The impact of pumpkin seed-derived silver nanoparticles on corrosion and cytotoxicity: a molecular docking study of the simulated AgNPs

ABSTRACT

Green-synthesized nanoparticles from pumpkin (Cucurbita pepo L.) seed extracts are economical and eco-friendly. Silver nanoparticles (AgNPs) and their selective cytotoxicity towards HCT116 and African Green Monkey Kidney, Vero cells were investigated. Chemical fingerprinting, heat stability, and 2D-images of nanoparticle size and morphology were determined using Fourier-transform infrared spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Transmission Electron Microscopy (TEM), and Scanning electron microscopy-energy dispersive X-ray (SEM-EDX) on AgNPs. UV-vis examination shows surface plasmons in the wide peak at 417 nm, indicating polydisperse nanoparticles. Small silver nanoparticles (AgNPs) below 2 µm demonstrated a rod-like form and a tendency to agglomerate. SEM-EDX element analysis and fingerprinting confirmed the material as AgNPs. TEM indicated that the nanoparticles were generally spherical or ellipsoidal, equally dispersed, and averaged 26.08 nm in diameter with negligible aggregation. The AgNPs are also stable at a temperature of 220°C, indicating that the green material is quite robust at 150°C to 400°C. According to cytotoxic studies, AgNPs are toxic to cancer cells (HCT 116 cells), however they have no effect on Vero cells. AgNPs and tubulin (TUB) domain active sites have a significant affinity, according to molecular docking analysis. In an electrochemical investigation, biogenic AgNPs effectively prevented mild steel from corroding in a 1.0 M HCl solution.

GRAPHICAL ABSTRACT

KEYWORDS:

• Cucurbita pepo L
• silver nanoparticles (AgNPs)
• anticorrosion
• cytotoxicity
• molecular docking

1. Introduction

Cancer happens when cells of a few body parts grow out of control and spread to other body parts. According, to the World Health Organization (2021), Malaysia has witnessed around 48,639 cancer cases since 2021, and this number is cases expected to quadruple by 2040. In Malaysia, cancer is the 2nd leading cause of death, and around 9.6 million deaths from cancer were reported globally in 2017. Cancer occurrence and mortality are spreading globally to the current date, early diagnosis and surgery have been the most common and popular treatments for most cancers (1). According to Varmus (2), molecular understanding of cancer tends to improve strategies for human cancer detection, monitoring, classification, and treatment as a result of advancements in the past ten years. Over 60% of the anti-cancer drugs currently being used are made from natural sources, such as plants to treat many diseases, including cancer. For instance, vinca alkaloids, taxanes, anthracyclines, and many others are naturally occurring anti-cancer drugs. Modified Nanotechnology medications and drug delivery systems have the potential to treat cancer, and some are being utilized clinically with promising results (3). Due to the pathophysiology of cancer, nano-medicine has a specific diameter range that enables it to be vaccinated without blocking the needle with capillary and is optimal for the delivery of targeted medication (4). In addition, Nobel metal nanoparticles have grown in strategic value recently due to their use in biology materials, chemistry, science, medicine, and biology. Metal nanoparticles, namely silver nanoparticles (AgNPs), are significant due to their exceptional physicochemical features, which include magnetic, optical, and antibacterial potentials (5). AgNPs are used as drug delivery vectors, antiseptics, and physical treatment agents in the field of medicine. AgNPs can induce oxidative stress, change the mitochondrial membrane, expedite cell death, and activate DNA destruction, as demonstrated by Sangour et al. (6). Furthermore, the current available cytotoxic drugs (via oral and parenteral) such as cisplatin and vincristine can inhibit the growth of tumors but they also cause adverse effects on normal human cells (7). This has attracted researchers to search for anticancer drugs or substances that have target specificity (targeted therapy) on cancer cells due to their specific target of destroying cancer cells only and not healthy cells (8). Interestingly, many researchers are focusing on the biosynthesis of nanoparticles (NPs) to synthesize a new potential anticancer treatment ascribed to the distinct physical and chemical properties of nanoparticles which have target specificity on cancer cells. Recently, silver nanoparticles (AgNPs) synthesized in an environmentally friendly way from various plant sources, such as pods from Acacia nilotica, leaves from Agaricus bisporus, Commiphora gileadensis, Adansonia digitata fruit extract, aqueous leaf extract from Rosmarinus officinalis, and leaf extract from Azadirachta indica, have been chacteraized using advanced spectroscopy and microscopy techniques and utilized for their antibacterial and anticancer properties (9, 10, 11, 12, 13). These AgNPs have been described using a variety of investigations, and they are effective against a range of bacterial strains and cancer cell lines (14, 15, 16). According to Gomes et al. (17), AgNPs using an eco-friendly substance like green synthesis Nanoparticles (NPs) have a potential cytotoxicity drug, however, due to concerns about toxicity stability, AgNPs are not frequently used in drug delivery nanosystems. Thus, this research highlighted the potential of AgNPs as cytotoxic agents due to their unique characteristics. Additionally, molecular docking between silver nanoparticles and tubulin (TUB) (PDB ID: 5NM5) was achieved. Tubulin is a cellular polymer of the microtubules and is involved in a variety of functions in the cell, including cell division, maintaining the cell's structure, and producing cytokines.

