Abstract

This study focuses on establishing a green, inexpensive, non-toxic alternative for treating cancers. This aim explores the synthesis and use of silver oxide nanoparticles (Ag2O/Ag NPs) using a green synthesis technique. Considering that there is a high death rate in cancer, breast cancer in particular, which is highlighted by a breast cancer cell line MCF-7, we have decided to use nanotechnology as a therapeutic tool. Conventional ways of coping with cancer symptoms, including radiotherapy, chemotherapy, and immunotherapy, even though they are the most effective, have the drawback of high costs and side effects. Instead of toxic chemicals, nanoparticles are gaining more interest due to their peculiarity of chemical and physical specifications, with Ag2O/Ag NPs pointing to a wide range of actions from anticancer effectiveness. Using the extract from the Ginkgo biloba plant, we achieved the green synthesis of Ag2O/AgNP via the new method widely adopted for environmental and health safety reasons. The nanoparticles were synthesized and characterized with SEM, HRTEM, EDX Spectroscopy, SEAD pattern, and DLS, and it was evident that the particles were spherical, homogeneously distributed in size, and compositively pure. Moreover, the Ag2O/Ag NPs were tested for their cytotoxic efficacy towards MCF-7 breast cancer cells, employing the MTT assay and the Hochst blue-fluorescent staining method, which demonstrated an effective dose-dependent toxicity that highlighted the possibility of such NPs as a candidate cancer therapeutic. Moreover, these nanoparticles were found to show antimicrobial activity, which helped to broaden their biomedical potential. The present research emphasizes the necessity of friendly-to-environment nanoparticle synthesis and provides an additional clue toward developing cancer treatments.

Highlights:

1. Develop green, cost-effective, non-toxic cancer treatment using Ag2O nanoparticles.
2. Synthesized Ag2O/AgNPs via Ginkgo biloba extract; tested anticancer efficacy.
3. Ag2O/AgNPs show dose-dependent cytotoxicity and antimicrobial potential.

Keywords: AgO ;Ag ; Capping agent; Cancer; Ginkgo biloba

Introduction

The most meaningful cause of death is cancer in our world, and breast cancer is the second major cause of death [1]. There is a collection of available therapies for cancer treatment comprising radiation, surgery, chemotherapy, immunotherapy, and hormone therapy. [2]. However, these approaches are expensive and have side effects, limiting their utilization. As a result, there is a need for practical, non-toxic, low-cost treatments with minimal side effects. Nanotechnology is one of the most important new research areas in current material science, and nanoparticles are employed in every science phase, influencing all aspects of human life. Nanoparticles, which have a size range of (1–100nm), are the most straightforward structure [3, 4]. These nanoparticles have therapeutic promise for various ailments due to their physicochemical features and qualities [5]. Silver oxide nanoparticles (Ag2ONPs) are a novel nanometal particle type widely applied in biology, medicine, and engineering [6]. Synthetic physiologically active Ag2ONPs, in particular, have shown a wide range of therapeutic potential, including anticancer, anti-fungal, antibacterial, anti-inflammatory, anti-oxidative, anti-oxidative, anti-diabetic action, and wound healing [6, 7]. Various approaches are available for synthesizing metal oxide nanoparticles, including biological, physical, and chemical. In the biological technique, nanoparticles are produced by the biosynthesis of plants, algae, yeast, bacteria, and fungi [8, 9]. The physical and chemical processes have various advantages, such as a narrow size range of nanoparticles. Still, the main disadvantages are low efficiency, costs and time, and toxic byproducts [10].

On the other hand, biological approaches have several advantages over conventional techniques: low cost, eco-friendly, no downstream processing, and excellent yields.

