Introduction

Cancer is the second leading cause of death worldwide, claiming around 9.6 million lives in 2018. Every year, almost 16% of all fatalities worldwide are caused by cancer [1]. The anticipated death toll from cancer in 2020 was 10 million individuals globally [2]. Medicinal plants have been used for centuries as both a foundation of modern medicine and a source of traditional cures. Medications derived from medicinal plants are gaining popularity due to their efficacy, cheap cost, and minimal risk of adverse effects [3].

Clove (Syzygium aromaticum L.) is used in conventional medicine for a variety of biological activities [4]. Clove phytocomponents consist of hydrocarbons, monoterpenes, phenolic, and sesquiterpenes. [5]. Eugenol, eugenol acetate, and β-caryophyllene are all found in clove oil [6]. Eugenol (4-allyl-2-methoxyphenol) is one of several active compounds with multiple biological benefits, such as antioxidant, carcinogenic, antibacterial, antifungal, and insecticidal properties [7]. Many studies have shown numerous molecular pathways for eugenol's cancer-curing actions. Eugenol has produced apoptosis, cell cycle arrest, proliferation, migration, angiogenesis, and metastasis in a variety of cancer cell lines [8,9,10,11].

Nanotechnology is an emerging field that combines the use of nanoscale biomaterials for a variety of pharmacological benefits [12, 13]. Polysaccharides found in marine seaweeds provide a cheap, sustainable source of tailored medicine delivery systems. Chitosan, an intriguing biopolymer among these polysaccharides, has a distinctive combination of properties, including biocompatibility and biodegradability [14, 15].

Silver nanoparticles are one of the most widely used nanomaterials due to their variety of applications as anti-bacterial, anti-cancer and anti-inflammatory effects [16, 17]. By using microorganisms, plant extracts, and fungi, silver nanoparticles may be synthesized without the need of hazardous chemicals [18,19,20,21]. Silver nanoparticles developed through green synthesis have excellent anti-cancer properties [22]. Combining the features indicated above to develop nano-hybrids of silver and chitosan is particularly promising for anti-cancer applications [23, 24].

The aim of this work is to synthesize nanoparticles encasing EGN from chitosan polysaccharide and silver, and to assess their anticancer potential using advanced pharmacological and computational methods.

Materials and Methods

Material

4,6-diamidino-2-phenylindole dihydrochloride (DAPI), acetic acid, annexin-V-FITC staining kit, chitosan, chloroform, dialysis membrane (10 K), dimethyl sulfoxide (DMSO), dulbeccos modified eagles medium (DMEM), ethanol, ethylene-diamine-tetra-acetic acid (EDTA), fetal bovine serum (FBS), formaldehyde, lactose, low melting point agarose (LMPA), lysozyme, phosphate-buffered saline (PBS) tablets (pH 7.4), polyvinyl alcohol (PVA), Pure EGN, silver nitrate (AgNO3), sodium chloride (NaCl), sodium hydroxide (NaOH), sulforhodamine B (SRB) dye, trichloroacetic acid (TCA), tris-base (pH 10.5), tri-sodium citrate (Na3C6H5O7), triton X-100, trypsin were acquired from Sigma-Aldrich. These compounds were utilized without additional purification.

Methods

Preparation of Hybrid Nanoparticles

According to earlier investigation [25], silver nitrate (AgNO3) was reduced using tri-sodium citrate (Na3C6H5O7) to produce AgNPs. To summarize, a 75 ml of AgNO3 (0.0015 M) was heated in ultrapure H2O to 90°C while continuously stirring (SH-3 Hot Plate Magnetic Stirrer Tullytown PA, United States). The AgNO3 solution was then slowly mixed with 15 ml of tri-sodium citrate (1.5% w/v). A successful AgNPs synthesis was indicated by a color shift from clear to a deep yellow after 20 min of continuous stirring [26]. Eugenol was dissolved in a 0.5:1 ethanol–water combination with Tween 80 (0.75% v/v) and added to 3 ml of Ch (750 mg/ml in 1% acetic acid) with continual stirring. The TPP (1.5 ml, 2.4 mM) was then added in a 2:1 (Ch-EGN:TPP, v/v) ratio to the Ch-EGN mixture and stirred for 3 h. After that, 5 ml of AgNPs in a 2:1 AgNP:Ch (v/v) ratio was added dropwise to the Ch-EGN mixture kept on a magnetic stirrer overnight. The obtained nanoparticles were lyophilized by putting them into a glass vial and freezing them for two hours at -40°C in a single chamber LSCplus Martin Christ™ Germany. The lyophilized powder was refrigerated at 4°C until further studies were conducted.

