Therapeutic Applications of Biostable Silver Nanoparticles Synthesized Using Peel Extract of Benincasa hispida: Antibacterial and Anticancer Activities

The purpose of this study was to fabricate biostable inorganic silver nanoparticles (AgNPs) using fresh peel (aqueous) extract of Benincasa hispida. A fast, robust, and eco-friendly approach was used for the synthesis of AgNPs, where bioactive components of peel extract of B. hispida acted as reducing and stabilizing agents. Synthesized AgNPs were characterized using a UV–Vis spectrophotometer, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and electron microscopy. The synthesized nanoparticles exhibited maximum absorption at 418 nm under the typical AgNPs surface plasmon resonance band range. They depicted a mean size of 26 ± 2 nm with a spherical shape. Their therapeutic prospective was determined by evaluating their antimicrobial and anticancer potential. The bio-synthesized silver nanoparticles exhibited strong antimicrobial activity with minimum inhibitory concentration (MIC 50) values of 14.5, 8.6, 6.063, and 13.4 μg/mL against Staphylococcus aureus (ATCC 25923), Micrococcus luteus (ATCC 14593), Escherichia coli (ATCC 25922), and Klebsiella pneumonia (ATCC 13883), respectively. The biosynthesized AgNPs showed potent in vitro cytotoxicity against human cervical cancer cell line with a half maximal inhibitory concentration (IC50) value of 0.066 μg/mL; however, no cytotoxic effect was observed on normal human primary osteoblasts cell line. This study explored B. hispida extract and confirmed its effectiveness as a promising source in producing AgNPs that could be employed for several therapeutic applications.


Introduction
In the group of inorganic metals, the use of silver (Ag) can be traced to the Neolithic era. Moyer first recorded the therapeutic use of Ag during the eighth century [1]. In recent times, the tunable photophysical attributes of silver nanoparticles (AgNPs) [2][3][4], their competent addressability by spectroscopic and optical techniques, and speedy advances in nanocrystallizations and fabrication [5] In this study, B. hispida aqueous peel extract was used to synthesize biostable spherical AgNPs that were further screened for their antibacterial potential against Staphylococcus aureus, Micrococcus luteus, Escherichia coli, and Klebsiella pneumonia. The biosynthesized AgNPs' cytotoxicity was evaluated on human cervical cancer cell line (HeLa) and normal human primary osteoblasts cell line. The AgNPs were characterized by UV-Vis spectroscopy, dynamic light scattering (DLS), FTIR, and electron microscopy. We report the potential of B. hispida-synthesized AgNPs for different therapeutic applications given its lesser side effects.

B. hispida (Aqueous) Peel Extracts Preparation
The peel of B. hispida was washed with tap water followed by distilled water until all the impurities were removed. Then, 20 g of peel was weighed and crushed separately in 50 mL of double distilled water with pestle mortar placed in the polypropylene molded tray filled with ice cubes to prevent denaturation of the proteins. Whatman filter paper (42) was used to filter this mixture before centrifuging it at 6000 rpm for 15 min (4 • C). Pellet was removed by collecting the supernatant in another centrifuge tube, and then the filtered peel extract was stored at cooling temperature for the synthesis of AgNPs.

Biosynthesis of AgNPs
Initially, 1 M AgNO 3 stock solution in double-distilled water was prepared before preparing working 1 mM AgNO 3 solution. Filtered aqueous peel extract of B. hispida was mixed with this AgNO 3 solution in equal ratio until 30 mL volume was reached in a 50 mL screw-capped tube and kept at 40 • C for 5 h. After incubation, the aqueous peel extract changed color from light green to reddish-brown and then was filtered with a 2 µm syringe filter. Absolute ethanol in twice the volume of the filtrate was used to precipitate and remove unbound proteins via centrifugation, followed by filtration. The filtered reaction mixture was kept at a cool temperature until characterization, antibacterial screening, and cytotoxicity assessment on cell lines. A systemic view of AgNPs synthesis is depicted in Figure 1.

