Facile Synthesis and Characterization of Chitosan Functionalized Silver Nanoparticles for Antibacterial and Anti-Lung Cancer Applications

In the treatment of bacterial contamination, the problem of multi-drug resistance is becoming an increasingly pressing concern. Nanotechnology advancements enable the preparation of metal nanoparticles that can be assembled into complex systems to control bacterial and tumor cell growth. The current work investigates the green production of chitosan functionalized silver nanoparticles (CS/Ag NPs) using Sida acuta and their inhibition efficacy against bacterial pathogens and lung cancer cells (A549). Initially, a brown color formation confirmed the synthesis, and the chemical nature of the synthesized NPs were examined by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) coupled with energy dispersive spectroscopy (EDS), and transmission electron microscopy (TEM). FTIR demonstrated the occurrence of CS and S. acuta functional groups in the synthesized CS/Ag NPs. The electron microscopy study exhibited CS/Ag NPs with a spherical morphology and size ranges of 6–45 nm, while XRD analysis demonstrated the crystallinity of Ag NPs. Further, the bacterial inhibition property of CS/Ag NPs was examined against K. pneumoniae and S. aureus, which showed clear inhibition zones at different concentrations. In addition, the antibacterial properties were further confirmed by a fluorescent AO/EtBr staining technique. Furthermore, prepared CS/Ag NPs exhibited a potential anti-cancer character against a human lung cancer cell line (A549). In conclusion, our findings revealed that the produced CS/Ag NPs could be used as an excellent inhibitory material in industrial and clinical sectors.


Introduction
Infectious diseases are the leading cause of premature death on a global scale [1,2]. At the same time, cancer is the second leading cause of early death globally [3]. Many infectious illnesses have developed resistance to commercial antibiotics and alternative remedies [4]. The growth of drug resistance has become a major concern for humans and also for pharma industries [5]. Thus, the search for new and efficient inhibitory drugs against microbial infections has intensified. Over the past few decades, nanoparticles (NPs) have demonstrated an ability to eradicate several drug-resistant infections and diseases [6]. Among the many metal and metal oxide NPs, Ag NPs have attracted interest due to their distinctive characteristics [7]. From ancient times, Ag and Ag-based compounds have been recognized for their bactericidal characteristics [8]. Ag NPs have been utilized to prevent microbiological contamination in the textile, cosmetic, and food sectors using

Extraction of S. acuta Leaf Samples
The harvested leaves were initially cleaned, dried for four days, and ground into a fine powder. A Soxhlet extractor was utilized to obtain an aqueous extract by combining 10 g of fine powder with 100 mL of distilled water. Then, the extracted substance was kept at 4 °C until further usage.

Synthesis of CS/Ag NPs
The synthesis of CS/Ag NPs was carried out by a green chemistry method, as previously reported by Nandana et al. [30], with some modifications. About 10 mL of the S. acuta extract and 10 mL of CS solution (0.1 g of CS in 10 mL of 1% acetic acid) were mixed with 80 mL of a 1 mM AgNO3 solution, and then the solution was agitated for 2 h at room temperature under a dark state. After this, the resulting colloidal solution was configured at 15,000 rpm for 10 min before being separated by washing with distilled water to remove unbound S. acuta compounds and CS in the synthesized CS/Ag NPs and dried at 80 °C.

Extraction of S. acuta Leaf Samples
The harvested leaves were initially cleaned, dried for four days, and ground into a fine powder. A Soxhlet extractor was utilized to obtain an aqueous extract by combining 10 g of fine powder with 100 mL of distilled water. Then, the extracted substance was kept at 4 • C until further usage.

Synthesis of CS/Ag NPs
The synthesis of CS/Ag NPs was carried out by a green chemistry method, as previously reported by Nandana et al. [30], with some modifications. About 10 mL of the S. acuta extract and 10 mL of CS solution (0.1 g of CS in 10 mL of 1% acetic acid) were mixed with 80 mL of a 1 mM AgNO 3 solution, and then the solution was agitated for 2 h at room temperature under a dark state. After this, the resulting colloidal solution was configured at 15,000 rpm for 10 min before being separated by washing with distilled water to remove unbound S. acuta compounds and CS in the synthesized CS/Ag NPs and dried at 80 • C.

