Green Synthesis of -Coated Silver Nanoparticles: Antimicrobial, Antioxidant, and Anticancer Potentials

Introduction

Cancer remains one of the leading causes of mortality worldwide, presenting a significant challenge to global health [1,2]. Traditional cancer therapies, such as chemotherapy, radiotherapy, and surgery, often come with severe adverse effects and limitations, including non-specific targeting, multidrug resistance, and damage to healthy tissues. Consequently, there is an urgent need for novel and more effective treatment strategies [3,4].

Cancer therapy has evolved significantly with the advent of nanotechnology, which offers innovative approaches to enhance treatment efficacy while minimizing adverse effects. Metallic nanoparticles, particularly silver nanoparticles (AgNPs), have garnered attention due to their unique properties, including their antimicrobial, antioxidant, and anticancer activities [1–4].

Lately, noble metal nanoparticles (NPs) have seen widespread use in biomedical applications, including drug delivery, diagnostics, and tissue engineering, capitalizing on their unique optoelectronic and physicochemical characteristics [5,6]. Silver nanoparticles find widespread application in the pharmaceutical industry for producing ointments and creams to prevent infections associated with burns and wounds [7,8].

The silver ion exhibits potent inhibitory effects against various microorganisms [9]. An eco-friendly alternative to traditional nanoparticle production methods involves biological synthesis, also known as green synthesis [10].

Silver nanoparticles (AgNPs) have received significant attention across diverse applications, including nanoelectronic devices, sensors, imaging contrast agents, filters, and antimicrobial agents, due to their commendable electrical conductivity, stability, optical attributes, and antimicrobial efficacy [10–12]. The recent focus on silver nanoparticles revolves around their dual role as antimicrobial agents and potential drug carriers in cancer treatment [8]. Use of AgNPs has expanded into cancer therapy, exhibiting noteworthy anticancer effects in various in vitro studies involving human cervical, lung, and breast cancer cells [13].

The effectiveness and practicality of silver nanoparticles (AgNPs) are closely tied to factors such as their composition, shape, size, and surface chemistry [14]. Various methods, such as chemical reduction, photochemical reduction, electron irradiation, evaporation-condensation, sonochemical, microwave processing, microemulsion, gamma irradiation, electrochemical reduction, and heat vaporization, can be utilized for the synthesis of silver nanoparticles [15,16].

Unfortunately, while offering simplicity and convenience, these processes often use toxic chemicals, posing risks in human contact applications [17]. Hence, there is a growing need to develop environmentally friendly biosynthesis methods that eliminate the use of harmful substances [10,17].

To address toxicity concerns in synthesis and biological applications, plants or their extracts have emerged as prominent contributors to the biosynthesis of silver nanoparticles [18].

The protective and reductive properties of various chemical constituents in plant extracts play a pivotal role in reducing silver ions, with extracellular AgNPs synthesized using different plant extracts as potential reducing agents [19]. Dracocephalum kotschyi (D. kotschyi), a medicinal herb known for its rich repository of phytochemicals, stands out as a compelling candidate for the coating of silver nanoparticles [20].

The global rise of antibiotic-resistant microorganisms necessitates the exploration of alternative antimicrobial agents. Silver nanoparticles have exhibited potent antimicrobial activity against a broad spectrum of pathogens, making them a viable candidate for combating infectious diseases. Concurrently, the antioxidant potential of nanoparticles gains significance in the context of addressing oxidative stress-related disorders [21]. The integration of D. kotschyi, with its known antioxidant properties, enhances the overall antioxidant capacity of the hybrid nanoparticles [22].

In our investigation, we pursued an environmentally friendly alternative to traditional chemical reduction methods by synthesizing silver nanoparticles (AgNPs) through the reduction of silver ions using extracts of D. kotschyi. We characterized the green-synthesized nanoparticles for size and charge through UV-Vis spectroscopy, transmission electron microscopy (TEM), Fourier-transform infrared (FTIR) spectroscopy, and dynamic light scattering spectroscopy (DLS). Furthermore, our research involves the synthesis of AgNPs and then the characterization of their morphologies and compositions. The study also delves into assessing the antioxidant, antimicrobial, and anticancer activities of the biosynthesized AgNPs.