Furthermore, natural processes are often known as green synthesis. As a result, using plant extracts to produce nanoparticles enables a straightforward one-step reduction approach with large-scale manufacturing [10]. The plant extract stabilized, capped, and reduced the agent [8]. Numerous studies have been published on the creation of nanoparticles from plant extracts with biologically significant properties; Fayyad et al. (2021) synthesized AgNPs using henna (Li) aqueous plant extract for antimicrobial activity [11], and Sarala et al. (2020) synthesized zinc ferrite NPs using aqueous extract of henna (Li) plant for anticancer properties against the human breast cancer type MCF-7 cell line [12]. The study conducted by Al-Sheddi et al. (2018) that used plant extract of Nepeta deflersiana components to synthesize AgNPs for anticancer activity against human (HeLA) cancer cells was done. [13]. Research by Gomathi et al. (2020) applied ethanolic extract from tamarind fruit shells for synthesizing AgNPs in human breast cancer type (MCF-7) cell lines [8]. In this article, the synthesis of Ag2O/Ag NPs in a green way via the ginkgo biloba plant extract as a capping and reducing agent is aimed to be done. Furthermore, ag0/Ag NPs structural and morphological analyses and their cytotoxic potential in the breast cell line (MCF-7) should also be studied. Many investigations conducted in 2020 displayed Ginkgo Biloba-AgNPs as a potent suppressor of cancer cell proliferation and apoptosis inducers mediated through intracellular ROS production and mitochondrial signaling pathways in cervical cancer cells [6]. Moreover, plant-assisted green synthesis of silver nanoparticles is efficient in 2020 and even better than chemical methods in this area [14].

Because of their unique properties, silver nanoparticles (AgNPs) have become a subject of interest by the recent advancements in nanotechnology for diverse applications ranging from biomedical engineering to environmental remediation. Nevertheless, conventional synthesis methods are typically carried out using harmful chemicals and taxing processes powered by energy that make them environmentally and health-harming. As an alternative, natural green synthesis from plant extracts has been developed, which offers a chance to utilize biocompatible and eco-friendly pathways for nanoparticle fabrication. Recent research has highlighted the wide range of green synthesis methods and their effectiveness. Selvam et al. (2025) produced AgNPs using sandalwood extract because of catalysis and antibacterial applications.[15]. In 2024, Lalsangpuii and co-workers demonstrated that generating AgNP from Mikania micrantha was possible for remarkable anticancer activities when tested against human lung adenocarcinoma [16]. These and some other research projects demonstrate a wide range of plant sources naturally extracted from ordinary sources, such as Ginkgo biloba and rare species, which end up with conjugated nanoparticles with diverse physical and functional features. While the above studies have demonstrated the nano-size status of AgNPs as a bright area, the practical aspect of the specific optimization of AgNPs as green-synthesized remains a cavity, especially in cancer-specific targeting of specific cell lines by controlling the physicochemical properties. The incorporation of Ginkgo biloba extract as a reducing and capping agent, as well as for fine-tuning nanoparticles for enhanced cytotoxicity against breast cancer (MCF-7) cells, is the primary purpose of our study, which is an attempt to fill the gap in there. The idea underlying our research is based on the possibility of the phytochemical composition of Ginkgo biloba to determine the AgNPs with unique surface chemical properties and structures, which are likely to have better biological activity. It is our goal to use the MCF-7 cell line as an avenue to contribute to the growing demand to combat breast cancer with more effective yet less toxic chemotherapeutic agents. Ultimately, our methodologic innovations aim to bypass the issues observed in the previous studies, including particle agglomeration and suboptimal biological activity, by carefully selecting the synthesis conditions and utilizing the specific biological constants of a given plant [17].

The research addresses two pressing issues in modern science: a greener strategy and the search for the answer to cancer cure. This research aims to dominate a greener way of silver nanoparticles (Ag NPs) synthesis by biologically utilizing the properties of Ginkgo Biloba, a plant, credited for its medicinal value for several decades. Conventional techniques mainly comprise harsh chemicals and energy-consuming processes, which result in environmental issues and health risks. However, using Ag2O/Ag NPs from Ginkgo Biloba could be a better option, as it provides a sustainable selective approach to harnessing the bioactive compounds for nanoparticle synthesis. Moving beyond the boundaries of green chemistry, the study goes on to the pharmaceutical field, investigating whether nanoparticles have anticancer potential. Silver nanoparticles have appeared as a promoters of cancer treatments thanks to their unique physicochemical characteristics. Thus, the study will examine the cytotoxic effects on cancer cells. Finally, this interdisciplinary study aims to shed light on the synergy between environmentally friendly synthesis techniques and innovative cancer therapeutics by giving a vision of a green future with a natural answer to global problems