Physical Characterization of EGN-ChAgNPs

Entrapment Efficiency

The percent entrapment efficiency (EE) was evaluated by slightly modifying the previously reported method [27]. The EGN-ChAgNPs (59.5 mg which is equivalent to 50 mg of EGN) was added in 5 mL of PBS (pH 7.4) and injected into a dialysis membrane before being agitated in 500 mL of PBS (pH 7.4) for 1 h at 37°C on a magnetic stirrer set at 100 rpm. The required volume of sample (5 mL) was removed after specified time interval (0 and 1 h) and centrifuged (Hettich EBA 200S, Hettich, Germany) at 8000 rpm (6153 × g) for 15 min. The centrifuge speed was optimized after testing the sample at various RPM (5000, 8000, and 11,000 RPM). The pellet did not develop at 5000 RPM, which might be attributed to the dilute disperse system and sluggish RPM. At 8000 RPM, the pellet weight was sufficient for further studies. The obtained free drug concentration was quite high at 11,000 RPM, which might be attributed to nanoparticle breaking. The supernatant was decanted and filtered (Nylon filter membranes pore size 0.45 μm, Sigma Aldrich, USA). Without further dilution, the absorbance of sample was taken at 285 nm using UV–visible spectrophotometer (Shimadzu UV 1900i, Tokyo, Japan).

The percent EE was determined using the following formula;

$$EE\;\left(\%\right)=\frac{\mathrm{Total}\;\mathrm{ENG}\;\mathrm{added}-\mathrm{EGN}\;\mathrm{in}\;\mathrm{supernatant}}{\mathrm{Total}\;\mathrm{EGN}\;\mathrm{added}}\times100$$

Transmission Electron Microscopy

The morphology of the EGN-ChAgNPs (10 µl) was examined by transmission electron microscopy (TEM; JEM-2100; JEOL; Tokyo, Japan; 200 kV) after being negatively stained with 2% uranyl acetate (w/v), dried for 15–20 min, and then placed onto a carbon-coated copper grid (300 mesh) [28].

Hydrodynamic Diameter and Zeta Potential Estimation

The hydrodynamic diameter was determined by dispersing EGN-ChAgNPs in double-distilled water. The sample's hydrodynamic diameter distribution and zeta potential were quantified using Malvern Zetasizer Nano ZS (Cambridge, UK) [29].

Drug Release Kinetics

Based on the previously described approach, we investigated the EGN release pattern and with the help of kinetic models [30]. Briefly, a 59.5 mg of EGN-ChAgNPs equivalent to 50 mg of EGN was dispersed in 5 mL of PBS (pH 7.4) and added into a dialysis membrane which was subsequently submerged in 500 mL of PBS (pH 7.4) containing 1.2 µg/mL of lysozyme placed on a magnetic stirrer (75 rpm, 37°C). At specified time interval, the UV–visible spectrophotometer (Shimadzu UV 1900i, Tokyo, Japan) was used to assess EGN release at a particular wavelength (285 nm). Release kinetic models were employed using the DDsolver tool to determine the release mechanism of EGN from ChAgNPs.

Stability Test of Nanoparticles

With slight adjustments, a previously described approach was used for stability experiments [31]. The synthesized AgNPs were kept in a controlled atmosphere (30°C ± 2°C and RH 65% ± 5%) provided by a HPP750 stability chamber (Memmert, GmbH, Bavaria, Germany) for a period of 6 months to study their stability according to ICH (International Council for Harmonization) guidelines. To avoid solvent evaporation, a type II glass container is utilized. The UV–vis method was adopted to evaluate the samples.

Pharmacological Characterization

Sulforhodamine B Assay

The SRB assay was used to determine the extent to which pure EGN and EGN-ChAgNPs inhibited cell proliferation in the MCF-7 cell line [32]. The 1 × 104 cells were spread out across 96-well plates and given 24 h to develop. During a 24-h incubation period, varied quantities of pure EGN and EGN-ChAgNPs were added to separate wells. After incubation, cells were fixed with 40% ice-cold trichloroacetic acid (TCA). Afterwards they were washed in PBS and left out to dry in the air. The cells were stained with SRB dye (0.4% w/v) for 30 min. The cells were then mixed with 100 µL of Tris base (10 mM, pH 10.5). At 565 nm, measurements were taken using an ELISA microplate reader ELx808™ (BioTek equipment, Winooski, Vermont, United States). The IC50 (µg/ml) was calculated using Prism 5.0 (GraphPad Software, San Diego, CA, USA).