Material and Methods

CHEMICALS AND REAGENTS:

Dracocephalum kotschyi was finely ground for use in this study. Various substances employed in this experiment included silver nitrate (AgNO₃), 1,1-diphenyl-2-picryl-hydrazyl (DPPH), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) (Sinopharm Chemical Reagent Co., Ltd Shanghai), China, Mueller-Hinton agar and broth, 1% penicillin-streptomycin solution, deionized water, ethanol 96%, Trypsin, phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 6538, Luria-Bertani broth (USB Corporation, Cleveland, OH, USA), agar (LAB M Limited, Bury, UK), HeLa cells were purchased from Pasteur Institute, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Master Mix for Real-Time PCR, PCR Purification Kit, cDNA Synthesis kit, Safe Stain, loading dye, Ladder, and other chemicals were purchased from Sigma Aldrich, India. All chemicals used in this experiment were of analytical grade.

SYNTHESIS OF SILVER NANOPARTICLES (AGNPS):

Silver nanoparticles were synthesized through a green synthesis method utilizing Dracocephalum kotschyi extract. Dried leaves of Dracocephalum kotschyi (10 g) were macerated in 100 mL of ethanol for 24 hours. The resulting extract was filtered to obtain a clear solution. Subsequently, 10 mL of the extract was slowly added dropwise to a 90 mL aqueous solution of 1 mM silver nitrate (AgNO₃) under continuous stirring at room temperature. The reduction of silver ions to nanoparticles was visually confirmed by a color change from pale green to dark brown, indicating the formation of AgNPs. The reaction mixture was centrifuged at 10 000 rpm for 15 minutes, and the resulting pellet was washed with deionized water, followed by drying at 60°C for further characterization.

ULTRAVIOLET-VISIBLE (UV-VIS) SPECTROSCOPY:

The optical properties of AgNPs were assessed using a UV-Vis spectrophotometer (Shimadzu UV-1800) in the wavelength range of 250–850 nm. The characteristic surface plasmon resonance (SPR) peak was recorded.

DYNAMIC LIGHT SCATTERING (DLS):

Dynamic light scattering was conducted with a Zetasizer analyzed size and zeta potential of AgNPs.

TRANSMISSION ELECTRON MICROSCOPY (TEM):

The morphology and size distribution of AgNPs were determined using a transmission electron microscope (JEOL JEM-2100) operated at 200 kV. A drop of the nanoparticle suspension was placed on a copper grid and air-dried before imaging.

FOURIER-TRANSFORM INFRARED SPECTROSCOPY (FTIR):

FTIR spectra were acquired using a Perkin-Elmer Spectrum Two FTIR spectrometer. The recorded spectra were analyzed to identify the functional groups present in both D. kotschyi extract and the synthesized AgNPs.

THERMOGRAVIMETRIC ANALYSIS:

The mass of samples (2–3 mg) was analyzed using a Perkin-Elmer TGA-7 thermogravimetric system, ranging from 50°C to 700°C at a heating rate of 10°C per minute in a nitrogen atmosphere.

X-RAY DIFFRACTION ANALYSIS:

XRD measurements were conducted using a Philips X’Pert-MPD diffractometer with Cu Kα radiation, covering the 2θ range from 5° to 80° at a scanning rate of 2°/min.

ANTIOXIDANT ACTIVITY ASSAYS BY QUANTIFICATION OF RADICAL SCAVENGING ACTIVITY (DPPH):

The antioxidant capacity of AgNPs was determined using the DPPH radical scavenging assay. DPPH radical scavenging assay assessed antioxidant activity, expressed as a percentage of radical scavenging activity using butylated hydroxytoluene (BHT) as a standard.

ANTIMICROBIAL ACTIVITY ASSAYS:

The antimicrobial potential of AgNPs was evaluated against gram-positive bacteria (Staphylococcus aureus), gram-negative bacteria (Escherichia coli) using agar cup and micro-broth dilution methods.

CELL CULTURE:

Human cancer cell lines (HeLa) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics.

MTT ASSAY:

The cytotoxicity of AgNPs was determined using the MTT assay. Cells were treated with various concentrations of AgNPs, and absorbance at 595 nm was measured after incubation.

ANALYZING THE RELATIVE EXPRESSION WITH REAL-TIME PCR:

Total RNA concentrations were determined by measuring the absorbance at 260 nm using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies). Real-time PCR was performed using complementary DNA as a template, gene-specific primers (GSP) designed through the web-based NCBI Primer-BLAST tool (refer to Table 1), and qPCR Master Mix on a StepOnePlus real-time PCR instrument. The reaction mixture comprised 10 μL Master Mix, 1 μL Forward Primer (FP), 1 μL Reverse Primer (RP), 1 μL cDNA, and 7 μL PCR-grade water. Samples underwent initial denaturation for 10 min at 95°C, followed by amplification for 20 s at 95°C (denaturation), 30 s at 59°C (annealing), and 50 s at 72°C (extension), with subsequent quantification. A melting curve analysis (95°C for 15 s, 60°C for 1 min, and 95°C for 15 s) was carried out to confirm product specificity. Each cDNA sample was subjected to the reaction in triplicate.