Methods

Experimental section:

Plant Extract Preparation Method :The Ginkgo Biloba plants in Iraq/Wasit were bought from local nurseries. After being gently scraped to remove dirt and other dust, it was cleaned with tap water and rinsed with distilled water. For optimal drying, the Ginkgo Biloba leaves were finely chopped and dispersed. To avoid microbial fermentation and the consequent loss of plant components, ginkgo biloba leaves were air-dried for ten days in a dry, shaded setting. The dried leaf sections are mechanically ground into tiny particles to make the plant tissues into powder. 2g of Ginkgo Biloba powder was dissolved in 100 ml of distilled water with a pH of 3.9 using a hot plate stirrer, then heated and stirred for an hour at 45 °C. The Ginkgo Biloba solution was vacuum filtered with filter paper, a side-arm flask, and a Buchner funnel. The resultant aqueous solution should be kept at room temperature for future use.

Ag2O/Ag NPs green synthesis: Silver oxide nanoparticles were synthesized utilizing the green-synthesis method, employing silver nitrate (AgNO3) obtained from , UK, and an extract from the Ginkgo Biloba plant. A 1 molar solution of silver nitrate (AgNO3) was prepared by dissolving AgNO3 in 100 ml of distilled water. The dissolution process was conducted at a temperature of 75 °C for a duration of 1 hour, with strong agitation at a speed of 700 rpm. Subsequently, a 100 ml aliquot of silver nitrate aqueous solution was delicately combined with 100 ml of Ginkgo Biloba plant, followed by an hour-long heating at 70 °C. The green synthesized Ginkgo Biloba forms a dark brown show in figure 1 , water-based colloidal mixture. To study and analyze the crystal structure, elemental composition, and surface morphology of Ginkgo Biloba, XRD, EDX, and TEM techniques were used. The final Ginkgo Biloba colloidal solution was sprayed on a glass substrate using a micropipette and a hot plate stirrer to produce a film layer. A temperature lower than 50 °C was used for the drop-casting process.

Anticancer Activity assay :The anticancer activity of colloidal Ag2O/Ag NPs was investigated using the MTT assay. The antibiotic-antimitotic solution was utilized in DMEM with 10% fetal bovine serum to cultivate the MCF-7 breast cancer cells. The MCF-7 cells were then grown in 96-well plates at a density of 1x104 cells/well and treated for 24 hours with various Ag2O/Ag NP dilations (0.5,0.25, 0.125, 0.0625, 0.03125, and 0.015625 per well) and grew in 5 percent CO2 at 37 °C. Following the incubation period, the MTT solution was applied to each well. For 4 hours, the plate was put in incubation under dark circumstances. The formazan crystals generated during the reaction were decomposed in 100μl of DMSO. A microplate reader was used to assess the spectrophotometric absorbance of the purple-blue formazan dye at 570 nm. Eq. (1) calculates the relative cell viability of breast cancer cells.

Figure 1.

The anticancer activity of the Ag2O/Ag NPs of the MCF‑7 live cancer cells was determined using Hochst blue-fluorescent staining.

Characterization: Physicochemical characterization of Ag2O/Ag NPs produced utilizing the PHILIPS X-Ray Diffractometer (XRD) PW 1730 model was studied. Bragg's angle intensity evaluation was used to assess the Ag2O/Ag NPs layer film's crystalline structure. The goal is to concentrate on copper (Cu) at 1.54060 °A, 30 mA, and 40 kV. The scan covers 10 to 80 by 2θº . The elemental composition of the Ag2O/Ag NPs layer film was determined using the Energy Dispersive Spectroscopy (EDX) technique using the TESCAN Mira3 model. The surface topography and particle size measurements were done using the Transmission electron microscopy (TEM) model PHILIPS CM30 /V=200KV. The Fourier transforms infrared spectroscopy (FTIR) indicated the (4000–400 cm-1) wavenumber range of the plant extract and the green generated Ag2O/Ag NPs colloidal solution using FTIR device model IRAffinity-1 from SHIMADZU