Genotoxicity Assessment (Comet Assay)

Comet test was used to determine the double-strand DNA (dsDNA) breakage as a part of genotoxicity evaluation utilizing an established procedure [33, 34]. The MCF-7 cell suspension (2 × 104 cells/well) was treated separately for 18 h with pure EGN and EGN-ChAgNPs. Cells treated with 1% LMPA were cultured and then placed on comet slides. The slides were then immersed in a lysis solution of 1% Triton-X, 100 mM EDTA, 2.5 M NaCl, 10 mM Trizma-X, and 10% DMSO at pH 10. The buffering solution (pH 13, 1 mM EDTA, and 300 mM NaOH) for the time-course study was kept in horizontal electrophoresis chambers with the samples. An alkaline buffer was used to unwind the DNA. After that, the slides were washed with CH3OH, and they were dried completely. Extracted DNA from the comet was analyzed using CaspLab 1.2.3b2 software.

Flow Cytometry Analysis

MCF-7 cells (1 × 104) were treated for 24 h with pure EGN and EGN-ChAgNPs. The cells were detached with trypsin and EDTA for 5 min at 37°C. To prevent cells from clumping together, the cell culture media was added gradually and cautiously. Cells were recovered in 100 µl of binding buffer for 15 min after being exposed to 500 µM H2O2. The cells were then treated for 15 min individually with annexin-V FITC fluorescent dye and propidium iodide (PI) before being exposed in an obscure environment.

The cells were examined by fluorescence-activated cell sorting (FACS) with an emission wavelength filter of 600 nm for PI and 545 nm for annexin-V-FITC using FL2-A channel. In one cycle, 10,000 harvested cells were evaluated using the CytoFLEX (Beckman-Coulter Life- Sciences, Brea, California, USA). The results were displayed as a cell cycle analysis graph [35].

Animal Studies

Albino female adult BALB/C mice (Avg. 30 g) were maintained in animal house of Bahauddin Zakariya University Multan (60,800), Pakistan. The mice were retained in a room with a temperature of 25°C, a light/dark cycle of 12 h, with unrefined meal and water available at all times. The stress of overcrowding was reduced for five albino mice by housing them in a steel mesh cage. The animals at Bahauddin Zakariya University in Multan were cared for in compliance with the standards established by the university's ethical committee for the use of animals in scientific research (235/PEC/2022). There were a total of five mice split evenly among four groups. A total of five female mice were each given an injection containing about 4 × 106 MCF-7 cells (100 µL). Over the course of 20 days, the tumor was allowed to grow to a volume of 50–100 mm3, which was measured with a digital Vernier caliper (BR1078, China) and the following equation.

$$V \left({mm}^{3}\right)={~}^{1}\!\left/ \!{~}_{2}\right.\times a{b}^{2}$$

Where:

V is the estimated tumor volume in cubic units (e.g., mm3), a is the longest diameter or length of the tumor, and b is the shortest diameter or width of the tumor. Mice groups and their respective treatments are shown in Table I.

Table I In Both Experimental and Control Groups of Mice, Pure EGN, EGN-ChAgNPs, and Cisplatin were Found to Demonstrate Anticancer Effects
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The percent tumor inhibition rate (TIR) was used to figure out how well each formulation worked against cancer.

$$\mathrm{TIR}\left(\%\right)=\frac{\mathrm{weight}\;\mathrm{of}\;\mathrm{tumor}\;\mathrm{in}\;\mathrm{sample}\;\mathrm{group}}{\mathrm{weight}\;\mathrm{of}\;\mathrm{tumor}\;\mathrm{in}\;\mathrm{control}\;\mathrm{group}}\times100$$

In Silico Molecular Investigation

Chemical Structure and ADME Analysis

PubChem database of NCBI was used to obtain the structure of EGN and evaluate its chemical and physical parameters. Subsequently, absorption, distribution, metabolism, and excretion (ADME) analysis was performed by SwissADME, which not only predicts the pharmacokinetic features of the query compound but also interprets its solubility, medicinal chemistry, and physicochemical properties [36].

Biological Target Prediction and Validation

For in silico analysis of EGN anti-cancer activity, it was mandatory to predict the potential target of EGN. Swiss Target Prediction, an online database which contained information of active sites information of more than 3,000 proteins. The software assess the query molecule and predicts likely protein targets on the basis of similarity with known ligands. UniProt Knowledge-base (UniProtKB), validated the selected biological target by authenticating its role in various cancers. Furthermore, protein–protein interactions (PPI) of the target macromolecule were visualized by STRING database [37]. The PPIs were observed by limiting the results to Homo sapiens along with a confidence level of < 0.05.

Molecular Docking

The docking of EGN with the intended protein was carried out using the FlexX feature of SeeSAR version 12.1.0. Aurora A, the intended protein, was obtained from the Protein Data Bank (PDB) with identification no. 5EW9 [38]. Binding site extension was performed in SeeSAR's binding site mode preceding the docking analysis. At least 10 distinct EGN postures were created by varying the target protein's active pocket residues. The construction of these poses is dependent on the incremental construction algorithm. The predicted affinities, torsion angles, clashes, and optibrium attributes were analyzed to determine the optimal position.

