Bimetallic nanoparticles green synthesis from litchi leaf extract: a promising approach for breast cancer treatment

Noble metal nanoparticles have demonstrated promising biomedical and nanomedicine applications, and their bimetallic equivalents from the green synthesis approach are expected to be more promising. This study concerns bimetallic nanoparticle’s synthesis, characterization, and structure-function analysis for their potential application in breast cancer. Silver core (SCNPs) and Gold core (GCNPs) bimetallic nanoparticles were synthesized using Litchi Chinesis leaf extract (LCLE) and characterized using various physio-chemical techniques. The results revealed the successful synthesis of SCNPs and GCNPs with distinct surface plasmon resonance peaks at 551 nm and 531 nm, hydrodynamic sizes of 66 nm and 53 nm, and Zeta potential values of −26.0 mV and −20.6 mV. XRD analysis confirmed the presence of silver and gold phases, while HR-TEM images revealed spherical shapes for SCNPs and heterogenous shapes for GCNPs. Both nanoparticles demonstrated dose and time-dependent inhibition of breast cancer cell growth, with GCNPs requiring a higher concentration than SCNPs at 48 h compared to 24 h. Cell cycle evaluation indicated a cell cycle arrest in the G2M phase for both nanoparticles, an impact on the S phase distribution, and reactive oxygen species (ROS) generation, further contributing to their antiproliferative effects.


Introduction
Breast cancer is a global health concern, accounting for a significant proportion of cancer cases and mortality in women [1].Despite advances in cancer therapy, current treatments such as radiation therapy, chemotherapy, and surgery have limitations and can cause severe side effects [2].Therefore, there is an utmost need to generate more specialized and targeted therapies for breast cancer.Over the years, various categories of chemotherapeutic drugs have been used to treat diverse cancers of the liver, breast, etc [3].Furthermore, the efficiency of these drugs is hindered by challenges like stability issues, restricted solubility, and target site delivery, including some of the undesired side effects [3,4].Recent attention is towards synthesizing newer agents at the nanoscale level to boost their target delivery, decrease their dosage, and limit the undesirable side effects [4][5][6].Nanotechnology is a promising and emerging area that provides a versatile platform for various biomedical applications [7].
Further, the National Cancer Institute (USA) has promoted research into possible medicinal plant extractbased agents with anticancer properties [8].Medicinal plants effectively treat various metabolic illnesses and malignancies [7].However, biologically or green-synthesized metallic nanoparticles from medicinal plant extracts have piqued researcher's curiosity to explore the potential of nanoparticle-mediated anticancer treatment [8].
Silver (Ag) is widely recognized as the most electrically and thermally conductive metal.However, silver nanoparticles (AgNPs) have also been vastly investigated in cancer research due to their potent cytotoxic properties [9].Emerging studies suggest that nanoparticle synthesis involves the utilization of various components of medicinal plants to achieve biocompatibility and offers the advantage of lower side effects compared to chemotherapeutic drugs [10].On the other hand, gold nanoparticles (AuNPs) exhibit distinctive surface characteristics such as surface plasmon resonance (SPR) and the ability to conjugate with other biological molecules, enabling surface modification for diverse biological applications [10].Numerous studies have elucidated the cytotoxic effects of AuNPs both in vitro and in vivo, with evidence suggesting their potential as anticancer agents through the induction of oxidative stress [11].
Further, recent evidence also indicates that synthesizing bimetallic silver and gold nanoparticles using biological or green synthesizing methods is a promising approach with potential in cancer therapy due to their unique physicochemical properties, use of non-hazardous chemical agents, and lower side effects [12].Recent evidence also suggests that bimetallic nanoparticle synthesis using biological molecules such as proteins, enzymes, or other biomolecules facilitates the formation of metallic nanoparticles with unique physic-chemical properties [13].
Green synthesis methods for nanoparticle synthesis involve using natural or green sources, such as extracts of various parts of plants that act as reducing agents and completely limit the use of chemically hazardous materials [13].This approach offers an acceptable and ecological alternative to conventional chemical methods that employ toxic chemicals and solvents [13,14].Bimetallic nanoparticles synthesized using green methods possess unique properties, including smaller size, increase surface area, and surface charge, which enable them to penetrate cancer cells effectively and induce cell death [14].Moreover, these nanoparticles can target cancer cells while cautious healthy cells, thereby reducing the side effects of conventional cancer chemotherapies [15,16].For instance, metallic nanoparticles of silver and gold synthesized using an extract of Melissa Officinalis and Mentha Longifolia exhibited potent anti-cancer effects against human breast cancer cells and mediated through the regulation of caspases, apoptosis, and cell proliferation [16][17][18].The evidence highlights their remarkable attributes, including nanoparticle biocompatibility, cellular uptake capabilities, and multifunctionality [19].Some studies have also explored the potential of bimetallic nanoparticles even as a medical drug carriage complex for breast carcinoma therapy and have the potential to prevail over drug resistance in breast carcinoma [20].
This study aimed to synthesize silver (SCNPs) and gold (GCNPs) bimetallic nanoparticles using Litchi Chinesis leaf extract (LCLE).These nanoparticles were then subjected to various characterization techniques, including DLS, x-ray diffraction, HRTEM, UV-visible spectroscopy, and Zeta potential analysis.In addition to characterization, the study investigated the possible potential anticancer activities of SCNPs and GCNPs against MCF-7 breast cancer cells.This investigation not only focused on the bimetallic nanoparticles synthetic protocol but also targeted to assess their efficacy in suppressing cancer cell proliferation, cell cycle regulation, and generation of reactive oxygen species (ROS), thereby offering potential opportunities for their utilization in cancer therapeutics.

