaT cells after BSO exposure was estimated by fluorescence spectrophotometer. As it is evident from Figure 3I that 10 and 20 mM BSO exposure to HaCaT cells induced significant increase in ROS levels (up to ~140 %), however, cells pre incubated with PEGylated CeNPs, circumvent the reactive oxygen species induced by BSO. PEGylated CeNPs abrogate the nuclear fragmentation and micronucleus formation: ENMs are shown to interact with the cell nucleus and induce damage to genetic material46. It has also been shown that particles smaller than 50 nm size (smaller than nuclear pores) could easily penetrate inside nucleus and due to their strong affinity with DNA, may induce DNA damage46. Therefore, we also estimated the DNA damaging potential of PEGylated CeNPs in keratinocyte cells. As expected, it was found that PEGylated CeNPs did not induce any DNA damage to mammalian cells, rather, these nanoparticles showed a protective effect against DNA damaging chemical such as BSO. Further, in order to assess the protective effect of PEGylated CeNPs on DNA damage, induced by BSO, we performed nuclear fragmentation and micronucleus formation assay, which are well-known methods for the evaluation of DNA damage47, 48. To clearly visualize the nuclear region cells were stained with DAPI. It is clearly evident from Figure 4 that cells pre incubated with PEGylated CeNPs (Figure 4B) did not show any nuclear fragmentation, however, cells exposed to BSO (Figure 4C) showed extensively fragmented nuclei (the punctated nuclei are marked by white arrow). Cells pre-incubated with PEGylated CeNPs followed by BSO exposure did not show any sign of fragmentation (Figure 4D). Further, to obtain a quantitative data, ~ 300 cell nuclei were counted from cells exposed to BSO with and without PEGylated 15 ACS Paragon Plus Environment
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CeNPs pre incubation. Figure 4E clearly show that there was a significant increase the number of in fragmented nuclei after the treatment of 10 and 20 mM BSO, whereas cells pre incubated with PEGylated CeNPs did not show much fragmentation. BSO is also known to induce nuclear fragmentation, which could be detected by micronucleus formation47. Cytochalasin-B blocked micronucleus (CBMN) formation is a well-known method to analyse the DNA aberration. To investigate this, we used two techniques, flow cytometry analysis and manual counting of cells having micronuclei under microscope. Data obtained from flow cytometry clearly depicts that micronucleus formation frequency was significantly enhanced (~ 2.0 fold) after BSO exposure, nonetheless, cells pre-treated with PEGylated CeNPs inhibited the micronucleus formation induced by BSO (Figure 5A and ESI: 5). Similarly, CBMN-based method also revealed that at 20 mM BSO concentration exposure, there was ~ 2.0 fold increase in micronucleus formation; however, cells pre-treated with PEGylated CeNPs, successfully, avoided the micronucleus formation (Figure 5B and C). This observation suggests that PEGylated CeNPs can scavenge ROS from cells even in the presence of BSO. Additionally, BSO exposure to fibroblasts leads to the increased levels of ROS, which causes DNA damage and chromosomal breaks 49. Further, it has been reported that during mice development, BSO exposure leads to the decrease in the synthesis of thiol based biomolecules due to oxidative stress 32. Likewise, Seager et al have also shown that BSO treatment significantly increases the micronucleus formation frequency and decreases the protection against oxidative stress; however, pre-treatment with known antioxidant, i.e. N-acetylcysteine reduces the frequency incells 47. Similarly, in our experiments, cells pretreated with PEGylated CeNPs also exhibited protection against BSO induced nuclear fragmentation and micronucleus formation demonstrating the antioxidant potential of CeNPs analogous to an antioxidant. PEGylated CeNPs oversee the expression of antioxidant proteins. Since, GSH is directly associated with the ROS scavenging, we studied the expression levels of genes encoding protein involved in oxidative stress defence. We chose TrxR and Prx6 as targets proteins, which are well 16 ACS Paragon Plus Environment