Optical Studies
UV-vis spectroscopy (JASCO-V-670) was subjected to observe the reaction mixture as it underwent the formation of CS/Ag NPs. The visible spectra of NPs were observed from 300 to 800 nm.

FTIR
The IR analysis was used to identify the molecules of chitosan and S. acuta that were accountable for the formation of NPs. Readings from the FTIR (FTIR-00585, PerkinElmer) spectrum were taken with a resolution of 4 cm −1 across the range of 400-4000 cm −1 .

SEM-EDS
The SEM was used to characterize the NPs' morphology (JEOL-Model JSM 6390 coupled with EDS). In order to prepare a SEM sample on a grid, a pinch of NPs was positioned on the grid then the surfaces of the sample were sputter-coated with carbon tape and then imaged. This experiment was carried out with a voltage of 10 kV applied to the accelerator.

TEM
The structure of synthesized NPs was observed using TEM. A very small pinch of NPs was placed on a grid, and NPs images were observed at 120 kV (JEOL-Japan).

Disc Diffusion Antibacterial Activity Assay
The bactericidal activity of CS/Ag NPs was assessed against K. pneumoniae and S. aureus using a disc diffusion assay [36]. Briefly, the chosen bacterial strains (10 5 CFU/mL) were spreaded on the MHA plates. Then, the empty discs were filled with various concentrations of CS/Ag NPs (10, 20 and 30 µg/mL) and allowed to dry for a few minutes. Then, coated discs were positioned on the dish and kept at 37 • C for 24 h to observe the activity.

AO/EtBr Dual Staining and Growth Curve Assay
The changes in the K. pneumonia and S. aureus cell membrane upon action with NPs were examined using an AO/EtBr staining technique, as described by Kumar et al. [37]. Briefly, 1 mL of overnight-grown pathogens were incubated with 20 µg/mL of NPs for 24 h at 37 • C. The cells without NPs were kept as a control. Then, the cells were cleaned with a buffer solution and further stained with 1 µL of AO/EtBr, and images of fluorochemical staining were attained using a Nikon Eclipse Ni-E Microscope with a Nikon DS-Ri2 digital camera. The digitized images were processed using Nikon's proprietary software NIS-Elements BR.

Growth Curve Analysis
The growth curve analysis of synthesized CS/Ag NPs was tested against the chosen pathogens (K. pneumoniae and S. aureus) [38]. Briefly, overnight cultures of K. pneumoniae and S. aureus were incubated with 20 µg/mL of CS/Ag NPs, and the O.D. was monitored at every 4 h interval up to 24 h.

Anticancer MTT Assay against A549 Cells
Human lung cancer cells were obtained from the National Centre for Cell Science (NCCS), Pune, India. Initially, the monolayer cell culture was trypsinized, and the cell amount was attuned to 1 × 10 4 cells/mL with 10% FBS medium. About 0.2 mL of the diluted cell suspension was added to each well of the 96-micro titer plate. When a partial monolayer formed after 24 h, the supernatant was flicked off, and the monolayer was washed once with a buffer. Different concentrations of CS/Ag NPs (20,40,60,80, and 100 µg/mL) being diluted in media were added and incubated for 24 h to determine the effect of CS/Ag NPs on cell viability. After that 10 µL of MTT (5 mg/mL) was added and further incubated for 4 h, then removed, and a further 200 µL of DMSO was added and thoroughly mixed to dissolve the dark blue crystals. Then, the absorbance was measured at 570 nm, and the percentage of viability was calculated using Formula (1). (1)

Apoptosis Induction Studies
Apoptosis causing ability of synthesized CS/Ag NPs was studied using a fluorescence microscopic dual staining method as previously described by Bharathi et al. [39]. About 300 µL of lung cancer cells were treated with the IC 50 concentration of CS/Ag NPs in a 16-well plate and incubated for 24 h. After that, the treated and untreated cells were washed with PBS and stained with 10 µL of an AO/EtBr mixture before the morphology changes were imaged using a fluorescence microscope (Nikon Eclipse Ni-E Microscope with a Nikon DS-Ri2 digital camera).