UV-Visible Spectroscopic Profile of Synthesized AgNPs
One of the most basic and essential techniques for the characterization of nanoparticles is UV-Vis spectroscopy performed by a UV-Vis spectrophotometer (UV-2400PC Series, Shimadzu, Japan). This technique exploits the color-changing property due to the reduction of metal salts to biosynthesized nanoparticles, thereby resulting in surface plasmon resonance (SPR), which can be analyzed and recorded by the UV-Vis spectrometer in the wavelength range of 200-800 nm. The graphs were prepared with Origin-Pro 8.5 software (Version 85E, OriginLab Corporation, Northampton, MA, USA) to accentuate the raw results of the UV-Vis spectroscopy [34].

Transmission Electron Microscopy (TEM)
TEM (TecnaiTM G2 Spirit BioTWIN, FEI, Hillsboro, OR, USA) was applied to depict the shape and size of synthesized AgNPs, and a single drop of AgNPs suspension was dried out on TEM copper grids before performing TEM analysis at 80 kV accelerating voltage [35].

Particle Size and Zeta Potential
Dynamic light scattering (DLS) analysis was used to determine the hydrodynamic radius by approximating the particle size. Both particle size and zeta potential of synthesized AgNPs were determined utilizing Zetasizer Nano-ZS (ZEN3600 Malvern Instrument Ltd., Malvern, UK) [36].

FTIR
To observe the functional group present on the synthesized AgNPs surface, FTIR analysis was performed using FTIR spectroscopy (PerkinElmer Inc., Waltham, MA, USA) via a full reflectance sampling tool and scanning by applying a transmission technique with above a 4000-650 cm −1 range wave number of 4 cm −1 resolutions [37].

UV-Visible Spectroscopic Profile of Synthesized AgNPs
One of the most basic and essential techniques for the characterization of nanoparticles is UV-Vis spectroscopy performed by a UV-Vis spectrophotometer (UV-2400PC Series, Shimadzu, Japan). This technique exploits the color-changing property due to the reduction of metal salts to biosynthesized nanoparticles, thereby resulting in surface plasmon resonance (SPR), which can be analyzed and recorded by the UV-Vis spectrometer in the wavelength range of 200-800 nm. The graphs were prepared with Origin-Pro 8.5 software (Version 85E, OriginLab Corporation, Northampton, MA, USA) to accentuate the raw results of the UV-Vis spectroscopy [34].

Transmission Electron Microscopy (TEM)
TEM (TecnaiTM G2 Spirit BioTWIN, FEI, Hillsboro, OR, USA) was applied to depict the shape and size of synthesized AgNPs, and a single drop of AgNPs suspension was dried out on TEM copper grids before performing TEM analysis at 80 kV accelerating voltage [35].

Particle Size and Zeta Potential
Dynamic light scattering (DLS) analysis was used to determine the hydrodynamic radius by approximating the particle size. Both particle size and zeta potential of synthesized AgNPs were determined utilizing Zetasizer Nano-ZS (ZEN3600 Malvern Instrument Ltd., Malvern, UK) [36].

FTIR
To observe the functional group present on the synthesized AgNPs surface, FTIR analysis was performed using FTIR spectroscopy (PerkinElmer Inc., Waltham, MA, USA) via a full reflectance sampling tool and scanning by applying a transmission technique with above a 4000-650 cm −1 range wave number of 4 cm −1 resolutions [37].

Antibacterial Activity of AgNPs
Antibacterial assessment of synthesized AgNPs was performed primarily by the disk diffusion technique [38]. However, after the selection of active AgNPs based on preliminary results, minimum inhibitory concentration (MIC) was evaluated by applying the microbroth dilution approach on the microtiter plate [39]. Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 13883), and Micrococcus luteus (ATCC 14593) bacterial strains were procured from the National Chemical Laboratory, India. Mid-log phase bacteria for each strain were harvested through centrifugation and washed with Na-PO 4 buffer before dilution to the desired concentration in a Luria-Bertani (LB) broth. Serial dilutions of AgNPs were performed with LB broth to attain the anticipated concentrations of 0.1 to 20 µg⁄mL in microtiter plates, and 5 × 10 4 colony-forming unit (CFU) of bacterial inoculum was added to each. MIC was determined as the minimum AgNPs concentration where growth inhibition was observed after overnight incubation of the microtiter plates at 37 • C.