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known to regulate the oxidative stress in cells, and studied the expression levels of each of these by western blotting. TrxR is a selenium containing protein that catalyses the reduction of the active site disulfides of thioredoxin, these plays a major role in regulating the redox status (reducing vs oxidizing environment) of the cells. As expected, cells pre-exposed to BSO triggered an increase in TrxR protein expression levels due to the oxidative stress, whereas, cells pre-exposed to PEGylated CeNPs displayed similar expression levels as untreated healthy control cells (Figure 6). Cells exposed to PEGylated CeNPs did not show any alteration in TrxR protein expression level than control cells. A quantitative estimation revealed that there was ~1.7 fold (compared to untreated control cells) increase in TrxR protein expression level in cells treated with 500 µM BSO. Additionally, we also followed the mRNA expression levels of TrxR in response to micro molar levels of BSO treatment with or without PEGylated CeNPs pre exposure (Figure 7). It was found that the level of TrxR mRNA was significantly increased during BSO exposure; however, cells pre incubated with PEGylated CeNPs showed decreases in mRNA levels with respect to control. This observation suggests that PEGylated CeNPs act as an antioxidant, which results in lower expression of TrxR mRNA due to the absence of ROS. Peroxiredoxin 6 (Prx6) is different from other five peroxiredoxins (1 to 5) in a manner that peroxiredoxin (1 to 5) uses thioredoxin as a physiological reductant50, 51, whereas, Prx6 utilizes glutathione52,
53
.Since, Prx6 is dependent on
cellular GSH levels, we chose this isoform for our study. Upon exposure of 500 µM BSO, the level of Prx6 protein in the cells was decreased to 0.6 fold (~40% decrease). However, PEGylated CeNPs, pre-treated cells did not alter the expression of Prx6 protein. Similarly, it was found that exposure of 500µM BSO decreases the Prx6 mRNA level up to 0.7 fold (~30%decrease), which does not alter by the pre-exposure of PEGylated CeNPs (Figure 7). This observation is in agreement with GSH levels in cells after BSO treatment (Figure 2C). Interestingly, HaCaT cells exposed to PEGylated CeNPs alone do not show any significant decrease in Prx6 mRNA expression. Since expression of TrxR and Prx6 proteins and mRNAs are linked with the level of cellular oxidative stress, and PEGylated CeNPs could reverse the deleterious effects of ROS, 17 ACS Paragon Plus Environment
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therefore, it can be concluded that PEGylated CeNPs could also oversee the expression levels of TrxR and Prx6 at mRNA and protein level. Discussion: PEGylated CeNPs are being considered for several applications based on their unique antioxidant properties. The work described in this paper demonstrates that PEGylated CeNPs can act as universal ROS scavenger in reduced concentration GSH, which is a dominant cellular antioxidant molecule in form of a reduced thiol. We also delineate that how these PEGylated CeNPs could mechanistically work as an antioxidant in the complex cellular milieu. It has been shown that CeNPs can scavenge reactive oxygen and nitrogen species , thus prevent the damage in cells 54. Since, depletion in cellular GSH level has been correlated with several disease conditions 33, therefore, if an inorganic antioxidant could act as a suitable alternative, it would be of a great interest. We have shown that BSO treatment leads to the decrease in cell viability that was successfully circumvented by PEGylated CeNPs. Further, in the quest for identifying the mechanism involved, we first substantiated the level of free radicals in cells upon BSO treatment, and it was found that PEGylated CeNPs pre-treatment suppressed the free radicals in the cytoplasm. This suggests that PEGylation CeNPs protect cells from the damage caused by ROS generated due to BSO exposure. Recently, it has also been shown that CeNPs are nearly homogenously present in the cytoplasm after internalization in HaCaT cells 55, which could be a possible reason for efficient ROS scavenging property shown by PEGylated CeNPs. The interaction of PEGylated CeNPs with inorganic salts and other components of cells and tissues could also be a considerable factor to influence the antioxidant nature. In this context, Singh et al11 and others 56 have shown that bare CeNPs show selective interaction with the inorganic phosphate anions but not with sulphate and carbonate anions, under in vitro experimental condition. However, PEGylation could avoid such an interaction and other protein adsorption/desorption processes over the nanoparticle surface23. Further, genotoxicity induced by nanomaterials have also been studied by several groups in detail and suggested that bare
