Evaluation of the Cytotoxicity and Oxidative Stress Response of CeO2-RGO Nanocomposites in Human Lung Epithelial A549 Cells

Graphene-based nanocomposites have attracted enormous interest in nanomedicine and environmental remediation, owing to their unique characteristics. The increased production and widespread application of these nanocomposites might raise concern about their adverse health effects. In this study, for the first time, we examine the cytotoxicity and oxidative stress response of a relatively new nanocomposite of cerium oxide-reduced graphene oxide (CeO2-RGO) in human lung epithelial (A549) cells. CeO2-RGO nanocomposites and RGO were prepared by a simple hydrothermal method and characterized by relevant analytical techniques. Cytotoxicity data have shown that RGO significantly induces toxicity in A549 cells, evident by cell viability reduction, membrane damage, cell cycle arrest, and mitochondrial membrane potential loss. However, CeO2-RGO nanocomposites did not cause statistically significant toxicity as compared to a control. We further observed that RGO significantly induces reactive oxygen species generation and reduces glutathione levels. However, CeO2-RGO nanocomposites did not induce oxidative stress in A549 cells. Interestingly, we observed that CeO2 nanoparticles (NPs) alone significantly increase glutathione (GSH) levels in A549 cells as compared to a control. The GSH replenishing potential of CeO2 nanoparticles could be one of the possible reasons for the biocompatible nature of CeO2-RGO nanocomposites. Our data warrant further and more advanced research to explore the biocompatibility/safety mechanisms of CeO2-RGO nanocomposites in different cell lines and animal models.


Introduction
Graphene is a single atom thick two-dimensional sheet of sp 2 hybridized carbon atoms with multifaceted properties, such as elasticity, flexibility, high conductivity, and ease of functionalization [1,2]. Due to these properties, graphene has attracted enormous interest in synthetic biology and nanomedicine, besides its applications in photonics, optoelectronics, and environmental remediation [3][4][5]. Despite the great potential of graphene in biomedical applications, its unmodified state has poor solubility in biological media, which makes nanomedicine study in this context complicated [6,7]. To overcome this problem, one of the most successful approaches has been the use of graphene derivatives such as graphene oxide (GO) and reduced graphene oxide (RGO). GO can be prepared by the oxidation of graphite, and RGO is the reduced form of GO [8]. GO and RGO exhibit excellent dispersibility and stability in physiological media because of their oxygenated functional groups [9].

Toxicity Experiments
Cells were treated for 24 h to CeO 2 NPs, RGO and CeO 2 -RGO nanocomposites at a concentration of 50 µg/mL. Cell viability was measured by MTT and neutral red uptake (NRU) assays, with some specific changes [30]. The MTT assay is based the principle that live cells have the ability to reduce MTT in blue formazan products that are dissolved in a solvent. The absorbance of this product was measured at 570 nm using a microplate reader (Synergy-HT Biotek, Winooski, VT, USA). In the NRU assay, live cells have ability to bind neutral red (NR) dye with lysosomes. The absorbance of the NR probe was determined at 540 nm using a microplate reader (Synergy-HT, Biotek). The lactate dehydrogenase (LDH) enzyme has the ability to oxidize lactate into pyruvate. The LDH enzyme resides in cytoplasm and leaks into a culture medium when the cell membrane is damaged. LDH leakage was measured by a BioVision kit (Milpitas, CA, USA), and the detailed procedure here has been reported in our earlier paper [31]. A propidium iodide (PI) probe was applied to determine the cell cycle phases using a flow cytometer (Coulter Epics XL/XI-MCL) via FL2 filter (585 nm) [32]. Caspase-3 enzyme activity was measured using a BioVision colorimetric kit. Mitochondrial membrane potential (MMP) was examined by a rhodamine-123 (Rh-123) probe [32]. The cationic fluorochrome Rh-123 binds to mitochondria of live cells in a membrane potential-dependent manner. The MMP level was measured by two different methods, namely, quantitative analysis by a microplate reader (Synergy-HT, Biotek) and qualitative examination by a fluorescent microscope (DMi8, Leica Microsystems, Wetzlar, Germany). A dichlorofluorescin diacetate (DCFH-DA) probe was employed to assess the intracellular level of reactive oxygen species (ROS), as reported earlier [32]. DCFH-DA passively enters the cells and reacts with ROS to form a fluorescent compound called dichlorofluorescein (DCF). The fluorescent intensity of DCF was determined by two different methods, namely, quantitative analysis by a microplate reader (Synergy-HT, Biotek) and qualitative assessment by a fluorescent microscope (DMi8, Leica Microsystems, Germany). Hydrogen peroxide (H 2 O 2 ) generation in cells was measured by a commercial kit (Sigma-Aldrich, St. Louis, Missouri, USA). The glutathione (GSH) level was estimated by Ellman's procedure, using 5,5-dithio-bis-nitrobenzoic acid (DTNB) [33]. Protein content was estimated by Bradford's method [34].

