Antimicrobial and Wound Healing Properties of FeO Fabricated Chitosan/PVA Nanocomposite Sponge

Diabetic and anemia-associated diabetic wounds increase the considerable morbidity and mortality in people, as reported by clinical studies. However, no anemia-associated diabetic wound dressing materials have been developed until now. Hence, this study aimed to develop a nanocomposite scaffold composed of chitosan (CS), poly (vinyl alcohol) (PVA), and phytogenic iron oxide nanoparticles (FeO NPs), for accelerated anemia-associated diabetic wound healing. The aqueous leaves extract of Pinus densiflora (PD) was utilized for the synthesis of iron oxide nanoparticles (FeO NPs). TEM and elemental analysis confirmed smaller size PD-FeO NPs (<50 nm) synthesis with the combination of iron and oxide. In addition, in vitro biological studies displayed the moderate antioxidant, antidiabetic activities, and considerable antibacterial activity of PD-FeO NPs. Further, the different concentrations of PD-FeO NPs (0.01, 0.03, and 0.05%) incorporated CS/PVA nanocomposites sponges were developed by the freeze-drying method. The porous structured morphology and the presence of PD-FeO NPs were observed under FE-SEM. Among nanocomposite sponges, PD-FeO NPs (0.01%) incorporated CS/PVA sponges were further chosen for the in vitro wound-healing assay, based on the porous and water sorption nature. Furthermore, the in vitro wound-healing assay revealed that PD-FeO NPs (0.01%) incorporated CS/PVA has significantly increased the cell proliferation in HEK293 cells. In conclusion, the CS/PVA-PD-FeO NPs (0.01%) sponge would be recommended for diabetic wound dressing after a detailed in vivo evaluation.


Introduction
Diabetes mellitus (DM) is a class of metabolic disorders caused by the impairment of metabolism in insulin secretion or resistance. In general, diabetes and its associated complications are rising eternally due to the modern lifestyle, including nutrition imbalance, less physical activity, and mental stress [1]. The diabetes condition alters the metabolic function of the kidney, which potentially causes anemia (iron deficiency) [2]. One of the clinical studies reports that iron deficiency can be caused even without anemia [3]. Iron is a major mineral that regulates the key metabolism, such as oxygen supply and energy through iron-containing proteins (myoglobin, hemoglobin, ferritin, and cytochromes), in the human body. Moreover, iron-containing proteins are involved in collagen metabolism through procollagen-proline dioxygenase [4]. Concurrently, iron overload (hemochromatosis), and chronic venous disease (CVD) causes severe complication through specific organ damage in liver, heart, kidney, leg veins, and skin [5]. Diabetes, anemia, and iron deficiency are intently correlated with diabetic wound healing [6][7][8]. Diabetic wound healing is a detrimental impediment in the management of wound healing because of the pathogenic macro and microvascular complications [9,10]. A recent pilot study revealed that 88% of patients' wounds had not healed completely, due to anemic and/or iron defects [11]. Hence, iron supplementation can improve the wound healing of patients who are affected with chronic

Results and Discussion
The present work reports the green synthesis of multifunctional FeO NPs using the Pinus densiflora (PD). We also analyze the PD-FeO NPs incorporated chitosan/PVA nanocomposite wound dressing sponge for anemia-associated diabetic wound healing. The detailed characterization and biological applications of PD-FeO NPs and CS/PVA-PD-FeO NPs sponges are discussed in the below sections.

TEM Analysis of PD-FeO NPs
TEM micrograph of PD-FeO NPs showed that the uniform size and spherical shape particles surrounded the organic layer, with sizes ranging from 20-50 nm ( Figure 1). The aggregation/agglomeration in PD-FeO NPs might be due to the capping of the bioactive molecules, such as polyphenols on the surface of particles, which form the strong bonding between the nanoparticles [32,33]. Similarly, an earlier study has reported that aqueous solution and neutral pH can increase the aggregation in FeO NPs [34,35]. Moreover, the elemental mapping analysis demonstrated the presence of iron and oxygen (Figure 1c-e), which is in accordance with an earlier report [36]. Furthermore, the presence of nitrogen and oxygen in PD-FeO NPs indicates the successful capping of phytomolecules.

