Graphene oxide-silver nanoparticle nanocomposites induces apoptosis in caprine fetal fibroblast cells via induction of oxidative stress CURRENT

Background: Graphene oxide (GO) has drawn much attention as excellent platform to which silver nanoparticles (AgNPs) can be anchored for the production of biomedical nanocomposites. Yet, the potential toxicity of GO-AgNPs nanocomposites to animal and human is complex to evaluate and remains largely unknown. Results: Our data indicated that GO-AgNPs caused cytotoxicity in dose-dependent manner. GO-AgNPs induced significant cytotoxicity by the loss of cell viability, production of reactive oxygen species (ROS), cell cycle arrest, increasing leakage of lactate dehydrogenase (LDH) and level of Malondialdehyde (MDA), increasing expression of pro-apoptotic genes and decreasing expression of anti-apoptotic genes. Conclusions: Taken together, our study demonstrated that GO-AgNPs potentially induce oxidative damage to DNA, which result in toxicity and cell apoptosis in caprine fetal fibroblast cell due to an increased generation of ROS. It strongly suggests that applications of GO-AgNPs nanocomposite in animal must be further evaluated.


Background
Nanotechnology is related to the controlled design, characterization, production and application of nano-sized materials, which are defined as small particles ranging in size between 1 and 100 nm. The increasing application of nanoparticles in microelectronics, cosmetics, ceramics, catalysts and food has made them widely present in our lives and attracts increasing investment from governments and industry around the world [1][2][3][4]. Currently, because of their unique physiochemical properties, a growing number of nanomaterials have been applied in the area of biomedicine, which emphasis on the diagnostic and therapeutic purposes, such as cancer diagnosis and therapy, nanocarriers for targeted delivery of drugs and genes, design of novel candidate nanoscale constructs for drug development [5][6][7][8][9][10]. Consequently, the bio-application and biosafety of the nanomaterials need to be further explored.
In recent decades, graphene and graphene related nanocomposite have attracted much attention both in the industry and scientific community due to their unique physiochemical and biological 4 properties [11][12][13]. Silver nanoparticles (AgNPs) are one of the most frequently used nanoparticles in a variety of biomedical applications [14]. Recent advances in nanotechnology have widened the potential combination of AgNPs with graphene-based nanocomposite, which offers a novel graphenesilver hybrid nanomaterials with unique functions in biomedical nanotechnology, and nanomedicine [15,16]. For example, AgNPs attaching on the surface of Graphene oxide (GO) sheets can prevent AgNPs from aggregating, allow a more controlled release of AgNPs + ions and lead to the increase antibacterial and anticancer activity [17,18]. Despite their wide applications in several areas, many evidences suggested that some nanomaterials accumulates in human and animal tissue, such as heart, kidney and other organs [19][20][21][22], which modifies the transcription of genes related to transport pathways, nuclear signaling, endocytosis, reproductive behavior and general defenses [23,24]. Thus, any potential nanotoxicity should be thoroughly and carefully evaluated for promoting its safe application as a clinical agent.
Nanotoxicity involves the understanding of adverse biological effects of nanoparticles using both in vitro and in vivo model systems such as cell, tissue, organ and organism [25]. Several studies have been dedicated to examine the effects of graphene and graphene-related nanomaterials in various cell culture systems, including HeLa, MCF-7, SKBR3, NIH3T3, epithelial lung carcinoma, primary mouse embryonic fibroblast, human breast cancer and ovarian cancer [15,[26][27][28]. Graphene and graphene related nanomaterials have been proved to lead to inflammatory response in the liver and kidney [29][30][31], genotoxicity and DNA damage [30,32], and adverse functional effects in the lungs, heart, intestine, and spleen [33][34][35].
Recently, due to rapid growing of nanotechnology, investigation of nanomaterials toxicity and their potential risks are attracting interests [24]. Although a wide spectrum of studies have been performed to determine the potential toxicity of graphene and graphene related nanomaterials in different cancer cell lines and small animal, such as rat and mouse, so far the effect of graphene oxide-silver nanoparticles (GO-AgNPs) nanocomposites on caprine fetal fibroblast cells remained to be illuminated. Therefore, the present study was conducted to investigate the effect of GO-AgNPs nanocomposites on caprine fetal fibroblast cells in vitro and to determine the underlying molecular mechanisms of their 5 cytotoxicity. To our knowledge, this is the first report in caprine fetal fibroblast cells to demonstrate cellular responses and functional aspects of GO-AgNPs .