Ligand Interaction Visualization

The Molecular Visualization Software Discovery Studio 2021 was used in order to investigate the positive interactions that exist between EGN and the amino acid residues that were located in the protein's binding pocket [39]. These beneficial interactions, such as hydrophobic and electrostatic interactions, as well as others, were indicated by dotted lines of various colors. After that, the hydrogen bond and dehydration energy (HYDE) score of the protein–ligand complex was computed using SeeSAR version 12.1.0 (BioSolveIT GmbH, Sankt Augustin, Germany, 2022).. A better understanding of how each atom of EGN contributes to the projected estimated affinity was gained by visualizing the relative contributions of the favorable and unfavorable atoms [40].

Statistical Analysis

An independent two-sample t-test (95%) was used to analyze the cell cycle analysis and genotoxicity assessment. The remaining results of the study were presented as mean and standard deviation (SD). However, Prism 5.0, SPSS 9.0 (SPSS Inc. Chicago, IL, USA), and Microsoft Excel 2010 (Microsoft Corp, Redmond, WA, USA) applications were used.

Results and Discussion

Entrapment Efficacy

According to Table II, the synthesized EGN-ChAgNPs exhibited an excellent entrapment efficiency of around 84 ± 7%. Results demonstrated that EGN was effectively entrapped inside the nanoformulation. Entrapment inside a nanocomposite was dependent on the type and interaction between the therapeutic agent and the nanocomposite itself [41]. Previous research has shown that itraconazole had a greater entrapment efficiency (94.56 ± 0.11%) inside chitosan embedded silver nanoparticles [42]. Nanocomposites based on chitosan had the potential to preserve the enclosed substance by shielding it from environmental hazards and increasing its solubility in water [43]. In another investigation, the chitosan silver encapsulating ability towards Calotropis procera extract was tested, and it was shown that nanocarrier displayed a greater percentage of effective encapsulation (%EE), which was around 77.12% [44]. The increased entrapment efficiency found with hybrid nanoparticles is consistent with the data that we uncovered.

Table II Physical Characterization of EGN-ChAgNPs
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One aspect that may improve EGN entrapment efficiency was the ideal amount of Ch used in nanoparticle synthesis. We optimized chitosan amount in formulation by using varying quantities (500, 750, 1000 mg) and tested for entrapment efficiency, zeta potential and hydrodynamic diameter. The optimal concentration was found to be 750 mg that had produced a stable dispersion (> 30), desired hydrodynamic diameter (< 200 nm) and excellent entrapment efficiency (> 80%).

Transmission Electron Microscopy

The evaluation of the surface morphology of EGN-ChAgNPs was carried out by the use of transmission electron microscopy (TEM). Nanoparticles have a spherical form with smooth edges when measured against a scale bar of 200 nm (Fig. 1a). Our findings were in line with those of previous investigations on the surface morphology of ChAgNPs at varying Ag concentrations. It was observed that spherical shaped nanoparticles were generated at low silver concentrations and were homogeneously dispersed in chitosan matrix [45]. A study reported previously demonstrated spherical shape of the hybrid chitosan silver nanoparticles, which validated our suggested EGN-ChAgNPs formulation [28].

Fig. 1
figure 1

Representation of physiochemical characterization of EGN-ChAgNPs, transmission electron microscopy analysis (a) and hydrodynamic diameter of EGN-ChAgNPs (b)

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Estimation of Hydrodynamic Diameter and Zeta Potential

When designing the size of particles, there were two primary considerations that need to be made: the first was that the particles need to be big enough to avoid being eliminated by the kidney [46], and the second was that the particles need to be small enough to avoid being phagocytosed and eliminated by the RES [47]. According to evidence, macromolecules with a molecular weight greater than 40 kDa and nanoparticles with a size range of 10 to 500 nm had the ability to travel through the capillary bed and consolidate in the interstitial region of a tumor to accomplish passive targeting [48]. A study had demonstrated that particles larger than 200 nm were more readily cleared by the body [49].

Figure 1(b) displayed the results of dynamic light scattering (DLS), which was used to establish that hybrid ChAgNPs incorporated EGN indicated the adequate hydrodynamic diameter of approximately 128 ± 14 nm. When it pertains to nanomedicines, the scale of the nanoformulation is of utmost significance because of the importance of its resemblance to bioactive components in order to produce therapeutic effects [50]. The hydrodynamic dimension is a separate measure used to determine the stability, dispersion, and biological uptake of nanoparticles in aqueous media [51].