Statistical Analysis
A one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test, was employed for the statistical analysis of the toxicity results. Here, p < 0.05 was assigned as statistical significance.

Statistical Analysis
A one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test, was employed for the statistical analysis of the toxicity results. Here, p < 0.05 was assigned as statistical significance.

Characterization of CeO2 NPs, RGO and CeO2-RGO Nanocomposites
To examine the crystal structure of the prepared samples, XRD patterns of the RGO, CeO2 NPs, and CeO2-RGO nanocomposites were recorded ( Figure 1). The XRD profile of RGO demonstrated an intense reflection plane (002) at 2θ = 24.92. The presence of another diffraction peak at 2θ = 42.97, which is attributed to the (100) plane, suggests the polycrystalline nature of the prepared RGO. The diffraction peaks in the XRD pattern of the prepared CeO2 NPs were also recorded at 2θ values of 28.87°, 33.32°, 47.68°, 56.57°, 59.37°, and 69.79°, which correspond to the (111), (200), (220), (311), (222) and (400) crystal planes, respectively. All the peaks were indexed to the pure cubic fluorite-type structure of CeO2 (JCPDS: 75-0076) [35]. In the XRD pattern of the CeO2-RGO nanocomposite, two additional peaks of RGO [(002) and (100)] were also found with all the peaks of pure CeO2 NPs ( Figure 1). These results indicated that CeO2 NPs were fairly anchored on RGO nanosheets. The average crystallite sizes of CeO2 NPs and CeO2-RGO nanocomposites were estimated by Scherrer's formula, using the prominent peak (111), and it was found to be around 18 and 16 nm for CeO2 NPs and CeO2-RGO nanocomposites, respectively. Our XRD data on RGO, CeO2 NPs, and CeO2-RGO nanocomposites were according to earlier reports [36,37]. X-ray photoelectron spectroscopy (XPS) was done to further analyze the chemical composition of CeO2-RGO nanocomposites. The presence of C (C1s), O (O1s), and Ce (Ce 3d) elements in the XPS analysis suggests that the CeO2-RGO nanocomposite was successfully prepared. The peaks that appeared at 888.9, 895.1, and 903.9 eV correspond to the components of Ce3d5/2, while the X-ray photoelectron spectroscopy (XPS) was done to further analyze the chemical composition of CeO 2 -RGO nanocomposites. The presence of C (C1s), O (O1s), and Ce (Ce 3d) elements in the XPS analysis suggests that the CeO 2 -RGO nanocomposite was successfully prepared. The peaks that appeared at 888.9, 895.1, and 903.9 eV correspond to the components of Ce3d5/2, while the remaining signals of Ce3d3/2 components could be seen at 906.9, 913.6 and 923.1 eV (Figure 2A) [35,38]. The XPS spectra of C1s given in Figure 2B contain a non-oxygenated aromatic sp 2 bonded carbon ring (C-C) Nanomaterials 2019, 9,1709 5 of 14 at 286.5 eV [39]. Figure 2C represents the O1s spectra of CeO 2 , with two intense peaks at 530.9 and 536.8 eV, linked with anionic oxygen in CeO 2 and the presence of the residual oxygen functional group in the RGO sheets, respectively [37]. Nanomaterials 2019, 9, x FOR PEER REVIEW 5 of 15 remaining signals of Ce3d3/2 components could be seen at 906.9, 913.6 and 923.1 eV (Figure 2A) [35,38]. The XPS spectra of C1s given in Figure 2B contain a non-oxygenated aromatic sp 2 bonded carbon ring (C-C) at 286.5 eV [39]. Figure 2C represents the O1s spectra of CeO2, with two intense peaks at 530.9 and 536.8 eV, linked with anionic oxygen in CeO2 and the presence of the residual oxygen functional group in the RGO sheets, respectively [37]. The morphologies of the CeO2 NPs, RGO, and CeO2-RGO nanocomposites were further characterized by SEM and TEM ( Figure 3A-F). Figure 2A,D represents the TEM and SEM morphology of CeO2 NPs, which show some level of agglomeration. The SEM and TEM images of RGO ( Figure 2B,E) indicate the formation of few layers of RGO, with visible wrinkles and a silky morphology because of their high aspect ratio. The SEM and TEM micrographs of CeO2-RGO nanocomposites ( Figure 2C,F) indicate that the CeO2 NPs were well anchored on the sheets of RGO. It is also noticeable from the SEM and TEM images that the CeO2-RGO nanocomposites still maintained layered structures, although some distinct wrinkles disappeared [36].  Figure 2C,F) indicate that the CeO 2 NPs were well anchored on the sheets of RGO. It is also noticeable from the SEM and TEM images that the CeO 2 -RGO nanocomposites still maintained layered structures, although some distinct wrinkles disappeared [36].