Results
Characterization of GO-AgNPs TEM analysis was conducted to confirm the structural and surface morphology of the GO-AgNPs composites. The size distribution of the GO-AgNPs was about 20 nm as shown in the image of TEM ( Fig. 1). AgNPs were uniformly distributed on the GO sheets. GO-AgNPs images clearly showed transparent, single-layer sheets containing flake-like wrinkles (Fig. 1). The wrinkled silk waves were presented on the GO sheets.

Effect Of Go-agnps On Caprine Fetal Fibroblast Cells Viability
The viability of caprine fetal fibroblast cells was determined by CCK-8 assay. As shown in Fig. 2, there were no significant differences in cell viability between control cells and those exposed to 1 µg/mL GO-AgNPs for 24 h, however, the viability of cells was significantly reduced when the concentration increased (4, 8, 12 and 16 µg/mL), suggesting that GO-AgNPs induced toxicity in caprine fetal fibroblast cells in a dose-dependent manner.

Effect Of Go-agnps On Cell Morphology
The morphologies of caprine fetal fibroblast cells after exposure to GO-AgNPs for 24 h were shown in Fig. 3. Caprine fetal fibroblast cells that had been exposed to 4 µg/mL and 8 µg/mL GO-AgNPs exhibited marked morphological changes, with obvious reduction in number of cells in the group of 8 µg/mL GO-AgNPs.

Effect Of Go-agnps On Reactive Oxygen Species (ros) Production
To study whether GO-AgNPs induced oxidative impact involving in the apoptosis, the intracellular ROS level in caprine fetal fibroblast cells was analyzed. As shown in Fig. 4, the level of intracellular ROS in caprine fetal fibroblast cells was significantly increased (P < 0.05) when the cells were treated with 4 and 8 µg/mL of GO-AgNPs for 24 h compared to the control group.

Effects Of Go-agnps On Apoptosis
An Annexin V/PI apoptosis kit was ued to quantify the percentage of caprine fetal fibroblast cells undergoing apoptosis and dying by flow cytometry. The results suggested that the GO-AgNPs induced significant apoptosis and dead in caprine fetal fibroblast cells (Fig. 5). 6 Effects Of Go-agnps On Superoxide Dismutase (sod) Production Effects of GO-AgNPs on the production of SOD in caprine fetal fibroblast cells were determined with SOD assay kit. As shown in Fig. 6, SOD activity decreased significantly (P < 0.05) in caprine fetal fibroblast cells treated with 4 µg/mL GO-AgNPs for 24 h when compared to the control group.

Effects Of Go-agnps On Malondialdehyde (mda) Production
The production of MDA in caprine fetal fibroblast cells was determined with the MDA assay kit after

Effects Of Go-agnps On Caspase-3 Activity
To confirm whether caspase-3 is involved in apoptosis of caprine fetal fibroblast cells induced by GO-AgNPs (4 and 8 µg/mL), caspase-3 activity was measured by the caspase-3 kit. The activity of caspase-3 in the 4 and 8 µg/mL group was significantly (P < 0.05) higher after treatment than that in the control group ( Fig. 9).

Effects Of Go-agnps On Gene Expression
The expression level of apoptotic genes was determined in the caprine fetal fibroblast cells treated with GO-AgNPs (4 and 8 µg/mL) for 24 h. The results showed that the level of caspase-3, Cyt-C, BAX, Smac and p53 were significantly (P < 0.05) upregulated in the GO-AgNPs treated groups, compared to the control group (Fig. 10). However, the level of BCL2 in the GO-AgNPs treated group was significantly (P < 0.05) downregulated, compared with the control group (Fig. 10).