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nanoparticles could potentially induce more toxicity than surface coated with PEG or other biocompatible molecules and polymers 57. In our study, PEGylated CeNPs did not show any sign of genotoxicity or nuclear fragmentation, studied by CBMN formation method. We also studied the alteration in expression of protein level in treated cells. Thioredoxin (Trx) plays an important antioxidant role in mammalian cells. Trx reduces the 2-cys Peroxiredoxin and during in this process, Trx itself gets oxidized51. Further, TrxR reverses the action and catalyses the reduction of oxidized Trx through NADPH dependent mechanism 58. As expected, increase in TrxR level occurs due to the decrease in GSH level after BSO treatment, suggesting the production of ROS in cells. However, in presence of PEGylated CeNPs, TrxR level is restored back to the normal level, probably due to the scavenging of the free radicals by PEGylated CeNPs. Since, after pretreatment with PEGylated CeNPs and BSO treatment, the level of GSH in cells does not improve, which suggest that the likely mechanism is that the ROS is are scavenged by internalized PEGylated CeNPs, therefore, the level of TrxR does not increase even during the low levels of GSH. Prx6 has direct correlation with the GSH; therefore, level of Prx6 decreases in relation with GSH after BSO treatment. Further, cells exposed to PEGylated CeNPs alone did not alter the expression of Prx6. Conclusion: PEGylated CeNPs show ROS scavenging activity even in the reduced concentration of known cellular antioxidant molecule, GSH. CeNPs prevented the free-radical insult induced by BSO and scavenged ROS homogeneously from the cell cytoplasm. PEGylated CeNPs also protected the cells from genotoxicity caused by BSO exposure. PEGylated CeNPs did not increase the expression of cellular antioxidant molecule (GSH) or proteins (TrxR, Prx6) suggesting that CeNPs itself interact with free radicals and neutralize their effect analogous to naturally-occurring enzymes. These results, combined with other increasing well documented evidence suggest that CeNPs efficiently scavenge multiple ROS in the biological system, further suggest that CeNPs is a promising unique nanomaterial for applications in biology and medicines.
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Acknowledgements: R. Singh would like to thank Department of Science and Technology, New Delhi for providing INSPIRE Senior Research Fellowship (SRF). The financial assistance for the Centre for Nanotechnology Research and Applications (CENTRA) by The Gujarat Institute for Chemical Technology (GICT) is acknowledged. The funding from the Department of Science and Technology - Science and Engineering Research Board (SERB) (Grant No.: ILS/SERB/201516/01) to Dr Sanjay Singh under the scheme of Start-Up Research Grant (Young Scientists) in Life Sciences is also gratefully acknowledged. This manuscript carries ILS communication number ILS-058. Conflict of Interest: Authors declare no conflict of interest. References: 1. Kuo, W. S.; Chang, Y. T.; Cho, K. C.; Chiu, K. C.; Lien, C. H.; Yeh, C. S.; Chen, S. J., Gold nanomaterials conjugated with indocyanine green for dual-modality photodynamic and photothermal therapy. Biomaterials 2012, 33, 3270-3278. 2. Jhaveri, A.; Deshpande, P.; Torchilin, V., Stimuli-sensitive nanopreparations for combination cancer therapy. J. Control Release 2014, 190, 352-370. 3. Akhtar, M. J.; Ahamed, M.; Alhadlaq, H. A.; Alrokayan, S. A.; Kumar, S., Targeted anticancer therapy: overexpressed receptors and nanotechnology. Clin. Chim. Acta. 2014, 436, 78-92. 4. Singh, A. V.; Khare, M.; Gade, W. N.; Zamboni, P., Theranostic implications of nanotechnology in multiple sclerosis: a future perspective. Autoimmune Dis. 2012, 160830. 5. Singh, A. V.; Sitti, M., Targeted Drug Delivery and Imaging Using Mobile Milli/Microrobots: A Promising Future Towards Theranostic Pharmaceutical Design. Curr. Pharm. Des. 2016, 22, 1418-1428. 6. Singh, A. V.; Vyas, V.; Montani, E.; Cartelli, D.; Parazzoli, D.; Oldani, A.; Zeri, G.; Orioli, E.; Gemmati, D.; Zamboni, P., Investigation of in vitro cytotoxicity of the redox state of ionic iron in neuroblastoma cells. J. Neurosci. Rural Pract. 2012, 3, 301-310. 7. Valko, M.; Morris, H.; Cronin, M. T., Metals, toxicity and oxidative stress. Curr. Med. Chem. 2005, 12, 1161-1208. 8. Karakoti, A. S.; Monteiro-Riviere, N. A.; Aggarwal, R.; Davis, J. P.; Narayan, R. J.; Self, W. T.; McGinnis, J.; Seal, S., Nanoceria as Antioxidant: Synthesis and Biomedical Applications. Jom (1989). 2008, 60, 33-37. 9. Rubio, L.; Annangi, B.; Vila, L.; Hernandez, A.; Marcos, R., Antioxidant and anti -genotoxic properties of cerium oxide nanoparticles in a pulmonary-like cell system. Arch. Toxicol. 2016, 90, 269-278. 10. Chen, J.; Patil, S.; Seal, S.; McGinnis, J. F., Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides. Nat. Nanotechnol. 2006, 1, 142-150. 11. Singh, R.; Singh, S., Role of phosphate on stability and catalase mimetic activity of cerium oxide nanoparticles. Colloids Surf. B Biointerfaces 2015, 132, 78-84.