The influence of chitosan on the size of AgNPs was significant due to its terminal glucosamine groups, since it worked as a capping and reducing agent in the synthesis step by transferring electrons to silver ions as an electron acceptor [52]. The current study showed an improvement over a previous study, in which hybrid chitosan silver nanoparticles were found to be 218.4 nm in size [53]. The zeta potential was utilized as a metric to assess the charge, stability, and dispersion potential, thereby verifying the stability of EGN-ChAgNPs. A higher absolute value of the zeta potential signifies greater stability of the system, whereas lower absolute values indicate a tendency of the colloids to agglomerate [54,55,56].

The zeta potential of the EGN-ChAgNPs was determined to be + 30.58 ± 6.92 mV and is presented in Table II. The confirmation of greater dispersion stability was attributed to the higher magnitude of zeta potential. The positive charge on the surface of nanoparticles suggested the existence of repulsive forces that might hinder the aggregation of nanoformulations, thereby preserving their stability. In addition, it was noteworthy that the cell membrane exhibited a slight negative charge, thereby facilitating the electrostatic adsorption of positively charged particles onto the membrane surface by cells [57]. Animal studies had shown that nanoparticles (NPs) with a modest positive charge may concentrate considerably at tumor sites after systemic administration [58].

Drug Release Kinetics

The pure EGN and EGN-ChAgNPs dissolution profile had been seen in Fig. 2(a). Pure EGN was dissolved in medium in 2 h. The kinetic model could not be developed since there were fewer data points owing to rapid dissolution. EGN's short biological half-life, which was believed to be 90 min, may be the cause of its rapid breakdown [59]. However, the release kinetics approach for EGN-ChAgNPs was employed to gain a deeper understanding of the drug releasing mechanism. Various kinetic models were utilized to investigate the nature of the release, including first order, zero order, Korsmeyer-Peppas, and Higuchi models. The R2 value had served as a metric for evaluating the kinetics and release mechanism of EGN from AgNPs.

Fig. 2
figure 2

Dissolution profile of EGN-ChAgNPs at 7.4 pH (a), stability analysis of AgNPs when initially synthesized (yellow line), tested for maximum wavelength after six months (red line) and green line (chitosan) appeared with no absorbance peak (b)

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Table II had presented an R2 value of 0.9641 obtained from zero-order analysis, which suggested that drug release was not influenced by concentration at a specific time. It was stated that this kind of kinetic model demonstrated controlled release of a specific substance from hybrid nanoparticles. The Korsmeyer-Peppas model’s higher R2 value (0.9780, n = 0.879) revealed the swelling and erosion, non-fickian case II type release of EGN from hybrid nanoparticles. In polymeric systems wherein the drug is disseminated or dissolved inside a polymer matrix or where the drug is chemically bound to the polymer, non-Fickian Case II release is frequently seen. A number of variables, including polymer erosion, expansion and unwinding as well as drug dissolution or diffusion within the polymer matrix, influence the release process [60].

The findings were in agreement with a previous investigation, wherein chitosan nanoparticles exhibited swelling characteristics during the release mechanism of a specific medication. The observed swelling phenomenon could potentially be attributed to the protonation of amino groups, leading to the generation of NH3+. This, in turn, may result in the formation of a gel-like structure through the retention of water [61].

Stability Test of Nanoparticles

The key sign that noble metal nanoparticles have been successfully biosynthesized is a change in the color of the solution from an apparently dull yellow to a yellowish-brown as silver ion (Ag +) is bio-reduced to metallic AgNPs [62]. The most important method for identifying the formation of metal nanoparticles and demonstrating surface metal plasmon resonance is UV–visible spectroscopy [63].

The UV–visible spectroscopy technique is the most crucial way to determine the creation of metal nanoparticles by revealing the occurrence of metal plasmon resonance at the surface [62]. A prominent signal at 429 nm indicates that the biosynthesized AgNPs have induced surface plasmon resonance (SPR) [63]. Furthermore, the deficiency of any peak in the range of 450 to 800 nm suggests the absence of agglomeration, which adds to the stability of bioassisted AgNPs [64]. The distinctive absorbance peak that exceeds 400 nm in the UV–vis spectrum is designated as a supporting index for AgNPs generation [26]. Figure 2(b) depicted the UV–vis absorption spectra of the biosynthesized AgNPs following a 6 months storage period to test the AgNPs’ stability under ICH guidelines [65].

When compared with the initially created AgNPs solution (Fig. 1b yellow line), no significant shift in the absorption peak or change in the color of the solution was noticed (Fig. 1b red line), suggesting that bioprepared AgNPs had extremely high stability over a long period of time. Our results were corroborated with previously reported studies on stability of biosynthesized AgNPs [31, 65].

