Green synthesis of bimetallic ZnO–CuO nanoparticles and their cytotoxicity properties

In this study, a simple and green strategy was reported to prepare bimetallic nanoparticles (NPs) by the combination of zinc oxide (ZnO) and copper oxide (CuO) using Sambucus nigra L. extract. The physicochemical properties of these NPs such as crystal structure, size, and morphology were studied by X-ray diffraction (XRD), field emission gun scanning electron microscopy (FEG-SEM), and transmission electron microscopy (TEM). The results suggested that these NPs contained polygonal ZnO NPs with hexagonal phase and spherical CuO NPs with monoclinic phase. The anticancer activity of the prepared bimetallic NPs was evaluated against lung and human melanoma cell lines based on MTT assay. As a result, the bimetallic ZnO/CuO NPs exhibited high toxicity on melanoma cancer cells while their toxicity on lung cancer cells was low.

www.nature.com/scientificreports/ side effects of current cancer therapy. Rapid and targeted penetration in cancer cells can prevent the spread of disease to other tissues, and thus control the cancer. Nanoparticles (NPs) have gained interest of scientists in the field of nanomedicine [29][30][31][32][33][34][35][36] . The therapeutic effects of NPs depend on the particle size, the culture time of the target cell, the amount of metal in the targeted cell, and their physicochemical properties [36][37][38][39] . On the other hand, bimetallic and multimetallic NPs have shown unique physicochemical properties with synergistic effects and high functionality 40 . These integrated NPs have more reactive sites, increased efficiency, and greater stability 41,42 . In recent decades, the antitumor activity of different biogenic and non-biogenic bimetallic NPs has been evaluated [43][44][45] . In 2019, Lomelí-Marroquín et al. synthesized silver/gold bimetallic NPs with the aim of reducing the nano metallic toxicity of silver, and evaluated their antitumor activity on melanoma cancer cells 46 . Pt/Pd bimetallic NPs were synthesized using Dioscorea bulbifera extract, and gold-silver bimetallic NPs were synthesized using Stigmaphyllon ovatum leaf extract; their anticancer activity against HeLa cells was also investigated 47,48 . In another study, the gold(i)-BODIPY-imidazole bimetallic complex demonstrated good antiproliferative activity against breast, colon, and prostate cancer 49 . In 2019, silver/palladium bimetallic NPs were synthesized using Terminalia chebula fruit extract, and their antitumor properties were evaluated against A549 50 . Additionally, biogenic copper and zinc oxide NPs were synthesized in 2018 and 2019, and their anticancer activities were evaluated against T98G human glomerular superficial cell and cervical cancer, respectively 51,52 . Typically, the synthesis of multimetallic and bimetallic NPs is costly and time-consuming, and may include toxic or hazardous substances. Metal oxide NPs such as zinc oxide (ZnO) and copper oxide (CuO) demonstrated diverse biological applications; these NPs were highly compatible with normal cells in the body. Biogenic ZnO NPs were eco-friendly synthesized using Cardiospermum halicacabum and Mangifera indica leaf extracts, and they illustrated good antitumor properties against A375 and A549 cells at a concentration of 50 μg/mL, respectively 4,53 .
In another study, CuO NPs were synthesized using a green method; these NPs inhibited mRNAII expression in A549 cancer cells and stimulated apoptosis 54 52 . Green synthesis of bimetallic ZnO/CuO using Sambucus nigra L. plant extract was illustrated, with the advantages of simplicity, cost-effectiveness, low temperature, eco-friendliness, desirable anticancer properties, and efficient cytotoxicity of biogenic ZnO/CuO NPs 51 . Using synergistic biological effects such as increased ROS production, Cu genotoxicity, and increased cancer cell apoptosis, these materials can be employed as effective therapeutic agents in the treatment of lung and melanoma cancer.