Cell Culture
The human cervical cancer cell lines (HeLa) and normal human primary osteoblasts cell lines were procured from the National Centre for Cell Science (NCCS), Pune, India. HeLa and primary osteoblast cells were grown as a monolayer in DMEM and Mac Coy's media, respectively, supplemented with 10% fetal bovine serum and 1% antibiotic. The cell lines were maintained and grown at 37 • C with a humidified atmosphere containing 5% of CO 2 .

Assessment of Cytomorphological Changes
Various concentrations of synthesized AgNPs were added to HeLa and human primary osteoblasts cells and incubated at 37 • C for 48 h (5% CO 2 ). An inverted phase-contrast microscope (Nikon ECLIPSE Ti-S, Tokyo, Japan) was used to perceive the gross changes in morphology after incubation.

Assessment of Cytotoxicity
Initially, both the cancerous and normal cell lines (1 × 10 4 cells/well) were added to a 96-well microtiter plate and incubated at 37 • C for 24 h. After incubation, various concentrations, i.e., 0.363, 0.176, 0.0922, 0.036, 0.0222, and 0.0162 µg/mL, of synthesized AgNPs were added in triplicates and incubated at 37 • C for 48 h. Then, the cytotoxicity was checked by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) cytotoxicity assay. The media of 96-well plates were discarded, followed by the addition of 50 µL of MTT dye (prepared at 5 mg/mL in phosphate buffer saline (PBS)) in each well and incubation at 37 • C for 4 h. Dimethyl sulfoxide (DMSO; 150 µL) was added to dissolve the formazan crystals. A microplate reader (BIORAD-680) was used to estimate the amount of reduced MTT by measuring the optical density (OD) at 570 nm with a reference filter of 655 nm [40]. Cell growth inhibition percentage was evaluated using the formula 100 − (A test − A blank )/(A control − A blank ) × 100, where test absorbance is A test , blank absorbance is A blank, , and control absorbance is A control .

Biosynthesis and Characterization of AgNPs
The AgNPs were synthesized by incubating 15 mL of B. hispida aqueous peel extract with 1 mM of AgNO 3 solution. We found that aqueous peel extract could synthesize AgNPs due to its reducing enzymes and capping agents like secondary metabolites that could synergistically reduce AgNO 3 (+1) oxidation state to Ag (0) oxidation state. The reaction mixture performed under identical control conditions in the absence of the aqueous peel extract did not show any changes in absorption, signifying the role of aqueous peel extract in the formation of AgNPs. Similarly, the incubation of the aqueous peel extract alone in double distilled water did not show any absorption peak(s) characteristic of AgNPs. A related approach for AgNPs biosynthesis was applied by using Mentha piperita leaf extract in 2018 [39,41].

UV-Visible Spectroscopic Profile of Synthesized AgNPs
UV-visible spectroscopic investigation was performed, and a characteristic maximum absorption centered at 418 nm was recorded as presented in Figure 2A, which was attributed to the surface plasmon resonance (SPR) band of the AgNPs.

Antibacterial Activity of AgNPs
We found that biogenic AgNPs synthesized by B. hispida aqueous peel extract shows a strong antibacterial activity against both Gram-positive and Gram-negative pathogenic bacterial strains. The MIC50 of AgNPs ( Figure 3) was evaluated against different pathogenic bacterial strains that included 14.5 μg/mL against S. aureus, 8.6 μg/mL against M. luteus, 6.063 μg/mL against E. coli, and 13.4 μg/mL against K. pneumoniae, indicating its broad-spectrum feature. However, we found that AgNPs were more effective against E. coli (Gram negative) and M. luteus (Gram positive) than other pathogenic strains [8]. Similar inhibition behavior was observed when AgNPs were synthesized by leaf extract of Carya illinoinensis [44]. It was proposed by several reports that the lower AgNPs

TEM
The high-resolution image ( Figure 2B) was acquired using the transmission electron microscope (TEM), which confirmed the average size of AgNPs as 26 ± 2 nm using a Gatan digital micrograph, and showed by the spherical form of AgNPs. The TEM micrographs did not expose the agglomeration of the as-synthesized AgNPs.