Various methods have been employed to reduce the corrosion rate of metals. These methods include techniques such as cathodic protection, protective coatings, heat treatment, alloying, use of inhibitors, and modification of the surrounding environment. Among these methods, the use of corrosion inhibitors (CIs) remains a cost-effective method of corrosion protection (18, 19, 20). A corrosion inhibitor, as defined by NACE International (21), is generally a chemical substance that, when added to the environment in low concentrations, is effective in reducing the rate of corrosion. Corrosion inhibitors are substances that can be added to a system to reduce the rate of corrosion. They do this by forming a thin passivating layer on the surface of the material. This layer acts as a barrier, preventing corrosive substances from penetrating the metal surface. As a result, it limits or decreases either the oxidation or reduction component of the redox corrosion system or renders dissolved oxygen ineffective as a corrosion catalyst (22). To investigate the effect of silver nanoparticles (AgNPs) derived from pumpkin seeds on corrosion, electrochemical polarization experiments were performed. These experiments were performed to evaluate the corrosion resistance of mild steel in the presence and absence of AgNPs.

2. Materials and methods

RPMI 1640 media were purchased from Nacalai Tesque. Whereas, fetal bovine serum (FBS), ethanol 70%, phosphate buffer saline (PBS), penicillin–streptomycin, accutase, deionized water, dimethyl sulfoxide (DMSO), and AlamarBlue® were purchased from Sigma Aldrich, UK. Pumpkin seeds were harvested in Jaipur, Rajasthan, India, in the Jagatpura market. The specimen was authenticated by the Department of Botany at Aligarh Muslim University and deposited in the departmental herbarium under voucher number 84/21. Chemicals and silver nitrate (AgNO3) were bought from Sigma. The remaining chemicals required for the synthesis of silver nanoparticles were all purchased locally.

2.1. Preparation of plant extract

Pumpkin seed extraction and AgNP biosynthesis were carried out at Jaipur National University in India. Double distillation of water was applied to wash the collected pumpkin seeds to detach the unwanted debris and air-dried them at room temperature. 100 mL of double-distilled water and 5.0 g of finely ground pumpkin seeds were combined in a 250 mL round flask and refluxed for 45 min. The extracts were dried and Whatman filter paper no.1 was used (23).

2.2. Biosynthesis of AgNPs

100 mL of a 1 mM concentrated AgNO₃ aqueous solution was prepared in an Erlenmeyer flask, and 90 mL of AgNO₃ aqueous solution was combined with 10 mL of pumpkin seed extract. The mixture was left at room temperature with constant stirring until the solution's color became apparent. The changes led to the appearance of a colorless solution to wine red, and the reduction of Ag + to Ag0 was the result. UV-spectroscopy analysis supported the formation of AgNPs (24).

2.3. Characterization of biogenic silver nanoparticles

The characterization of AgNPs was determined to confirm the characterization of the NPs with literature before conducting a cytotoxicity assay to assess the functional aspects of synthesized particles (25), as well as to confirm its structural characterization with previous literature (26). Several analytical methods, including Scanning Electron Microscope (SEM), Thermogravimetric Analysis (TGA), Energy Dispersive X-ray (EDX), and Fourier Transform Infrared Spectroscopy (FTIR) were used to conduct the characterization analysis.

2.3.1. UV-Vis spectroscopy

Ultraviolet–visible (UV-vis) spectrum of the synthesized silver nanoparticles was examined in the range of 300–800 nm using a UV-Vis spectrophotometer (UV-2450, Shimadzu, Japan) with a resolution of 1 nm to investigate the conversion of Ag⁺ to Ag⁰. For background correction, double-distilled water was employed as a blank reference.

2.3.2. X-ray powder diffraction (XRD)

The pellets were redispersed in sterile double-distilled water for 10 min before being centrifuged at 10,000 rpm for 15 min, air dried at 50 °C in an oven, and examined by XRD (Pan Analytical, X-pert Pro, Netherlands) with a Cu-Kα radiation source over a 2θ scan range of 20-80° on the instrument operating at a voltage of 45 kV and a current of 40 mA. The grain size of biogenic silver nanoparticles was calculated using Scherrer's equation after evaluating the XRD pattern. $D=(\mathrm{k\lambda }/\beta \mathrm{cos\theta })$where D, β, and λ, θ, and K signify average crystallite size, line broadening (half-maximal width of the peak in radians), X-ray wavelength, Braggs angle, and constant (geometric factor = 0.94).

2.3.3. Scanning electron microscopy – energy dispersive X-ray (SEM-EDX)

SEM analysis of synthesized AgNPs was done using TESCAN VEGA, where the instrument was equipped with a Thermo EDX attachment. AgNPs were placed on sample stubs with carbon tape, which provided excellent adhesion and conductivity (27). The sample was coated with gold to prevent charging of the surface to increase the thermal conduction that eases the scanning electron microscope to observe the sample, and to provide a homogeneous surface for analysis and imaging (28).

2.3.4. Transmission electron microscopy (TEM)

Transmission electron microscopy (TEM) was used to determine the shape and size of AgNPs. The size of biogenic silver nanoparticles and their morphology were studied using a TEM (100 W infrared lamp, JEOL JSM-2100F) at a 200 kV accelerating voltage.

