Green Synthesis and Comparison of Biological Properties of Ag-NPs as a Metal Nanoparticle with ZnO-NPs as a Metal Oxide Nanoparticle and their Applications

Aim The aim of study was synthesized both silver- and zinc-oxide nanoparticles utilizing the Peganum harmala smoke extract (PHSE) bio-platform to evaluate their cytotoxicity on different types of human cancer cell lines and study their antioxidant, and antiangiogenic potentials. The Ag and ZnO nanoparticles were produced utilizing the green-synthesize method applying PHSE bio-platform. After characterization by XRD, DLS, FESEM, and FTIR methods, MTT assay was used for evaluation toxicity of nanoparticles. ABTS, DPPH, FRAP and ROS for antioxidant capacity, CAM and qPCR for antiangiogenesis effects of nanoparticle’s were used


Introduction
Nanotechnology is one of the emerging branches of science with wide application in the eld of biomedicine [1]. Size-dependent quantum effects alter the physicochemical and biological properties of nanomaterial relative to their bulk counterparts. The small size of nanoparticles and the presence of a higher percentage of atoms on their surface make it possible to interact with biomolecules both on the surface and inside the cells of the body and can leading to achieve methods for diagnosing and treating cancer [2].
Nanotechnology has taken an important step in improving the quality of anti-cancer drugs by producing nano drugs and targeting tumor cells [3]. One of the important elds of study in biomedical sciences is the evaluation of pharmacological properties of nanomaterial [4]. Silver and ZnO-NPs have recently attracted considerable attention due to their bene cial and potential effects on human health. The use of plant extracts and natural products as reducing and stabilizing agents in the synthesis of these nanoparticles (green synthesis) can increase clinical e ciency and reduce the effects of toxicity and ine ciency of nanoparticles synthesized by chemical and physical methods [5]. In various studies, the antioxidant [6,7], anti-cancer [8,9], antimicrobial [10,11] and anti-angiogenesis [12,13] activities of these nanoparticles have been investigated and con rmed.
NPs can lead to cell death by increasing the intracellular reactive oxygen species (ROS), followed by increased oxidative stress and in ammatory products such as cytokines [14,15]. ROS are produced as byproducts of oxygen metabolism in the body and play several physiological roles, such as cellular signaling. However, some factors such as environmental stressors help increase ROS production and subsequently cause an imbalance in production and its elimination by the antioxidant system.
Antioxidants play an important role in the body's defense against the damaging effects of free radicals and ROS. An imbalance between antioxidants and ROS causes oxidative stress and subsequent cell damage [16]. Although an increase in ROS in healthy cells can cause carcinogenesis, however, the increase in ROS and subsequently damage to biological macromolecules and the initiation of apoptosis is one of the most important mechanisms for the removal of cancer cells in chemotherapy [17]. Despite the inhibitory effect of ZnO and Ag nanoparticles on free radicals in vitro [6,7], their role in the production of oxidative stress following the increase in ROS in cancer cells has been shown in various studies [18,19]. Cancer cells have a negative charge on their surface due to the high percentage of anionic phospholipids and certain groups of charged proteins and carbohydrates on their surface, and this feature can cause electrostatic interaction of nanoparticles with a positive surface charge to cancer cells as target cells [20,21]. Exposure of cancer cells to nanoparticles activates cellular defense mechanisms to minimize damage, and if the stimulation of ROS production within the cell by nanoparticles is greater than the cell antioxidant defense capacity, the cells undergo apoptosis [14,15]. Other effective mechanisms of nanoparticles in inhibiting cancer cells include inhibition of angiogenesis and their anticancer effects. Studies have shown that cells in tumor tissue create new blood vessels, by secreting stimulatory factors. Therefore, the initial growth of the tumor and its continued growth depends on adequate blood supply to the tumor tissue, and inhibition of blood ow can prevent further tumor growth [22]. Various studies have con rmed the inhibitory effect of silver [13] and ZnO-NPs [12] on angiogenesis.
However, some studies have reported the pro-angiogenesis effects of ZnO-NPs [23].
In current survey, for the rst time, Pecan smoke extract was used to synthesize ZnO and Ag-NPs by green way. After characterizing and con rming the synthesis of nanoparticles, their antioxidant, cytotoxic and anti-angiogenesis effects were investigated and compared.