Results and Discussion
The present work reports the green synthesis of multifunctional FeO NPs using the Pinus densiflora (PD). We also analyze the PD-FeO NPs incorporated chitosan/PVA nanocomposite wound dressing sponge for anemia-associated diabetic wound healing. The detailed characterization and biological applications of PD-FeO NPs and CS/PVA-PD-FeO NPs sponges are discussed in the below sections.

TEM Analysis of PD-FeO NPs
TEM micrograph of PD-FeO NPs showed that the uniform size and spherical shape particles surrounded the organic layer, with sizes ranging from 20-50 nm (Figure 1). The aggregation/agglomeration in PD-FeO NPs might be due to the capping of the bioactive molecules, such as polyphenols on the surface of particles, which form the strong bonding between the nanoparticles [32,33]. Similarly, an earlier study has reported that aqueous solution and neutral pH can increase the aggregation in FeO NPs [34,35]. Moreover, the elemental mapping analysis demonstrated the presence of iron and oxygen (Figure 1c-e), which is in accordance with an earlier report [36]. Furthermore, the presence of nitrogen and oxygen in PD-FeO NPs indicates the successful capping of phytomolecules.

Zeta Potential and Particle Size Analysis
Zeta potential and particle size analysis revealed an average size of 136.6 ± 0.36 nm, with a polydispersity index (PDI) of 0.122 ± 0.01 for PD-FeO NPs (Figure 2A). The size differences between TEM and the particle size analyzer depend on the analysis condition. Particle size analysis exhibited the hydrodynamic particle size because the analysis was done in the aqueous medium [37]. Further, PD-FeO NPs exhibited the zeta potential of −29.6 ± 1.0 mV ( Figure 2B), which indicated its good stability. The good zeta potential and smaller particles could induce the cell receptor binding and cellular uptake ability [38]. Moreover, the size and zeta potential of the FeO NPs core was influenced by the capping of phytomolecules [39,40].

Zeta Potential and Particle Size Analysis
Zeta potential and particle size analysis revealed an average size of 136.6 ± 0.36 nm, with a polydispersity index (PDI) of 0.122 ± 0.01 for PD-FeO NPs (Figure 2A). The size differences between TEM and the particle size analyzer depend on the analysis condition. Particle size analysis exhibited the hydrodynamic particle size because the analysis was done in the aqueous medium [37]. Further, PD-FeO NPs exhibited the zeta potential of −29.6 ± 1.0 mV ( Figure 2B), which indicated its good stability. The good zeta potential and smaller particles could induce the cell receptor binding and cellular uptake ability [38]. Moreover, the size and zeta potential of the FeO NPs core was influenced by the capping of phytomolecules [39,40].

ATR-FTIR Spectroscopy Analysis
The functional properties and the interaction of PD aqueous extracts, PD-FeO NPs, CS, CS/PVA, and CS/PVA-PD-FeO NPs (0.01%), were studied through the ATR-FTIR spectroscopy method (Figure 3). PD aqueous extract showed strong broad peaks at 3290 cm −1 , corresponding to O-H stretching, and medium peaks at 2921 cm −1 , due to C-H stretching. The medium band at 1604 cm −1 is attributable to C=C stretching. The peaks at 1391 cm −1 are a result of the O-H bending, and medium peaks at 1248 cm −1 are a result of C-N stretching. The strong peaks at 1040 cm −1 can be attributed to the C-O stretching of

Zeta Potential and Particle Size Analysis
Zeta potential and particle size analysis revealed an average size of 136.6 ± 0.36 nm, with a polydispersity index (PDI) of 0.122 ± 0.01 for PD-FeO NPs (Figure 2A). The size differences between TEM and the particle size analyzer depend on the analysis condition. Particle size analysis exhibited the hydrodynamic particle size because the analysis was done in the aqueous medium [37]. Further, PD-FeO NPs exhibited the zeta potential of −29.6 ± 1.0 mV ( Figure 2B), which indicated its good stability. The good zeta potential and smaller particles could induce the cell receptor binding and cellular uptake ability [38]. Moreover, the size and zeta potential of the FeO NPs core was influenced by the capping of phytomolecules [39,40].