Statistical Analysis
The experimental investigation, including testing for antibacterial and anticancer activities, was performed in triplicates, and the findings were reported as the mean ± standard deviation. The statistical significance of the differences were determined using a p-value of 0.05.

Formation of CS/Ag NPs
The green production of CS/Ag NPs was carried out using S. acuta leaves extract ( Figure 1b). Previously, Uysal et al. [27] reported that S. acuta contained various phytochemical properties such as hydroxybenzoic, hydroxycinnamic and acylquinic acids, hydroxycinnamoyl tartarates, flavonoids, cinnamic acid amides, alkaloids, and amino acids. The presence of these phytocompounds in S. acuta and CS might have acted as reducing and capping agents for the synthesis of CS/Ag NPs [39]. The development of NPs was established by the color change from yellow to brown ( Figure 1c). The development of this brown color was well known to be emerged from SPR vibrations of nano-CS/Ag [40]. Similarly, various phytocompounds of Myristica fragrans [41], Muntingia calabura [42], and Rumex nervosus [43] mediated and synthesized Ag NPs and exhibited brown color formation. The mechanism of this chain network formation by CS and phyto-compounds with Ag is shown in Figure 1d. The active phyto-compounds presented in S. acuta and CS acted as ligation agents. The existence of long pair of e − , free -NH 2 , and O-H groups in CS and phytocompounds of S. acuta might have ligated with Ag 2+ and developed an Ag-CS-ellagate complex. Then, this network system naturally led to a nucleation process that went into reverse micellization, which further rooted the reduction of Ag ions (Ag + ) to nano Ag [30,44,45].

Optical Studies
The UV-vis spectroscopy investigation of CS/Ag NPs exhibited an absorbance peak at 464 nm and thus supported the synthesis of CS/Ag NPs (Figure 2a). According to reports, the UV absorbance peak around 400-480 nm was a distinct property of nano-Ag [46]. Similarly, CS-Aegle marmelos entrapped Ag NPs exhibited UV-vis absorbance at 420 nm [47]. The UV-visible spectrum of CS-decorated Ag NPs was prepared using Piper betle exhibited an absorption peak at 430 nm [35]. Recently, Ag NPs were synthesized using CS, and seaweed showed the UV-vis absorbance peak to be around 425 nm [48].

FTIR-Functional Groups Investigation
The attraction of active groups and the development of end product CS/Ag NPs were studied using FTIR. The IR analysis of CS/Ag NPs is depicted in Figure 2b. The FTIR of NPs showed a typical primary N-H band at 3328 cm −1 , C=C vibration at 2118 cm −1 , N-H band at 1645 cm −1 , C-H bending at 1393 cm −1 , C-N band at 1280 cm −1 , C-O stretching at 1011 cm −1 and a C-I band at 608 cm −1 . The obtained N-H and C-O functional groups could be derived from -NH2 groups and acetylated parts of CS [49]. Other groups, namely C=C, C-H, C-N, and C-I, may be derived from the phyto-extract of S. acuta. Moreover, certain band vibrations around 600-400 cm −1 could be accredited to the presence of metal (Ag) in the synthesized CS/Ag NPs. The presence of these bio-active derivatives might have acted as a reductant for the formation of final CS/Ag NPs [36]. Figure 3a shows the FE-SEM of CS/Ag NPs. FE-SEM images showed that the NPs had a spherical morphology. Ag NPs were synthesized using various plant extracts, which showed a spherical-shaped morphology [50,51]. Further, EDS was performed to analyze the elemental composition of CS/Ag NPs (Figure 3b). The elemental spectra of CS/Ag NPs exhibited a peak of silver (Ag), which supported the formation of Ag NPs. The other peaks of C, N, and O were detected due to the emission of X-rays from the -NH2 and de-acetylated groups of CS. The elemental percentage in the synthesized CS/Ag NPs is given in Table 1

