One-pot synthesis of bimetallic Cu/ZnO nanoparticles using tea leaves (Camellia sinensis) for anticancer applications

Introduction

Nanotechnology has become a transformative force in various sectors, particularly in the biomedical field, where it has introduced revolutionary methods for diagnosing and treating severe medical conditions such as cancer. The unique physiochemical, electrical, catalytic, and optical properties of nanomaterials have significantly expanded their utility, garnering substantial interest across multiple disciplines including sewage treatment, food technology, optoelectronics, and cosmetics [1][2][3][4][5][6][7][8] . Within the diverse array of nanoparticles, bimetallic nanoparticles (NPs) stand out due to their unique structural and morphological characteristics that enhance their performance in various applications compared to their monometallic counterparts [9,10] . Historically, the synthesis of nanoparticles has predominantly relied on chemical methods that use various hazardous chemical precursors. These methods not only pose significant environmental risks due to their toxicity but also often lead to colloidal instability in the nanoparticles produced. This instability can severely restrict the usability of nanoparticles, especially in sensitive applications like those in clinical and biomedical settings [11,12] . As a response to these challenges, biological or green synthesis methods have been developed. These methods leverage natural reducing agents, such as plant extracts, to facilitate the synthesis process, making it more sustainable, rapid, and environmentally friendly. Such green synthesis approaches offer a less toxic, more cost-effective alternative to conventional chemical synthesis methods [13][14][15] .

In the process of green synthesis, two aqueous metal precursor solutions are typically mixed with a plant extract containing phytochemicals, which act as reducing and stabilizing agents. These phytochemicals efficiently convert metal ions into metal nanoparticles. The reduction potential of the metal ions plays a critical role, with ions having a stronger reduction potential being reduced faster than others, which can lead to the formation of a core-shell structure within the nanoparticles [1,16] . This research utilizes Camellia sinensis, commonly known as tea leaves, revered for its antitumor, antimicrobial, and antioxidant properties. The presence of potent phytochemicals like polyphenols, which include flavonoids and catechins, underscores the plant's potential for nanoparticle synthesis. The efficacy of tea leaves in the synthesis of bimetallic nanoparticles has been underexplored, making this study particularly novel [1719] . This research paper presents a novel one-pot synthesis approach using Camellia sinensis tea leaves as both a reducing and capping agent to produce biologically active bimetallic Cu/ZnO nanoparticles. This methodology not only highlights the potential of environmentally benign processes in nanoparticle synthesis but also probes the viability of these nanoparticles as a safe, effective treatment for cancer. The Cu/ZnO nanoparticles synthesized in this study exhibited promising biological activity, suggesting their potential applications in a wide array of fields. A schematic representation of the synthesis method is illustrated in Figure 1, providing a visual guide to this innovative synthesis process.

Materials and Methods

The materials used in this study include Camellia sinensis (Tea leaves), Copper Sulphate (CuSo 4 ) and zinc sulphate (ZnSO 4 ), ethanol, and deionized water. The Tea leaves were collected from a local source (Mumbai) and washed thoroughly with deionized water to remove any impurities. The leaves were then dried in an oven at 60 °C for 24 hours and ground to a fine powder using a mortar and pestle [20] . DI water was used to extract the phytochemicals from the powdered leaves. Silver nitrate and zinc sulphate were used as precursors for the synthesis of bimetallic Cu-ZnO nanoparticles. Deionized water was used as a solvent for the synthesis process. All the chemicals used in the study were of analytical grade and purchased from Sigma Aldrich, INDIA.

Preparation of Tea leaves Extract

The preparation of plant extract from Camellia sinensis (Tea leaves) was carried out using the following procedure: 1. The dried Tea leaves were ground to a fine powder using a mortar and pestle. 2. 10 g of the powdered leaves were mixed with 100 mL of water in a round-bottom flask. 3. The mixture was refluxed at 70 °C for 1 hours with continuous stirring. 4. The extract was then filtered using Whatman filter paper and the filtrate was collected in a beaker. 5. The filtrate was concentrated using a rotary evaporator under reduced pressure at 40 °C. 6. The resulting concentrated extract was used as a reducing agent for the synthesis of Cu-ZnO nanoparticles.

Preparation of Bimetallic Cu-ZnO NPs

The synthesis of bimetallic Cu-ZnO nanoparticles using tea leaves extract as a reducing agent was carried out using the following procedure: 1. 8 mM Copper sulphate and 6 mM zinc sulphate were separately prepared in deionized water. 2. 10 mL of each solution were mixed together in a roundbottom flask under magnetic stirring at room temperature.