ATR-FTIR Spectroscopy Analysis
The functional properties and the interaction of PD aqueous extracts, PD-FeO NPs, CS, CS/PVA, and CS/PVA-PD-FeO NPs (0.01%), were studied through the ATR-FTIR spectroscopy method (Figure 3). PD aqueous extract showed strong broad peaks at 3290 cm −1 , corresponding to O-H stretching, and medium peaks at 2921 cm −1 , due to C-H stretching. The medium band at 1604 cm −1 is attributable to C=C stretching. The peaks at 1391 cm −1 are a result of the O-H bending, and medium peaks at 1248 cm −1 are a result of C-N stretching. The strong peaks at 1040 cm −1 can be attributed to the C-O stretching of

ATR-FTIR Spectroscopy Analysis
The functional properties and the interaction of PD aqueous extracts, PD-FeO NPs, CS, CS/PVA, and CS/PVA-PD-FeO NPs (0.01%), were studied through the ATR-FTIR spectroscopy method (Figure 3). PD aqueous extract showed strong broad peaks at 3290 cm −1 , corresponding to O-H stretching, and medium peaks at 2921 cm −1 , due to C-H stretching. The medium band at 1604 cm −1 is attributable to C=C stretching. The peaks at 1391 cm −1 are a result of the O-H bending, and medium peaks at 1248 cm −1 are a result of C-N stretching. The strong peaks at 1040 cm −1 can be attributed to the C-O stretching of primary alcohol. PD-FeO NPs exhibited strong and broad peaks at 3214 cm −1 and 1599 cm −1 , corresponding to O-H stretching and C=C stretching, respectively. The peaks at 1360 cm −1 and 1023 cm −1 are responsible for the O-H bending and medium C-N stretching of amine, respectively. The CS exhibited strong and broad peaks at 3262 cm −1 , which can be attributed to intermolecular O-H stretching, and the medium peaks observed at 2877 cm −1 are a result of C-H stretching. Amide I and amide II were observed at 1637 cm −1 and 1546 cm −1 , corresponding to C=O stretching and N-H bending of chitosan [19,41]. Further, the deformation of the CH 3 band was observed at 1406 cm −1 and 1373 cm −1 [42]. The PVA exhibited strong broad peaks at 3435 cm −1 and 2949 cm −1 , corresponding to the O-H intermolecular, intramolecular hydrogen bond, and C-H stretching of alkyl groups, respectively. The peaks at 1653 cm −1 are a result of C=C stretching, and 1421 cm −1 can be Antibiotics 2021, 10, 524 5 of 20 attributed to the CH 2 group [43]. The intermolecular hydrogen bond O-H and C-H stretching at 3275 cm −1 and 2910 cm −1 was formed respectively in the CS/PVA composites. The shifting of the major peaks was noted when compared to CS alone. Further, the minor shift was observed in the amide I and amide II bands of chitosan at 1639 cm −1 and 1553 cm −1 . The uses of poly(vinyl alcohol) in the scaffold would help to improve the mechanical strength. Moreover, the fabrication of PD-FeO NPs did not exhibit significant changes on CS/PVA composites, because PD-FeO NPs was encapsulated by CS/PVA. Alongside this, the concentration of PD-FeO NPs was lower (0.01%).
amine, respectively. The CS exhibited strong and broad peaks at 3262 cm , which can be attributed to intermolecular O-H stretching, and the medium peaks observed at 2877 cm −1 are a result of C-H stretching. Amide I and amide II were observed at 1637 cm −1 and 1546 cm −1 , corresponding to C=O stretching and N-H bending of chitosan [19,41]. Further, the deformation of the CH3 band was observed at 1406 cm −1 and 1373 cm −1 [42]. The PVA exhibited strong broad peaks at 3435 cm −1 and 2949 cm −1 , corresponding to the O-H intermolecular, intramolecular hydrogen bond, and C-H stretching of alkyl groups, respectively. The peaks at 1653 cm −1 are a result of C=C stretching, and 1421 cm −1 can be attributed to the CH2 group [43]. The intermolecular hydrogen bond O-H and C-H stretching at 3275 cm −1 and 2910 cm −1 was formed respectively in the CS/PVA composites. The shifting of the major peaks was noted when compared to CS alone. Further, the minor shift was observed in the amide I and amide II bands of chitosan at 1639 cm −1 and 1553 cm −1 . The uses of poly(vinyl alcohol) in the scaffold would help to improve the mechanical strength. Moreover, the fabrication of PD-FeO NPs did not exhibit significant changes on CS/PVA composites, because PD-FeO NPs was encapsulated by CS/PVA. Alongside this, the concentration of PD-FeO NPs was lower (0.01%).