Collection of chemicals
Hydrochloroauric acid (HAuCl 4 .3H 2 O) and Silver Nitrate (AgNO 3 ) were obtained from M/s Sigma Aldrich Chemicals.The Litchi Chinensis fresh leaves were gathered from the botanical garden at Panjab University, Chandigarh, India.

Preparation of Litchi Chinensis leaf extract
Litchi Chinensis leaves were cleaned thoroughly with faucet H 2 O and then surface sterilized with Deionized water to detach any grime particles.The leaves were dried in the sunlight and crushed.After that, 50 ml of DI water and 3 gm of leaf powder were simmered for few min.Further, the solution was filtered using Whatman filter (paper No. 1) and stored at 4 °C for later use.The schematic presentation for the preparation of LCLE is shown in figure 1.

Synthesis of bimetallic nanoparticles 2.3.1. Synthesis of AgNPs and AuNPs
AgNPs and AuNPs were synthesized using LCLE, as reported earlier [21,22].AgNO 3 (1 mM) and HAuCl 4 .3H 2 O (1 mM) solutions were put on a magnetic plate for stirring.The LCLE (15 μl) solution was added to each AgNO 3 and HAuCl 4 .3H 2 O solution and stirred continuously.The color of the mixture changes to red wine and brownish yellow within 15 min, which shows the synthesis of AuNPs and AgNPs, respectively.

Synthesis of bimetallic nanoparticles
The same synthesis procedures for reducing metallic salt were utilized for pure metallic NPs [21,22] to cover the surface of the generated metal nanoparticles, which were serving as a kernel, with another metal as a shell.The nanoparticles (AuNPs and AgNPs) prepared in the above section 2.3.1 were used for the further bimetallic synthesis of silver-coating gold nanoparticles (GCNPs) and gold-coating silver nanoparticles (SCNPs).For SCNPs, fresh solutions of HAuCl 4 .3H 2 O (1 mM) were prepared, and AgNPs were added and stirred vigorously on a magnetic stir plate.The color of the solution converts to blue, indicating the formation of bimetallic SCNPs.In a similar way, GCNPs were synthesized.The fresh AgNO 3 (1 mM) solution was developed and dropwise addition of AuNPs into mixture of AgNO 3 was done.The color of the mixture alter into brick red, which indicates the formation of GCNPs [23].

Characterization of bimetallic nanoparticles 2.4.1. Uv-visible spectroscopy
It was used to obtain absorption spectra of bimetallic nanoparticles, and the samples were scanned in the 400 nm-800 nm range for further analysis.UV-visible spectral experiments were performed using the LABINDIA UV-3000 + spectrometer to record surface plasmon resonance (SPR).

High-resolution transmission electron microscopy
For the ultrastructural analysis of synthesized bimetallic NPs, HRTEM (Hitachi H-7500, Japan) working at 120 kV (voltage) and outfitted with a CCD camera is employed.This device can magnify objects up to 0.6 million times in the maximum resolution mode (0.36 nm) with an operating voltage range of 40-120 kV.TEM was used to measure the size and form of bimetallic nanoparticles.