2.3.5. Fourier transform infrared spectroscopy (FTIR)

Fingerprinting of the chemical structure of AgNPs from pumpkin seeds was conducted using FTIR-ATR at the wavelength range of 4000-400 cm⁻¹. This was done to determine the functional groups of AgNPs. AgNPs were placed on ATR crystals to obtain FTIR spectra. For the light spectrum to transmit, the sample was embedded with an automatic presser and compressed with a clamp.

2.3.6. Thermogravimetric analysis (TGA)

TGA was performed to determine the approximate sample component proportion, decomposition temperature, and nanoparticle residues. The thermal properties of the AgNPs were observed using TGA at the heating rate of 10 °C/min from 50 °C until reached 850 °C. Oxygen gas was used as gas flow in TGA to analyze the thermal properties of the sample.

2.4. Preparation of chemical solutions for cell culture

The complete culture media, phosphate buffer saline, and cryopreservation solution were prepared for cell maintenance.

2.4.1. Preparation of complete medium (RPMI 1640)

Five mL of penicillin–streptomycin (100 mg/mL) and 10% of fetal bovine serum (FBS) were added into RPMI 1640 of 500 mL supplemented with L-glutamine and transferred to an empty culture flask. The complete media were stored in 4°C of a refrigerator.

2.4.2. Preparation of phosphate buffer saline (PBS)

Phosphate buffer saline (PBS) 2 tablets (Sigma Aldrich, UK) was used. Then 400mL of deionized water was added and set until the tablet dissolved in the water. PBS solution was autoclaved to obtain sterile buffer saline and was stored in a refrigerator at 4°C.

2.4.3. Preparation of cryopreservation solution

A cryopreservation solution of 1 mL was prepared using 100 µL DMSO and 900 µL of resuspended cells with the ratio of 1:10. The cryopreservation solution was then stored in a refrigerator of −80°C.

2.5. Cell culture

HCT 116 cancer cells and Vero cells were cultivated before the cytotoxicity assay of AgNPs using AlamarBlue® assay. The cells were kept in RPMI 1640 media (as mentioned in section 3.5.1). Then, HCT 116 cells were incubated in a 5% CO₂ atmosphere at a temperature of 37°C.

2.5.1. Cell thawing

Both cancer and normal cells were taken out from the −80°C of a refrigerator. The cells in the cryovial were thawed a 37°C of the water bath for 1 min. The thawed cells were transferred into 15 mL falcon tubes and then 4 mL of complete culture media was added. The tube containing the media and cells was centrifuged at 2000rpm for 5 min at 4°C. After removing the supernatant, the pellets were resuspended in 2 mL of culture media, which was transferred into two medium-sized culture flasks (T-25), each having 1 mL of resuspended cells. The culture flask was incubated in an environment of 5% CO₂.

2.5.2. Subculture

The pellet was obtained from a trypsinization procedure where the medium was discarded and PBS was added to wash the cells two times to remove any Fetal Bovine Serum (FBS) in the culture media. Then, 1 mL of accutase was added to detach the cells of the adherent surface incubated in a CO₂ incubator for 5 min and resuspended with 1 mL complete medium. Each of the 2 ml suspensions was transferred into a new 25 cm³ culture flask (T-25) which was filled with another 4 mL of a complete RPMI medium. The cells were incubated in a 37°C CO₂ incubator provided with 5% CO₂.

2.5.3. Cryopreservation

Before moving on to the next phase, the previously described trypsinization procedure was carried out in a condition where the cells have reached confluency. Colon cancer and Vero cells were resuspended in 1 mL of complete media. The following step involves resuspending 900 mL of cell suspension in 100 mL of pre-warmed dimethyl sulfoxide (DMSO) in a cryovial that was parafilm-sealed. The date, cell name, and passage number were noted on the cryovial for future reference. The cryovials were frozen for at least one night in a refrigerator set at −20°C before moving to a refrigerator set at −80°C to achieve a progressive transition in the ultra-low temperature required to preserve the cells.

2.5.4. Cell counting

The number of cells for HCT 116 and Vero Cell Lines were counted using a hemocytometer and Trypan blue to view the cell viability. The viable cells in four portions (1, 2, 3, and 4) of the grid were counted and the calculations were performed using the formula below: $\mathrm{Cell}\mathrm{number}={\displaystyle \frac{\begin{array}{rl}& \mathrm{average}\mathrm{number}\mathrm{of}\mathrm{cells}\mathrm{in}4\mathrm{grids}\mathrm{corners}\\ & \times \mathrm{Dilution}\mathrm{factor}\end{array}}{10000\mathrm{cells}/\mathrm{mL}}}$

2.5.5. Cell seeding

Each HCT 116 and Vero cells (1x 10⁴) were seeded separately in 96 well plates to ensure standardization. The quantity per ml of the desired cells can be identified using the following formula: $Y(\mathrm{mL})={\displaystyle \frac{\mathrm{Desirednumberofcells}(\mathrm{cells})}{\mathrm{Cellsobtained}(\mathrm{cells}/\mathrm{mL})}}$Y = Volume of the medium and cell suspension required that contain the desired number of cells to be seeded in each well.