XRD Spectrum of CS/PVA-PD-FeO NPs
The crystalline properties of CS, PVA, CS/PVA, PD-FeO NPs, and PD-FeO NPs (0.01%) incorporated CS/PVA, were studied through XRD analysis, as shown in Figure 4A,B. The XRD pattern of PD-mediated synthesized FeO NPs was agreed to standard Fe 3 O 4 (JCPDS:   [44,45]. The CS showed its respective crystalline peaks at 2θ angle 10.32 • and 19.94 • . Similarly, sigma chitosan was reported as the two crystalline peaks at 10.1 • and 19.8 • [46]. We also found that sigma chitosan showed major crystalline peaks at 10.0 • and 20.1 • [47]. Another polymer, PVA, also exhibited the two major crystalline peaks at 2θ angle 11.6 • and 19.7 • , and the obtained crystal peaks were consistent with earlier reports [48]. The CS blended PVA exhibited the two crystalline peaks at 8.  Figure 4B). In addition, earlier reports found that Fe 3 O 4 nanoparticle crystallinity was not changed by the chitosan coating [50].

SEM
The structural morphology of CS, CS/PVA, and PD-FeO NPs (0.01% and 0.03%) incorporated CS/PVA sponges are shown in Figure 5A-D. SEM analysis displayed that CS sponges have a well-interconnected, honey-comb structure, with thin-walled morphology, compared to CS/PVA and CS/PVA-PD-FeO NPs. A dense inter-walled, microporous structure was observed in CS/PVA sponges, which is attributable to the strong hydrogen bonding between the polymer [51]. The 0.01% of PD-FeO NPs incorporated CS/PVA sponges displayed significant dense microporous structured morphology, but this was not observed in CS and CS/PVA sponges. Further, the presence of PD-FeO NPs on the surface was observed under higher magnification ( Figure 5(Ciii)). However, when increasing the concentration of PD-FeO NPs to more than 0.01%, the interconnected porous structure of sponges was highly interrupted ( Figure 5(Di-iii)). Moreover, the higher concentration of PD-FeO NPs may influence the physical nature of CS/PVA sponges through cross-linking. Accordingly, an earlier study evidenced that the topology and pore size of CS/PVA hydrogel were significantly changed while loading cerium oxide nanoparticles [25].