Dynamic light scattering (DLS) and Zeta potential
The colloidal nanoparticle solution was measured in a quartz cuvette to calculate the particle size distribution and potential charge.For bimetallic NPs size distribution and surface charge, experiments were performed using a Malvern zetasizer (ZEN3600).The molecules in a colloidal solution are in random thermal motion called Brownian motion.

X-ray diffraction (XRD)
X' Pert PRO was used to record the XRD pattern of SCNPs and GCNPs.XRD is an experimental technique used to investigate all aspects of solid crystal structure, including geometry, identification of unknown materials, lattice constants orientation of single crystals, defects, and stresses preferred orientation of polycrystals.By drop casting, the film was placed on clean microscope slides.The scanning speed was set at 5 0 /min, and the range selected to 2Ө = 35 0 to 80 0 .

Cell culture and treatment
The current study used the MCF-7 cell line.The MCF-7 cell lines were grown in RPMI 1640 with FBS (10%) in CO 2 (5%) at 37 °C.The cells were treated with variable quantities of bimetallic GCNPs and SCNPs for 24 h and 48 h, respectively.The concentrations were discovered in accordance with IC 50 and evaluated by cell viability assay.

Cell viability assay
In a 96-well plate, the cells were planted at 0.1 × 10 5 (m/v) per well.After 24 h, the solution was take out, and the cells were treated with variable concentrations of bimetallic GCNPs and SCNPs for 24 or 48 h using (10 μl-40 μl) from the prepared drug solution.A wide range of concentrations was prepared, and triplicates were used for all the drug concentrations corresponding with vehicle and control groups (Untreated group) wells.After exposure, 50 ml of MTT solution (5 mg ml −1 in PBS) was put into the each well.The plates were then covered in aluminium foil to shield them from light and incubated in an incubator for an additional 4 h at 37 °C in a CO 2 (5%).To make crystal soluble, 100 μl of DMSO was put into the each well after the solution was discarded.The IC 50 values were then determined from the graph using the absorbance at 570 nm (TECAN, Austria).

Cell cycle analysis
In this study, MCF-7 cells were cultured at 0.3 × 10 6 cells (m/v) in a 6-well plate and exposed to GCNPs and SCNPs at a concentration of 40 μl for 24 h.The cells were exposed, rinsed with PBS, and centrifuged at 1000 rpm for five minutes.The cells were then fixed in ethanol (70% ) for 12 h at 4 °C, and the ethanol was extracted by centrifuging the sample for 5 min at 2000 rpm.The frozen cells were then given two PBS washes before being given ribonuclease (RNase) treatment by adding 50 μl of a 100 g ml −1 RNase stock in PBS to each tube.The cells were resuspended in 200 μl of propidium iodide solution at a concentration of 50 g ml −1 in PBS after RNase treatment, and then incubation were done at 37 °C for few minutes.By quantifying the quantity of DNA that was propidium iodide-labeled in the fixed cells, flow cytometry was then used to examine the cell cycle distribution.

Estimating ROS
In this investigation, bimetallic GCNPs and SCNPs were applied to MCF-7 cells planted in a 12-well plate at 0.1 × 10 6 cells/well for 24 h.Immediately following treatment, the cells were trypsinized, and the supernatant was collected.The cells were rinsed two times with PBS and a cell pellet was obtained by centrifuging.Next, the cell pellets were stained with 1 μl of 2,7-dichloro-fluorescein diacetate (DCFH-DA) in 1 mg ml −1 PBS.DCFH-DA is a fluorescent dye that measures the cellular levels of reactive oxygen species (ROS).After staining, the cells were anaylised by flow cytometry to calculate the extent of ROS production.Flow cytometry analysis was performed using a flow cytometer that measured the fluorescence intensity of the stained cells.To calculate the ROS levels in the cells, the flow cytometer data was examined using the appropriate software.

Statistical analysis
In this study, a Student's t-test was employed (considering significant p value < 0.05) to analyze the experimental data and compare the cell viability percentages of the group treated with bimetallic nanoparticles with the control group.