Discussion
Nanotechnology has become an indispensable field tool to develop various kinds of nanoparticles with unique properties [36]. As an efficient support material, graphene sheets can disperse and stabilize silver nanoparticles preventing their agglomeration, which open up a way for the nanomaterial development. Therefore, the combination of graphene and AgNPs based nanocomposites has been widely produced to enhanced antibacterial and anticancer activity [37]. This kind of nanomaterial can easily enter cells, thus affect the physiology of organisms, which may have the potential toxicity both on human and animal health or ecosystems. Therefore, the adverse effects of GO-AgNPs composites have been considered as a major limitation for its broad applications. Numerous studies have proved the toxicological effects of GO-AgNPs nanocomposite on animal and human normal cells [21,37,38].
In the present study, a GO-AgNPs nanocomposite was synthesized using quercetin and was confirmed its surface and structural morphology, and the uniform distribution of AgNPs on the GO sheets with TEM, and investigated its toxic effect on caprine fetal fibroblast cells.
In order to evaluate the effect of GO-AgNPs on caprine fetal fibroblast cell viability, cells were treated with differnet concentrations of GO-AgNPs for 24 h. The data showed that 4 or 8 µg/mL GO-AgNPs reduced cell growth, viability and induced morphological changes in a concentration-dependent manner. In our previous study, GO-AgNPs significantly decreased the human ovarian cancer cell viability with an IC50 of 5 µg/mL [39], which is lower than that in the present study, suggesting that caprine fetal fibroblast cells are less sensitive to GO-AgNPs than human cancer or mouse cells. Similar results were reported that 5 µg/mL rGO-Ag nanocomposite did not induce cytotoxicity in human normal cells (CHANG cell) but could slightly induce toxic effect on HepG2 cells, however, when the concentration of rGO-Ag increased up to 25 µg/mL, cell viability significantly decreased [38]. The loss of cell viability by graphene materials may depend on many factors, such as the surface chemistry charge, size and physicochemical properties of the materials.
To investigate whether GO-AgNPs inducing the cytotoxicity was the cause of cell apoptosis, caprine fetal fibroblast cells were analyzed with Annexin V/PI double labeling assay. The data by flow cytometry showed that GO-AgNPs induced more apoptosis and inhibited cell proliferation compared to that of control group. Similarly, the level of caspase-3 was higher in GO-AgNPs treated groups, which confirms the results of apoptosis. Oxidative stress inducing ROS is one of the proposed toxicological 8 mechanisms of various nanomaterials such as Ag or Ag-graphene nanocomposites, can cause mitochondrial damage, and initiation of lipid peroxidation [39][40][41]. In current study, the production of ROS in caprine fetal fibroblast cells was evaluated. After treatment with 4 and 8 µg/mLGO-AgNPs for 24 h, the level of ROS increased significantly at a 1.4-and 1.8-fold in caprine fetal fibroblast cells. The present data suggests that the prodution of ROS is a common toxicity mechanism of GO-AgNPs for animal cells.
The incrseased level of MDA is generally considered to imply the cell injury. Human cells treated with AgNPs and GO showed significantly increased levels of MDA [42,43]. Assessing the release of intracellular LDH in cell, which is the reslut from the breakdown and alteration in the permeability of the plasma membrane, is one of marker for estimating cytotoxicity [16,41]. Previous studies reported that the combination of GO with AgNPs could significantly increase the intracellular production of MDA [15,44]. The present data indicated that the mechanism of increased level of MDA in GO-AgNPstreated caprine fetal fibroblast cells may be due to the strong hydrophobic interactions with the cell membrane and ROS formation, which influenced the viability and proliferation of cells.
The apoptosis in cell is a highly conserved mechanism, and ROS is an important factor in the apoptotic process [45]. ROS induced by nanomaterial could result in nuclear DNA damages and leakeage of lipids, proteins and carbohydrates in the cell. In the present study, apoptosis was observed in GO-AgNPs-treated caprine fetal fibroblast cells. ROS production and lipid peroxidation induced by GO-AgNPs affected cellular redox homeostasis and decrease antioxidant levels, which were evaluated by the SOD, which act as an active oxygen free radical scavenger that can combat the accumulation of ROS and reduce the oxidative injury. The SOD activity has a relationship with the antioxidative ability and also can be used as indicative of mitochondrial activity [46,47]. The present data showed that the level of SOD significantly decreased in GO-AgNPs-treated caprine fetal fibroblast cells, which corresponded with the high rates of apoptotic cells.
Anti-apoptotic and apoptotic genes play an important role in cell death and survival. A study reported that GO-AgNPs can cause oxidative damage, leakage of LDH, and enhance expression of apoptotic genes, thus lead to mitochondrial dysfunction and trigger apoptosis [48]. All apoptotic pathways appear to terminate in the activation of the caspase family of proteases [49]. Moreover, oxidative stress is reported to cause the mitochondrial translocation of the pro-apoptotic protein of Bax, increase the total expression of Bax and also down-regulate the expression of the anti-apoptotic protein of BCL-2 [50]. The present data suggested that GO-AgNPs up-regulated the expression apoptotic genes such as caspase-3, Bax, Smac and c-myc, and down-regulated anti-apoptotic genes such as Bcl-2.