Pharmacological Analysis

Sulforhodamine B Assay

The SRB assay was utilized to evaluate the cytotoxicity of both pure EGN and EGN-ChAgNPs on MCF-7 cells Fig. 3(a). MCF-7 cells were exposed to various quantities of pure EGN and EGN-ChAgNPs for a duration of 24 h. The IC50 values for pure EGN and EGN-ChAgNPs were estimated to be 30.72 ± 4.91 and 14.87 ± 5.34 µg/ml, respectively (Table II).

Fig. 3
figure 3

The pharmacological characterization represents cell death and cell viability percentage of pure EGN and EGN-ChAgNPs nanoparticles (a), Comet assay shows DNA damage in MCF-7 cells treated with pure EGN (b), cisplatin (c), and EGN-ChAgNPs (d)

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An earlier investigation reported the anticancer properties of gold (Au) and silver (Ag) nanoparticles conjugated with EGN, on two distinct cancer cell lines, namely HCT116 and MCF-7, across varying concentrations. The EGN-AuNPs exhibited a significantly higher potency, approximately 18 times greater than that of EGN alone, in inducing cytotoxic effects in both cancer cell lines [66]. Likewise, the encapsulation of EGN in magnetic nanoparticles resulted in the highest inhibition percentage in HeLa cells [67].

Genotoxicity Analysis

DNA damage was verified using the comet test. DNA damage was primarily validated by measuring tail moment (TM), olive tail moment (OTM), and tail DNA as a fraction of total tail DNA. The damaged DNA is transferred to the tail end. The experiment was conducted on MCF-7 cells that were subjected to pure EGN treatment as depicted in Fig. 3(b). Additionally, cisplatin was employed in the assay, as illustrated in Fig. 3(c), along with EGN-ChAgNPs (Fig. 3d). Table III presents the outcomes of various parameters utilized in the comet assay.

Table III Analysis of Different Parameters of Comet Assay Treated with Cisplatin, EGN-ChAgNPs and Pure EGN on MCF-7 Cell Line
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The results indicated that EGN-ChAgNPs caused greater DNA damage in MCF-7 cells when compared to pure EGN. The distance travelled by the distorted DNA segments during electrophoresis is represented by a comet tail length (L-Tail). Greater DNA damage is indicated by longer tail lengths, whereas minor or no damage is indicated by shorter tail lengths [68]. Our findings indicated that the L-Tail of EGN-ChAgNPs had a higher numeric value (90) than cisplatin (88), which might be attributed to the bigger L-Head (211) of the comet treated with EGN-ChAgNPs. The Olive tail moment gives a numerical value to the level of DNA damage in a cell. A larger score implies more DNA damage, whereas a lower value indicates less damage [69]. Our research showed that pure EGN had the lowest numerical value of OTM [6], whereas EGN-ChAGNPs had an OTM value that was comparable to cisplatin (11 and 13), respectively.

The comet-like structure formed by DNA fragments migrating out from the nucleus is referred to as the tail movement. The degree of the breakdown of DNA found inside the cells is determined by the amount of tail movement during the comet assay. Less damage results in shorter or missing tails, whereas higher degrees of DNA damage cause greater tail length and intensity. The tail movement in the current investigation revealed similar quantitative information (09) for both cisplatin and EGN-ChAgNPs, whereas it has the lowest value for pure EGN (06), indicating that pure EGN has reduced efficacy. According to previous study, the concentration of damaged DNA tails was reduced in MCF-7 cells treated with Withaferin-A Loaded nanosponges compared to those treated with standard and pure withaferin-A [70].

Flow Cytometry Analysis

Flow cytometry experiment was used to examine the progression of cell arrest data presented in Fig. 4. Earlier techniques provided qualitative confirmation of cancer cell death; the flow cytometry assay provided quantitative validation of these analysis [71]. Figure 4(a) demonstrates that a higher proportion of MCF-7 cells persisted in the G0/G1 phase following treatment with the control agent. Conversely, when incubated with pure EGN, the cells exhibited a higher rate of mortality, as evidenced by the higher values of sub-G1 (3.51%) and lower values for G0/G1 (74.9%), as illustrated in the histograms Fig. 4(b). While incubation with EGN-ChAgNPs increased cell mortality, as seen by the greater values of sub-G1 (8.72%) and lower values of G0/G1 (67.9%) in the histograms Fig. 4(c). The findings of our study had aligned with those of a prior investigation, which demonstrated that fenugreek protein hydrolysates possess anti-proliferative properties against colorectal cancer cells. Specifically, these hydrolysates were observed to impede cell proliferation by facilitating intrinsic apoptosis [72].