Materials
ABTS, DPPH, MTT, ferric tripyridyl triazine (Fe 3+ TPTZ), Dimethyl sulfoxide (DMSO), Zinc acetate, silver nitrate were purchased from Merck Co. The human cancer cell lines including PC3, A2780, HepG2, and HFF were purchased from ferdowsi university of Mashhad, Iran. All the materials needed for cell culture were bought from Sigma Aldrich.

PHSE extraction
The dried Peganum harmala seeds (1000 g) were burned at 80 C in a 1000-mL distilling ask. The oxygen gas was injected 1 puff per 5 minutes during burning. Then, the smoke components were collected by cooling and distilling by a condenser tube. N-Hexane was used as the collector solvent then, the Peganum harmala smoke extract (PHSE) was dried.

Zn-oxide nanoparticles synthesis
Zinc acetate were added at 9 gr concentration in 20 ml distilled water (DW) and mixed for 1 h at 40 ° C.
After adding the PHSE (20 mg), 1M NaOH solution was added drop wiseto the mixture. In order to form a white precipitate of nanoparticles, the resulting mixture was placed on the heater for 2 to 3 hours. Sediment (ZnO-NPs) was collected by centrifugation (8000 rpm, 20 min) and then dried at 80 ° C for 2 hours.

Ag-NPs synthesis
In order to synthesize silver nanoparticles, 0.85 g of silver nitrate was added to 500 ml of DW and after adding 20 mg of the PHSE, it was placed on the stirrer (3 h at 37 ° C). The NPs were centrifuged and dried at 80 ° C for 2 [24,25].

Identi cation of Ag and ZnO-NPs
The size and dispersion index (PSI) of NPs were evaluated by DLS method and then XRD assay was used to evaluate the crystal structure and phase of pure Ag and ZnO-NPs. The FTIR spectroscopy was applied to verify the NPs' formation, features, and interactions [26]. The size and morphology of NPs were evaluated by FESEM. For this purpose, a drop of nanoparticles (1mg / 10mL distilled water) was sprayed on a special grade and after drying and coating, it was examined microscopically.

Cell Culture
The PC3, A2780, and HepG2 as cancer cells and HFF as normal cells were seeded at the 8 x 10 4 cells/cm 2 density in special cell culture media (RPMI or DMEM) at 37 C. The media was supplemented by FBS, streptomycin, and penicillin in the required proportion.

MTT assay
For this purpose, 5 x 10 3 cells in each well of 96-well plates were cultured for 24 h and after that, the cells were exposed with serial doses of NPs. After 48 h the old culture media of each treating group was refreshed with a fresh media supplemented with MTT (0.5 mg/mL). The cells were incubated (3 h at The antioxidant potential of Ag and ZnO nanoparticles was assessed by determining their ability to reducing the ABTS free radicals [27]. Brie y, ABTS solution was made by adding potassium persulfate and ABTS in a proportion of 2.45 mM: 7 mM in distilled water (1:1). The solution was incubated for 16 h at room temperature (RT) and diluted with DW. Absorbance of the diluted solution was recorded at 0 .700 in 734 nm wavelength. Then, the diluted solution of ABTS was mixed with a range of nanoparticles' concentrations with equal volume and incubated for 1h in dark conditions at RT; nally, the absorbance of ABTS was read at 734 nm.

DPPH assay
For this, an ethanolic solution of DPPH (1mg/16ml) was made and mixed with a range of nanoparticles' different concentrations in equal volume. Then, the solution was incubated for 30 min in dark at room temperature and, the samples absorbance was read at 517 nm. The antioxidant activity of NPs was measured by using the following equation:

FRAP assay
The antioxidant potential of Ag and ZnO nanoparticles was studied by detecting their reductive potential for producing ferrous from ferric ions. According to the Benzie and Strain protocol [28], the solution called FRAP was prepared by mixing tripyridyltriazine (TPTZ), HCL, acetate buffer, and FeCl 3 .6H 2 O in a proportion of 10 ml: 40 mM: 300 mM: 20 mM at 37°. Finally, different doses of nanoparticles (1, 2, 3, 4, 5, and 6 mg/mL) were mixed with the FRAP solution in a ratio of 20: 280 µl. The standard curve was prepared regarding the FeSO 4 absorbance of the several standard doses at 593 nm [29]. Then, after 30 min incubation in FRAP solution, the absorbance of both ZnO-and Ag-NPs was recorded. The FRAP values were expressed as the produced ferrous ions (mg) for each concentration of nanoparticles (mg). All the measurements were carried out in triplicate manner.