3. 8 mL of Tea leaves extract was added drop-wise to the above mixture and the reaction mixture was stirred for 24 hours at room temperature. 4. The color of the reaction mixture changed from colourless to dark brown, indicating the formation of Cu-ZnO nanoparticles. The synthesized nanoparticles were centrifuged at 10,000 rpm for 10 minutes to remove any unreacted precursors and plant extract. upon solubilization and is directly proportional to the number of viable cells in the culture. The optimal wavelength for absorbance is 570 nm, but any filter that absorbs between 550 and 600 nm may be used. The MTT assay is a safe, sensitive, and reliable indicator of cell viability and is preferred over other endpoint viability assays, such as the radioactive 3H-thymidine incorporation assay. Besides having a lengthy and tedious protocol, the health hazards of working with radioactive materials are well recognized, as are the accompanying economic considerations required for the appropriate handling and disposal of radioactive waste. MTT is cell-permeable. The net positive charge of MTT allows it to readily penetrate the intact plasma membranes of viable cells. Other tetrazolium salts, such as MTS, XTT, and WST-1, are negatively charged compounds and unable to cross the plasma membrane. MTT is a salt, so high levels may be toxic to cells. Should the stock concentration of MTT be too high, cells may be disturbed and reduce viability readings. In such high concentrations, the cytotoxicity of formazan can also pose experimental difficulties when the culture media must be removed from the well-plate. MTT is soluble in water (10 mg/mL), ethanol (20 mg/mL), buffer salt solutions (5 mg/mL), and culture media (5 mg/mL) [21][22][23] .

The principle of the MTT assay

The principle behind the MTT assay is that only viable cells have active metabolism and can convert MTT to purple formazan, which has an absorbance maximum at 590 nm.

The amount of formazan produced can be measured using a spectrophotometer and is proportional to the number of viable cells [24][25][26][27] . When cells die, they lose their metabolic activity and are unable to convert MTT resulting in no visible color change.

Results and Discussion Dynamic Light Scattering (DLS)

The size of nanoparticles is measured using DLS, and their stability in suspension over time is also assessed. Figure 5 shows the size distribution of the prepared nanoparticles. The size distribution and polydispersity index (PDI) of Cu-ZnO NPs were detected by DLS analysis. The results have indicated the presence of smaller particles of measures 6-8 nm. Z-average size and PDI for the synthesized nanoparticles were observed to be 12.1 nm and 0.39 respectively [35] . Figure 6 shows the zeta potential distribution for Cu-ZnO NPs. The zeta potential value for the synthesized nanoparticles was found to be -46 mV. Copper-zinc oxide nanoparticles (Cu/ZnO NPs) in comparison to plant extracts, zinc sulfate (ZnSO 4 ), and copper sulfate (CuSO 4 ) across the wavelength range of 200 nm to 600 nm. The absorption peak observed for the plant extract at approximately 260 nm suggests the presence of phenolic compounds or other UV-absorbing organic molecules, which typically exhibit strong absorption in this region due to π-π* transitions. The Cu/ZnO nanoparticles demonstrate a broad and intense absorption band from about 200 nm to over 300 nm. This broadening can be attributed to the quantum confinement effects and the interband transitions within the nanoparticles. In contrast, the absorption spectra for ZnSO 4 and CuSO 4 show minimal activity in this region, with the latter displaying a subtle absorption feature near 800 nm, indicative of the d-d transitions of Cu(II) ions in an octahedral coordination. Figure 8 represents FT-IR spectrum of Cu/ZnO nanoparticles synthesized using tea leaf extract, several characteristic peaks are evident, each associated with different components of the composite material. The broad peak at 3403 cm⁻¹ is indicative of O-H stretching vibrations, typical of hydroxyl groups present in water molecules or organic compounds such as phenols, which are abundant in tea extracts. This suggests the presence of residual moisture or bonded hydroxyl groups from the biological material used in synthesis. The peak at 1614 cm⁻¹ can be attributed to the C=O stretching vibrations commonly found in carboxylic acids or amides, or it could also represent N-H bending in primary amines. This band may also reflect the interaction of organic components from the tea extract with the metal oxide surface, possibly indicating the binding or coordination of organic molecules to the metal sites. The 1034 cm⁻¹ band is likely associated with C-O stretching vibrations in alcohols, ethers, or esters, which are typical functional groups in plant extracts. This peak highlights the incorporation of organic moieties from the tea leaf extract into the nanoparticle matrix.

