Biogenic Preparation, Characterization, and Biomedical Applications of Chitosan Functionalized Iron Oxide Nanocomposite

: Chitosan (CS) functionalization over nanomaterials has gained more attention in the biomedical ﬁeld due to their biocompatibility, biodegradability, and enhanced properties. In the present study, CS functionalized iron (II) oxide nanocomposite (CS/FeO NC) was prepared using Sida acuta leaf extract by a facile and eco-friendly green chemistry route. Phyto-compounds of S. acuta leaf were used as a reductant to prepare CS/FeO NC. The existence of CS and FeO crystalline peaks in CS/FeO NC was conﬁrmed by XRD. FE-SEM analysis revealed that the prepared CS/FeO NC were spherical with a 10–100 nm average size. FTIR analyzed the existence of CS and metal-oxygen bands in the prepared NC. The CS/FeO NC showed the potential bactericidal activity against E. coli , B. subtilis , and S. aureus pathogens. Further, CS/FeO NC also exhibited the dose-dependent anti-proliferative property against human lung cancer cells (A549). Thus, the obtained outcomes revealed that the prepared CS/FeO NC could be a promising candidate in the biomedical sector to inhibit the growth of bacterial pathogens and lung cancer cells.


Introduction
Nanomaterials have received significance in developing new and different industrial applications due to their unique shape, size, and high surface area to volume ratio [1][2][3]. In biomedical industries, various approaches have been used to develop a new path to control and prevent microbial infections using novel nanoparticles (NPs). Many literature studies have demonstrated the significant bactericidal properties of NPs against bacterial pathogens [4][5][6]. However, antibacterial resistance is the key problem in the biomedical field, increasing the severity of bacterial infections that can indicate the requirement of novel antibacterial agents against those drug-resistant and severe infection-causing pathogens. Previous reports revealed that the metallic NPs and NCs could be better alternative antibacterial agents like natural and synthetic antibiotics [7][8][9][10][11]. Different NPs and NCs have been utilized in the textile coating, cosmetics, food, and paint industries, which interestingly showed a high prospective to solve the antibiotic resistance problem [12][13][14][15][16][17].
Currently, NPs and NCs synthesized using plants by green chemistry route have been received the focus of interest for their various applications in biological, pharmaceutical, and food industries, because of the unique physicochemical properties, less toxic, eco-friendly, economic, and easy scale-up [18][19][20][21]. Iron (II) oxide nanoparticles (FeO NPs) are typically interesting in modern green nanotechnology because of their superparamagnetic properties, high magnetic susceptibility, superior catalytic and bactericidal properties [22,23]. Additionally, researchers have been focusing on the functionalization of CS over FeO NPs to enhance the physio-chemical and biological properties [24].
Among the various natural biopolymers, several researchers have focused on chitosan as a potential functional biomaterial for its biocompatibility, biodegradability, non-toxicity, and bacteriostatic properties [25]. Previous reports demonstrated the chitosan's utilization for surface functionalization of metal and metal oxide NPs [26,27]. Apart from the antibiotic properties, several reports demonstrated the anti-cancer and antioxidant properties of CS and chitosan-functionalized NPs [28,29]. Many studies have revealed that the CS functionalized biomaterials can be prepared as a nanostructured anti-cancer drug carrier [30]. From this significance, the present study aimed to prepare CS/FeO NC using the Sida acuta plant. S. acuta belongs to the family of Malvaceae and has many pharmacological properties [31]. Further, the prepared CS/FeO NC was characterized and assessed its bactericidal and anti-proliferative properties.

S. acuta Leaf Extraction
Freshly collected leaf parts of S. acuta were cleaned with tap water and dried. About 1 g of dried leaf sample was mixed with 100 mL of distilled water and heated at 100 • C for 15 min, and then the extracted solution was filtered and used for the biogenic preparation of CS/FeO NC ( Figure 1).