SEM
The structural morphology of CS, CS/PVA, and PD-FeO NPs (0.01% and 0.03%) incorporated CS/PVA sponges are shown in Figure 5A-D. SEM analysis displayed that CS sponges have a well-interconnected, honey-comb structure, with thin-walled morphology, compared to CS/PVA and CS/PVA-PD-FeO NPs. A dense inter-walled, microporous structure was observed in CS/PVA sponges, which is attributable to the strong hydrogen bonding between the polymer [51]. The 0.01% of PD-FeO NPs incorporated CS/PVA sponges displayed significant dense microporous structured morphology, but this was not observed in CS and CS/PVA sponges. Further, the presence of PD-FeO NPs on the surface was observed under higher magnification ( Figure 5(Ciii)). However, when increasing the concentration of PD-FeO NPs to more than 0.01%, the interconnected porous structure of sponges was highly interrupted ( Figure 5(Di-iii)). Moreover, the higher concentration of PD-FeO NPs may influence the physical nature of CS/PVA sponges through crosslinking. Accordingly, an earlier study evidenced that the topology and pore size of CS/PVA hydrogel were significantly changed while loading cerium oxide nanoparticles [25].    Figure 6A. The porosity of various sponges is demonstrated, including CS at 90.57 ± 1.42%, CS/PVA at 82.6 ± 1.61%, CS/PVA FeO NPs (0.01%) at 81.78 ± 0.77%, CS/PVA FeO NPs (0.03%) at 62.72 ± 1.71%, and CS/PVA FeO NPs (0.05%) at 54.88 ± 2.22%. Among them, CS sponges showed the highest porosity, followed by CS/PVA, and 0.01% of PD-FeO NPs incorporated CS/PVA sponges (p < 0.001). However, the higher concentration of PD-FeO NPs (0.03 and 0.05%) incorporated sponges exhibited a decreased porosity when compared to CS/PVA-PD-FeO NPs (0.01%) (p < 0.05). In our previous study, we found that direct incorporation of silver nanoparticles inhibited a well inter-connected porous structure in fungal chitosan sponges [18]. Highly porous material in wound dressing would help gaseous exchange, excess wound exudate absorption, and maintenance of moist environments [52]. Moreover, moist wound healing supports re-epithelialization and inhibits secondary infections like diabetic condition [53].

Water Absorption
The water absorption behavior of CS, CS/PVA, and the different concentrations of PD-FeO NPs incorporated CS/PVA sponges were determined from 0 to 6 h ( Figure 6B). The Chitosan (CS) sponge alone showed a higher water absorption (%) in the 1st h at 2318.28 ± 65.05%, and the water absorption was increased up to 2511 ± 51.71% at 6th h. CS blended PVA sponges demonstrated water absorption at 2163.04 ± 56.56% (1st h), and 3358.91 ± 21.21% (6th h) which is higher than the CS sponge alone, while showing lesser porosity compared to CS. Although the incorporation of PD-FeO NPs (0.03% and 0.05%) in CS/PVA sponges significantly decreased the water absorption after 1 h interval, the 0.01% of PD-FeO NPs incorporation maintained the water absorption capacity constantly. The maximum % of water absorption was found in CS/PVA-PD-FeO NPs (0.01%) at 2855.55 ± 83.80% (6th h). However, 0.03% and 0.05% of PD-FeO NPs incorporated CS/PVA sponges were considerably degraded at the 4th h of the experiment. The water absorption nature highly depends on the properties of the material in composite sponges [54]. an advantage for the anemia-associated diabetic wound healing process because it avoids iron overload mediated toxicity. However, in vitro iron release may not precisely define the in vivo conditions. Figure 7 shows the estimated iron release (%) from CS/PVA-PD-FeO NPs (0.01%) at different time intervals (0 to 24 h). The rate of PD-FeO NPs release was significantly increased with the increase in time (p < 0.05). The rate of PD-FeO NPs release was 6.5 ± 0.49% at the 1st h and 14.25 ± 0.26% at the 24th h. Overall, the in vitro release pattern indicates the sustained release of iron with a lower percentage until 24 h. It was demonstrated that CS/PVA-PD-FeO NPs negatively impacted the iron release at the pH of 7.4, due to the gelling property of PVA blended chitosan. The lower iron release from nanocomposites is an advantage for the anemia-associated diabetic wound healing process because it avoids iron overload mediated toxicity. However, in vitro iron release may not precisely define the in vivo conditions.

Biological Activities
Biological activities of PD-FeO NPs were determined through antioxidant, antidiabetic, and antibacterial assays. Further, antibacterial, cell viability, and cellular internalization of CS/PVA-PD-FeO NPs composites were evaluated using standard in vitro assays. Moreover, the in vitro wound healing potential of CS/PVA-PD-FeO NPs composites was evaluated in HEK-293 cells. The detailed results and discussions are presented in the following sections.