FTIR-Functional Groups Investigation
The attraction of active groups and the development of end product CS/Ag NPs were studied using FTIR. The IR analysis of CS/Ag NPs is depicted in Figure 2b. The FTIR of NPs showed a typical primary N-H band at 3328 cm −1 , C=C vibration at 2118 cm −1 , N-H band at 1645 cm −1 , C-H bending at 1393 cm −1 , C-N band at 1280 cm −1 , C-O stretching at 1011 cm −1 and a C-I band at 608 cm −1 . The obtained N-H and C-O functional groups could be derived from -NH 2 groups and acetylated parts of CS [49]. Other groups, namely C=C, C-H, C-N, and C-I, may be derived from the phyto-extract of S. acuta. Moreover, certain band vibrations around 600-400 cm −1 could be accredited to the presence of metal (Ag) in the synthesized CS/Ag NPs. The presence of these bio-active derivatives might have acted as a reductant for the formation of final CS/Ag NPs [36]. Figure 3a shows the FE-SEM of CS/Ag NPs. FE-SEM images showed that the NPs had a spherical morphology. Ag NPs were synthesized using various plant extracts, which showed a spherical-shaped morphology [50,51]. Further, EDS was performed to analyze the elemental composition of CS/Ag NPs (Figure 3b). The elemental spectra of CS/Ag NPs exhibited a peak of silver (Ag), which supported the formation of Ag NPs. The other peaks of C, N, and O were detected due to the emission of X-rays from the -NH 2 and de-acetylated groups of CS. The elemental percentage in the synthesized CS/Ag NPs is given in Table 1. Similar to our study, Nandhana et al. reported on the presence of Ag, N, C, and O in the green synthesized CS/Ag nanocomposite using rutin. Further, elemental mapping analysis confirmed the random distribution of observed elements in the synthesized NPs (Figure 3e,f).   Further, the morphology and size of produced NPs were analyzed using TEM. The TEM images of synthesized CS/Ag NPs are shown in Figure 4a-c. TEM showed the particle sizes varied from 6 to 45 nm, and it also showed the synthesized NPs to have a spherical morphology with Ag crystallinity. Similar to our study, gallic acid-chitosan-modified Ag NPs exhibited a spherical-shaped morphology [52].

Disc Diffusion Bactericidal Assay
The antibacterial properties of CS/Ag NPs were investigated against K. pneumoniae and S. aureus. The synthesized NPs inhibited potential and concentration-dependent inhibitory activities against the tested bacterial pathogens. Figure 5a

TEM Analysis
Further, the morphology and size of produced NPs were analyzed using TEM. The TEM images of synthesized CS/Ag NPs are shown in Figure 4a-c. TEM showed the particle sizes varied from 6 to 45 nm, and it also showed the synthesized NPs to have a spherical morphology with Ag crystallinity. Similar to our study, gallic acid-chitosan-modified Ag NPs exhibited a spherical-shaped morphology [52].

TEM Analysis
Further, the morphology and size of produced NPs were analyzed using TEM. T TEM images of synthesized CS/Ag NPs are shown in Figure 4a-c. TEM showed the pa cle sizes varied from 6 to 45 nm, and it also showed the synthesized NPs to have a sph ical morphology with Ag crystallinity. Similar to our study, gallic acid-chitosan-modif Ag NPs exhibited a spherical-shaped morphology [52].

Disc Diffusion Bactericidal Assay
The antibacterial properties of CS/Ag NPs were investigated against K. pneumon and S. aureus. The synthesized NPs inhibited potential and concentration-dependent hibitory activities against the tested bacterial pathogens. Figure 5a