ROS assay
ROS is naturally produced in cells as a byproduct of oxygen metabolism. Increasing the amount of ROS due to environmental pressures causes damage to macromolecules and cellular structures and causes oxidative stress. In this study, due to the high sensitivity of A2780 cells to both nanoparticles, this cell line was used to evaluate the amount of intracellular ROS. For this, cells were cultured in 96-well plates and 48 h after treatment with various doses of NPs were prepared according to the protocol of the ROS kit, and nally the uorescence intensity was measured (excitation 480-500 nm and emission 510-550 nm) [30].

CAM assay
The anti-angiogenesis potential of NPs was analyzed by performing the CAM assay. Brie y, 64 fertilized Ross chicken eggs were divided into 8 group (N=8). The eggs were stored at 37 C for 48 hours in 55-70% humidity. Then, a 1-cm 2 window was cut on the shell of eggs and sealed by sterilized para n. Samples were treated with various doses of NPs on the eighth day as following treatment plan:

Gene expression pro le
The antiangiogenic activity of both NPs was measured on the chorioallantoic membrane tissue of the treated eggs. For this purpose, chorioallantoic membranes were separated from the eggs and pulverized in separate groups with liquid nitrogen. Then, using the bioteck kit, their total RNA was extracted and the amount of RNA in each group was evaluated by Nano drop method. Then a certain volume of RNA was used to synthesize cDNA. The resulting cDNA with sybergreen, speci c primer and deionized distilled water to evaluate the expression of VEGF and VEGFR as angiogenesis genes was examined by qPCR device (Bio-Rad CFX96).

Statistical study
For the cam method, image J software was used and then the data obtained from different experiments were analyzed with SPSS software version 21. One way ANOVA and LSD test was used to evaluate the signi cance level of the results. The less than 0.05 p-values were assigned as the signi cant levels.

Identi cation of Ag-NPs
The Z-average and PDI values of Ag-NPs were estimated 82.42 nm and 0.25, respectively, which make Zaverage as a reliable size index regarding Stetefeld's theory (Fig. 1A). The theory demonstrates that less than 0.7 PDI values refer to the mono-distribution condition [32]. Also, the XRD algorithms show the crystal features Ag-NPs (Fig. 1B). The individual pecks of Ag-NPs crystals are observable at 32 and 54 2θdegrees. FE-SEM micrographs of Ag-NPs indicate its pseudo spherical morphology, support the DLS data and approve their estimated D mean number values (Fig. 1C). attributed to the bonding of oxygen from the O-H groups [33]. The FTIR data (Fig. 1D) indicate that the biological molecules could possibly be involved for synthesis and stabilization of Ag-NPs.

Characterization of ZnO-NPs
The DLS data exhibited the formation of ZnO-NPs with a size of 163.05 nm and PDI of 0.25. Since colloidal particles with dispersion index of 0-0.7 are introduced as particles with uniform dispersion, so ZnO-NPs can be considered as monodisperse particles (Fig. 1A). FESEM results con rm the presence of spherical particles with a rough appearance and size of about 100 nm, which is consistent with the data from DLS (Fig. 2B)

The cytotoxicity of Ag-NPs
The effect of concentration-dependent toxicity of silver nanoparticles against cancer cell lines compared to normal is shown in Fig. 3. As shown in the diagram (Fig. 3A), prostate cancer cells show the highest resistance to treatment, while ovarian and liver cancer cells are highest sensitive to treatment. Ag-NPs reduced only HepG2 and A2780 cancer cells in lower than its IC 50 doses for normal HFF cells (Fig. 3B).
Therefore, the safe toxic doses (STD) of Ag-NPs are de ned at lower the 19.23 µg/ml.