Antioxidant Properties
The antioxidant potential of PD aqueous extract and PD-FeO NPs was evaluated by DPPH and ABTS + radical scavenging antioxidant assays ( Figures S1 and S2). The PD aqueous extract exhibited significant radical scavenging activity in both DPPH and ABTS + radicals, compared to PD-FeO NPs. The maximum tested concentration of 1 mg/mL of PD aqueous extract and PD-FeO NPs showed the percentage of DPPH radical scavenging activity at 75.86 ± 1.3 and 49.11 ± 2.5, respectively. Similarly, the percentage of ABTS radical scavenging activity of PD aqueous extract (1 mg/mL) and PD-FeO NPs (1 mg/mL) was found at 94.86 ± 0.11 and 48.51 ± 0.98, respectively. Ascorbic acid has shown substantial radical scavenging activity against both DPPH and ABTS radicals. The earlier studies have shown that Fe NPs exhibited lower antioxidant activity than Rhus and safflower aqueous extract [55] which supports the present results. Moreover, these extracts augment wound healing in the diabetic condition through inhibition of oxidative stress [56].

Antidiabetic Activity
The inhibition of digestive enzymes, such as α-glucosidase and α-amylase, has considerably attenuated the higher blood glucose level in diabetic conditions [57,58], hence the PD aqueous extract and PD-FeO NPs evaluated in vitro α-amylase and α-glucosidase enzyme inhibitory activity (Figures S3 and S4). Like the antioxidant activity, PD aqueous extract (1 mg/mL) exhibited higher digestive enzyme inhibitory activity than the PD-FeO NPs (1 mg/mL). The percentages of α-amylase inhibitory activity of PD aqueous extract and PD-FeO NPs were found at 20.71 ± 4.8 and 18.78 ± 3.14, respectively. In addition, PD aqueous extract and PD-FeO NPs exhibited the percentage of α-glucosidase activity at 79.29 ± 0.48 and 27.01 ± 5.01, respectively. However, PD aqueous extract and PD-FeO NPs significantly inhibit α-glucosidase activity, compared to αamylase. Accordingly, Sesamum indicum seeds extract mediated synthesis of iron nanoparticles significantly inhibits the α-amylase comparatively less than acarbose [59].

Antibacterial Activity
Microbial population is common in open wounds, but infective conditions have a detrimental effect on the normal healing process [60]. PD-FeO NPs showed moderate antibacterial activity against tested pathogens, such as B. cereus, S. aureus, E. coli, and S. enterica. The minimum inhibitory concentration (MIC) was observed in S. aureus (250 µg/mL) while not detected in other tested bacteria (Table 1). Accordingly, earlier studies indicated that FeNPs and FeO NPs have moderately inhibited bacterial growth [61][62][63]. But, CS/PVA incorporated PD-FeO NPs (0.01%) composite sponges showed significant inhibitory activity against all the tested pathogens with MIC (Table 1). Furthermore, the significant zone of inhibition was observed in CS/PVA-PD-FeO NPs (0.01%) composite sponges treated B. cereus (22 ± 2 mm), S. aureus (21 ± 1 mm), E. coli (20 ± 2 mm), and S. enterica (22 ± 1.5 mm) (Table 1; Figure S5). However, moderate inhibitory activity was observed in CS/PVA-PD-FeO NPs and compared to the standard antibiotic tetracycline hydrochloride. The antimicrobial activity of chitosan is highly dependent on the degree of deacetylation, molecular weight, and solubility [64]. The blending of PVA in the nanocomposite turns the chitosan into hydrophilic nature, which exerts antimicrobial properties [65]. To understand the detailed mechanism of the PD-FeO NPs incorporated CS/PVA nanocomposite treated, control bacterial cells was observed in HR-TEM analysis (Figure 8). The untreated S. aureus and E. coli control bacterial samples maintained the cocci and rod morphology with intact cytoplasm, which seemed to be normal. In contrast, the CS/PVA-PD-FeO NPs treated S. aureus cell wall significantly damaged and cytoplasmic leakages were observed, indicated by yellow arrows (Figure 8). Clear morphological changes were observed in the nanocomposite treated E. coli cells. The yellow arrows indicate the nanoparticle accumulation and internalization on/onto the cell wall of E. coli. The nanocomposites were initiated by the bacterial cell death through structural destabilization and a cytoplasmic leakage mechanism, identified in HR-TEM analysis. Further, the flow cytometry analysis confirmed the considerable bacterial death in nanocomposite treated S. aureus (78.6%) and E. coli (76.17%), compared to non-treated control cells (Figure 9). Both Syto-9 and PI are referred to as the nuclear stain. Syto-9 could stain both live and dead cells, but PI stained only the cells with damaged membranes [66]. Based on the antibacterial results, CS/PVA-PD-FeO NPs could be the potent antibacterial dressing material that helps to heal wounds by preventing microbial growth.