Disc Diffusion Bactericidal Assay
The antibacterial properties of CS/Ag NPs were investigated against K. pneumoniae and S. aureus. The synthesized NPs inhibited potential and concentration-dependent inhibitory activities against the tested bacterial pathogens. Figure 5a,b depicts a well diffusion plate experiment with various concentrations (10, 20, and 30 µg/mL) of CS/Ag NPs. Against all the pathogens examined, clear zones of bacterial suppression appeared around the CS/Ag NPs embedded discs. At an increasing concentration of NPs, the bactericidal ZOI increased. The ZOI for K. pneumoniae was found to be 9 ± 0.5 mm for 10 µg/mL, 11 ± 0.8 mm for 20 µg/mL and 13 ± 0.2 for 30 µg/mL of CS/Ag NPs, whereas S. aureus exhibited 10 ± 0.2 mm for 10 µg/mL, 11 ± 0.6 mm for 20 µg/mL, and 13 ± 1 mm for 30 µg/mL. Synthesized NPs exhibited equal activity for both tested pathogens. The results of NPs' action could change depending on the bacteria's cell wall and membrane structure [36]. Moreover, nano-scale CS possessed a significant bactericidal and broadrange antibacterial action against bacterial infections [53]. Similar to our study, CS-Ag NPs coated linen fabrics showed their potential antibacterial activity against E. coli and S. aureus [54].
the CS/Ag NPs embedded discs. At an increasing concentration of NPs, the bactericidal ZOI increased. The ZOI for K. pneumoniae was found to be 9 ± 0.5 mm for 10µg/mL, 11 ± 0.8 mm for 20µg/mL and 13 ± 0.2 for 30 µg/mL of CS/Ag NPs, whereas S. aureus exhibited 10 ± 0.2 mm for 10 µg/mL, 11 ± 0.6 mm for 20 µg/mL, and 13 ± 1 mm for 30 µg/mL. Synthesized NPs exhibited equal activity for both tested pathogens. The results of NPs' action could change depending on the bacteria's cell wall and membrane structure [36]. Moreover, nano-scale CS possessed a significant bactericidal and broad-range antibacterial action against bacterial infections [53]. Similar to our study, CS-Ag NPs coated linen fabrics showed their potential antibacterial activity against E. coli and S. aureus [54].

Dual Fluorescent Staining
Furthermore, the antibacterial efficacy of produced CS/Ag NPs against K. pneumoniae and S. aureus was validated by utilizing a fluorescent-based live/dead cell test. The fluorescent microscopic pictures of the control and NPs treated cells are shown in Figure 5cf. The untreated cells fluoresced green due to the existence of live cells, whereas treated cells fluoresced red, confirming their death. Fluorescent AO is a green dye that stains both live and dead bacteria, whereas EtBr is a red dye that exclusively stains dead bacteria. The EtBr penetrates dead cells via the cell membrane and reduces the green fluorescent color of AO [55]. Accordingly, the green color denotes living bacterial cells, whereas the red hue signifies dead bacterial cells. The obtained results revealed that produced NPs had a bacteriostatic impact against the tested bacterial pathogens. Further, the growth curve analysis revealed that CS/Ag NPs were able to prevent the growth of K. pneumoniae and S. aureus and also showed delayed growth ( Figure 6).

Dual Fluorescent Staining
Furthermore, the antibacterial efficacy of produced CS/Ag NPs against K. pneumoniae and S. aureus was validated by utilizing a fluorescent-based live/dead cell test. The fluorescent microscopic pictures of the control and NPs treated cells are shown in Figure 5c-f. The untreated cells fluoresced green due to the existence of live cells, whereas treated cells fluoresced red, confirming their death. Fluorescent AO is a green dye that stains both live and dead bacteria, whereas EtBr is a red dye that exclusively stains dead bacteria. The EtBr penetrates dead cells via the cell membrane and reduces the green fluorescent color of AO [55]. Accordingly, the green color denotes living bacterial cells, whereas the red hue signifies dead bacterial cells. The obtained results revealed that produced NPs had a bacteriostatic impact against the tested bacterial pathogens. Further, the growth curve analysis revealed that CS/Ag NPs were able to prevent the growth of K. pneumoniae and S. aureus and also showed delayed growth ( Figure 6).
A potential mechanism for the bacterial suppression of CS/Ag NPs is shown in Figure 5g. One of the two fundamental processes that may be responsible for the inhibition action of Ag-based NPs is the breakdown of cell walls and membrane destruction (1). The attraction and interaction between NPs and bacteria begin with Ag adhesion to the plasma membrane, which causes membrane structural alterations, resulting in membrane depolarization, disruption of permeability, and disruption of cell wall integrity. As a consequence of depolarization, the internal material of bacterial cells escapes into the surrounding environment, leading to the death of the cells. The second approach involves the creation of reactive oxygen species (ROS), which can include superoxide, hydroxyl Polymers 2023, 15, 2700 9 of 13 radicals, and hydrogen peroxides, among other things. These limit the development of bacteria by binding to the genetic elements and proteins that are found on their cells [56][57][58]. A potential mechanism for the bacterial suppression of CS/Ag NPs is shown in Figure  5g. One of the two fundamental processes that may be responsible for the inhibition action of Ag-based NPs is the breakdown of cell walls and membrane destruction (1). The attraction and interaction between NPs and bacteria begin with Ag adhesion to the plasma membrane, which causes membrane structural alterations, resulting in membrane depolarization, disruption of permeability, and disruption of cell wall integrity. As a consequence of depolarization, the internal material of bacterial cells escapes into the surrounding environment, leading to the death of the cells. The second approach involves the creation of reactive oxygen species (ROS), which can include superoxide, hydroxyl radicals, and hydrogen peroxides, among other things. These limit the development of bacteria by binding to the genetic elements and proteins that are found on their cells [56][57][58].