Biogenic Preparation of CS/FeO NC
CS (0.5 g) was dissolved into 1% (v/v) acetic acid (50 mL) followed by the addition of 0.5 M iron (II) sulfate (25 mL) and S. acuta leaf extract (25 mL), and vigorously stirred under 60 • C for 30 min. Further, the resultant was centrifuged at 12,000 rpm for 10 min and air-dried at 80 • C to obtain the final CS/FeO NC.

Biogenic Preparation of CS/FeO NC
CS (0.5 g) was dissolved into 1% (v/v) acetic acid (50 mL) followed by the addition of 0.5 M iron (II) sulfate (25 mL) and S. acuta leaf extract (25 mL), and vigorously stirred under 60 °C for 30 min. Further, the resultant was centrifuged at 12,000 rpm for 10 min and air-dried at 80 °C to obtain the final CS/FeO NC.

Antibacterial Assay
Spectrophotometer turbidimetry technique was used to analyze the antibacterial activity of CS/FeO NC. S. aureus, B. subtilis, and E. coli were used as test bacterial pathogens. Bactericidal properties of CS/FeO NC were evaluated in nutrient broth using the optical density (570 nm) measurement method. The bacterial concentration was diluted to 10 5 CFU/mL. CS/FeO NC was added into the bacterial suspension at a 50 μg/mL concentration and incubated overnight. The bacteriostatic rate (%) was calculated as follows Equation (1)

Antibacterial Assay
Spectrophotometer turbidimetry technique was used to analyze the antibacterial activity of CS/FeO NC. S. aureus, B. subtilis, and E. coli were used as test bacterial pathogens. Bactericidal properties of CS/FeO NC were evaluated in nutrient broth using the optical density (570 nm) measurement method. The bacterial concentration was diluted to 10 5 CFU/mL. CS/FeO NC was added into the bacterial suspension at a 50 µg/mL concentration and incubated overnight. The bacteriostatic rate (%) was calculated as follows Equation (1), (1)

Antiproliferative Activity of CS/FeO NC
Antiproliferative property of prepared CS/FeO NC was evaluated by 3-[4, 5-96 dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, as demonstrated previously by Seetharaman et al. (2017) [32]. Lung cancer cells (A549) were procured from NCCS, Pune, India, and grown in a medium containing fetal bovine serum (10%) and antibiotics (1%) in a CO 2 incubator under standard conditions. The grown cells (100 µL) at a density of 1 × 10 3 were loaded on a culture plate and incubated at 37 • C with 5% CO 2 overnight. Then, various concentration of CS/FeO NC (10, 25, 50, 100, 150 and 200 µg/mL) were added into respective wells and re-incubated. After that, the media was replaced with 10 µL of MTT (5 mg/mL) and again incubated. Further, the MTT solution was removed, and 100 µL of DMSO was added and incubated. The absorbance value was measured using a BioRad-680 plate reader at 570 nm, and the viability percentage was calculated Equation (2).
Viability (%) = Treated cells/Control cells × 100 (2) where C is the control cell's value, and T is the value of the treated cell. Furthermore, inducing apoptosis by CS/FeO NC against A549 cells was studied by AO/EtBr fluorescent staining technique, as demonstrated previously by Bharathi et al. [33].

Statistical Analysis
All in vitro tests were performed three independent times in triplicates (n = 3), and the obtained data were represented as mean ± standard deviation. The significance level was analyzed at p < 0.05.

Fabrication of CS/FeO NC
Biogenic conversion of iron (II) sulfate to CS/FeO NC was initiated by mixing of FeSO 4 and CS with S. acuta aqueous leaf extract, which also stimulated the color change from yellowish to black, and thus supported the development of CS/FeO NC (Figure 2(Aa)). Similarly, Boudouh et al. (2021) [34] documented the exhibition of black color during the fabrication of FeO NPs. Secondary metabolites present in S. acuta leaf extract and free amino, and hydroxyl groups existing in CS might have joined with ferrous ions (Fe II ) and formed Fe-ellagate conjugate. Simultaneously, the formed conjugate leads to the nucleation that goes into reverse micellization, which later causes the reduction of Fe + to nano-FeO [35].
Viability (%) = Treated cells/Control cells × 100 (2) where C is the control cell's value, and T is the value of the treated cell. Furthermore, inducing apoptosis by CS/FeO NC against A549 cells was studied by AO/EtBr fluorescent staining technique, as demonstrated previously by Bharathi et al. [33].