Particle Size and Zeta Potential
A dynamic light scattering microscope (DLS) and zeta potential of AgNPs were adopted to characterize the physicochemical characteristics of the prepared nanoparticles. The prepared AgNPs had an average size within the nanosize range ( Figure 2C). The zeta potential of the prepared AgNPs was −28 mV ( Figure 2D), suggesting higher stability of the particles. The synthesized AgNPs were found to be stable with no aggregation when stored at room temperature. The electrostatic repulsive forces among nanoparticles avoid their agglomeration in aqueous suspension and prevent them from approaching each other. In this study, the DLS-estimated size was larger than the TEM size. DLS estimates the hydrodynamic diameter of the inorganic core and the solvent layer attached to the particle, whereas TEM provides information about the size of the inorganic core alone without the hydration layer. Thus, the size obtained by DLS will always be larger than the actual size estimated by TEM [42,43].

FTIR
The FTIR analysis of synthesized AgNPs in Figure 2E depicts an existing peak focused at 1643.24 cm −1 , i.e., uniqueness of amide C=O groups. A medium and wide shoulder for the amide I linkage and amide II band was observed at 1537.02 cm −1 . N-H twist and carboxyl stretch in the protein amide bond were found to be responsible for the presence of amides bands I and II that are capped or surface-modified on AgNPs [41]. The N-H stretch vibration peak was observed at 3296.5 cm −1 ; however, this vibration is susceptible to hydrogen bond strength with no dependence on backbone confirmation. Moreover, the alcohol and ether group (C-O-C/C-OH) C-O stretch [41], along with (aliphatic amine) the C-N stretch vibration, showed a peak at 1081.8 cm −1 . The alkynes C≡C stretched vibration because numerous secondary metabolites showed a peak at 2127.39 cm −1 . Peaks at 3756.013 and 3868.76 cm −1 were observed for free (O-H) hydroxyl on the terminus.

Antibacterial Activity of AgNPs
We found that biogenic AgNPs synthesized by B. hispida aqueous peel extract shows a strong antibacterial activity against both Gram-positive and Gram-negative pathogenic bacterial strains. The MIC 50 of AgNPs ( Figure 3) was evaluated against different pathogenic bacterial strains that included 14.5 µg/mL against S. aureus, 8.6 µg/mL against M. luteus, 6.063 µg/mL against E. coli, and 13.4 µg/mL against K. pneumoniae, indicating its broad-spectrum feature. However, we found that AgNPs were more effective against E. coli (Gram negative) and M. luteus (Gram positive) than other pathogenic strains [8]. Similar inhibition behavior was observed when AgNPs were synthesized by leaf extract of Carya illinoinensis [44]. It was proposed by several reports that the lower AgNPs potential against Gram-positive bacteria was due to differences in cell walls [44][45][46][47]. A thick peptidoglycan layer in Gram-positive bacteria prohibited the entry of AgNPs into the cytoplasm, and a higher AgNPs concentration is required to inhibit the growth of Gram-positive than Gram-negative bacteria [44,48]. Silver (Ag) has been applied for countering spoilage and infections since archaic times. The mechanism of inorganic metallic nanoparticles against pathogenic microbes is significant for achieving synergistic effects with natural compounds. In broad-spectrum applications, the hypothetical mechanism of cytotoxicity exhibited by metallic nanoparticles occurs via releasing reactive oxygen species (ROS) [49,50]. However, its slow oxidation and Ag+ ions release reveal it as the strongest biocidal agent or molecule against pathogenic micro-organisms. It was widely reported that the loss of sub-cellular materials because of pit creations in the cell membrane is a feature of the bactericidal action of AgNPs. This action of AgNPs is augmented by the inhibition of respiratory chain dehydrogenases, subsequently affecting cell growth. Some phospholipids and proteins might work jointly to trigger membrane breakdown, causing death and cell decomposition [49]. The pathogenic G+ bacteria react differently after exposure to inorganic metal nanoparticles [23]. The antibacterial mechanism shows that AgNPs attack both primary and secondary (α-helix) cell wall structures by creating a link with the cell wall peptide and glycan, prompting the formation of pits. AgNPs form a link with N-acetylglucosamine and N-acetylmuramic acid (β-1/4 bonds), leading to obliteration of their interaction and liberating them to the surroundings [51].