Cytotoxicity
As we can see in Figure 4A, the CeO2 NPs were not toxic to A549 cells up to the concentration of 100 µg/mL. The non-cytotoxic nature of CeO2 NPs against human breast cancer (MCF-7) and human fibroblast (HT1080) cells was also observed in our previous work [21]. However, RGO was found to induce dose-dependent cytotoxicity in A549 cells in the concentration range of 25-100 µg/mL ( Figure  4B). Below the concentration of 25 µg/mL, RGO was also non-cytotoxic. Our results were according to other recent reports that demonstrated the cytotoxic potential of RGO in different types of human cells [6,7]. However, Bengtson et al. [13] reported that GO and RGO did not produce cytotoxicity and genotoxicity to murine lung epithelial (FE1) cells. These contrasting results could be due to the use of different cell lines, different preparation methods, and the manipulation of physicochemical properties. Interestingly, CeO2-RGO nanocomposites did not induce cytotoxicity to A549 cells in all selected concentration ranges (1-100 µg/mL) ( Figure 4C).

Cytotoxicity
As we can see in Figure 4A, the CeO 2 NPs were not toxic to A549 cells up to the concentration of 100 µg/mL. The non-cytotoxic nature of CeO 2 NPs against human breast cancer (MCF-7) and human fibroblast (HT1080) cells was also observed in our previous work [21]. However, RGO was found to induce dose-dependent cytotoxicity in A549 cells in the concentration range of 25-100 µg/mL ( Figure 4B). Below the concentration of 25 µg/mL, RGO was also non-cytotoxic. Our results were according to other recent reports that demonstrated the cytotoxic potential of RGO in different types of human cells [6,7]. However, Bengtson et al. [13] reported that GO and RGO did not produce cytotoxicity and genotoxicity to murine lung epithelial (FE1) cells. These contrasting results could be due to the use of different cell lines, different preparation methods, and the manipulation of physicochemical properties. Interestingly, CeO 2 -RGO nanocomposites did not induce cytotoxicity to A549 cells in all selected concentration ranges (1-100 µg/mL) ( Figure 4C). The toxicity of GO and RGO has been observed in different types of cells, including human breast cancer (MCF-7), liver cancer, and (HepG2) and lung (A549) cells, all with significant cytotoxicity detected at a concentration of around 50 µg/mL [10,12]. Hence, we have chosen one concentration (50 µg/mL) of CeO2 NPs, RGO, and CeO2-RGO nanocomposites for further experiments to explore the toxicity mechanisms of these nanoscale materials. The A549 cells were exposed for 24 h to 50 µg/mL of CeO2 NPs, RGO, and CeO2-RGO nanocomposites, and cytotoxicity was assessed by MTT, NRU, and LDH assays. As we can see in Figure 5A,B, the CeO2 NPs did not reduce cell viability. However, cell viability was significantly decreased in the RGO-treated group as compared to the control group. Interestingly, compared to the RGO group, in the CeO2-RGO nanocomposites group, cell viability was significantly increased to the level that was not much less than the CeO2 NPs or control groups ( Figure 5A,B). Cytosolic enzyme LDH leakage into the culture medium is an indicator of cell membrane damage. Our results demonstrated that CeO2 NPs did not cause LDH leakage, however RGO significantly induced LDH leakage in the culture medium of A549 cells. Besides, compared to RGO group, in the CeO2-RGO nanocomposite group, leakage