2.6. Cytotoxicity assay

An Alamar blue assay was performed to ascertain the cytotoxicity effect of AgNPs from pumpkin seed extract in inhibiting HCT 116 and Vero Cells.

2.6.1. Determination of the cytotoxicity effect of pumpkin seeds derived AgNPs in inhibiting HCT 116 and vero cells

The sample was measured out and placed in an Eppendorf tube with 1 mL of distilled water.

The AgNPs were diluted using a 2-fold dilution method with 0.01% DMSO into five diverse concentrations (Figure 1): 1000, 500, 250, 125, and 62.5 µg/mL. A fixed number of Vero cells was prepared as the negative control. The following formula was used to calculate the various concentrations: $M_1{V}_1=M_2{V}_2$M₁= Stock solution

Figure 1. (2-fold dilution method) using serial dilution.

V₁= Volume (stock) that needs to be filled in each well

M₂= The final and highest concentration from the journal

V₂= Total volume in each well, (90 µL cells + media) + (10 µL treatment) + (10 µL AlamarBlue® reagent)

Different concentrations of AgNPs were used to treat Vero cells and cancer cells. Each well received ten µL of different AgNPs concentrations, and the 96-well plates were incubated at 37°C and at 5% CO₂ for 24 h and 48 h with the 96-well plate, used to perform the treatment utilizing AgNPs derived from pumpkin seeds.

2.6.2. Alamarblue® assay

The cytotoxicity of AgNPs on HCT 116 and Vero cells was assessed using the AlamarBlue® assay. AlamarBlue® (Sigma-Aldrich) indicator dye changes fluorescence and color with the reduction of resazurin (opaque blue) to resorufin (fluorescence pink) by metabolically active cells. In 96-well plates, the cells were seeded (104 cells/well) and incubated overnight. The AgNPs were treated in the cells in each well at concentrations of 1000, 500, 250, 125, and 62.5 µg/mL. A fluorescence absorbance multi-plate reader was assessed to evaluate the fluorescence values with the excitation wavelengths of 570 nm and emission wavelengths of 590 nm. The conversion of the dimly fluorescent substance resazurin into fluorescence pink indicated the presence of living cells.

2.7. Statistical analysis

Data are interpreted as the mean ± SEM of four replications. Control and treated cells were compared by manipulating one-way ANOVA, and differences between groups were considered significant when p values ≤ 0.05. One-way ANOVA statistical analysis was used to analyze the cytotoxicity impact of AgNPs on both HCT 116 and Vero cells and results were expressed as a percentage of cell viability. GraphPad Prism 5.0 was used to analyze the significant difference in cytotoxic effects of HCT 116 and Vero cells after the treatments with AgNPs using AlamarBlue® assay.

2.8. Molecular docking study

The receptor (PDB ID: 5NM5) used in the docking study was downloaded from the RCSB PDB. The complexation of colchicine with the active amino acids of the receptor revealed the location of the active site of the receptor. Before docking, the receptor was prepared to remove heteromolecules and the water molecule from the protein using Discovery Studio (29). Silver nanoparticles were constructed using silver crystallographic information with Material Studio. The constructed AgNPs structure was minimized, optimized, and dynamically simulated using a COMPASS (condensed phase optimized molecular potential for atomistic simulation) force field to ascertain the proper silver packing and orientation in the geometric structure and was saved in pdb format. In order to obtain the docking results, the prepared receptor and the AgNPs’ energy-minimized structure were both added to the HADOCK server. The best docking model from the results was then used to study the interaction analysis between amino acids and NPs using Discovery Studio.

2.9. Preparation of steel samples and corrosion testing solutions

2.9.1. Samples compositions and preparation

The mild steel samples under examination are made up of a specific metal alloy with the chemical makeup (in weight percent) as follows: 0.21% Carbon (C), 0.38% Silicon (Si), 0.05% Manganese (Mn), 0.05% Sulfur (S), 0.09% Phosphorus (P), 0.01% Aluminum (Al), and the rest is Iron (Fe) (30). Each sample of mild steel was cut to a size of 20 mm × 20 mm. In order to make it simpler to suspend and retrieve each sample from the corrosive conditions, a 3 mm hole was drilled into each one. The specimens were mechanically polished in advance of corrosion testing with emery papers, a fine-grained abrasive. Specific emery papers used in the corrosion study are mentioned in the literature (31, 32). Prior to the experiment, the samples were degreased with ethanol, rinsed with acetone, and cleaned with distilled water to remove contaminants and avoid beginning corrosion. The samples were polished and cleaned before being dried and kept in a desiccator to stop moisture buildup (33).

2.9.2. Preparation of corrosion environment

To create corrosion conditions, prepare a mixture by mixing a solution of biosynthesized silver nanoparticles with 1.0 M hydrochloric acid. Silver nanoparticles (AgNPs) solutions at different concentrations were prepared, including a control solution without AgNPs (0 µg/ml), and four experimental solutions containing 10, 20, 40, and 80 µg/ml AgNPs, respectively. These solutions were prepared by diluting 100 g/ml stock solutions with distilled water. Five separate containers were then prepared. Each container contained a sample of mild steel. In each container, an equal measure of hydrochloric acid (HCl) solution was added. Each container was then filled with silver nanoparticle (AgNP) solutions of varying concentrations. The solutions were mixed extensively. Notably, the control solution lacked any AgNPs content.