UV-Visible Spectroscopy

For the inorganic components, the sharp peak at 470 cm⁻¹ is characteristic of Zn-O stretching vibrations, a fundamental feature of zinc oxide nanoparticles. This peak confirms the formation of ZnO in the nanocomposite. Although copper itself does not exhibit distinct IR-active peaks due to its metallic nature, the presence of copper may influence the electronic environment of the zinc oxide, potentially affecting the intensities and positions of the zinc oxide peaks. These spectral features collectively provide evidence of the successful synthesis of Cu/ZnO nanoparticles using tea leaf extract, where the extract not only acts as a reducing agent but also imparts functional organic groups that are integrated into the nanoparticle structure, potentially influencing their physicochemical properties The scanning electron microscopy (SEM) (Fig9) image reveals distinct morphologies of copper (Cu) and zinc oxide (ZnO) nanoparticles. The Cu nanoparticles, identifiable by their dense, spherical appearance, are uniformly dispersed across the substrate. These particles exhibit the typical characteristics of metallic nanoparticles with their compact and homogenous structure. In contrast, the ZnO nanoparticles display a unique flower-like morphology, with multiple nanostructures resembling petals emanating from a central point. This intricate configuration enhances the surface area of the ZnO nanoparticles, which is advantageous for applications that require high surface reactivity [38] .

Investigation of Antimicrobial Activity

The antibacterial activity of various substances, namely Madagascar periwinkle extract, copper sulphate, and zinc sulphate as well as bimetallic Cu-ZnO NPs was evaluated against both Gram-positive and Gram-negative bacteria. The findings demonstrated that bimetallic Cu-ZnO NPs, at a concentration of 1000 µg/ml, exhibited significant antibacterial effects against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Enterococcus faecalis, resulting in inhibition zones measuring 16.4 mm, 15.9 mm, 27.3 mm, 20.7 mm, and 22.8 mm, respectively. Additionally, Bacillus subtilis displayed the highest sensitivity to bimetallic Cu-ZnO NPs, with a minimum inhibitory concentration (MIC) of 15.32 µg/ml, while Enterococcus faecalis exhibited the lowest sensitivity, with an MIC of 250 µg/ml. Furthermore, the MIC values of bimetallic Cu-ZnO NPs against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were determined as 62.5 µg/ml, 125 µg/ml, and 62.5 µg/ml, respectively. On the other hand, Madagascar periwinkle extract displayed limited antibacterial activity, specifically against Escherichia coli and Bacillus subtilis. Neither copper sulphate nor zinc sulphate exhibited any antibacterial effects against the tested bacterial strains.

Conclusion

In this study, we have successfully demonstrated the one-pot synthesis of bimetallic Cu-ZnO nanoparticles using tea leaf extract, highlighting its dual role as both a reducing and stabilizing agent. This approach aligns with the growing trend toward utilizing natural plant extracts in nanoparticle synthesis, offering a green and sustainable alternative to traditional chemical synthesis methods. The inherent advantages of using plant extracts include their low toxicity, environmental friendliness, and the availability of a diverse range of naturally occurring reducing agents, which facilitate the reduction of metal ions to their nanoparticulate forms.

The synthesized Cu-ZnO nanoparticles were extensively characterized through several analytical techniques to ascertain their physicochemical properties. Ultraviolet-Visible (UV-Vis) spectroscopy confirmed the successful formation of nanoparticles, while Dynamic Light Scattering (DLS) provided insights into their size distribution, revealing an average particle size of 6-8 nm. Furthermore, Fourier Transform Infrared Spectroscopy (FTIR) was employed to identify functional groups and verify the chemical composition, confirming the presence of characteristic bonds associated with both the metal oxides and organic constituents from the tea leaf extract.

Importantly, the nanoparticles displayed excellent stability and biocompatibility, criteria that are essential for biomedical applications. The biological activity of the Cu-ZnO nanoparticles was evaluated against various microbial and cancer cell lines, demonstrating potent antimicrobial activity against both Gram-positive and Gram-negative bacteria. This broad-spectrum antibacterial effectiveness suggests that Cu-ZnO nanoparticles could be further developed as antibacterial agents in medical and environmental applications. The mechanism of action underlying the antibacterial activity of bimetallic Cu-ZnO NPs appears to involve several factors. Electrostatic interactions may cause damage to the bacterial cell membrane, disrupt proteins and enzymes, generate reactive oxygen species (ROS), and induce oxidative stress. The NPs may also bind to proteins, disrupting cellular homeostasis, including the electron transport chain and signal transduction pathways, potentially leading to genotoxic effects. Moreover, the rough external surface of Cu-ZnO NPs may contribute to cell wall damage, allowing the nanoparticles to penetrate the bacterial plasma membrane and exert toxic effects on the bacteria. Moreover, the anticancer properties of these nanoparticles were assessed through cytotoxicity studies against cervical cancer (HeLa) cells. The results were promising, showing a significant reduction in cell viability, with only 7% of the cells remaining viable at the highest concentration tested (100 µg/ml). This indicates the potential of Cu-ZnO nanoparticles as effective anticancer agents, particularly in the treatment of cervical cancer.

6.Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Acknowledgement

7.Conflicts of interestThe authors have no conflicts of interest to declare.

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