of the LDH enzyme was significantly reduced to a level that was almost close to the CeO2 NPs or the control group ( Figure 5C). These results indicated that CeO2-RGO nanocomposites induce much less cytotoxicity to A549 cells then RGO. The toxicity of GO and RGO has been observed in different types of cells, including human breast cancer (MCF-7), liver cancer, and (HepG2) and lung (A549) cells, all with significant cytotoxicity detected at a concentration of around 50 µg/mL [10,12]. Hence, we have chosen one concentration (50 µg/mL) of CeO 2 NPs, RGO, and CeO 2 -RGO nanocomposites for further experiments to explore the toxicity mechanisms of these nanoscale materials. The A549 cells were exposed for 24 h to 50 µg/mL of CeO 2 NPs, RGO, and CeO 2 -RGO nanocomposites, and cytotoxicity was assessed by MTT, NRU, and LDH assays. As we can see in Figure 5A,B, the CeO 2 NPs did not reduce cell viability. However, cell viability was significantly decreased in the RGO-treated group as compared to the control group. Interestingly, compared to the RGO group, in the CeO 2 -RGO nanocomposites group, cell viability was significantly increased to the level that was not much less than the CeO 2 NPs or control groups ( Figure 5A,B). Cytosolic enzyme LDH leakage into the culture medium is an indicator of cell membrane damage. Our results demonstrated that CeO 2 NPs did not cause LDH leakage, however RGO significantly induced LDH leakage in the culture medium of A549 cells. Besides, compared to RGO group, in the CeO 2 -RGO nanocomposite group, leakage of the LDH enzyme was significantly reduced to a level that was almost close to the CeO 2 NPs or the control group ( Figure 5C). These results indicated that CeO 2 -RGO nanocomposites induce much less cytotoxicity to A549 cells then RGO.

Apoptosis
Apoptotic cell death is a highly regulated process for the removal of irreparable damaged cells to maintain tissue homeostasis [40]. The process of apoptosis can be triggered by several factors, including nutrient deficiency, growth factors, and environmental contaminants [41]. GO and RGO have shown potential to induce apoptosis in human cells [7,18,19,26]. Ali et al. [18] also reported that Ag-doped RGO triggered apoptosis in human hepatic normal and carcinoma cells. Hence, we further examined the apoptotic potential of the prepared materials in A549 cells. Cell cycle phases were analyzed in A549 cells after exposure for 24 h to 50 µg/mL of the CeO2 NPs, RGO, and CeO2-RGO nanocomposites. Cells with damaged DNA are accumulated in the G1 (gap1), S (DNA synthesis, or in G2/M (gap2/mitosis) phases for DNA repair. However, cells with irreparable DNA damage are directed to undergo apoptosis and are anticipated to accumulate in the sub-G1 phase [42]. Our flow cytometer data indicated that RGO induced apoptosis in A549 cells, however, the apoptotic response of the CeO2-RGO nanocomposites was indeed much lower than RGO. The CeO2 NPs also did not induce apoptosis in A549 cells. Cells accumulated in all phases of the cell cycle were almost similar in the control and CeO2 NPs groups. However, cell accumulation in sub-G1 phase of the RGO group was significantly higher (6.88%) than those of the control group (4.16%) (p < 0.05) ( Figure 6A). Interestingly, the number of cells accumulated in the sub-G1 phase in CeO2-RGO nanocomposites was much lower (4.32%) than the RGO group (6.88%) and was almost similar to the control group (4.16%) ( Figure 6A). The caspase-3 enzyme also plays a significant role in apoptotic pathway. Our results demonstrate that the activity of caspase-3 enzyme is similar in both the CeO2 NPs and the control group. However, caspase-3 enzyme activity was significantly higher in the RGO group than that of control group. Interestingly, we observed that the activity of the caspase-3 enzyme in the CeO2-RGO nanocomposite group was much lower than in RGO and was almost similar to the control group ( Figure 6B).