2.9.3. Electrochemical polarization measurement

An electrochemical study was carried out at room temperature using the AUTOLAB potentiostat device, and the data were evaluated and visualized using GPES (General Purpose Electrochemical Software), EC-Lab 10.4, and Origin2024 (Trial version) software. A typical three-electrode cell configuration was employed, where metal samples (20 mm²) were inserted in resins. The samples inserted were used as the working electrode, and a platinum electrode and a saturated silver/silver chloride electrode were used as the counter and reference electrodes, respectively. The electrolyte was a solution of 1.0 M hydrochloric acid (HCl) with varying concentrations of silver nanoparticles (AgNPs). The open circuit corrosion potential was observed for thirty minutes until a stable potential was reached. Polarization experiments were conducted by altering the potential of the working electrode and monitoring the resulting current over time. Examine the Tafel polarization over an anodic potential range of −1250 mV to +250 mV with a scan rate of 1.0 mVs-1 to assess the effect of the inhibitor solution on the corrosion behavior. Corrosion current density can be calculated by extrapolating the linear component of the Tafel curve's anodic and cathodic segments to the corrosion potential axis. Corrosion rate and inhibition efficiency (IE%) can be calculated from corrosion current densities. Surface coverage (θ) and inhibitor efficiency (IE%) can also be determined by applying equations (1-2) (34, 35, 36, 37). (1) $\mathrm{Surfacecoverage}(\theta )=I_{\mathrm{corr}}^0-I_{\mathrm{corr}}/I_{\mathrm{corr}}^0$(1) (2) $\mathrm{Inhibitorefficiency}(\mathrm{IE}\text{\%})=I_{\mathrm{corr}}^0-I_{\mathrm{corr}}/I_{\mathrm{corr}}^0\times 100$(2)

I⁰corr and Icorr represent the corrosion current densities in the absence and presence of inhibitors, respectively.

3. Results and discussion

3.1. UV-vis spectroscopic analysis

The results of ultraviolet–visible spectroscopy confirmed the successful formation of nanoparticles and their stability in an aqueous colloidal solution. The color change of an aqueous solution containing nanoparticles is attributed to the excitation of surface plasmon phenomena in silver nanoparticles. The change in color of an aqueous solution indicates the reduction of silver ions into silver metal, leading to the formation of silver nanoparticles. During the experiment, the shape and size of as-prepared silver nanoparticles were examined using UV-Vis spectroscopy of aqueous suspensions recorded after solution cooling. The UV-Vis of the extract was well documented in the literature (23). The UV-vis results revealed a broad absorbance peak at 417 nm (Figure 2), indicating that the nanoparticles were polydisperse.

Figure 2. The UV-Vis spectrum of biogenic silver nanoparticles derived from pumpkin seeds.

3.2. Analysis of powder X-ray diffraction (PXRD) pattern

The crystalline nature of the nanoparticles derived from the aqueous extract of pumpkin seeds was identified using X-ray crystallography recorded over a scanning range of 2θ = 20°–80°. The XRD pattern of biogenic AgNPs (Figure 3(a)) demonstrates a limited number of peaks, but all peaks are significant and well correlated with samples in the Joint Committee on Powder Diffraction Standards database (JCPDS). The XRD pattern has four peaks at 38.24°, 44.47°, 64.69°, and 77.59° that correspond to the (111), (200), (220), and (311), which are all well related to the face-centered cubic (FCC) crystal structure of silver (JCPDS, No. 04-0783). Also, the sharp diffraction peaks in the XRD pattern suggest that the biosynthesized silver nanoparticles are crystalline.

Figure 3. (a) XRD pattern of biosynthesized silver nanoparticles and (b) TEM image of biosynthesized silver nanoparticles.

The XRD crystallite (grain) size of the synthesized nanoparticles was calculated using the intense peak (111), which is relatively higher than the typical values in the XRD pattern and is generally responsible for particle size. The Full Width at Half Maximum (FWHM) value of the intense peak was calculated, and the obtained value was used to find out the particle size with the Scherrer equation. The biogenic silver nanoparticles’ grain size was noticed to be approximately 25 nm.

3.3. TEM characterization of biogenic AgNPs

The silver nanoparticle-droplet-coated film was used to collect TEM micrographs of the nanoparticles in order to determine their morphology, shape, and size. The particles are spherical or oval and evenly distributed, with maximum diameters in the 26.08 nm range and no significant clumping, as seen in the TEM images in Figure 3(b). The size observed by TEM analysis was found to be roughly correlated with the size obtained using the Scherrer equation, leading to the formation of silver nanoparticles derived from the use of aqueous extracts of pumpkin seeds in our present study. Close examination reveals that the silver nanoparticles are surrounded by a cloudy, thin layer of another material, which is thought to be the organic material found in aqueous extracts and that caps the nanoparticles.

3.4. Scanning electron microscope-energy dispersive X-Ray (SEM-EDX)

The structure and size of the biosynthesized AgNPs were examined using SEM analysis at various magnifications (24). At magnifications of 5000x and 10,000x, SEM analysis revealed aggregated nano-silver particles with rod-shaped morphology (38). Figure 4 proves that the rod-shaped with 1-5 µm-sized green synthesized AgNPs were produced. The aggregation might be the cause of the bigger AgNPs. It is well known that nanoparticles and the phenomenon of accumulation frequently occur together (39). Our results are similar to Bélteky et al. (40), who demonstrated that high aggregation formation in AgNPs samples reduced their cytotoxic effect.