UV-visible spectroscopy analysis
The synthesized nanoparticles were anaylsed using UV-visible spectroscopy.Figure 2 reveals the formation of SCNPs and GCNPs bimetallic nanoparticles with surface Plasmon absorption peaks observed at 551 nm and 531 nm, respectively [24,25].The alteration in color of the solution to colorless to blue and light yellow to red for SCNPs and GCNPs, respectively, indicates the reduction of metal ions into metal nanoparticles.The LCLE serves as both a reducing and a capping agent.

Dynamic light scattering (DLS)
DLS is a non-destructive spectroscopic analysis approach that allows determining particle size from 1-1000 nm suspended in a liquid.The size distribution of synthesized bimetallic was observed using dynamic light scattering.DLS measures the intensity of light in a specific direction.Figure 3 represents the graphical distribution of the size of SCNPs/GCNPs in the 40 nm-110 nm range.The average hydrodynamic size of SCNPs and GCNPs was recorded to be 66 nm and 53 nm.

Zeta potential
Zeta potential is a significant factor influencing the stability of nanoparticle colloidal dispersion.It measures the strength of electrostatic repulsion between charged nanoparticles.The larger value of zeta potential for colloidal nanoparticles, the smaller the aggregation and the higher the nanoparticles' stability.Zeta potential determines the particle's surface charge (figure 4) at −26.0 mV and −20.6 mV for SCNPs and GCNPs.The findings show that the nanoparticle surfaces were negatively charged, revealing their good colloidal nature.They are distributed uniformly in the medium due to the inherent repulsion between the particles [24].The stability of nanoparticles is correlated with the amount of negative charge on their surface.The feasibility of negative charge on bimetallic nanoparticles could be due to the capping agent of leaf extract components [21].The standard deviation is 5.28 mV and 6.36 mV for SCNPs and GCNPs.

High-resolution transmission electron microscopy (HRTEM)
The shape and size of synthesized bimetallic nanoparticles revealed by HRTEM images.Figure 6 shows the HRTEM images of SCNPs and GCNPs.From images, it was observed that SCNPs were spherical and GCNPs were spherical, triangular, and hexagonal.The bimetallic nanoparticles have particle sizes of 20 nm-80 nm.The average particle size was 47 and 41 nm for SCNPs and GCNPs, respectively.

Cell viability assay
The IC 50 values for GCNPs at 24 h and 48 h were 40 μl and 6 μl, respectively, indicating that a higher concentration was needed at 24 h to achieve the same level of growth inhibition as at 48 h (figure 7(A)).In contrast, for SCNPs, the IC 50 values were 12 μl at 24 h and 6 μl at 48 h, demonstrating a decrease in the concentration required for 50% growth inhibition over time (figure 7(B)).At the 48 h, both GCNPs and SCNPs showed similar potency, as indicated by their identical IC 50 values of 6 μl.However, at the 24 h time, GCNPs required a significantly higher concentration (40 μl) for 50% growth inhibition, whereas SCNPs achieved the same level of inhibition with a lower concentration (12 μl).These results suggest that both GCNPs and SCNPs effectively inhibited the growth of MCF-7 cells in a dose-dependent and time-dependent manner.However, there were differences in their response patterns.GCNPs showed a decreased effectiveness over time, as evidenced by the higher IC 50 value at 24 h compared to 48 h.On the other hand, SCNPs exhibited enhanced  effectiveness over time, with a lower IC 50 value at 48 h compared to 24 h.These findings highlight the timedependent effects and differential responses of GCNPs and SCNPs on the growth of MCF-7 cells.While GCNPs showed reduced effectiveness over time, SCNPs displayed sustained or improved effectiveness, making them a potentially more promising candidate for inhibiting MCF-7 cell growth over an extended duration.Recent studies have provided insights into the cytotoxic effects of nanoparticles on MCF-7 breast cancer cells.In the first study, Ag/Au nanoparticles (NPs) were investigated, revealing that they exhibited a cytotoxicity rate of 60% on MCF-7 cell lines at a concentration of 50 mg ml −1 [27].Our study focused on silver-coated nanoparticles (SCNPs) and gold-coated nanoparticles (GCNPs).We found that at 48 hs, 50% cell death was achieved using 6 μl of SCNPs and 12 μl of GCNPs, indicating their effectiveness in inhibiting MCF-7 breast cancer cell growth.Another study explored the synthesis of nanoparticles derived from Terminalia plants, where the IC 50 values for these bimetallic nanoparticles ranged from 0.18 to 93.73 μg ml −1 [28].Notably, the study also suggested a potential cytotoxic effect of these nanoparticles on MCF-7 cells, highlighting their potential as cytotoxic agents against breast cancer cells.
Moreover, the statistical analysis revealed a notable difference (P < 0.002 and p < 0.01) between the twotime durations for both GCNPs and SCNPs, indicating that the duration of treatment influenced the inhibitory effect of the bimetallic nanoparticles on MCF-7 cell growth.These findings suggest that both GCNPs and SCNPs could be potential candidates for further development as anticancer agents for breast cancer treatment.The importance of these results lies in the potential of bimetallic nanoparticles as an alternative treatment for breast cancer.Due to their capacity to precisely target cancer cells, lower systemic toxicity, and increase anticancer efficacy, nanoparticles have been a promising area of research in cancer therapy [29,30].Additionally, the growing body of literature on the use of bimetallic nanoparticles in cancer treatment and highlight the potential of these nanoparticles as a promising avenue for further research and development.