STATISTICAL ANALYSIS:

All experiments were performed in triplicate, and the data were subjected to analysis using one-way analysis of variance (ANOVA) with a significance level of P<0.01. The results are expressed as mean±standard deviation.

Results

Characterization of Silver Nanoparticles (AgNPs)

BIOSYNTHESIS OF AGNPS AND CHARACTERIZATION BY UV-VIS SPECTROSCOPY: A noticeable color transformation was noted, transitioning from pale green to dark brown. The color intensity exhibited a rise corresponding to an increase in the incubation time. To check the power of synthesized AgNPs by Dracocephalum extract, light absorption was determined at 260 nm. The results show that with the increase in the concentration of AgNPs, the amount of synthesized AgNPs increases at a fixed concentration of the extract, and the rise in time also increases the biosynthesis for a certain period. The formation of AgNPs was confirmed by the appearance of a surface plasmon resonance peak at 418 nm (Figure 1).

DYNAMIC LIGHT SCATTERING (DLS) ANALYSIS: The Dynamic Light Scattering (DLS) method provided insights into the hydrodynamic size and surface charge of silver nanoparticles (AgNPs) in an aqueous colloidal environment. Figure 2 shows that, while no aggregation was observed, the approximate size of the synthesized nanoparticles was 40 to 90 nm. DLS confirmed an average particle size of 43 nm with a zeta potential of −23 mV, indicating good stability.

TRANSMISSION ELECTRON MICROSCOPY (TEM): TEM analysis revealed that the AgNPs displayed a predominantly spherical morphology with a narrow size distribution. The average particle size was determined, showing the uniformity achieved through the green synthesis method. Transmission electron microscopy (TEM) images depicting the biosynthesized silver nanoparticles (AgNPs) were presented in Figure 3. TEM images revealed spherical AgNPs with an average diameter of 43 nm.

FOURIER-TRANSFORM INFRARED SPECTROSCOPY (FTIR) ANALYSIS OF AGNPS: The Fourier-transform infrared spectroscopy (FTIR) spectra of the silver nanoparticles (AgNPs) validated the potential engagement of various functional groups in the biosynthesis and stabilization of the nanoparticles. FTIR spectra showcased distinct peaks corresponding to functional groups present in D. kotschyi extract, such as hydroxyl and carbonyl groups. The shifts and intensities of these peaks indicated strong interactions between the bioactive molecules of the plant extract and the AgNPs, affirming successful coating and stabilization (Figure 4). FTIR spectra indicated the presence of phenolic compounds in the extract, acting as reducing and capping agents for AgNPs.

THERMOGRAVIMETRIC ANALYSIS (TGA): The TGA curve of the synthesized AgNPs in the temperature range of 0°C to 700°C is given in Figure 5. Pure zinc nanoparticles up to 700°C increased temperature by nearly 10% and showed no significant weight loss. It has considerable thermal stability due to the absence of volatile compounds in the pure nanoparticle. The presence of organic groups of Dracocephalum extracts on the surface of biosynthesized nanoparticles caused an increase in weight loss in the range of 200°C to 300°C following the loss of water molecules absorbed by the particles.

X-RAY DIFFRACTION (XRD) ANALYSIS: The X-ray diffraction (XRD) spectrum of the biosynthesized silver nanoparticles (AgNPs) is illustrated in Figure 6, showing 5 peaks on the 2θ range. Distinct XRD patterns were observed at 24.7, 30.1, 43.2, 57.1, and 62.7.

ANTIMICROBIAL ACTIVITY OF BIOSYNTHESIZED AGNPS:

The antimicrobial activity of the synthesized silver nanoparticles (AgNPs) was assessed by cultivating colonies of Escherichia coli (E. coli) ATCC 10536 and Staphylococcus aureus (S. aureus) ATCC 6538. The antimicrobial potential of AgNPs was evaluated against pathogenic microorganisms is given in Table 2 and Figure 7. AgNPs demonstrated strong antibacterial activity against both E. coli and S. aureus, with inhibition zones measuring 15±0.5 mm and 17±0.5 mm, respectively, at the highest concentration tested (200 μg/mL).