Figure 4. The SEM image at (a) 5000x magnification and (b) 10 000x magnification showing aggregate and rod shapes.

Thermo EDX attachment equipped with SEM instrument (TESCAN VEGA) has been used to conduct EDX analysis. As seen in Figure 5, the Ag atoms had strong signals in the Ag nanoparticles’ EDX profile about 90.90% of Ag and 9.10% of O₂. The loss of Ag ions using Cucurbita pepo L. extract results in the presence of crystalline AgNPs, as is evident from the EDX pattern (41). The present study's EDX analysis, which confirmed the existence of AgNPs, primarily revealed the highest signal energy peaks of Ag atoms are in the 3-3.5 keV range.

Figure 5. A higher proportion of Ag signals were detectable in the energy-dispersive X-ray (EDX) spectrum.

3.5. Fourier transform infrared spectroscopy

The absorbance range of different functional groups is shown in Figure 6(a), along with the set of bonding that the functional groups have achieved. The peaks at the wavelength of 3240 cm⁻¹, 1291 cm⁻¹, 1040 cm⁻¹, 715 cm⁻¹, and 500 cm⁻¹ were noticed. The results of FTIR analysis in this study show peaks at 3240 cm⁻¹ assigned to O-H stretching of biomolecules. This functionality could be a part of alcohol and phenols in biomolecules. The aqueous extract was also analyzed using FTIR, revealing a complex band pattern suggestive of diverse biomolecules. By comparing the IR frequencies with those found in the literature, specific functional groups associated with various classes of biomolecules were identified (42, 43). Based on phytochemical screening and extraction from aqueous extracts, biomolecules such as flavonoids and phenolic are important molecules present in aqueous extracts and play an important role in the reduction and capping of nanoparticle synthesis. IR frequencies in the FTIR spectrum also supported the presence of the aforementioned molecules. Some peaks, especially for C–H stretching vibration around 2800–2990 cm⁻¹, were clothed by a broad OH peak which could be likely part of COOH. However, the C–H bending at 1291 cm⁻¹ yielded information about the functionality of alkanes in the bimolecular skeleton. Weaker bands of C–H stretching were found at 715 cm⁻¹. The stretching vibration of the C–O group was attributed to the stretch, sharp, and strong absorption band seen in the spectra at 1040 cm⁻¹ characterizing the presence of ether in the extract. The stretch and sharp band at 500 cm⁻¹ in the spectra corresponded to Ag-O which was similar to Al-Zahrani et al. (44)’s statement where a band in the dimension of 500–600 cm⁻¹ corresponded to Ag–O vibration. Ag⁺ is bio-reduced to AgNPs by functional groups like OH and C–O groups that were present in this sample.

Figure 6. (a) FTIR analysis of AgNPs synthesized by pumpkin seed extracts and (b) Tentative mechanism for the synthesis of AgNPs from pumpkin seed extract.

Pumpkin and its seeds have been shown in earlier investigations and spectroscopic studies to be a rich source of various bioactive chemicals such as carotenoids, flavonoids, and phenolic acids, as well as potassium and vitamin C (23, 45). Flavonoids have been found in the aqueous portion of seed extracts. Biomolecules involved in the formation of silver nanoparticles as reducing and protective agents, as shown in the graphic illustration of the proposed mechanism (Figure 6(b)).

3.6. Thermogravimetric analysis

TGA analysis was performed to demonstrate the thermal characteristics of the AgNPs and their stability towards increased temperature (Figure 7). AgNPs subjected to thermogravimetric analysis (TGA) revealed a three-state decomposition pattern. In the first zone, the decrease in weight is 12%, at 49°C which is attributed to the desorption of water or solvent evaporation. Secondary weight loss was degraded dramatically with a percentage of about 40% at 120°C due to the evaporation of organic compounds from AgNPs and the decomposition of tertiary weight loss was about 9% at 450°C (46).

Figure 7. Thermogravimetric analysis (TGA) of AgNPs.

3.7. Effect of AgNPs on cell viability

Based on the % cell viability of green synthesized AgNPs in Figure 8(a) the effect of AgNPs was evaluated in vitro against HCT 116 colon cancer cells at different concentrations (1000, 500, 250, 125, and 62.5) µg/mL. In this study, we emphasize that pumpkin seed-derived AgNPs showed a toxicity effect on HCT 116 cancer cells with minimal inhibition cells of 40%, at the highest concentrations of 1000 µg/mL and 500 µg/mL on 24 h of incubation. This finding is supported by Alharbi and Alsubhi (47), where the authors stated that AgNPs have the potential to act as toxic to cancerous cells. IC₅₀ value of AgNPs was not determined because AgNPs only induce 40% of the cytotoxicity effect on HCT 116 at 24 and 48 h incubation. It is possible to hypothesize that substances that are attached to AgNPs’ surface during chemical synthesis procedures contribute to the level of toxicity in living cells (39). This was due to impurities such as other trace elements (Au, Na, Si, and C) present in the pumpkin seeds extract that showed in EDX spectrum analysis. Green synthesis techniques use phytochemicals acting as a non-toxic reducing agent, resulting in AgNPs with minimal toxicity to cancerous cells (39). Furthermore, Jabeen et al. (48), mentioned that in vivo cytotoxicity and genotoxicity of green synthesized AgNPs pose minimal toxicity and are biologically compatible with chemically produced AgNPs.