Statistical Analysis
All in vitro tests were performed three independent times in triplicates (n = 3), and the obtained data were represented as mean ± standard deviation. The significance level was analyzed at p < 0.05.

Fabrication of CS/FeO NC
Biogenic conversion of iron (II) sulfate to CS/FeO NC was initiated by mixing of FeSO4 and CS with S. acuta aqueous leaf extract, which also stimulated the color change from yellowish to black, and thus supported the development of CS/FeO NC ( Figure  2(Aa)). Similarly, Boudouh et al. (2021) [34] documented the exhibition of black color during the fabrication of FeO NPs. Secondary metabolites present in S. acuta leaf extract and free amino, and hydroxyl groups existing in CS might have joined with ferrous ions (Fe II ) and formed Fe-ellagate conjugate. Simultaneously, the formed conjugate leads to the nucleation that goes into reverse micellization, which later causes the reduction of Fe + to nano-FeO [35].

XRD Analysis
As shown in Figure 2B, the obtained 2θ peaks around 28.  [38]. The obtained other peaks at 2θ = 21.0 and 26.0 may be exhibited due to the CS crystallization over CS/FeO NC and consequently revealed the establishment of organic/inorganic NC. The other peaks may be raised from organic phases in the CS/FeO complex [26]. Further, the crystalline size of CS/FeO NC was calculated using Scherrer's equation, as described previously by [36], and was found to be 18 nm.

FE-SEM and EDS Analysis
FESEM analyzed the morphological features of prepared CS/FeO NC. The FE-SEM revealed that the prepared NC was spherical with an average size of 10-100 nm (Figure 4a,b). Following our study, Pérez-Beltrán et al. (2021) [44] demonstrated that the SEM investigation of FeO NPs displayed a spherical shape. The elemental configuration of CS/FeO NC was identified by EDS analysis. EDS showed the peaks of Fe (45.90%) together with O (39.50%), C (7.13%), Na (1.40%), and S (6.07%) (Figure 4c), which obviously confirms the development of CS/FeO NC [45]. Similarly, Bibi et al. (2019) [36] reported the detection of Fe, O, and C elements in the synthesized FeO NPs using P. granatum.

Bactericidal Activity
The bactericidal property of prepared CS/FeO NC was analyzed using the broth dilution technique. As shown in Figure 5A, CS/FeO NC showed remarkable bactericidal properties against B. subtilis, S. aureus, and E. coli. S. acuta leaf extract exhibited potential bacteriostatic inhibition against the chosen pathogens, and CS showed moderate inhibition activity. The bactericidal activity was higher in the case of CS/FeO NC compared to that of S. acuta and CS. However, prepared CS/FeO NC showed higher ZOI against E. coil than B. subtilis and S. aureus. Similarly, Das et al. (2020) [46] demonstrated that the synthesized FeO NPs exhibited potential bactericidal against S. aureus, P. vulgaris, and P. aeruginosa. The FeO NPs synthesized using leaf extract of A. spinosus exhibited bactericidal activity against E. coli and B. subtilis [47]. The bactericidal activity of CS/FeO NC may be due to cell membrane disruption by the generation of ROS, which causes cell death [33].

Bactericidal Activity
The bactericidal property of prepared CS/FeO NC was analyzed using the broth dilution technique. As shown in Figure 5A, CS/FeO NC showed remarkable bactericidal properties against B. subtilis, S. aureus, and E. coli. S. acuta leaf extract exhibited potential bacteriostatic inhibition against the chosen pathogens, and CS showed moderate inhibition activity. The bactericidal activity was higher in the case of CS/FeO NC compared to that of S. acuta and CS. However, prepared CS/FeO NC showed higher ZOI against E. coil than B. subtilis and S. aureus. Similarly, Das et al. (2020) [46] demonstrated that the synthesized FeO NPs exhibited potential bactericidal against S. aureus, P. vulgaris, and P. aeruginosa. The FeO NPs synthesized using leaf extract of A. spinosus exhibited bactericidal activity against E. coli and B. subtilis [47]. The bactericidal activity of CS/FeO NC may be due to cell membrane disruption by the generation of ROS, which causes cell death [33].