ANTIOXIDANT ACTIVITY OF THE BIOSYNTHESIZED AGNPS BY DPPH RADICAL SCAVENGING ASSAY:

The antioxidant activity of the biosynthesized silver nanoparticles (AgNPs) was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, as depicted in Figure 8. Table 3 shows the amount of antioxidant IC50 of AgNPs in comparison with the control (BHA). Figure 9 shows the antioxidant effect of synthesized AgNPs and BHA on DPPH free radical inhibition. The noteworthy antioxidant potential of silver nanoparticles (AgNPs) was determined through the DPPH radical scavenging assay, yielding an IC50 value of 255.3 μg/mL (Table 3). The AgNPs exhibited significant antioxidant activity, with an IC50 value of 40 μg/mL, comparable to BHT.

MTT ASSAY: Each of the wells contained a specific number of cells treated with silver nanoparticles (AgNPs) at concentrations of 20, 10, 5, 2.5, and 1.25 nM for 24 and 48 hours (Figure 8). The cell viability (%) of the HeLa cell line (human cervical cancer cells) following treatment with the biosynthesized silver nanoparticles was assessed through the MTT assay. The cell viability (%) after exposure to different concentrations of AgNPs is illustrated in Figure 8. There was a significant difference in comparing the results obtained from the study of the number of living cells (cell viability) treated with different amounts of AgNPs, and Dracocephalum extract 24 and 48 hours after the treatment with the control. The study’s results showed that the cell viability of HeLa cancer cells decreased with increasing drug concentration and treatment time. The MTT assay revealed a dose-dependent decrease in HeLa cell viability upon treatment with AgNPs. The IC50 value was 50 μg/mL, indicating potent cytotoxic effects.

APOPTOSIS DETECTION:

Figure 10 shows the apoptosis rate of HeLa cells in the group treated with Dracocephalum extract and group treated with AgNPs was investigated using Annexin V-FITC/PI staining. The apoptosis rate in both treatment groups was significant (P<0.001) and was more than in the control group.

The outcome depicting live cells (Annexin V−, PI−), necrotic cells (Annexin V−, PI+), early apoptotic cells (Annexin V+, PI−), and late apoptotic/dead cells (Annexin V+, PI+) is illustrated in Figure 10.

ANALYZING THE RELATIVE EXPRESSION RESULTS OF GENES WITH REAL-TIME PCR:

The data must be normalized first to analyze the results obtained from the RT-PCR technique. For this purpose, we applied the difference between the Ct of the group treated with AgNPs and the extract with the Ct of the control group. For this purpose, the Ct of the control group was subtracted from the Ct of the group treated with AgNPs or extract, and the resulting normalized data is called ΔCt. The best way to express the number of gene changes in one sample compared to another is fold change, which means the expression of the tested gene in the samples of the treated group has decreased or increased many times compared to the control group, so we multiplied the obtained ΔΔCt by −1. Therefore, the higher the ΔΔCt, the higher the expression. Considering that ΔΔCt and dAC are based on a logarithm based on base 2, we had to convert them into a linear form. 2-ΔΔCt indicates the fold change of each sample.

According to Figure 11, it can be seen that the expression of the Bax gene as one of the apoptosis reagents increased, and the Bcl-2 gene as one of the anti-apoptotic reagents decreased significantly (P<0.05) compared to the control group, indicating the induction of apoptosis.

Figure 11 shows that the expression of VEGF and KDR genes, as marker genes, had a significant (P<0.05) decrease in angiogenesis in cancer cells compared to the control group. It induces the reduction of angiogenesis and effectively reduces the growth of cancer cells.

Discussion

The environmentally friendly synthesis of silver nanoparticles (AgNPs) using D. kotschyi extract presents a sustainable alternative to traditional chemical methods, aligning with current trends in green nanotechnology. The comprehensive characterization of the synthesized AgNPs confirms their uniform size distribution and stability, which are crucial for biomedical applications. Recent studies corroborate the advantages of biosynthesized AgNPs, highlighting their reduced toxicity and enhanced biocompatibility compared to chemically synthesized counterparts [23,24].

This study highlights the efficacy of D. kotschyi extract as a reducing and capping agent in the biosynthesis of AgNPs. The green synthesis approach not only mitigates environmental and health hazards associated with chemical methods but also endows the nanoparticles with enhanced biological properties. The significant antibacterial, antioxidant, and anticancer activities of the synthesized AgNPs suggest their potential application in biomedical fields [23,24].

The synthesized silver nanoparticles coated with D. kotschyi extract represent a novel approach in nanomedicine, capitalizing on the synergistic properties of metallic nanoparticles and bioactive compounds from medicinal plants. The comprehensive characterization of the nanoparticles through UV-Vis spectroscopy, TEM, and FTIR not only confirms their successful synthesis but also highlights their uniform size distribution and stability, which are critical factors for their potential biomedical applications.