Apoptosis
Apoptotic cell death is a highly regulated process for the removal of irreparable damaged cells to maintain tissue homeostasis [40]. The process of apoptosis can be triggered by several factors, including nutrient deficiency, growth factors, and environmental contaminants [41]. GO and RGO have shown potential to induce apoptosis in human cells [7,18,19,26]. Ali et al. [18] also reported that Ag-doped RGO triggered apoptosis in human hepatic normal and carcinoma cells. Hence, we further examined the apoptotic potential of the prepared materials in A549 cells. Cell cycle phases were analyzed in A549 cells after exposure for 24 h to 50 µg/mL of the CeO 2 NPs, RGO, and CeO 2 -RGO nanocomposites. Cells with damaged DNA are accumulated in the G1 (gap1), S (DNA synthesis, or in G2/M (gap2/mitosis) phases for DNA repair. However, cells with irreparable DNA damage are directed to undergo apoptosis and are anticipated to accumulate in the sub-G1 phase [42]. Our flow cytometer data indicated that RGO induced apoptosis in A549 cells, however, the apoptotic response of the CeO 2 -RGO nanocomposites was indeed much lower than RGO. The CeO 2 NPs also did not induce apoptosis in A549 cells. Cells accumulated in all phases of the cell cycle were almost similar in the control and CeO 2 NPs groups. However, cell accumulation in sub-G1 phase of the RGO group was significantly higher (6.88%) than those of the control group (4.16%) (p < 0.05) ( Figure 6A). Interestingly, the number of cells accumulated in the sub-G1 phase in CeO 2 -RGO nanocomposites was much lower (4.32%) than the RGO group (6.88%) and was almost similar to the control group (4.16%) ( Figure 6A). The caspase-3 enzyme also plays a significant role in apoptotic pathway. Our results demonstrate that the activity of caspase-3 enzyme is similar in both the CeO 2 NPs and the control group. However, caspase-3 enzyme activity was significantly higher in the RGO group than that of control group. Interestingly, we observed that the activity of the caspase-3 enzyme in the CeO 2 -RGO nanocomposite group was much lower than in RGO and was almost similar to the control group ( Figure 6B). The mitochondrial membrane potential (MMP) level is a hallmark of apoptosis [43]. MMP loss is also an indicator that cells are going to die through the apoptotic pathway. Hence, we further examined the MMP level in A549 cells against CeO2 NPs, RGO, and CeO2-RGO nanocomposites exposure at a concentration of 50 µg/mL for 24 h. The quantitative data demonstrated that the MMP level in CeO2 NPs and control group was similar. However, in RGO group, MMP level was significantly lower as compared to the control group. In addition, the MMP level in CeO2-RGO nanocomposites were much higher than those of the RGO group and were almost close to the control group ( Figure 7A). Fluorescent microscopy data also suggested that the brightness of the Rh-123 probe was almost similar in the CeO2 NPs, CeO2-RGO nanocomposite, and control groups, while much lower in the RGO group ( Figure 7B). These results suggest that the CeO2-RGO nanocomposite induced much less of a apoptotic response in A549 cells than those of the RGO nanosheets.  The mitochondrial membrane potential (MMP) level is a hallmark of apoptosis [43]. MMP loss is also an indicator that cells are going to die through the apoptotic pathway. Hence, we further examined the MMP level in A549 cells against CeO 2 NPs, RGO, and CeO 2 -RGO nanocomposites exposure at a concentration of 50 µg/mL for 24 h. The quantitative data demonstrated that the MMP level in CeO 2 NPs and control group was similar. However, in RGO group, MMP level was significantly lower as compared to the control group. In addition, the MMP level in CeO 2 -RGO nanocomposites were much higher than those of the RGO group and were almost close to the control group ( Figure 7A). Fluorescent microscopy data also suggested that the brightness of the Rh-123 probe was almost similar in the CeO 2 NPs, CeO 2 -RGO nanocomposite, and control groups, while much lower in the RGO group ( Figure 7B). These results suggest that the CeO 2 -RGO nanocomposite induced much less of a apoptotic response in A549 cells than those of the RGO nanosheets. The mitochondrial membrane potential (MMP) level is a hallmark of apoptosis [43]. MMP loss is also an indicator that cells are going to die through the apoptotic pathway. Hence, we further examined the MMP level in A549 cells against CeO2 NPs, RGO, and CeO2-RGO nanocomposites exposure at a concentration of 50 µg/mL for 24 h. The quantitative data demonstrated that the MMP level in CeO2 NPs and control group was similar. However, in RGO group, MMP level was significantly lower as compared to the control group. In addition, the MMP level in CeO2-RGO nanocomposites were much higher than those of the RGO group and were almost close to the control group ( Figure 7A). Fluorescent microscopy data also suggested that the brightness of the Rh-123 probe was almost similar in the CeO2 NPs, CeO2-RGO nanocomposite, and control groups, while much lower in the RGO group ( Figure 7B). These results suggest that the CeO2-RGO nanocomposite induced much less of a apoptotic response in A549 cells than those of the RGO nanosheets.