Result and Discussion

The x-ray diffraction at grazing incidence angle (GIXRD) technique was utilized to investigate the crystal structure of an Ag2O/Ag NPs layer film. This technique studied the sample's surface layer rather than the substrate material (i.e., glass). A surface-sensitive diffraction method limits penetration into bulk materials while maximizing intensity using a small incident angle X-ray beam. The polycrystalline Ag2O/Ag NPs layer film's X-ray diffraction patterns are represented in Figure.1

Figure 2.X-ray diffraction (XRD) patterns of Ag2O/Ag NPs layer film

The most muscular three X-ray diffraction patterns 2-theta at 32.889˚, 38.103˚, and 55.011˚can be well-matched with miller indices (111), (020), and (202) crystal planes of silver oxide in cubic phase can be well-matched with Crystallography Open Database (COD) card number 96-101-0487 (cubic) Ag2O. On the other side, 2-theta at 44.54˚, 64.59˚, and 65.69˚ can be well-matched with miller indices (200), (202), and (131) crystal planes of silver in the cubic phase can be well-matched with (COD) card number 96-500-0219 (cubic) Ag. Table 1. Provides a detailed analysis of the crystal structure of the layer film formed by green synthesis Ag2O/Ag NPs. The crystallite size was determined using the Debye-Scherer equation (Eq. 2), and the average length of the three most prominent peaks was approximately 20nm.

Figure 3.

D refers to the crystallite size in nanometers, λ refers to the wavelength of the X-ray Cu-K radiation, which is 1.54060 °A, β refers to the full width at half maximum in radians, θ refers to the Bragg's diffraction angle in radians. K refers to the shape factor of 0.94 [11]. The spacing (d) between the crystalline layers was calculated using Eq. (3).

Figure 4.

Here, n denotes the sequence of the diffraction peak [11].

The Ag2O/Ag NPs layer film's components are identified using an x-ray method known as (EDX). Ag2O/Ag NPs contain various elements, each with a distinct atomic structure, causing it to emit a spectrum with a particular collection of peaks. The energy-dispersive X-ray spectrum of the greenly synthesized Ag2O/Ag NPs is shown in Figure. 2, along with a quantitative result corresponding to oxygen (O) and silver (Ag) elements and the element's normalized concentration expressed as an atomic weight (A%) and a proportion of weight (W%). Owing to surface plasmon resonance (SPR), strong and medium AgLα and AgLβ absorption peaks of silver (Ag) nanocrystals were seen at (2.970 and 3.170 KeV). Further, a slight absorption peak of the oxygen element (Okα) has been observed at 0.50 KeV only. This outcome can also correspond with the references of the theoretical model. [18].

Figure 5.EDX spectrum and quantitative results of Ag2O/Ag NPs.

The green-synthesized nanoparticle layer film samples' surface morphology and particle size were evaluated using field emission-scanning electron microscopy (FE-SEM) imaging. Figure 3 shows the surface morphology of Ag2O/Ag NPs samples with four different magnifications. , The average particle size of the Ag2O/Ag NPs was 39.1 nm, and the FE-SEM image revealed accumulations and uniform dispersion with semi-spherical shape morphologies.

Figure 6.FE-SEM images of Ag2O NPs layer film surface morphology and particle size at (A) 25 kx,(B) 50 kx, (C) 100 kx, and (D) 200 kx.