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12. Pirmohamed, T.; Dowding, J. M.; Singh, S.; Wasserman, B.; Heckert, E.; Karakoti, A. S.; King, J. E.; Seal, S.; Self, W. T., Nanoceria exhibit redox state-dependent catalase mimetic activity. Chem. Commun (Camb). 2010, 46, 2736-2738. 13. Heckert, E. G.; Karakoti, A. S.; Seal, S.; Self, W. T., The role of cerium redox state in the SOD mimetic activity of nanoceria. Biomaterials 2008, 29, 2705-2709. 14. Korsvik, C.; Patil, S.; Seal, S.; Self, W. T., Superoxide dismutase mimetic properties exhibited by vacancy engineered ceria nanoparticles. Chem. Commun (Camb). 2007, 1056-1058. 15. Li, Y.; He, X.; Yin, J. J.; Ma, Y.; Zhang, P.; Li, J.; Ding, Y.; Zhang, J.; Zhao, Y.; Chai, Z.; Zhang, Z., Acquired superoxide-scavenging ability of ceria nanoparticles. Angew. Chem. Int. Ed. Engl. 2015, 54, 18321835. 16. Heckman, K. L.; DeCoteau, W.; Estevez, A.; Reed, K. J.; Costanzo, W.; Sanford, D.; Leiter, J. C.; Clauss, J.; Knapp, K.; Gomez, C.; Mullen, P.; Rathbun, E.; Prime, K.; Marini, J.; Patchefsky, J.; Patchefsky, A. S.; Hailstone, R. K.; Erlichman, J. S., Custom cerium oxide nanoparticles protect against a free radical mediated autoimmune degenerative disease in the brain. ACS Nano 2013, 7, 10582-10596. 17. Estevez, A. Y.; Erlichman, J. S., The potential of cerium oxide nanoparticles (nanoceria) for neurodegenerative disease therapy. Nanomedicine (Lond) 2014, 9, 1437-1440. 18. Ciofani, G.; Genchi, G. G.; Liakos, I.; Cappello, V.; Gemmi, M.; Athanassiou, A.; Mazzolai, B.; Mattoli, V., Effects of cerium oxide nanoparticles on PC12 neuronal -like cells: proliferation, differentiation, and dopamine secretion. Pharm. Res. 2013, 30, 2133-2145. 19. Nelson, B.; Johnson, M.; Walker, M.; Riley, K.; Sims, C., Antioxidant Cerium Oxide Nanoparticles in Biology and Medicine. Antioxidants 2016, 5, 15. 20. Xu, C.; Qu, X., Cerium oxide nanoparticle: a remarkably versatile rare earth nanomaterial for biological applications. NPG Asia Mater. 2014, 6, e90. 21. Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S.; Yan, X., Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2007, 2, 577-583. 22. 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. 23. Karakoti, A. S.; Singh, S.; Kumar, A.; Malinska, M.; Kuchibhatla, S. V.; Wozniak, K.; Self, W. T.; Seal, S., PEGylated nanoceria as radical scavenger with tunable redox chemistry. J. Am. Chem. Soc. 2009, 131, 14144-14145. 24. Townsend, D. M.; Tew, K. D.; Tapiero, H., The importance of glutathione in human disease. Biomed. Pharmacother. 2003, 57, 145-155. 25. Herzenberg, L. A.; De Rosa, S. C.; Dubs, J. G.; Roederer, M.; Anderson, M. T.; Ela, S. W.; Deresinski, S. C.; Herzenberg, L. A., Glutathione deficiency is associated with impaired survival in HIV disease. Proc. Natl. Acad. Sci. U S A. 1997, 94, 1967-1972. 26. Traverso, N.; Ricciarelli, R.; Nitti, M.; Marengo, B.; Furfaro, A. L.; Pronzato, M. A.; Marinari, U. M.; Domenicotti, C., Role of glutathione in cancer progression and chemoresistance. Oxid. Med. Cell Longev. 2013, 972913. 27. Singh, A. V.; Mehta, K. K.; Worley, K.; Dordick, J. S.; Kane, R. S.; Wan, L. Q., Carbon nanotubeinduced loss of multicellular chirality on micropatterned substrate is mediated by oxidative stress. ACS Nano 2014, 8, 2196-2205. 