The confirmation of AgNPs formation was evidenced by the emergence of a dark brown color, characteristic of AgNPs in solution owing to their surface plasmon resonance (SPR) [25,26]. In our investigation, the identified absorption peak at 428 nm validated the biosynthesis of Ag nanoparticles.

The TEM image revealed that the AgNPs were in close physical proximity but dispersed with a consistently uniform distance between particles. Earlier investigations, such as the biosynthesis of silver nanoparticles by Memecylon edule leaf extract, also reported a circular morphology [27]. Our findings align with various studies that have documented similar XRD patterns in biosynthesized AgNPs [11,28].

The potent biological activities observed can be attributed to the inherent properties of AgNPs and the bioactive compounds from D. kotschyi. The antimicrobial action is likely due to the disruption of bacterial cell membranes by AgNPs, leading to cell death. The antioxidant activity is facilitated by the phenolic compounds, which scavenge free radicals. The cytotoxic effect on HeLa cells may be mediated through the induction of apoptosis, as suggested by the observed decrease in cell viability.

The antimicrobial potential of the silver nanoparticles is particularly noteworthy, showing efficacy against both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. The ability to inhibit the growth of a diverse range of microorganisms underscores their potential as broad-spectrum antimicrobial agents. This is especially relevant in the context of the escalating global challenge of antibiotic resistance, where alternative antimicrobial strategies are urgently needed. Recent research has also emphasized the role of nanoparticle size, shape, and surface charge in determining antimicrobial efficacy [29,30].

The observed antioxidant activity of the silver nanoparticles further enhances their appeal for therapeutic interventions. The concentration-dependent scavenging of DPPH radicals and the dose-dependent increase in total antioxidant capacity reveal their potential to mitigate oxidative stress, a hallmark of various diseases, including neurodegenerative disorders and cardiovascular conditions. This aligns with recent findings that highlight the significance of nanoparticle-mediated antioxidant defense mechanisms in biological systems [31,32].

The integration of D. kotschyi extract appears to contribute to this antioxidant activity, as suggested by FTIR analysis, opening avenues for applications in oxidative stress-related pathologies. Kharat and Mendhulkar (2016) investigated the antioxidant activity of synthesized nanoparticles through a DPPH assay, noting the antioxidant potential of photosynthesized nanoparticles [33]. They proposed that photosynthesized NPs have the potential to act as effective free radical scavengers. The findings strongly advocate for the utilization of AgNPs as valuable natural antioxidants to preserve health against oxidative stress associated with degenerative diseases. Antioxidant assessment is crucial for AgNPs prior to their application in in vivo models and human contexts.

Silver nanoparticles (AgNPs) exhibit promising applications in therapeutic effectiveness, including wound care, skin cancer, and breast cancer [28]. These nanoparticles demonstrated excellent anticancer activity against the HeLa cell line, with comparable IC50 values to those reported in previous studies [34–36].

In cancer therapy the cytotoxic effects of the silver nanoparticles on HeLa cells present a promising foundation for targeted anticancer strategies. The induction of apoptosis, as evidenced by flow cytometry, hints at the potential molecular mechanisms underlying the observed cytotoxicity. Understanding these mechanisms will be pivotal for the development of targeted therapies with minimal off-target effects. Some studies have reported similar findings, where biosynthesized AgNPs exhibit selective cytotoxicity towards cancer cells while sparing normal cells, underscoring their therapeutic potential [23,37].

The findings of this study open up exciting possibilities for the integration of silver nanoparticles coated with D. kotschyi extract into various biomedical applications. However, it is imperative to acknowledge the limitations of the current study, primarily its reliance on in vitro models. Further investigations using in vivo models are essential to validate the safety and efficacy of these nanoparticles in a more physiologically relevant context. Moreover, elucidating the specific bioactive compounds responsible for the observed activities and understanding their interactions with the nanoparticles would provide crucial insights. This knowledge could inform the design of tailored nanomaterials with optimized properties for specific therapeutic applications. While the in vitro results are promising, further investigations using in vivo models are essential to validate the safety and efficacy of AgNPs in a physiological context. Identifying specific bioactive compounds in D. kotschyi responsible for these effects could inform the design of optimized nanomaterials for therapeutic applications. Future research should also focus on the mechanistic insights of nanoparticle interactions at the molecular level, which could pave the way for targeted and effective biomedical applications.

Conclusions

The present study successfully demonstrates the synthesis of silver nanoparticles using D. kotschyi extract, showing significant antimicrobial, antioxidant, and anticancer properties. The integration of green synthesis principles with plant-derived compounds is a sustainable approach in nanomedicine. Future research should focus on in vivo validation and mechanistic insights to realize the full potential of these nanoparticles in biomedical applications.