Conclusion
Previously, we described a simple and environmentally friendly method to synthesize GO-AgNPs nanocomposite, which exhibited enhanced cytotoxicity to human cancer cells (SH-SY5Y) compared with that of GO at a very low concentration [39]. In the present study, the cytotoxic potential and molecular pathway of the GO-AgNPs nanocomposite were evaluated in caprine fetal fibroblast cells. Synthesis And Characterization Of Go-agnps GO-AgNPs nanocomposites were synthesized using the biomolecule quercetin as described previously [39]. Briefly, 50 mg GO was dispersed in 30 mL water and sonicated for 60 min. One mM AgNO 3 were dissolved in 15 mL water in a 500 mL round-bottom flask. 30 mL of the GO dispersion was added, followed by addition of 5 mL of aqueous 1 mM quercetin, and then stirred at 60 °C for 12 h. The resultant mixture was washed and centrifugated three times with water. The size and shape were observed under a transmission electron microscope (TEM; HT7800, Hitachi High-Technologies Corporation, Tokyo, Japan).
Cell Culture 10 Caprine fetal fibroblast cells were isolated from 70-day old fetuses that were recovered surgically from a Boer goats as previously described [51]. After removal of the head and internal organs, the remaining tissues were dissociated into small pieces using scissors and digested with 0.25% trypsin

Cell Viability Assay
The cell viability was assessed by using an in vitro cell-counting assay kit (CCK-8; Rockville, MD, USA) as described previously [40]. Caprine fetal fibroblast cells were seeded on 96-well or 6-well plate and The cell suspension was determined by flow cytometry to analyze the apoptotic rate.

Measurement Of Ros Production 11
Dichlorodihydroflfluorescein diacetate (DCFH-DA) was used to detect intracellular ROS induced by different concentrations (0, 4 and 8 µg/mL) of GO-AgNPs [40]. Caprine fetal fibroblast cells were incubated in 10 µM DCFH-DA for 30 min at 37℃. The cells were rinsed with PBS twice, and then the intracellular accumulation of ROS was measured by flow cytometry (Beckman-Coulter, USA).

Measurement Of Total Sod Enzyme Activity
The SOD activity in caprine fetal fibroblast cells was detected following the the SOD assay kit (Beijing Solarbio Science & Technology, beijin, China) [40]. Cells were treated with 0, 4 and 8 µg/mL of GO-AgNPs for 24 h. Cells were washed with PBS twice, and lysed with lysis buffer on the ice. The lysates were then centrifuged for 15 min. Then, the supernatant was analsysed with a U-vis spectrophotometer (Nanodrop, Thermo, USA) at 550 nm.  Table 1). The PCR cycle was as follows:

Measurement Of Mda Production
initial denaturation at 95 °C for 30 s, followed by 41 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 s. RT-qPCR was performed independently four times. The target genes were quantified by the delta-delta Ct method using CFX manager V1.1 software (Bio-Rad Laboratories). Normalization was performed using β-actin as the reference gene.