Fig. 4
figure 4

The flow cytometry analysis of control (a), pure EGN (b) and EGN-ChAgNPs (c) treated MCF7 cells. Percentage of cell population in cell cycle treated with control, pure EGN and EGN-ChAgNPs (d)

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In Vivo Studies

In order to verify the results of the in vitro research, an in vivo anticancer investigation was carried out using female albino mice. The survival rates of mice given cisplatin (0.02 mg/kg), pure EGN (0.05504 mg/kg), and EGN-ChAgNPs (0.02676 mg/kg) are shown in Fig. 5(a & b). The outcomes were evaluated against the gold standard of cisplatin. Compared to the pure EGN treated group, the EGN-ChAgNPs (0.02676 mg/kg) treated group showed a greater percentage of survivors. In the untreated group, all animals perished within 6 days, but in the EGN and EGN-ChAgNPs groups, the last animal died on days 9 and 10, respectively. These findings were slightly inconsistent with those of Almalki and Naguib (2021), who found that pure EGN dramatically improves survival rates, from 20% in the untreated group to 100% [73].

Fig. 5
figure 5

In vivo percent survival rate (a) and percent reduction in tumor weight (b)

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The group that received EGN-ChAgNPs exhibited a greater reduction in tumor weight percentage on the tenth day following administration, in comparison to the group that received solely pure EGN treatment. The in vivo cytotoxicity of pure EGN against tumor cells was augmented through its conversion into hybrid nanoparticles. Prior research had corroborated our present study’s findings regarding the advantages of nanoparticles in facilitating controlled and targeted drug delivery, thereby augmenting treatment efficacy across various cancer therapies.

Molecular Docking

Ligand Structure Retrieval and ADME Analysis

The 2D structure of EGN was retrieved from PubChem (PubChem CID: 3314) as shown in Fig. 6(a). According to its physical and chemical properties, EGN is a colorless or pale yellow liquid with pungent taste. In addition, it darkens upon exposure to air, decomposes upon heating to emit irritating fumes and is freely soluble in benzene [74].

Fig. 6
figure 6

A 2-dimensional structure of EGN (2-methoxy-4-prop-2-enylphenol) (a), a string network elucidating the protein–protein interaction of Aurora kinase A (AURKA) with other proteins. The color of lines is depicting the type of evidence of interaction between proteins. (light green: textmining; black: co-expression; pink: experimentally determined; light blue: curated databases) (b), representation of active site residues in Aurora kinase A after extending the active site to 30 residues. These residues include Leu139, Gly140, Gly142, Lys143, Asn146, Val147, Leu159, Ala160, Leu161, Lys162, Leu194, Leu210, Glu211, Tyr212, Ala213, Pro214, Leu215, Gly216, Thr217, Arg220, Lys258, Glu260, Asn261, Leu262, Leu263, Leu264, Ala273, Phe275, Trp277, and Ser278 (c), the docked complex containing the EGN (pink) docked in the active pocket of Aurora Kinase A (purple) (d)

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The EGN is considered to be a viable target for drug development due to its adherence to the established criteria for druggability. According to these criteria, the TPSA should be greater than 20 Å and less than 130 Å, rotatable bonds should be less than 9, and molecular weight should be from 150 to 500 g/mol. Whereas, the log Kp should be negative to prevent the skin permeation. As the skin permeability of a compound decreases by increase in the negative value of log Kp, which is directly correlated with the size of the compound (molecular size) [75, 76]. Additionally, the value synthetic accessibility should be lower (ranges from 1 to 10) as it is essential for selecting a druggable compound. It predicts the ease to synthesize a compound in vitro which will be more difficult when value of synthetic accessibility is higher [77].

The pharmacokinetic properties of EGN by SwissADME predicted that EGN has a molecular weight of 164.20 g/mol, 12 hydrogen bond acceptors, 3 rotatable bonds, 2 hydrogen bond acceptors and 1 hydrogen bond donor. The topological surface area (TPSA), molar refractivity, consensus log P and log Kp was predicted to be 29.46 Å, 49.06 m3 mol−1, 2.25, and -5.69 cm/s. It has high gastrointestinal absorption, no PAIN alerts and is synthetically accessible. Moreover, EGN is non-toxic as shown in Table IV.

Table IV ADME Analysis of EGN by SwissADME
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Druggable Target Prediction and Validation

The structure of the EGN was used as query to predict the target proteins from SwissADME. Total 32 target proteins were predicted based on the structural similarity of EGN with their reported ligands. Among these, serine/threonine-protein kinase Aurora-A was selected after validating its role in various cancers from UniProtKB (https://www.uniprot.org/uniprotkb/O14965/entry#expression). It is specifically upregulated in breast, colon, cervical, ovarian and neuroblastoma cell lines [78]. Owing to this, protein–protein interactions were determined by STRING which predicts that Aurora Kinase A interacts with targeting protein for Xklp2 (TPX2), disks large-associated protein 5 (DLGAP5), protein aurora borealis (BORA), and inner centromere proteins (INCENP). Other proteins include transforming acidic coiled-coil-containing protein 3 (TACC3), polo like kinase 1 (PLK1), cell division cycle protein 20 homolog (CDC20), baculoviral IAP repeat containing 5 (BIRC5), tumor antigen p53 (TP53) and MYCN proto-oncogene (MYCN). Aurora Kinase A interacts with most of the protein by phosphorylating them and regulated the protein that are either involved in cell division or tumor suppression as represented in Fig. 6(b).