The transmission electron microscopy (TEM) technique is used to study and investigate the shape and particle size of the nanoparticles more accurately than FE-SEM. Figure 3 d shows the distribution of particle sizes of Ag2O/Ag NPs. The nanoparticles range from 12.64 to 115.9 nm, averaging particle size of (20.71 nm). In contrast, Figure. 4(a-c) TEM images show the Shape and Aggregation State of Ag2O/Ag NPs at three different magnifications. TEM images can show whether nanoparticles are dispersed individually or tend to aggregate. Aggregation can significantly affect the biological activity of nanoparticles, including their ability to be taken up by cells and their toxicity profile. The TEM images reveal that the nanoparticles are encased in a capping layer from the Ginkgo biloba plant extract. This capping layer is significant because it suggests that compounds from the plant extract are binding to the surface of the nanoparticles, potentially influencing their stability, solubility, and biocompatibility. The presence of this layer indicates that the plant extract not only acts as a reducing agent to form the nanoparticles but also as a stabilizing agent that controls their growth and prevents aggregation.

Figure 7.The transmission electron microscopy (TEM) with three various magnifications: (a) 50 nm, (b) 100 nm, (c) 200 nm, (d) particle size distribution of Ag2O/Ag NPs.

The 400–4000 cm-1 wavenumber range of the FTIR spectrum was utilized to identify the main functional groups and chemical bonds of Ginkgo Biloba plant extract and green synthesized Ag2O/Ag NPs colloidal solution. FTIR analysis confirms that the phytochemical constituents of Ginkgo Biloba extract are encapsulating the surface of Ag2O/Ag nanoparticles.

Figure. 5 shows the Ginkgo Biloba plant extract and Ag2O/Ag NPs colloidal solution FTIR spectrum. Ginkgo Biloba shows a medium and broad peak at 3503.2 cm-1 and 3331.3 cm-1 due to stretching vibration of the hydroxyl group H-bonded (O-H stretch) and amine stretching vibration (N-H stretch), respectively. A broad and robust peak at 2897.4 cm-1 is due to sp2 alkenes (C-H stretch). Two minor peaks at 1599.39 cm-1 and 1525.02 cm-1 are due to carbonyl and aromatic groups of (C=O and C=C stretch), respectively.

Figure 8.Ginkgo Biloba plant extract (Blue spectrum) and Ag2O/Ag NPs colloidal solution (Red spectrum).

A sharp and medium peak at 1028.38 cm-1 is attributed to carbohydrates (C-O bending). Minor peaks appeared between (783.4-725.3) cm-1 in the fingerprint region, representing carbohydrates, aromatic, and alkyne of C-O and C-H bending. On the other hand, Ag2O/Ag NPs showed shifting in the FTIR spectra, with medium and broad peaks appearing at 3521.4 cm-1, 3339.7 cm-1, and 3017.65 cm-1 due to the hydroxyl group (O-H stretch) H-bonded and stretching vibration of amine (N-H stretch), and sp2 alkenes (C-H stretch), respectively. Minor peaks at 1642.25 cm-1 of C=O stretching were attributed to the carbonyl group. Strong and little peaks appearing at 1361.5 cm-1 and 1060.2 cm-1 are due to carbohydrates and aldehyde groups of C-O and C-H binding, respectively. In the fingerprint region, Ag2O/Ag NPs peaks obtained at 555.9 - 515.1 cm-1 represent the Ag-O mode of vibration.

The anticancer activity of the green synthetic form Ag2O/Ag NPs was evaluated using the MTT assay against human breast cancer (MCF7) cells within the dilations range (from 0.5 to 0.015625). The association between the relative cell viability of the cancer (MCF7) cell line and different dilations of the green synthesized Ag2O/Ag NPs is shown in Figure. 6-a.

According to the chart findings, colloidal Ag2O/Ag NPs demonstrate considerable toxicity at greater dilations (doses), steadily dropping from 0.5 to 0.015625. On the other hand, the viability of breast cancer (MCF7) cells increased steadily from 37.08 percent to 96.88 percent, which means that the amount of surviving breast cancer cells is inversely proportional to the Ag2O/Ag NPs dilations. In addition, prior studies have revealed that many nanoparticles have dose-dependent cytotoxicity [1-3].