Figure 8. The effect of AgNPs on Cell Viability after 24 and 48 h of incubation period; (a) the cell viability of HCT 116 cancer cells and (b) the cell viability of Normal Vero cells. One-way ANOVA (Dunnett post-test) analysis was carried out, and ** indicates a P < 0.05 significance difference in concentration from the control.

Based on the % cell viability of green synthesized AgNPs in Figure 8(b) shows the effect of AgNPs against Vero cells. In our study, AgNPs exhibited non-toxic to the Vero cell line after 24 and 48 h of incubation. AgNPs indicated no detrimental impacts on the Vero cell line as stated by Zhong et al. (8) that anticancer drugs or substances that had target specificity (targeted therapy) on cancer cells due to their specific target in killing cancer cells only and not normal cells. Furthermore, this statement is supported by Jini et al. (49), where the author stated that AgNPs in the structures of spherical does not cause toxicity in the A549 cells.

3.8. Molecular docking analysis

The constructed structure of the silver nanoparticles was energy minimized and optimized using the COMPASS force field available in the Forcite module with Material Studio. Figure 9 depicts the optimized system with multiple energy components, including potential (−6454.615 kcal/mol), non-bonded energy (−6523.786 kcal/mol), and kinetic energy (207.858 kcal/mol), followed by the total energy (−6351.522 kcal/mol) of silver nanoparticles. The energy plot reveals that non-bonded energy and potential energy have been combined, implying that they have roughly the same energy in kcal/mol. Kinetic energy is produced in greater quantities than other types of energy. The well-optimized structure of silver nanoparticles was simulated at picoseconds so that the atoms of AgNPs could be packed in a crystallite in a well-organized way (Figure 9(b)). Throughout the process, the system was determined to be equilibrated. After energy minimization and optimization, followed by simulation, the resulting structure was saved in PDB format for the next operation of molecular docking of AgNPs with tubulin protein.

Figure 9. (a) Total energy distributions for AgNPs system, and (b) simulate NP as a function of time and temperature.

The as-synthesized silver nanoparticle was molecular docked in an attempt to explain the observed cytotoxic activity as well as the possible underlying interaction mechanism of action involving the tubulin domain and nanoparticles. Tublin, or microtubules, play an important role in cellular function. Chemicals or nanoparticles ranging in size from 1 to 100 nm could easily disrupt microtubule function, making this a promising approach for new chemotherapeutic agents. The best-docked model of protein-NPs with a docking score of −98.68 and Ligand rmsd (Å) 99.66 was chosen for visualization to understand nonbonding interactions on the surface of silver nanoparticles (Figure 10). Silver nanoparticles were introduced into the active site of the tubulin domain through a docking procedure (Figure 10(a,b)), which contains an active amino acid pocket (Figure 10(c)). Figure 10 illustrates that pocket amino acids such as GLN11, THR65, SER170, ARG213, THR215, and TYR216 interact with silver atoms in AgNPs in an unbound manner, resulting in antiproliferative activity by enhancing mitotic arrest and cell apoptosis via stabilization or destabilization of microtubules (50).

Figure 10. Molecular docking of receptors (a), with NPs (b), the best docking model (c), and non-bonding interactions of amino acids with NPs (d).

3.9. Corrosion studies

The three-electrode cell configuration is a more advanced corrosion cell setup. It is made up of three essential parts: the working electrode, which is the metal that corrodes, the counter electrode, which is typically made of platinum wire, and the reference electrode (Figure 11(a)). A potentiostat, a device responsible for regulating the potential applied to the working electrode, is also included in the three-electrode cell. The onset and end potentials of the mild steel sample were recorded as −1250 and +250 mV, respectively. However, corrosion occurs in the potential range of −575 mV to −350 mV. The Tafel polarization curves used to demonstrate the corrosion behavior of the metallic samples are displayed in Figure 10(b). The anodic and cathodic Tafel slopes (βa and βc, respectively) and the corrosion current density (Icorr) were determined by extrapolating linear portions of the curves to the corrosion potential (Ecorr). The values are shown in Table 1, together with other relevant data including the inhibition efficiency. The curve (Figure 11(b)) is divided into two sections: anodic and cathodic. The metal is oxidized in the anodic region, while it is reduced in the cathodic region. The corrosion potential is defined as the point where the anodic and cathodic currents are equal. Figure 11(b) depicts the polarization curves of the applied potential vs current density for mild steel corrosion in the presence and absence of silver nanoparticles. Figure 11(b) shows that the introduction of inhibitor leads to a greater shift of the cathodic curve towards lower current densities compared to the anodic curve. These changes can be attributed to the reduction of the hydrogen evolution reaction rate on the low-carbon steel surface due to the adsorption of corrosion inhibitor molecules to the metal surface (51). The shift of the Ecorr value has been reported to classify a compound as an anodic or cathodic type inhibitor. If the Ecorr value shifts by more than 85 mV towards the anode or cathode, the inhibitor is classified as an anodic or cathodic type. Otherwise, the inhibitor is classified as mixed (52, 53).