The cytotoxicity of ZnO-NPs
The results MTT assay showed that ZnO-NPs are able to inhibit all three cancer lines in dose-dependent way (Fig. 4A). No inhibitory effect against normal cells was reported at the concentrations studied. Examination and comparison of IC 50 showed that nanoparticles inhibited A2780, HepG2 and PC3 cells with moderate concentrations of 11.65, 47.36 and 49.96 µg/mL, respectively (Fig. 4B). Thus, A2780 cell showed the highest sensitivity to ZnO-NPs treatment compared to other cells. Figure 5A demonstrations the potential of Ag-NPs to reduce iron ions compared to Feso4 as a standard antioxidant. According to the Fig, the FRAP value increases with increasing concentration of Ag-NPs and its highest value (0.14) is observed at a concentration of 4 mg / mL Ag-NPs. Therefore, it can be said that nanoparticles at a concentration of 4 mg / mL 0.1 Feso4 have the ability to reduce iron ions. The concentration-dependent inhibitory effects of Ag-NPs against ABTS and DPPH free radicals are shown in Fig. 5B. As can be seen, nanoparticles are able to inhibit 50% of ABTS free radicals at a concentration of about 200 µg / mL, while the median concentration for inhibiting DPPH free radicals is about 707.3 µg / mL. Therefore, the results show higher inhibitory effects of nanoparticles on ABTS free radicals compared to DPPH. Evaluation of changes in intracellular ROS in Ag-NPs treatment was measured by adsorption method (5C). The results showed a signi cant increase in the amount of intracellular ROS in the treated cells with a concentration of 2 mg / mL. As the concentration of the treatment increases, the amount of intracellular ROS increases dramatically, this con rms the role of Ag-NPs in inducing cell death by increasing intracellular ROS and causing oxidative stress damage [30].

Antioxidant capacity of Zno-NPs
The reducing power of iron ions in the presence of ZnO-NPs compared to Feso4 was evaluated by FRAP method. The results showed that nanoparticles at the highest concentration are able to inhibit iron ions with a frap value of about 0.1 (Fig. 6A). The inhibitory effect of ZnO-NPs on ABTS and DPPH free radicals was evaluated to evaluate the antioxidant power of ZnO-NPs. As shown (Fig. 6B), increasing the concentration of ZnO-NPs increases the inhibition rate and the highest percentage of inhibition for ABTS free radicals is reported at a concentration of 2 mg / mL ZnO-NPs of about 4%. While at similar concentrations, about 34.25% of DPPH free radicals were inhibited. These results con rm the moderate inhibitory potency of ZnO-NPs against DPPH free radicals. One of the most important features of anticancer compounds is their ability to induce intracellular ROS, followed by cell damage and induction of cell death. In this study, intracellular ROS changes were evaluated in cells treated with different concentrations of ZnO-NPs. The results, showed a signi cant increase in ROS in treated cells with concentration of 8, 16 and 32µg/mL (Fig. 6C). Figure 7A shows the images of the treated area in the experimental groups compared to the control. As can be seen, the images clearly show the reduction of blood vessels in the treated groups with 250 and 500 µg/mL of Ag-NPs. Examination of the mean number of blood vessels in control laboratory control and samples treated with a concentration of 125 µg/mL Ag-NPs not show a statistically signi cant difference (P>0.05). However, with increasing the concentration to 250 (*P<0.05) and 500 µg/ml (***P<0.001), a signi cant decrease in the number of blood vessels is observed. Signi cant reduction in blood vessel length was also observed only in treated samples with concentrations of 250 (**P<0.01) and 500µg/mL (***P<0.001). Examination of fetal height and weight in experimental groups compared to control shows that only in samples treated with a concentration of 500µg/mL there is a signi cant reduction (*P<0.05) (Fig. 7B). Examination of alterations in the expression of angiogenesis-related genes in Ag-NPs-treated samples showed that the VEGFR gene was signi cantly reducing in all concentrations studied, while the expression of the VEGF gene at both concentrations of 125 and 250µg/mL is approximately constant and similar to the control group, while in the samples treated with a concentration of 500µg/mL, its expression was signi cantly reduced (Fig. 7C).

Anti-angiogenesis properties of Zno-NPs
Microscopic changes of vessels in nanoparticle-treated samples compared to the control are shown in Fig. 8A. As can be seen, the amount of blood vessels in the experimental groups is almost similar to the control sample. To further evaluate the effect of nanoparticles on angiogenesis, mean length and number of blood vessels were evaluated with Imag J software and the results exhibited an increase in the average number of blood vessels in the two treated groups 500 (*P<0.05) and 1000µg/mL (**P<0.01). While the average length of blood vessels showed an increase only in 1000µg/mL (**P<0.01) treated group. An increase in the mean length (**P<0.01) and weight (*P<0.05) of the embryos during the increase in vascular factors was observed only in samples treated with 1000µg/mL (Fig. 8B). In order to evaluate the NPs effect on angiogenesis at the molecular level, the expression of VEGF and VEGFR genes was evaluated by QPCR method. As can be seen, at two concentrations of 250 and 500µg/mL, a signi cant decrease in the expression of genes associated with angiogenesis is observed, but with increasing the concentration of treatment to 1000 µg / mL, the expression of genes increases signi cantly (Fig. 8C). Considering the alteration in the expression of angiogenesis-related genes and the results of CAM test, it can be said that the effect of ZnO-NPs on angiogenesis was dose-dependent. In such a way that at low concentrations at the molecular level, it inhibits angiogenesis and by increasing the concentration, its effect on angiogenesis is reversed. Therefore, in order to use these nanoparticles in studies related to cancer treatment, it is important to pay attention to the concentration used.