Oxidative Stress
The underlying mechanisms of nanoscale materials toxicity are not completely delineated. Oxidative stress generation, through which nanoscale materials exert toxicity, is now being studied by investigators [44]. Nanoscale materials might induce oxidative stress, either by the generation of pro-oxidants or by the reduction of antioxidants. A higher production of intracellular ROS or the depletion of antioxidants may lead to the oxidative damage of cell biomolecules [45]. ROS such as the superoxide anion (O 2 •− ), hydroxyl radical (HO•), and hydrogen peroxide (H 2 O 2 ) serve as signaling molecules in the process of apoptosis [46]. The GO-ZnO nanocomposite was found to induce oxidative stress mediated toxicity in MCF-7 cells [47]. In this study, we examined several parameters of oxidative stress in A549 cells against CeO 2 NPs, RGO, and CeO 2 -RGO nanocomposite when exposed to said particles for 24 h. The quantitative data demonstrated that the intracellular level of ROS in the CeO 2 NP group was almost similar to that of the control group, but significantly higher than the RGO group ( Figure 8A). Interestingly, in the CeO 2 -RGO nanocomposite group, the ROS level was significantly lower than the RGO group and was not statistically different from the control group. Similar to the quantitative data, fluorescent microscopy images demonstrated that the brightness of the DCF probe (marker of ROS level) in CeO 2 NPs and the CeO 2 -RGO nanocomposite was almost close to the control group, but the brightness of DCF in the RGO group was much higher than that of the control group ( Figure 8B). RGO group also showed low cell density because several cells died in 24 h exposure time of RGO. Besides, we assessed the H 2 O 2 level in A549 cells against CeO 2 NPs, RGO, and CeO 2 -RGO nanocomposite exposure for 24 h. As we can see in Figure 8C, the H 2 O 2 level in the CeO 2 NP group was not different from the control group. However, the H 2 O 2 level in RGO was significantly higher in comparison to the control and CeO 2 NP groups. Again, an interesting result was that the H 2 O 2 level in the CeO 2 -RGO nanocomposites was significantly lower than the RGO group and was not statistically different from the control group ( Figure 8C). In similarity, Qiang et al. [26] observed that Pb 2+ reduced the cytotoxicity, oxidative stress, and apoptotic response of GO through the regulation of the GO morphology. The equilibrium between pro-oxidant generation and their scavenging by antioxidant molecules and enzymes is a delicate phenomenon. The antioxidant molecule glutathione (GSH) plays an important role in cellular defense against oxidant injury. Several redox modulating enzymes, including thiol reductases and peroxidase, rely on the intracellular GSH pool as their source of reducing equivalents [48]. Intracellular GSH depletion has also been linked with either the induction or stimulation of apoptosis [49]. In this study, we observed that RGO significantly decreased the GSH level as compared to the control group. Furthermore, the GSH levels in CeO 2 -RGO nanocomposites were significantly higher than in the RGO group and were not statically different from the control group ( Figure 8D). Here, we found a very interesting result, namely, that CeO 2 NPs significantly increase GSH levels in A549 cells as compared to the control. The GSH replenishing potential of CeO 2 NPs was also reported in our previous work [21]. Some other studies have also reported the antioxidative potential of CeO 2 NPs [50,51]. This could be one of the possible reasons behind the biocompatible nature CeO 2 -RGO nanocomposite to A549 cells as compared to pure RGO. This study highlights the role GSH in the replenishing potential of CeO 2 NPs when converting toxic RGO into biocompatible CeO 2 -RGO nanocomposites. However, the elucidating the underlying mechanisms through which CeO 2 NPs convert cytotoxic RGO nanosheets into biocompatible CeO 2 -RGO nanocomposites still remains a daunting task.