Cell cycle analysis
The cell cycle analysis showed that in the control samples, the majority of cells were in the G0-G1 phase (76.2%), followed by the G2M phase (16.6%) and the S phase (6.6%) (figure 8(A)).Only a small fraction of cells were in the Sub G1 phase (0.7%).After treatment with GCNPs, there was a slight decrease in the G0-G1 population (69.1%), along with a corresponding increase in the G2M phase (21.6%) and the S phase (8.9%).In contrast, the Sub G1 population remained unchanged (0.7%) (figure 8(B)).Similarly, treatment with SCNPs resulted in a decrease in the G0-G1 population (68.4%) and an increase in the G2M phase (22.2%) and the S phase (8.3%), with a slightly higher proportion of cells in the Sub G1 stage (1.1%) (figure 8(C)).These results suggest that both GCNPs and SCNPs can induce a cell cycle arrest in the G2M stage, consistent with the observed inhibitory effect on MCF-7 breast cancer cell growth.However, the distribution of cells in the S phase with GCNPs and SCNPs was also affected, indicating that both the bimetallic nanoparticles may interfere with DNA replication and cell division.The slight increase in the Sub G1 population may also suggest some level of apoptosis or cell death.The results of flow cytometry analysis in this study are significant because they provide insight into the mechanisms by which bimetallic nanoparticles exert their inhibitory effect on MCF-7 breast cancer cell growth.By analyzing the distribution of cells during various stages of cell cycle, it is possible to identify specific cell cycle checkpoints that may be affected by the treatment with GCNPs and SCNPs [31].The finding that both GCNPs and SCNPs induce a cell cycle arrest in the G2M phase is particularly significant because this phase is critical for the proper progression of the cell cycle and DNA repair [32].Disrupting this phase can lead to DNA damage, apoptosis, or mitotic catastrophe, ultimately resulting in cancer cell death [32].Moreover, the observed effect on the S phase distribution suggests that the bimetallic nanoparticles may also interfere with DNA replication and cell division, which are crucial for cancer cell survival and proliferation.This further supports the potential utility of bimetallic nanoparticles as anticancer agents for treating breast cancer.These findings highlight their potential as targeted therapeutic agents against breast cancer, leading to reduced cell viability and potential induction of apoptosis.Overall, our study provides valuable insights into the cytotoxic mechanisms of GCNPs and SCNPs, paving the way for further exploration of nanoparticle-based treatments for breast cancer.