Anti-Proliferative Property of CS/FeO NC
The anti-proliferative property of prepared CS/FeO NC was determined by MTT assay against A549 cells. The obtained result revealed that the CS/FeO NC was a concentration-dependent inhibitor for A549 cells ( Figure 5B). In our study, 16% inhibition was seen

Anti-Proliferative Property of CS/FeO NC
The anti-proliferative property of prepared CS/FeO NC was determined by MTT assay against A549 cells. The obtained result revealed that the CS/FeO NC was a concentrationdependent inhibitor for A549 cells ( Figure 5B). In our study, 16% inhibition was seen at 10 µg/mL and 94% inhibition was found to be at 200 µg/mL concentration. Prepared CS/FeO NC induced 50% (IC 50 ) of growth suppression property at the concentration of 82 ± 0.50 µg/mL. Similar to our study, biosynthesized silver nanoparticles from Caulerpa taxifolia also showed the concentration-dependent anti-lung cancer activity [48]. In addition, the A549 cell proliferation was observed using fluorescence microscopy. Fluorescent microscopic observation of AO/EtBr stained A549 cells is shown in Figure 6a,b. The control cells showed green color, and CS/FeO NC treated cells exhibited red color. The observed, red-colored bodies revealed the cell damage, membrane blebbing, and the presence of apoptotic bodies. Similarly, recent studies demonstrated the anti-cancer properties of FeObased nanomaterials against various cancer cells [49,50]. The antiproliferative property of CS/FeO NC depends on Fe ions permeability into cells, which induces cell damage, nuclear fragmentation, and apoptosis.

Anti-Proliferative Property of CS/FeO NC
The anti-proliferative property of prepared CS/FeO NC was determined by MTT assay against A549 cells. The obtained result revealed that the CS/FeO NC was a concentration-dependent inhibitor for A549 cells ( Figure 5B). In our study, 16% inhibition was seen at 10 μg/mL and 94% inhibition was found to be at 200 μg/mL concentration. Prepared CS/FeO NC induced 50% (IC50) of growth suppression property at the concentration of 82 ± 0.50 μg/mL. Similar to our study, biosynthesized silver nanoparticles from Caulerpa taxifolia also showed the concentration-dependent anti-lung cancer activity [48]. In addition, the A549 cell proliferation was observed using fluorescence microscopy. Fluorescent microscopic observation of AO/EtBr stained A549 cells is shown in Figure 6a,b. The control cells showed green color, and CS/FeO NC treated cells exhibited red color. The observed, red-colored bodies revealed the cell damage, membrane blebbing, and the presence of apoptotic bodies. Similarly, recent studies demonstrated the anti-cancer properties of FeO-based nanomaterials against various cancer cells [49,50]. The antiproliferative property of CS/FeO NC depends on Fe ions permeability into cells, which induces cell damage, nuclear fragmentation, and apoptosis.

Conclusions
This study has employed an eco-friendly and green chemistry way for the CS/FeO NC preparation. The development of CS/FeO NC has been analyzed by UV-vis, XRD, FE-SEM, EDS, and FTIR. Prepared CS/FeO NC showed remarkable bactericidal properties against E. coli, B. subtilis, and S. aureus. However, CS/FeO NC exhibited higher bactericidal properties against E. coli than other pathogens due to the permeability nature of bacterial cells. Furthermore, CS/FeO NC has efficiently suppressed the growth of A549 cells in a concentration-dependent way and stimulated the apoptosis in A549 cells. In conclusion, this study demonstrated that the eco-friendly prepared CS/FeO NC using S. acuta leaf could be used as a novel biomaterial to inhibit pathogens and lung carcinoma cells.