Altogether, our data demonstrate that RGO induces toxicity in A549 cells. However, CeO 2 -RGO nanocomposites showed good biocompatibility toward A549 cells. The biocompatible nature of CeO 2 -RGO nanocomposites can be utilized for their potent application in a number of biomedical fields, such as drug delivery and biosensing. Earlier studies have also suggested the biomedical application of graphene-based nanocomposites. For instance, Wang et al. [52] observed that gold (Au)-RGO nanocomposites can effectively carry the anticancer drug doxorubicin inside human liver cancer (HepG2) cells. Au-RGO nanocomposites can also be used for the detection of micro-RNA [53]. The equilibrium between pro-oxidant generation and their scavenging by antioxidant molecules and enzymes is a delicate phenomenon. The antioxidant molecule glutathione (GSH) plays an important role in cellular defense against oxidant injury. Several redox modulating enzymes, including thiol reductases and peroxidase, rely on the intracellular GSH pool as their source of reducing equivalents [48]. Intracellular GSH depletion has also been linked with either the induction or stimulation of apoptosis [49]. In this study, we observed that RGO significantly decreased the GSH level as compared to the control group. Furthermore, the GSH levels in CeO2-RGO nanocomposites were significantly higher than in the RGO group and were not statically different from the control group ( Figure 8D). Here, we found a very interesting result, namely, that CeO2 NPs significantly increase GSH levels in A549 cells as compared to the control. The GSH replenishing potential of CeO2 NPs was also reported in our previous work [21]. Some other studies have also reported the antioxidative potential of CeO2 NPs [50,51]. This could be one of the possible reasons behind the biocompatible nature CeO2-RGO nanocomposite to A549 cells as compared to pure RGO. This study highlights the role GSH in the replenishing potential of CeO2 NPs when converting toxic RGO into biocompatible CeO2-RGO nanocomposites. However, the elucidating the underlying mechanisms through which CeO2 NPs convert cytotoxic RGO nanosheets into biocompatible CeO2-RGO nanocomposites still remains a daunting task.
Altogether, our data demonstrate that RGO induces toxicity in A549 cells. However, CeO2-RGO nanocomposites showed good biocompatibility toward A549 cells. The biocompatible nature of CeO2-RGO nanocomposites can be utilized for their potent application in a number of biomedical Quantitative data are presented as mean ± SD of three independent experiments (n = 3). * Indicates a significant difference from the control group (p < 0.05). # Indicates a significant difference from the RGO group (p < 0.05). RGO: Reduced graphene oxide; CeO 2 : Cerium oxide; NPs: Nanoparticles; ROS: Reactive oxygen species; H 2 O 2 : Hydrogen peroxide; GSH: Glutathione.

Conclusions
CeO 2 -RGO nanocomposites and RGO were prepared and characterized by XRD, XPS, SEM, and TEM. The cytotoxic and apoptotic responses of RGO and CeO 2 -RGO nanocomposites were explored in A549 cells. We observed that RGO significantly induce cytotoxicity (cell viability reduction and LDH leakage), apoptosis (cell cycle arrest and MMP loss), and oxidative stress (ROS generation and GSH depletion) in A549 cells. However, CeO 2 -RGO nanocomposites did not cause a statistically significant change in toxicity to A549 cells as compared to the control group. Oxidative stress experiments suggested that the GSH replenishing potential of CeO 2 NPs could be one of the potential mechanisms for the biocompatible nature the CeO 2 -RGO nanocomposite. Our encouraging data suggest that CeO 2 -RGO nanocomposites are a safe material at the cellular level. This study further provokes more advanced research for the development of graphene-based nanocomposites for their application in biomedicine.