The medicinal plant black elderberry with the scientific name of Sambucus nigra L. from the Caprifoliaceae family was collected from the forest of Mazandaran, Iran. This plant contains different types of secondary metabolites including glycosidic compounds, terpenoids, phenolics, tannins, anthocyanins, etc. In traditional medicine, S. nigra was utilized to treat bone fractures, kidney diseases, respiratory diseases, and eczema 58 . Generally, green and eco-friendly synthetic strategies are deployed instead of conventional industrial manufacturing methods to eliminate/prevent hazardous materials and reduce expensive/complex instruments and materials. These eco-friendly methods have some benefits compared to the conventional physicochemical approaches such as simplicity, rapidness, cost-effectiveness, low toxicity, and environmentally friendliness. In this study, bimetallic ZnO/CuO NPs have been synthesized using S. nigra L. extract via a green and simple method. The synthesized bimetallic NPs have been characterized by X-ray diffraction (XRD), field emission gun scanning electron microscopy (FEG-SEM), and transmission electron microscopy (TEM) combined with energy dispersive analysis of X-rays (EDX) and selected area electron diffraction (SAED). Additionally, the anticancer activity of these NPs have been evaluated on human melanoma cell line (A375) and human lung cancer (A549).
Green synthesis of ZnO/CuO NPs. Fresh shoots of S. nigra shrub were sterilized with 5% NaOCL and their surface moisture was removed at 25 °C. The plant material was dried with an electric grinder to turned into a soft powder. Then, 7 mL of deionized water was added to 1 g of plant powder, and it was shaken overnight at 25 °C. The prepared mixture was brewed at 100 °C for 10 min. Finally, the plant extract was separated by filter paper and centrifugation. To support the reproducibility, voucher specimens for the described plants www.nature.com/scientificreports/ were deposited in University public herbarium. The S. nigra Fresh shoots were collected under the licence of the collection of plant or seed specimens in the University in accordance with applicable institutional, national, and international rules and legislation. It was verified by the Iranian Botanical Survey, whose voucher specimen number was 1400/8 deposited at the Department Pharmacognosy, Kerman University. To synthesize bimetallic NPs, 1.6 g of Zn(CH 3 COO) 2 was added to 100 mL of extracts at 70 °C. After dissolving the zinc salt with a strainer, 0.8 g of CuCl 2 was added to the mixture. The pH of the solution was adjusted to 8 by adding 1 mol/L of NaOH. The mixture was sterilized at 70 °C for 3 h. The ensuing NPs were washed 3 times with deionized water and dried at 90 °C in an oven. Finally, the synthesized NP powder was calcined at 400 °C for 6 h.
Characterization of NPs. XRD analysis was performed with X'PertPro device from Panalytical Holland Company with Anod material Cu (1.54 Å, 40 kV, 30 mA) within a 2θ range of 10° to 80° in order to confirm the expected crystal structure of hexagonal ZnO and monoclinic CuO nanocrystals. The size, surface morphology, and elemental analysis of synthesized NPs were studied by FEG-SEM (microscope Sigma VP; ZEISS, Germany) and EDX analyses (EDX detector from Oxford Instruments Company; UK). The NP analysis at higher magnifications was performed by means of TEM (microscope Tecnai G2 Spirit Twin; FEI, Czech Republic). The TEM microscopy was performed at accelerating voltage of 120 kV and yielded not only standard bright-field images (TEM/BF) of the individual NPs, but also EDX spectra (TEM/EDX; EDX detector from EDAX; USA) and electron diffraction patterns (TEM/SAED; verification of crystalline structure from XRD). Assessment of anticancer activity. The effect of bimetallic NPs was evaluated on the susceptibility of A375 and A549 cell lines using MTT assay. After 24 h of culturing cancer cells in microplates, 100 μL of the ZnO/ CuO bimetallic NPs in the concentrations of 1, 10, 100, 500, and 1000 μg/mL were added to each well. Treated plates with ZnO/CuO bimetallic NPs and Doxorubicin with 4 and 8 μg/mL (as positive controls) were incubated at 37 °C and 5% CO 2 for 72 h. Then, 20 μL of MTT solution was added to the treatments and after 4 h of incubation, 100 μL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the absorbance was measured at 490 nm by an enzyme-linked immunosorbent assay (ELISA) reader (BioTeks Elx 800). The survival rate (%) was calculated by the following formula:

Culturing of cells.
The inhibitory concentration needed for 50% cytotoxicity (IC 50 ) was assessed using the Probit analysis and plotting the level of inhibition versus the concentration.

Statistical analysis.
All experiments were carried out in triplicate. Data were analyzed using ANOVA followed by unpaired Student's t-test. P-values of < 0.05 were considered statistically significant.
P-values of < 0.01 were considered statistically significant.
Ethical statement. We confirm that all methods were carried out under in vitro condition.

Characterization of ZnO/CuO bimetallic NPs. XRD analysis.
The crystalline structure of the zinccopper bimetallic NPs calcined at 400 °C is illustrated in Fig. 1. The prominent peaks observed in 2θ = 32, 34.5, 37, 47.5, 57, 63 and 69° confirmed the crystalline structure and hexagonal phase of zinc oxide NPs (JCPDS card 01-080-0075). The peaks in the 2θ = 36, 39, 49, 66.5 and 68.5° confirmed the crystal structure and monoclinic copper oxide phase (JCPDS card 01-080-1917) 59 . As can be observed in the Fig. 1, the number of clear diffraction and peak intensities of ZnO NPs are higher than of CuO NPs. This indicates that ZnO NPs have higher percentage in the structure of zinc-copper bimetallic NPs and high degree of crystallinity. The peak of low crystallization of CuO NPs is due to the coating role of ZnO NPs on them 60 .
SEM analysis. The surface morphologies, particle sizes and EDX measurements of the bimetallic NPs are shown in Fig. 2. Consequently, Fig. 2a (Fig. 2c).
TEM analysis. The detailed analysis of ZnO/CuO bimetallic NPs by TEM (Fig. 3) showed the NPs in thin layer deposited on carbon film (TEM/BF; Fig. 3a), confirming their crystalline structures (TEM/SAED; Fig. 3b,c) and their elemental composition (TEM/EDX; Fig. 3d www.nature.com/scientificreports/ lic NPs were mostly isometric, tended to form agglomerates during the drying process, and exhibited quite broad particle size distribution (particle diameters from tens to hundreds of nm). The TEM/SAED diffractogram (Fig. 3b) exhibited quite low diffraction intensities. This could be attributed to the fact that relatively low concentration of small particles was deposited on carbon film and the nanoparticle diffractions were hidden in amorphous carbon background. After careful background correction, the comparison of radially averaged TEM/SAED diffraction pattern with theoretically calculated powder XRD of hexagonal ZnO and monoclinic CuO nanocrystals showed quite good agreement (Fig. 3c), confirming the expected crystalline structure. The processing of TEM/SAED patterns and calculation of theoretical power XRD patterns was described in our previous studies 30,31 . The TEM/EDX spectrum was dominated by peaks of Cu (~ 11%), Zn (~ 8%), O (~ 25%) and C (~ 37%), while the concentrations of all other elements (Mg, Ca, Cl, Si and P) were below 1%. The high concentration of carbon was due to the fact that the NPs were deposited on the supporting carbon film. The presence of  Cytotoxicity of the NPs. The in vitro cytotoxic effects of bimetallic ZnO/CuO NPs on A375 and A549 are shown in Fig. 4. The cytotoxicity of untreated cells and doxorubicin (4 and 8 µg/mL) was evaluated as negative and positive control, respectively. The index of anti-proliferative activity of ZnO/CuO bimetallic NPs on A375 cells IC 50 was 41 µg/mL while this value for A549 cells was 785 µg/mL. Figure 4 shows that these NPs are not very toxic against A549 cancer cells while their toxicity is higher than the evaluated positive control (doxorubicin). These NPs showed remarkable cytotoxic effects towards A375 cancer cells, and at a concentration of 500 µg/mL, the number of cancer cells has reached zero (Fig. 4).

Discussion
The potential therapeutic effects of trace elements against cancers made the metal and metal oxide nanomaterials promising candidates for cancer treatment and diagnosis. Trace elements in the body such as zinc (Zn), copper (Cu), iron, iodine, fluoride, chromium, selenium, manganese and molybdenum are involved in the structure  www.nature.com/scientificreports/ of metalloenzymes, proteins, immune function DNA production, enzyme function, hormones and antioxidant function. Absorption and accumulation of Cu by cancer cells can cause the angiogenesis of tumors, tumor growth, and proliferation. Since, there is a competition in the uptake of Cu and Zn in cells, the serum levels of them have been evaluated in a variety of cancers 62,63 . Interestingly, changes in serum levels of Cu to Zn play vital roles in the diagnosis and progression of lung cancer; therefore, the increasing serum Cu levels to Zn has led to the spread of malignant lung tumors 62 . However, serum Cu levels in patients with melanoma are not varied from those in healthy individuals 64 . In this study, the bimetallic ZnO/CuO NPs were successfully synthesized in one-step using S. nigra extract. Therefore, we showed the promising potentials of this plant extract for the green, eco-friendly, and cost-effective fabrication of ZnO/CuO NPs. The synthesis of NPs using conventional physicochemical methods may include the application of expensive devices.materials, toxic/hazardous precursors, high temperatures and pressures, and complicated steps 60 . The formation of NPs were confirmed by XRD, FEG-SEM, and TEM. The synthesized NPs had hexagonal and monoclinic crystalline structures. The presence of Zn, Cu, and O ions in NPs were verified by EDX analysis. The MTT assay shows the anti-cancer activity of these bimetallic NPs against cancer A549 and A375 cancer cells. Notably, the high toxicity of positive control on A549 cancer cells compared to the prepared ZnO/CuO NPs can be related to the presence of Cu in the synthesized bimetallic ZnO/CuO NPs. However, the bimetallic NPs had a favorable effect on the mortality of A375 cancer cells. According to Zarghami et al. 65 , there was a direct relationship between Cu levels and telomerase gene expression in A549 cancer cells. The enzyme telomerase is active in all cancer cells, and the level of telomerase activity is directly related to the progression of A549 cells. With increase in amount of Cu, the higher activity of this enzyme is occurred, which can result in the development and proliferation of the lung tumors. However, Zn levels do not play a critical role in the progression of this disease 65 . Also, these green-synthesized NPs can be employed for the diagnosis and treatment of other cancer cell lines.

Conclusions
A simple and inexpensive method was established for the green and eco-friendly synthesis of bimetallic ZnO/ CuO NPs using S. nigra extract as a reducing and stabilizing agent. The high purity of these bimetallic NPs as well as the monoclinic and hexagonal crystalline structures of ZnO and CuO NPs were confirmed by XRD and TEM/SAED diffractograms. The ZnO and CuO NPs with different shapes were existed in the product where the diameter of these NPs ranged approximately from 20 to 130 nm. These bimetallic NPs exhibited low toxicity on A549 cancer cells while their toxicity on A375 cancer cells was dose-dependent and the survival of cancer cells was reduced via an increase in the concentration of the bimetallic NPs. It can be concluded that the serum level of trace elements in different cancer cells is different, and according to the serum level of each element, an appropriate treatment strategy can be deployed for the relevant cancer.