Estimation of ROS
The study evaluated the effect of bimetallic gold-core nanoparticles (GCNPs) and silver-core nanoparticles (SCNPs) on the generation of reactive oxygen species (ROS).After 24 h of treatment with 40 μl of GCNPs and SCNPs, the ROS levels were measured and compared to the control group (figure 9(A)).The results indicated that both GCNPs and SCNPs exhibited high ROS levels, which suggests that these nanoparticles could induce cell death (figures 9(B) and (C)).Reactive oxygen species are essential in controlling many cellular processes, including cell death [33][34][35].GCNPs and SCNPs can generate ROS, which in turn induces significant cell death and contributes to their anti-proliferative effects.The ability of these nanoparticles to generate ROS may be attributed to their distinct physicochemical properties, which include their size, shape, and surface charge.Some studies have disclosed the potential of gold and silver nanoparticles to induce ROS generation and inhibit proliferation in various cell types [36].The mechanism by which these nanoparticles induce ROS generation is not fully understood.Still, it is believed to involve the generation of free radicals and activating intracellular signaling pathways toward apoptosis [37].Overall, the results of this study suggest that bimetallic gold-core nanoparticles and silver-core nanoparticles can inhibit proliferation by generating ROS and show anticancer effects.This finding is significant because ROS generation controls cellular processes, including cell death [37].These results provide valuable insights into the anticancer effects of GCNPs and SCNPs, indicating their potential as excellent candidates for further research and development in cancer therapeutics utilising nanoparticles.

Conclusion
In conclusion, bimetallic nanoparticles have disclosed the potential of the Litchi leaf extract in obtaining 47 nm and 41 nm SCNPs and GCNPs, respectively, as active anticancer agents.XRD reveals that bimetallic nanoparticles were crystalline.TEM results show that particles were spherical, triangular, and hexagonal in shape.The negative Zeta potential results indicate that the nanoparticles were highly stable and could efficiently interact and be uptaken by the cells.Further, these bimetallic nanoparticles also exhibit potential anticancer effects against MCF-7 breast cancer cells.Both GCNPs and SCNPs effectively inhibited the growth of MCF-7 cells, though their response patterns differed over time.GCNPs showed reduced effectiveness at 24 h compared to 48 h, requiring a higher concentration for growth inhibition at 24 h.In contrast, SCNPs displayed sustained or improved effectiveness, requiring a lower concentration at 48 h.These results reveals that SCNPs have the potential to be a more promising candidate for inhibiting MCF-7 cell growth over an extended duration.Flow cytometry evaluation showed that the nanoparticles induced cell cycle arrest in the G2M phase and affected the distribution of cells in the S phase, indicating interference with DNA replication and cell division.Further, both types of nanoparticles generated high levels of ROS, which may contribute to their antiproliferative and anticancer effects.These findings suggest that bimetallic nanoparticles could be a promising avenue for future research and development as an alternative potential treatment for breast cancer.Using nanoparticles as a drug delivery system could also reduce systemic toxicity and enhance the chemotherapeutic drug efficacy, highlighting their potential for cancer therapy.Overall, these results indicate the future use of bimetallic nanoparticles in cancer treatment and provide essential insights into their mechanistic aspects by performing extensive explorations in preclinical and clinical settings.

Figure 1 .
Figure 1.The schematic representation for preparation of LCLE.

Figure 3 .
Figure 3.The DLS size measurement in intensity distribution for (a) SCNPs and (b) GCNPs.

Figure 7 .
Figure 7.The inhibitory effect of bimetallic nanoparticles on MCF-7 cell growth.MCF-7 cells were treated with increasing concentrations of (A) gold-core nanoparticles (GCNPs) or (B) silver-core nanoparticles (SCNPs) for 24 or 48 h, and cell viability was measured using the MTT assay.The IC 50 values of GCNPs and SCNPs after 24 and 48 h of treatment are shown in the graphs.The * P < 0.002 (GCNPs) and #P < 0.01 (SCNPs) compared to the control groups.

Figure 8 .
Figure 8. Cell cycle analysis of MCF-7 cells treated with GCNPs and SCNPs after 24 h.(A) Representative histogram of cell cycle distribution in control cells.(B) Representative histogram of cell cycle distribution in cells treated with GCNPs.(C) Histogram of cell cycle distribution in cells treated with SCNPs.The percentage of cells in each phase of the cell cycle (G0-G1, S, G2M, and Sub G1) is indicated.

Figure 9 .
Figure 9.The effect of bimetallic gold-core nanoparticles (GCNPs) and silver-core nanoparticles (SCNPs) on the generation of reactive oxygen species (ROS) and proliferation inhibition.(A) Comparison of ROS levels in the control group for 24 h.(B) Inhibition of cell proliferation and increased levels of ROS activity treated with GCNPs (C) Representative images of cell proliferation inhibition and increased ROS activity treated with SCNPs.The data indicate that both GCNPs and SCNPs exhibit high ROS level activity up to 10 5 folds and inhibit cell proliferation.