Discussion
The genotype complexity and poor prognosis of cancer are considered as the main barriers in its treatment. The use of nanotechnology as a practical eld to develop effective methods in the diagnosis and treatment of cancer has been considered by many researchers today [35]. Metal and metal oxides NPs such as Ag and ZnO-NPs play a prominent role in biomedical applications [36]. In current study, Pecan smoke extract (PHSE) was used as a biological platform for the green synthesis of ZnO and Ag-NPs, and then the biological effects of nanoparticles were investigated and compared.
The use of biological systems such as plant extracts and metabolites as reducing and stabilizing agents for the fabrication of NPs is called the green synthesis method, which has been successfully used in cancer treatment approaches and is an effective step in eliminating the disadvantages of physical and chemical methods [37].
In this study, smoke from burning Peganum Harmala seeds was used to green synthesize of silver (ps: 25.99 nm and PDI: 0.25) and zinc oxide (ps: 55.72 nm and PDI: 0.25) nanoparticles. Harmala is a native plant of Iran and belongs to the Zygophyllaceae family [38,39]. In the roots and seeds of this plant, alkaloid compounds, beta-carbolines such as harmin, Harman, quinazoline and etc. are found [40,41] and its anti-cancer, antimicrobial, anti-in ammatory and antioxidant effects have been reported in previous studies [42]. Due to the growing burden of cancer worldwide, different types of metal NPs have been fabricated through the green approach. For example, Zinc oxide-nanoparticles have been fabricated by various types of bio-platforms such as Laurus nobilis [43], Deverra tortuousa [44], Garcinia mangostana fruit [45], and Cucumis melo inodorus rough shell extracts [46]. Also, there are several types of plant extracts that have been utilized for synthesizing Ag-NPs including Amphipterygium adstringens [47], Centella Asiatica [48], and Ficus benghalensis prop root extracts [49]. In a study in 2015, Ag-NPs with a size of 5 to 25 nm from the aqueous extract of Caulerparacemosa were prepared by green method [50].. In another study, Ag-NPs were green synthesized using the sumac aqueous extract with an average size of 35nm [51] which is comparable to the Ag-NPs synthesized in the present study.
Similar to the present study, in a study conducted in 2021, globular nanoparticles with 55 nm were fabricated by the green method. In this study, Arthrospira platensis was used as a reducing agent for the formation of NPs [52].
In this study, the toxicity of Ag-NPs and ZnO-NPs against three cancer cell lines was compared with HFF cells. The most sensitive and resistant cell lines to treatment with Ag-NPs were HepG2 and PC3 with IC 50 about1.95 and above of 40 µg/mL, respectively, while in the treatment with ZnO-NPs the highest and lowest resistance to treatment in HFF and A2780 cells with IC 50 higher than 62 µg/mL and 11.65 µg/mL (respectively) was observed. Since ZnO-NPs did not show any toxic effect on normal cells at the studied concentrations, it can be said that ZnO-NPs are safer compared to Ag-NPs. Many studies similar to our study have shown the toxicity of various NPs including Ag-NPs and ZnO-NPs on cancer cells.
A study in 2018 showed higher cytotoxic effects of Ag-NPs synthesized from Artemisia tournefortiana extract against HT-29 (IC 50 =40.71 mg/mL) compared to the HEK293 (IC 50 =61.38 mg/mL) as a normal cells [53]. In another study was reported the strongly cytotoxic effects of Ag-NPs synthesized by aqueous extract of Rubia tinctorum against HepG-2 cells (IC 50 : 6µg/mL) compared to the HFF (IC 50 : 100µg/mL) as a normal cells [54].
