During the last decades, rGO and AgNPs deserved an enormous academic interest and a significant attention especially as apoptotic agents (Zhang et al., 2011; Zhang et al., 2020; ). rGO is a compatible material to stabilize and disperse AgNPs since it is distinguished with the abundant oxygenated functional groups and large specific surface area (Ali et al., 2020). There were different methods that used reducing chemical agents in the synthesis of rGO/AgNC such as poly(N-vinyl-2-pyrrolidone) (Hu et al., 2013) and ammonia (Chook et al., 2012). Bao et al. (2011) prepared the rGO/AgNC using AgNO3 as a salt precursor, hydroquinone as the reductant, and citrate as the stabilizer. Pasricha et al. (2009) synthesized the rGO/AgNC under alkaline conditions using sodium borohydride as a reducing agent to Ag+. In addition, different physical methods were used in synthesis the rGO/AgNC such as Zainy et al. (2012) who studied the preparation of rGO/AgNC via rapid thermal treatment. They used silver acetate as a silver precursor and GO served as a substrate for the AgNPs that treated at 1000°C in a furnace for 20 s under an ambient atmosphere. On the other hand, the green synthesis approach of nanomaterials possesses minimum toxicity, lower-cost, lower reaction temperature and lower reaction time compared to the other physical and chemical methods (Awwad et al., 2020). Gurunathan et al. (2015) reported the synthesis of rGO/AgNC in three separated steps starting with synthesis of AgNPs using Tilia amurensis leaves extract, reduction of GO by T. amurensis leaves extract and then mixing the rGO with AgNO3 to synthesized rGO/AgNC. Thus, the exploitation of green synthesized silver nanomaterials had an increasing interest in the elaboration of safe bioactive biomaterials in addition to their distinguished properties, such as antimicrobial activity, antiviral and antiangiogenesis action (Patil et al., 2019; Galvez et al., 2021). The present work provided a one-step green method for the synthesis of rGO/AgNC by dual bioreducing of GO and Ag in the presence of sunlight and revealed promising effect of this nanocomposite in of cancer treatment. In addition, it explored the in vivo toxicity of this nanocomposite, and revealed a moderate effect on mice liver and kidney.
The main parameters of rGO/AgNC are its purity, shape and size which control its biological activities (Nel et al., 2006). The obtained data by UV-Vis spectroscopy, FT-IR, XRD and TEM studies confirmed the crystalline nature of AgNPs and strong interactions between the AgNPs and rGO. The TEM micrographs showed the transparent and sheet-like structure of rGO/AgNC embedded with spherical shaped and well-dispersed AgNPs. These observations matched with that of Cobos et al. (2020) study in which the obtained GO/AgNC sheets via the in situ method through the simultaneous reduction of AgNO3 and GO using L-ascorbic acid showed flexible sheets, paper-like structures morphologies of GO with few layers. The dark areas showed the thick stacking nanostructure of several GO and rGO/AgNC layers. The lower opaque areas designate much thinner sheets of a GO and rGO/AgNC layers indicating to their delamination. This exfoliation might increase the surface area of the synthesized materials (Stobinski et al., 2014). The resulting rGO/AgNC in the presented study showed the decorated AgNPs had an average size of 8–17 nm on the rGO sheets. Chook et al. (2012) fabricated GO/AgNC using microwave irradiation with 40.7 ± 7.5 nm of AgNPs on the GO sheets. Yun et al. (2013) prepared GO/AgNC with deposited AgNPs with an average size of 2 to 4 nm.
There are many hypotheses for mechanisms of rGO/AgNC formation but the more likely is the AgNPs were bound to GO through the collaboration of the Ag+ with the oxygenized functional groups on the GO surface such as the hydroxyl, epoxy and carboxylic groups (Faria et al., 2012). El-Dein et al. (2021) confirmed the presence of proteins in the biosynthesis of AgNPs by E. coli D8 as stabilizing and capping agents for AgNPs. The protein capping agents around the AgNPs might interact with GO-oxygenated groups as de Faria et al. (2014) supposed that as a probable mechanism of the biosynthesis of GO/AgNC which may be similar to our involved mechanism for the synthesis of rGO/AgNC.
Ehrlich carcinoma has a closeness with human tumors and is considered the most sensitive type to chemotherapy. EAC cells are undifferentiated cancer that are primordially hyperdiploid and does not have tumor-specific transplantation antigen. In addition, they have rapid proliferation, short life span as well as high transplantable capability (Ozaslan et al., 2011). In the present study, rGO/AgNC reduced EAC cell count in vitro in a dose-dependent manner. The potential in vivo toxicity of nanomaterials is always considered as a great concern for using in the biomedicine applications. The formation of solid tumors and the ascites elevate weight and abdominal circumference in mice and decrease the survival time (Ninomiya et al., 2009). The treatment with some chemicals decreased the amount of ascitic fluid without notably inspiring the number of tumor cells (Sugiura, 1958). In this study, treating Ehrlich carcinoma bearing mice with rGO/AgNC at the dose of 10 mg/kg for 7 days could prolong the survival for more than 60 days, while the rGO/AgNC-untreated ones were all died within 3 weeks. In addition, treatment with rGO/AgNC restored body weight, abdominal circumference due to the reduction of the carcinoma cell viability and consequently of the ascitic fluid volume.
The penetration and accumulation of AgNPs in the treated mice were confirmed using histological and TEM examinations of liver and kidney. One of the main functions of the liver is to remove hazard compounds from the blood and transform those to suitable chemical forms that can be excreted by the kidney. Therefore, liver and kidney are the most prominent targets of nanoparticles. However, the observation of AgNPs in both organs infers the release of silver nanoparticles from the rGO/Ag nanocomposite. rGO/AgNC is formed of two parts: the rGO scaffolds and the decorated AgNPs. Polycationic rGO scaffolds could interact with the cell membrane negatively charged components (Ruiz-Herrera et al., 2006). It was reported that this interaction leads to the transposition of the potassium ions on the cell surface and losing of ionic equilibrium, which prompts the further efflux of potassium ions from the cell. This efflux leads to the hyperpolarization of the plasma membrane. It was affirmed that the plasma membrane hyperpolarization resort to the cell to increase the uptake of cations to balance the membrane potential (Peña et al., 2013). Vazquez-Muñoz et al. (2014) confirmed the gradual release and the spontaneous ionization of the Ag+ from AgNPs. These ions penetrate the cell throughout a cationic influx. After Ag+ entering the cells, different Ag related toxic effects may result as observed in this work. This observed toxic effects of AgNPs are in agreement with that of many previous reports (Hajipour et al., 2020; Elsharawy et al. 2020).