MTT Assay
The anticancer property of CS/Ag NPs was assessed against human lung cancer cells using a MTT assay. The obtained findings exhibited a significant decrease in the growth of A549 cells (97.2%) when the concentration of CS/Ag NPs increased. CS/Ag NPs induced a 50% growth defeat property at a concentration of 34.5 ± 0.5 µg/mL. Similarly, Murugesan et al. [47] reported that the synthesized Ag NPs using Gloriosa superba exhibited potential anticancer activity against A549 cells. Similar findings were reported by Priya et al. [59], who observed the dose-dependent anticancer activity of biogenic CS-Ag NPs against hepatocellular carcinoma cells. The biogenic synthesized Ag NPs entrapped with CS showed anticancer activity against HeLa cells [47]. It was reported that the anticancer properties of Ag NPs were dependent on the morphology, size, and reducing agents of the NPs.

AO/EtBr Fluorescent Assay
Nearly all unicellular creatures undergo apoptosis, which is a type of planned cell death. A helpful strategy for the therapy of cancer is the induction of apoptosis [60]. Using a fluorescence microscope, the apoptosis cells were separated from one another by their orange or red-colored bodies. Fluorescence microscope images revealed that the prepared CS/Ag NPs could induce apoptosis in treated A549 cells. The control cells displayed a green color, and the treated cells showed a red color, thus supporting the induction property of CS/Ag NPs (Figure 7a,b). The appearance of a red hue indicated the presence of apoptotic bodies and displayed cell shrinkage as well as membrane blebbing. In most cases, the morphological and biochemical alterations in such cell shrinkage, membrane blebbing, membrane unity, nuclear fragmentation, and cytoplasmic condensation contributed to cell death by initiating the apoptotic pathway. This process was responsible for

MTT Assay
The anticancer property of CS/Ag NPs was assessed against human lung cancer cells using a MTT assay. The obtained findings exhibited a significant decrease in the growth of A549 cells (97.2%) when the concentration of CS/Ag NPs increased. CS/Ag NPs induced a 50% growth defeat property at a concentration of 34.5 ± 0.5 µg/mL. Similarly, Murugesan et al. [47] reported that the synthesized Ag NPs using Gloriosa superba exhibited potential anticancer activity against A549 cells. Similar findings were reported by Priya et al. [59], who observed the dose-dependent anticancer activity of biogenic CS-Ag NPs against hepatocellular carcinoma cells. The biogenic synthesized Ag NPs entrapped with CS showed anticancer activity against HeLa cells [47]. It was reported that the anticancer properties of Ag NPs were dependent on the morphology, size, and reducing agents of the NPs.