Cytotoxic Effect of AgNPs on HeLa and Primary Osteoblasts
The toxicity of AgNPs was investigated on HeLa and primary osteoblasts cell line using the MTT cell proliferation assessment. The cell viability was screened at different AgNPs concentrations (0.363, 0.176, 0.0922, 0.036, 0.0222, and 0.0162 μg/mL; Figure 4). The outcome showed that HeLa cell lines (Figure 3) quickly lost their viability when incubated with AgNPs with the concentrations range from 0.0116 to 0.156 μg/mL. However, it was not significantly decreased when AgNPs were applied at the increased concentration range of 0.156 to 0.313 μg/mL. These AgNPs showed less toxicity against normal primary osteoblasts cell lines, demonstrating their higher acceptable biological limits. The IC50 value obtained in HeLa cells was 0.066 μg/mL. Similarly, Melia-azedarachand Sargassum-wightii-biosynthesized AgNPs showed dose-dependent cytotoxicity against HeLa cells [52]. However, a marked decrease in glutathione and increased lipid peroxidation, leading to elevated oxidative stress, were suggested as two reasons for AgNPs' antiproliferative action on HeLa cells [53]. In other studies, selective cytotoxicity against cancer cells was observed for AgNPs synthesized by walnut extract and lignin extracted from wheat [54,55]. The reason for cancer selectivity was attributed to the higher AgNPs uptake by cancerous cells compared to normal cells, presumably due to atypical metabolism and increased rate of proliferation [54,56]. In our study, Silver (Ag) has been applied for countering spoilage and infections since archaic times. The mechanism of inorganic metallic nanoparticles against pathogenic microbes is significant for achieving synergistic effects with natural compounds. In broad-spectrum applications, the hypothetical mechanism of cytotoxicity exhibited by metallic nanoparticles occurs via releasing reactive oxygen species (ROS) [49,50]. However, its slow oxidation and Ag+ ions release reveal it as the strongest biocidal agent or molecule against pathogenic micro-organisms. It was widely reported that the loss of sub-cellular materials because of pit creations in the cell membrane is a feature of the bactericidal action of AgNPs. This action of AgNPs is augmented by the inhibition of respiratory chain dehydrogenases, subsequently affecting cell growth. Some phospholipids and proteins might work jointly to trigger membrane breakdown, causing death and cell decomposition [49]. The pathogenic G+ bacteria react differently after exposure to inorganic metal nanoparticles [23]. The antibacterial mechanism shows that AgNPs attack both primary and secondary (α-helix) cell wall structures by creating a link with the cell wall peptide and glycan, prompting the formation of pits. AgNPs form a link with N-acetylglucosamine and N-acetylmuramic acid (β-1/4 bonds), leading to obliteration of their interaction and liberating them to the surroundings [51].

Cytotoxic Effect of AgNPs on HeLa and Primary Osteoblasts
The toxicity of AgNPs was investigated on HeLa and primary osteoblasts cell line using the MTT cell proliferation assessment. The cell viability was screened at different AgNPs concentrations (0.363, 0.176, 0.0922, 0.036, 0.0222, and 0.0162 µg/mL; Figure 4). The outcome showed that HeLa cell lines (Figure 3) quickly lost their viability when incubated with AgNPs with the concentrations range from 0.0116 to 0.156 µg/mL. However, it was not significantly decreased when AgNPs were applied at the increased concentration range of 0.156 to 0.313 µg/mL. These AgNPs showed less toxicity against normal primary osteoblasts cell lines, demonstrating their higher acceptable biological limits. The IC 50 value obtained in HeLa cells was 0.066 µg/mL. Similarly, Melia-azedarach-and Sargassum-wightii-biosynthesized AgNPs showed dose-dependent cytotoxicity against HeLa cells [52]. However, a marked decrease in glutathione and increased lipid peroxidation, leading to elevated oxidative stress, were suggested as two reasons for AgNPs' antiproliferative action on HeLa cells [53]. In other studies, selective cytotoxicity against cancer cells was observed for AgNPs synthesized by walnut extract and lignin extracted from wheat [54,55]. The reason for cancer selectivity was attributed to the higher AgNPs uptake by cancerous cells compared to normal cells, presumably due to atypical metabolism and increased rate of proliferation [54,56]. In our study, capping of natural components of B. hispida on synthesized AgNPs could be attributed to the synergistic effect on cancer cells and reduced toxicity on a normal cell. It was observed that herbal natural compounds show selective toxicity against abnormal cells [54,57]. Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 13 capping of natural components of B. hispida on synthesized AgNPs could be attributed to the synergistic effect on cancer cells and reduced toxicity on a normal cell. It was observed that herbal natural compounds show selective toxicity against abnormal cells [54,57].  The findings of our study suggested that B. hispida-biosynthesized AgNPs have broad-spectrum antibacterial potential along with anticancer potential against human cervical cancer cells. However, the outcomes of the study should be explored to develop into multipotent therapeutic agents against different diseases.