Cell Viability
Biocompatible and less toxic materials can be considered as an ideal wound dressing. Hence, the cell viability of CS, PD-FeO NPs, and CS/PVA-PD-FeO NPs treated HEK-293 cells were evaluated by WST assay ( Figure S6) and fluorescent microscopy-based assays ( Figure 10). According to the results, the tested samples do not show significant cytotoxicity in HEK-293 cells up to the tested concentration (1 mg/mL). PD-FeO NPs treated cells showed an IC50 concentration at 780 ± 20 µg/mL. An earlier study indicates that any tested concentration of iron oxide nanoparticle did not cause toxicity in HUVEC cells up to 24 h incubation, but toxicity was found at 48 h incubation [67]. In the case of CS and CS/PVA-PD-FeO NPs (0.01%), treated samples were not observed to have half of the minimum inhibitory concentration (IC50). Several studies reported that chitosan and polyvinyl alcohol showed substantial cell proliferation and a biocompatible nature, which suggests the existence of primary hydroxyl, acetyl, and amine functionalities and its ratio [68][69][70][71][72]. Fur-

Cell Viability
Biocompatible and less toxic materials can be considered as an ideal wound dressing. Hence, the cell viability of CS, PD-FeO NPs, and CS/PVA-PD-FeO NPs treated HEK-293 cells were evaluated by WST assay ( Figure S6) and fluorescent microscopy-based assays ( Figure 10). According to the results, the tested samples do not show significant cytotoxicity in HEK-293 cells up to the tested concentration (1 mg/mL). PD-FeO NPs treated cells showed an IC 50 concentration at 780 ± 20 µg/mL. An earlier study indicates that any tested concentration of iron oxide nanoparticle did not cause toxicity in HUVEC cells up to 24 h incubation, but toxicity was found at 48 h incubation [67]. In the case of CS and CS/PVA-PD-FeO NPs (0.01%), treated samples were not observed to have half of the minimum inhibitory concentration (IC 50 ). Several studies reported that chitosan and polyvinyl alcohol showed substantial cell proliferation and a biocompatible nature, which suggests the existence of primary hydroxyl, acetyl, and amine functionalities and its ratio [68][69][70][71][72]. Further, AO/EB fluorescence staining showed a greater number of viable cells in various samples treatment, but in PD-FeO NPs alone, treatment showed fewer apoptotic cells (Figure 10). Similarly, fewer dead cells were observed in PI staining. The CS, CS/PVA, and CS/PVA-PD-FeO NPs treated cells have shown considerable mitochondrial membrane potential (∆Ψm) when compared to the control cells. Then, reactive oxygen species (ROS) production was not observed in the treatment and control cells, which means that the samples did not induce the oxidative stress ( Figure 10). Hence, our results conclude that CS/PVA incorporated PD-FeO NPs did not cause any cytotoxicity, and they improve cell viability. Moreover, it can be hypothesized that the fabricated composite decreases the inflammatory cytokines and oxidative enzymes and increases the anti-inflammatory cytokines.