Figure. 6 (b-h) displays fluorescence microscopy pictures demonstrating the vitality of cancer (MCF7) cells using Hochst blue-fluorescent staining. Images (c-f) give the cancer cell viability treated with Ag2O/Ag NPs at different dilations: 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.015625, respectively, in comparison to image (b) of control cancer cell viability (no treatment). The rise in the dilations of the Ag2O/Ag NPs was associated with a steady decrease in cancer cell viability. As a result, the initial finding in Figure. 6-a is affirmed. The reference [1] agrees with this result. The anticancer action of Ag2O/Ag NPs is related to producing reactive oxygen species and silver ions. Ag2O/Ag NPs directly communicate with cell membranes, causing cell membrane collapse, rupture, and death. Reactive oxygen species can affect mitochondrial respiration, disequilibrium, and cellular redox damage in mitochondria.[19]

The tabulated results of the relative cell viability (%) for breast cancer cells treated with different dilutions of green synthesized Ag2O/Ag NPs are as follows:

Figure 9.(a) MTT assay chart gives the relative cell viability of breast cancer vs Ag2O/Ag NPs at different dilations, (b-h) Blue-fluorescent staining of cancer (MCF‑7) cell viability.

A statistical analysis using ANOVA was performed to test for significant differences in cell viability between the different dilutions. The p-value obtained from the ANOVA test is 0.0, indicating statistically significant differences in the relative cell viability among the treatment groups, including the control group. This result supports the hypothesis that the anticancer activity of the green synthesized Ag2O/Ag NPs is dose-dependent, with higher toxicity observed at higher concentrations.

The analysis data of the anticancer doze of Ag2O/Ag nanoparticles (NPs) on human breast cancer (MCF7) cells showed time- and dose-dependent toxicity. This data demonstrates that as the dilution of Ag2O/Ag NPs increases (from 0.015625 to 0.5), the viability of breast cancer cells is reduced drastically, between 96.88% at the lowest concentration and 37.08% at the highest, respectively. Such behavior demonstrates the murderous or poisonous activity of nanoparticles toward cancer cells, as cell death will increase in line with the rise in concentrations of nanoparticles.

A striking discovery exhibiting a P-value of 0.0 through the ANOVA test calls into question a strong link between the anticancer activity and concentrations of the Ag2O/Ag NPs. This finding is critical for evaluating the remedial mechanism of Ag2O/Ag NPs where beginning with the right dosage can increase the impact of the anticancer effect but reduce the side effects that may occur on the healthy cells.

A previous study confirmed nanoparticles' dose-dependent toxicity, which is consistent with the results of this study.

To determine the selective cytotoxicity of Ag-nano particles fabricated from Scutellaria multicaulis stem on breast cancer cells (Z. Gharari et al., 2022), researchers explored an application of green synthesis. Their data demonstrate that certain plant extracts can promote anticancer characteristics of the synthesized nanoparticles, thereby forming a saving-as-an approach to cancer therapy.[20]

Conclusion

The green strategy has synthesized Ag2O/Ag NPs using Ginkgo biloba plant extract. XRD produced the hexagonal Ag2O with a packed shape and size of 20 nm. I have obtained the data by EDX, and the plots show that AgLα and AgLβ appear to be absorbed strongly and moderately at 2.97 and 3.17 KeV of silver (Ag) nanocrystals, accordingly. Similarly, the X-ray spectrum shows a reduced absorption of OKα at 0.5keV, indicating the presence of oxygen (O). TEM technique showed the surface topography of Ag2O/Ag nanoparticles is semi-spherical with an average particle size of ⁓20nm, and the capping layer of Ginkgo Biloba plant extract encased the Ag NPs, indicating that the plant extract works as a capping layer, molding the nanoparticles during their growth. The principal functional group peaks of the Ginkgo Biloba plant extract were shifted in the spectrum of the FTIR compared to Ag2O/Ag NPs. In the fingerprint region, Ag2O/Ag NPs peaks obtained at 555.9 - 515.1 cm-1 represent the Ag-O mode of vibration. The results demonstrated excellent anticancer efficacy using the green synthesized Ag2O/Ag NPs against human breast cancer-type MCF-7 cells.

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