AO/EtBr Fluorescent Assay
Nearly all unicellular creatures undergo apoptosis, which is a type of planned cell death. A helpful strategy for the therapy of cancer is the induction of apoptosis [60]. Using a fluorescence microscope, the apoptosis cells were separated from one another by their orange or red-colored bodies. Fluorescence microscope images revealed that the prepared CS/Ag NPs could induce apoptosis in treated A549 cells. The control cells displayed a green color, and the treated cells showed a red color, thus supporting the induction property of CS/Ag NPs (Figure 7a,b). The appearance of a red hue indicated the presence of apoptotic bodies and displayed cell shrinkage as well as membrane blebbing. In most cases, the morphological and biochemical alterations in such cell shrinkage, membrane blebbing, membrane unity, nuclear fragmentation, and cytoplasmic condensation contributed to cell death by initiating the apoptotic pathway. This process was responsible for the death of cells. [61,62]. The apoptosis induction property of NPs depended on the permeability of Ag ions into the cancer cells, which stimulated cell damage, and DNA fragmentation and, thus spontaneously led to apoptosis [63].
the death of cells. [61,62]. The apoptosis induction property of NPs depended on the permeability of Ag ions into the cancer cells, which stimulated cell damage, and DNA fragmentation and, thus spontaneously led to apoptosis [63].

Conclusions
Silver is one of the most often utilized metals that is accessible and has a diverse variety of applications. In biomedical applications, the nanoform of Ag, also known as Ag NPs, has provided a novel structure for particles. The synthesis of Ag NPs using chemical methods is both expensive and harmful; thus, efforts have been made to utilize biological sources such as plants, bacteria, and algae in order to cut down on both costs and toxicity. In the present investigation, S. acuta performed outstandingly both as a reducing and capping agent. The production of CS/Ag NPs using the S. acuta aqueous leaf extract has been presented as an eco-friendly and simple green technique. To our knowledge, this is the first report on the synthesis of CS/Ag NPs using S. acuta without any additional chemicals and reagents. The size of the prepared CS/Ag NPs ranged from 6 to 45 nm and had a spherical form. The occurrence of CS in prepared CS/Ag NPs was validated by EDS and FTIR analyses. Significant antibacterial activity was exhibited by the synthesized CS-Ag NPs against K. pneumonia and S. aureus pathogens. In addition, the MTT and fluorescentbased assay confirmed its anti-cancer capabilities with the apoptosis induction property of synthesized CS/Ag NPs against the human lung cancer cell line (A549). The potential antibacterial and anticancer properties of CS/Ag NPs could be due to the synergetic properties of both CS and Ag NPs. Thus, we believe that the CS/Ag NPs synthesized using S. acuta have the potential to be employed as NPs in clinical sectors to reduce the growth of bacterial and also cancer cells.

Conclusions
Silver is one of the most often utilized metals that is accessible and has a diverse variety of applications. In biomedical applications, the nanoform of Ag, also known as Ag NPs, has provided a novel structure for particles. The synthesis of Ag NPs using chemical methods is both expensive and harmful; thus, efforts have been made to utilize biological sources such as plants, bacteria, and algae in order to cut down on both costs and toxicity. In the present investigation, S. acuta performed outstandingly both as a reducing and capping agent. The production of CS/Ag NPs using the S. acuta aqueous leaf extract has been presented as an eco-friendly and simple green technique. To our knowledge, this is the first report on the synthesis of CS/Ag NPs using S. acuta without any additional chemicals and reagents. The size of the prepared CS/Ag NPs ranged from 6 to 45 nm and had a spherical form. The occurrence of CS in prepared CS/Ag NPs was validated by EDS and FTIR analyses. Significant antibacterial activity was exhibited by the synthesized CS-Ag NPs against K. pneumonia and S. aureus pathogens. In addition, the MTT and fluorescent-based assay confirmed its anti-cancer capabilities with the apoptosis induction property of synthesized CS/Ag NPs against the human lung cancer cell line (A549). The potential antibacterial and anticancer properties of CS/Ag NPs could be due to the synergetic properties of both CS and Ag NPs. Thus, we believe that the CS/Ag NPs synthesized using S. acuta have the potential to be employed as NPs in clinical sectors to reduce the growth of bacterial and also cancer cells.