Molecular Docking Analysis

Subsequent to validation the 3D structure of Aurora Kinase A was obtained from PDB having a PDB ID of 5EW9. The selected protein has a resolution of 2.18 Å and consists of 271 amino acids with a sole chain (chain A) [79]. The protein was uploaded in protein mode functionality of SeeSAR in which the protein was extracted with natural ligand to automatically choose the binding site. After standard docking with the co-crystalline ligand in the docking mode, protein–ligand complex was shifted to binding site mode for extending the binding site residues. The most druggable automatically selected binding site had 0.53 DoGSiteScorer, 25 amino acid residues, 0.73 hydrophobicity, 371.52 Å2 solvent accessible surface, and 554.90 Å3 volume. The amino acid residues in the binding site were extended from 25 to 30 amino acid residues as shown in Fig. 6(b).

The EGN was then docked to generate poses in the extended binding site of Aurora Kinase A as shown in Fig. 6(c). The results interpreted that Aurora kinase A-EGN complex has greater estimated affinity than the Aurora kinase A-co-crystalline ligand complex. Moreover, the EGN optimally interacts with the binding site of Aurora kinase A with no torsion, intra-clashes observed as elucidated in Fig. 6(d).

Protein–Ligand Interactions

Protein–ligand interactions visualized by Discovery studio predicts that EGN interacts favorably with the Aurora Kinase A via conventional hydrogen bond, carbon hydrogen bond, alkyl and π- alkyl interactions. The Glu211 and Ala213 of the receptor form the conventional hydrogen bonds with the H22 and O2 of the EGN having a bond length of 1.80 and 2.86 Å, respectively (Fig. 7a). In addition, the carbon hydrogen bond interaction exists between H19 of EGN and oxygen of Ala213 with a bond length of 2.80 Å. A total of 4 π- alkyl interactions are formed between the EGN and receptor binding site. The aromatic ring of EGN develops π- alkyl interactions with three different amino acid residues namely Val147, Ala160 and Leu263. The last π- alkyl intermolecular bond is attributed to the interaction of C11 of the terminal methyl group of EGN with the aromatic ring of Tyr212, which was placed at a distance of 5.34 Å from C11. Moreover, Leu139 and Leu263 interacts with the C11 and C12 of EGN via alkyl bond, respectively as indicated in Fig. 7(b). The HYDE scoring visualized by SeeSAR interprets that C3, C4, C7, C9, C11, C12 and O2 of the EGN have greater contribution in the overall predicted estimated affinity of EGN. The more negative the energy values; the highest will be the HYDE scoring and the atom will be more contributing to the overall affinity. Among these, C9 has the highest HYDE scoring that is -6.8 kJ/mol as shown in Fig. 7(c). It means that among all the atoms of EGN, C9 is significantly responsible for the binding affinity of EGN with aurora kinase A active pocket.

Fig. 7
figure 7

It is indicating that EGN has greater estimated affinity than the co-crystalline ligand of Aurora Kinase A after standard docking with the FlexX functionality of SeeSAR (a). A 2D representation of protein ligand interactions, in which the intermolecular interactions are represented by dotted line (Green dotted lines: conventional hydrogen bond; Pink dotted lines: alkyl and π- alkyl interactions; light blue: carbon hydrogen bond) (b). A 3D representation of protein–ligand interaction of EGN (dark pink) with the active site residues of Aurora kinase A (purple). The HYDE scoring of EGN atoms that have major share in the overall predicted estimated affinity of EGN (c)

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Conclusion

In this work, the hydrodynamic diameter, zeta potential, transmission electron microscopy, electron emission, and release kinetics of EGN-ChAgNPs were measured and characterized. EGN-ChAgNPs were tested for their anticancer effects on MCF-7 cells in vitro using SRB, genotoxicity and flow cytometry analysis. Female albino mice were utilized for in vivo anticancer tests to corroborate our in vitro results. Compared to the pure EGN group, the EGN-ChAgNPs (0.02676 mg/kg) treated group showed a better percentage of survival and tumor weight loss to a greater extent. EGN-ChAgNPs were synthesized in a cost-effective and environmentally friendly manner and shown substantial anticancer activity against breast cancer cells. The HYDE score showed that EGN had contributed more to its predicted affinity. In conclusion, ChAgNPs proved to be a prototype to deliver other naturally occurring medicinal chemicals in cancer therapy.