28. Halliday, G. M.; Damian, D. L.; Rana, S.; Byrne, S. N., The suppressive effects of ultraviolet radiation on immunity in the skin and internal organs: implications for autoimmunity. J. Dermatol. Sci. 2012, 66, 176-182. 29. Xia, T.; Kovochich, M.; Liong, M.; Madler, L.; Gilbert, B.; Shi, H.; Yeh, J. I.; Zink, J. I.; Nel, A. E., Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2008, 2, 2121-2134. 21 ACS Paragon Plus Environment
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30. Arya, A.; Sethy, N. K.; Singh, S. K.; Das, M.; Bhargava, K., Cerium oxide nanoparticles protect rodent lungs from hypobaric hypoxia-induced oxidative stress and inflammation. Int. J. Nanomedicine 2013, 8, 4507-4520. 31. Griffith, O. W., Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. J. Biol. Chem. 1982, 257, 13704-13712. 32. Reliene, R.; Schiestl, R. H., Glutathione depletion by buthionine sulfoximine induces DNA deletions in mice. Carcinogenesis 2006, 27, 240-244. 33. Meister, A., Glutathione deficiency produced by inhibition of its synthesis, and its reversal; applications in research and therapy. Pharmacol. Ther. 1991, 51, 155-194. 34. Suzuki, H.; Toyooka, T.; Ibuki, Y., Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis. Environ. Sci. Technol. 2007, 41, 3018-3024. 35. Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55-63. 36. Borenfreund, E.; Puerner, J. A., Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 1985, 24, 119-124. 37. Wan, C. P.; Myung, E.; Lau, B. H., An automated micro-fluorometric assay for monitoring oxidative burst activity of phagocytes. J. Immunol. Methods 1993, 159, 131-138. 38. Rabelo, L.; Monteiro, N.; Serquiz, R.; Santos, P.; Oliveira, R.; Oliveira, A.; Rocha, H.; Morais, A. H.; Uchoa, A.; Santos, E., A lactose-binding lectin from the marine sponge Cinachyrella apion (Cal) induces cell death in human cervical adenocarcinoma cells. Mar. Drugs 2012, 10, 727-743. 39. Fenech, M., The in vitro micronucleus technique. Mutat. Res. 2000, 455, 81-95. 40. Nusse, M.; Kramer, J., Flow cytometric analysis of micronuclei found in cells after irradiation. Cytometry 1984, 5, 20-25. 41. Babitha, K. K.; Sreedevi, A.; Priyanka, K. P.; Sabu, B.; Varghese, T., Structural characterization and optical studies of CeO2 nanoparticles synthesized by chemical precipitation. Indian Journal of Pure & Applied Physics (IJPAP). 2015, 53, 596-603. 42. Zamiri, R.; Abbastabar Ahangar, H.; Kaushal, A.; Zakaria, A.; Zamiri, G.; Tobaldi, D.; Ferreira, J. M., Correction: Dielectrical Properties of CeO2 Nanoparticles at Different Temperatures. PLoS One 2015, 10, e0131851. 43. James, M. A., Biological Responses to Materials. Annual Review of Materials Research 2001, 31, 81-110. 44. Zucker, R. M.; Daniel, K. M., Detection of TiO2 nanoparticles in cells by flow cytometry. Methods Mol. Biol. 2012, 906, 497-509. 45. Armstrong, J. S.; Steinauer, K. K.; Hornung, B.; Irish, J. M.; Lecane, P.; Birrell, G. W.; Peehl, D. M.; Knox, S. J., Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. Cell Death Differ. 2002, 9, 252-263. 