Conclusions
This paper described a green and environmentally-friendly method to produce AgNPs in large amounts. The formation of AgNPs with B. hispida aqueous peel extract was observed by the color Inhibition Concentration (μg/ml) HeLa and primary osteoblast cells with 70% confluence were kept with AgNPs in incubation for 48 h. The phase-contrast microscopy pictures ( Figure 5B) revealed alterations in the HeLa cell morphology. The maximum number of HeLa cells ( Figure 5C) showed notable shape variations (changing into circular), condensation of cytoplasm, membrane integrity failure, clumping of cells, and inhibition of cell growth. In contrast, insignificant morphological variations were observed in primary osteoblast cells compared to the control untreated normal cells. The synergistic effect of camptothecin and AgNPs on HeLa cells showed an augmented oxidative stress level and expression of the pro-apoptotic gene and increased disruption of membrane permeability as compared with camptothecin alone [58]. Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 13 capping of natural components of B. hispida on synthesized AgNPs could be attributed to the synergistic effect on cancer cells and reduced toxicity on a normal cell. It was observed that herbal natural compounds show selective toxicity against abnormal cells [54,57]. HeLa and primary osteoblast cells with 70% confluence were kept with AgNPs in incubation for 48 h. The phase-contrast microscopy pictures ( Figure 5B) revealed alterations in the HeLa cell morphology. The maximum number of HeLa cells ( Figure 5C) showed notable shape variations (changing into circular), condensation of cytoplasm, membrane integrity failure, clumping of cells, and inhibition of cell growth. In contrast, insignificant morphological variations were observed in primary osteoblast cells compared to the control untreated normal cells. The synergistic effect of camptothecin and AgNPs on HeLa cells showed an augmented oxidative stress level and expression of the pro-apoptotic gene and increased disruption of membrane permeability as compared with camptothecin alone [58]. The findings of our study suggested that B. hispida-biosynthesized AgNPs have broad-spectrum antibacterial potential along with anticancer potential against human cervical cancer cells. However, the outcomes of the study should be explored to develop into multipotent therapeutic agents against different diseases.

Conclusions
This paper described a green and environmentally-friendly method to produce AgNPs in large amounts. The formation of AgNPs with B. hispida aqueous peel extract was observed by the color Inhibition Concentration (μg/ml) The findings of our study suggested that B. hispida-biosynthesized AgNPs have broad-spectrum antibacterial potential along with anticancer potential against human cervical cancer cells. However, the outcomes of the study should be explored to develop into multipotent therapeutic agents against different diseases.

Conclusions
This paper described a green and environmentally-friendly method to produce AgNPs in large amounts. The formation of AgNPs with B. hispida aqueous peel extract was observed by the color change of B. hispida aqueous peel extract to brownish-yellow. The color changes indicated that AgNPs had produced the desired average nanometer size. B. hispida peel extract aqueous solution acted as a reducing and stabilizing agent. These biogenic AgNPs exhibited significant dose-dependent antibacterial and anticancer potentials. However, further investigations are warranted to assess the toxicity details and the mechanism associated with the antibacterial and anticancer action of the biosynthesized AgNPs. Nevertheless, the outcomes of the present study provide a broad AgNPs-based platform for various therapeutic applications in the near future.