In a study was conducted in 2021 on the fabricated ZnO-NPs by Swertia chirayita leaf extract revealed the toxic effects against HCT-116 and Caco-2. The nanoparticles showed a strongly cytotoxic potency against HCT-116 and Caco-2 cell lines, but less so against the normal HEK-293 cell line [55]. Also, in a study in 2019, Mahdizadeh et al. have reported the toxicity of ZnO-NPs from Cucumis melo on MCF7 (40 µg/mL) and Tubo (20 µg/mL) cells after 24 h [46]. ROS increasing, although under natural conditions causes diseases related to oxidative stress such as cancer, but in the treatment of cancer is used as one of the applied strategies in chemotherapy [17]. Studies show that increasing the amount of ROS can lead to the release of cytochrome C and activation of apoptotic pathway by changing the permeability of the mitochondrial membrane [56]. Our ndings show the antioxidant effect of ZnO and Ag-NPs in vitro and its pro-oxidant effects inside the cancer cell. Our results exhibited a signi cant antioxidant capability for Ag-NPs for ABTS (IC 50 :150.07µg/mL) and DPPH (IC 50 : 707.33µg/mL) free radicals (Fig. 5). In other words, ZnO-NPs appeared as a weak radical scavenger compared with Ag-NPs and glutathione antioxidant potential.
In 2019, Ahmadi et al reported the inhibitory effect of ZnO-NPs against ABTS and DPPH free radicals with IC 50 about of 31.2 and 60µg/mL [57], which shows a higher antioxidant power compared to our study.
Similar to the present study, in a 2015 study, the inhibitory effects of Ag-NPs from Cassia roxburghii on ABTS free radicals (IC 50 : 140 µg/mL) and their reducing power on iron ions (FRAP of 2.64 (Mg −1 )) were investigated and con rmed [58]. Also, in a study in 2017, 23-95% of ABTS free radical scavenging capacity and 8.8 Mg −1 FRAP activities of Ag-NPs were reported [59]. The results of many studies show that NPs increase ROS production in various cancer cells [22]. Similar to the present study, in a 2020 study, the anti-proliferative and pro-oxidant effects of Ag-NPs from Beta vulgaris L were demonstrated in human hepatic cancer cells (HUH7) by ROS induction[60]. Lee et al. reported the potential to stimulate ROS production in human keratinocytes treated with ZnO-NP. They showed that the mechanism of nanoparticle toxicity is related to ROS production, oxidative stress and apoptosis Angiogenesis is one of the most important features of tumor cells that help their survival and reducing angiogenesis plays the main role in preventing the growth of cancer cells and in ammation [22]. The results of this survey con rmed the reduction of angiogenesis in the treatment with different concentrations of Ag-NPs in CAM and qPCR assay. Investigation of the effect of ZnO-NPs on angiogenesis showed that, they suppress angiogenesis by down-regulating VEGF and VEGFR gene expression at 0 to 500µg/mL concentrations and act as a pro-angiogenic compound at>500µg/mL doses. In many studies consist with the present investigation, the anti-angiogenesis effects of nanoparticles have been investigated and con rmed [64, 65] .
For example, a reduction in angiogenesis by the CAM method was reported in a 2015 study by examining the mean number and length of blood vessels in samples treated with Ag-NPs [13]. Also, the inhibitory effect of Ag-NPs on VEGF expression in the mouse matrigel plug [64] and retinal endothelial cells [66] has been reported in previous studies.
Various investgations have shown the anti and pro angiogenesis potential of ZnO-NPs. In a study in 2019, the anti-angiogenesis effects of ZnO-NPs from Hyssops o cinalis L were reported using the CAM assay [12]. Ahtzaz et al reported the pro-angiogenic effects of ZnO-NPs in 2017 using the CAM method [23]. In a study by Oikawa et al., The inhibitory effect of ZnO-NPs on the formation of blood vessels by reducing the expression of VEGF and VEGFR was reported [67] which is comparable to our results.

Conclusion
According to the results, Ag-NPs were exhibited stronger antioxidants capacity than ZnO-NPs, also, the higher cytotoxic effect of Ag-NPs have been showed compared to ZnO-NPs by MTT method. In addition, Ag-NPs showed signi cant anti-angiogenesis effects by reducing VEGF and VEGFR genes expression. Considering the survival of cancer cells and suppression of angiogenesis, higher anti-cancer activity of Ag-NPs was detected compared to ZnO-NPs.