46. Singh, S.; Sharma, A.; Robertson, G. P., Realizing the clinical potential of cancer nanotechnology by minimizing toxicologic and targeted delivery concerns. Cancer Res. 2012, 72, 5663-5668. 47. Seager, A. L.; Shah, U. K.; Mikhail, J. M.; Nelson, B. C.; Marquis, B. J.; Doak, S. H.; Johnson, G. E.; Griffiths, S. M.; Carmichael, P. L.; Scott, S. J.; Scott, A. D.; Jenkins, G. J., Pro-oxidant induced DNA damage in human lymphoblastoid cells: homeostatic mechanisms of genotoxic tolerance. Toxicol. Sci. 2012, 128, 387-397. 48. Liang, C.-H.; Wang, G.-H.; Liaw, C.-C.; Lee, M.-F.; Wang, S.-H.; Cheng, D.-L.; Chou, T.-H., Extracts from Cladiella australis, Clavularia viridis and Klyxum simplex (Soft Corals) are Capable of Inhibiting the Growth of Human Oral Squamous Cell Carcinoma Cells. Marine Drugs 2008, 6, 595.
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49. Vafa, O.; Wade, M.; Kern, S.; Beeche, M.; Pandita, T. K.; Hampton, G. M.; Wahl, G. M., c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function: a mechanism for oncogene-induced genetic instability. Mol. Cell 2002, 9, 1031-1044. 50. Rhee, S. G.; Kang, S. W.; Chang, T. S.; Jeong, W.; Kim, K., Peroxiredoxin, a novel family of peroxidases. IUBMB Life 2001, 52, 35-41. 51. Hofmann, B.; Hecht, H. J.; Flohe, L., Peroxiredoxins. Biol Chem. 2002, 383, 347-364. 52. Manevich, Y.; Fisher, A. B., Peroxiredoxin 6, a 1-Cys peroxiredoxin, functions in antioxidant defense and lung phospholipid metabolism. Free Radic Biol Med. 2005, 38, 1422-1432. 53. Manevich, Y.; Feinstein, S. I.; Fisher, A. B., Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with pi GST. Proc. Natl. Acad. Sci. U S A. 2004, 101, 3780-3785. 54. Karakoti, A.; Singh, S.; Dowding, J. M.; Seal, S.; Self, W. T., Redox -active radical scavenging nanomaterials. Chem. Soc. Rev. 2010, 39, 4422-4432. 55. Singh, S.; Kumar, A.; Karakoti, A.; Seal, S.; Self, W. T., Unveiling the mechanism of uptake and subcellular distribution of cerium oxide nanoparticles. Mol. Biosyst. 2010, 6, 1813-1820. 56. McCormack, R. N.; Mendez, P.; Barkam, S.; Neal, C. J.; Das, S.; Seal, S., Inhibition of Nanoceria’s Catalytic Activity due to Ce3+ Site-Specific Interaction with Phosphate Ions. The Journal of Physical Chemistry C 2014, 118, 18992-19006. 57. Karakoti, A. S.; Das, S.; Thevuthasan, S.; Seal, S., PEGylated Inorganic Nanoparticles. Angewandte Chemie. International Edition. 2011, 50, 1980-1994. 58. Karlenius, T. C.; Tonissen, K. F., Thioredoxin and Cancer: A Role for Thioredoxin in all States of Tumor Oxygenation. Cancers (Basel) 2010, 2, 209-232.
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Figure 1. PEGylated CeNPs exhibiting SOD mimetic activity are efficiently internalized by HaCaT cells. (A) SOD mimetic activity of CeNPs suspended in water (aq.) and DMEM: F12 was measured by the reduction of ferri-cytochrome C by super-oxides and measuring the absorbance at 550 nm. (B) Internalization of PEGylated CeNPs in HaCaT cells, exposed for 6 hrs, was measured by flow cytometer. The increase in SSC intensity with nanoparticle concentration has been plotted as fold uptake. Data expressed as standard error (SE) calculated from three (n=3) independent experiments. *p