Figure 11. (a) Corrosion cell and three-electrode cell configurations, and (b) Potentiodynamic polarization curves of mild steel immersed in 1.0 M hydrochloric acid (HCl) solution in the absence and presence of inhibitor.

Table 1. Corrosion parameters studied with and without inhibitor, obtained from polarization measurements.

Inhibitors	Conc (µg/ml)	Icorr (mA/cm^2)	-Ecorr (mV) (SCE)	βc (mV/dec)	βa (mV/dec)	θ	CR mmpy	% I.E.
Blank	0	228.0	457.0	83.4	55.2	–	2.67	–
AgNPs	10	49.2	439.2	94.4	72.7	0.784	0.576	78.4
	20	38.0	446.7	92.1	69.3	0.833	0.440	83.3
	40	32.7	440.0	123.3	84.8	0.856	0.383	85.6
	80	25.3	441.6	109.1	86.3	0.88.9	0.296	88.9

In the present study, the maximum shift of the Ecorr value was approximately 16 mV (ΔEcorr = Ecorr(inhibitor) – Ecorr(blank), indicating the inhibitor was a mixed type and did not affect the reaction mechanism. Since the inhibitor physically blocks active sites on the metal surface, it is expected that corrosion will be inhibited because there will be less surface area available for corrosion (54). The most effective inhibition of mild steel corrosion occurs when 80 µg/ml of inhibitor is added to a 1.0 M solution of hydrochloric acid (HCl). This happens as a result of the inhibitor molecules coating the metal's surface, preventing the metal from reacting with the acid. The findings of this study were found to be consistent with the existing literature (33). Table 1 indicates that inhibitors considerably reduced the corrosion rate (Icorr), which influenced the anode and cathode reactions. This suggests that the silver nanoparticles in the solution prevented hydrogen from developing and metals from dissolving. Different concentrations of AgNPs were applied to assess their role as an anti-corrosion agent.

The results showed that the addition of AgNPs as inhibitors reduced the corrosion current density and shifted the cathodic potential towards more positive values, indicating a decrease in the rate of corrosion. The inhibition efficiency increased with increasing concentration of AgNPs, reaching a maximum of 88.9% at a concentration of 80 μg/ml. The anodic and cathodic Tafel slopes also decreased with increasing concentrations of AgNPs, indicating a decrease in the rate of anodic and cathodic reactions involved in the corrosion process. The decline in the Icorr value at higher concentrations is ascribed to the adsorption of inhibitor molecules on the surfaces of the metallic samples. The findings show that the corrosion of mild steel in a 1.0 M HCl solution may be effectively controlled by using a biogenic nanoparticle inhibitor. In this study, the greatest significant inhibitory impact on mild steel corrosion was reported when 80 µg /ml of the inhibitor was added to a 1.0 M hydrochloric acid solution. This highlights the importance of biogenic nanoparticle inhibitors in fighting corrosion in hostile chemical environments. A higher surface coverage (θ) of the inhibitor molecules on the metal surface results from an increase in inhibitor concentration. Because the inhibitor molecules work better to prevent corrosion when they are present in greater quantities, the increase in surface coverage (θ) leads to an improvement in the efficiency of inhibition (55). Additionally, ‘mmpy’ in Table 1 refers to the material loss rate or corrosion rate (MLR or CR) (56), which is stated in millimeters per year (mm/yr) using a metric that accurately reflects the material's loss of thickness from corrosion. This statistic quantifies the variation in corrosion observed at varying AgNPs inhibitor concentrations. It was found that the corrosion process slowed down as the inhibitor concentrations increased (33).

4. Conclusions

In this study, results revealed that pumpkin-derived AgNPs are less than 2 µm in size and naturally occur as agglomerates. Additionally, the AgNPs fingerprinting and SEM-EDX element analysis agreed with earlier studies, indicating that the sample had indeed been identified as AgNPs. AgNPs are also stable at a temperature of 220°C, speculating that the green material is quite robust at 150°C to 400°C. Based on cytotoxic results AgNPs did not inhibit Vero cells but were toxic to cancer cells. Pumpkin seed-derived AgNPs were found to be non-toxic to normal cells but have shown cytotoxicity effect on HCT 116 cells. In light of the fact that our research IC₅₀ was unclear and unreachable, further study is necessary. A morphological study using FE-SEM is suggested to obtain the detailed nanostructure image (nm) of AgNPs in further studies. More research needs to be carried out to examine the pumpkin seed-derived AgNP's mode of action to identify the effective doses that can inhibit the cancer cells in humans. Non-bonding interactions were predicted to result from molecular docking between receptors and NPs. In the future, clinical trials and in vivo techniques must be undergone to alter AgNPs’ drug delivery process. The corrosion resistance of mild steel in the presence and absence of pumpkin seed-derived silver nanoparticles (AgNPs) was tested using electrochemical polarization measurements. The results of the study showed that AgNPs boosted the corrosion resistance of mild steel.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was funded by the Researchers Supporting Project Number (RSP2024R339) at King Saud University, Riyadh 11451, Saudi Arabia.