Cellular Glucose Uptake
The cell viability and regulation of cellular glucose uptake behavior of PD-FeO NPs, CS, CS/PVA-PD-FeO NPs (0.01%) treated IR-HepG2 cells are shown in Figure 11A,B. The cell viability showed that treatment did not induce any cell toxicity when compared to control observed in AO/EB staining. Further, cellular glucose uptake results showed that CS/PVA-PD-FeO NPs treatment attenuated the insulin resistance, thereby increasing the cellular glucose uptake (78.75 ± 1.7%) in IR-HepG2 cells, when compared to the nontreated IR-HepG2 cells (glucose uptake at 42.52 ± 1.2%). The positive control metformintreated cells showed a cellular glucose uptake of 72.46 ± 2.4%, which is lesser than CS/PVA-PD-FeO NPs. Similarly, earlier work has reported that superparamagnetic iron oxide nanoparticles (SPION) significantly reduce the blood glucose level in diabetic rats more so than metformin [73]. In addition, SPION was found to regulate the expression of obesity and type-2 diabetic-related genes in human preadipocyte cells [74]. In the present study, the individual treatment of CS and PD-FeO NPs have induced the cellular glucose uptake at 69.07 ± 1.8% and 61.83 ± 3.81%, respectively ( Figure 11B). The earlier studies supported the fact that chitosan oligosaccharides and Korean red pine (Pinus densiflora)

Cellular Glucose Uptake
The cell viability and regulation of cellular glucose uptake behavior of PD-FeO NPs, CS, CS/PVA-PD-FeO NPs (0.01%) treated IR-HepG2 cells are shown in Figure 11A,B. The cell viability showed that treatment did not induce any cell toxicity when compared to control observed in AO/EB staining. Further, cellular glucose uptake results showed that CS/PVA-PD-FeO NPs treatment attenuated the insulin resistance, thereby increasing the cellular glucose uptake (78.75 ± 1.7%) in IR-HepG2 cells, when compared to the non-treated IR-HepG2 cells (glucose uptake at 42.52 ± 1.2%). The positive control metformin-treated cells showed a cellular glucose uptake of 72.46 ± 2.4%, which is lesser than CS/PVA-PD-FeO NPs. Similarly, earlier work has reported that superparamagnetic iron oxide nanoparticles (SPION) significantly reduce the blood glucose level in diabetic rats more so than metformin [73]. In addition, SPION was found to regulate the expression of obesity and type-2 diabetic-related genes in human preadipocyte cells [74]. In the present study, the individual treatment of CS and PD-FeO NPs have induced the cellular glucose uptake at 69.07 ± 1.8% and 61.83 ± 3.81%, respectively ( Figure 11B). The earlier studies supported the fact that chitosan oligosaccharides and Korean red pine (Pinus densiflora) show substantial antidiabetic activity through reduction of blood glucose level [29,75].  Figure 12E,F), while other treatments and control did not observe any iron content ( Figure 12A-D). An early study reports that iron oxide and doxorubicinloaded chitosan nanoparticles successfully internalized in glioblastoma cells, where iron oxide nanoparticles internalized depending on the concentration observed by Prussian blue staining [37]. The cellular iron oxide nanoparticle uptake significantly enhanced the neurite outgrowth in PC12 cells [76].   Figure 12E,F), while other treatments and control did not observe any iron content ( Figure 12A-D). An early study reports that iron oxide and doxorubicin-loaded chitosan nanoparticles successfully internalized in glioblastoma cells, where iron oxide nanoparticles internalized depending on the concentration observed by Prussian blue staining [37]. The cellular iron oxide nanoparticle uptake significantly enhanced the neurite outgrowth in PC12 cells [76].

In Vitro Wound Healing Assay
In the in vitro wound-healing assay, we evaluated cell migration and proliferation of mechanically created wounds with and without sample treatment. The different intervals, (0, 12, 24, 48 h) versus sample treatment, (PD-FeO NPs, CS, and CS/PVA-PD-FeO NPs), and control, are shown in Figure 13A. To evaluate the effects of CS/PVA-PD-FeO NPs, the wound area (%) was calculated after 48 h ( Figure 13B). The cell migration was seen in 24 h treatment of PD-FeO NPS, CS, CS/PVA-PD-FeO NPs, when compared to control, which had lesser cell migration. Among them, CS/PVA-PD-FeO NPs showed maximum wound area contraction at 48 h (Figure 13(Aiv)), followed by CS, PD-FeO NPs, and control ( Figure 13(Ai-iii)). The biocompatible chitosan and PVA provide sustained drug release and metal nanoparticle delivery thereby enhanced cell proliferation and augmented angiogenesis, leading to wound contraction and reduction in cytotoxicity. For example, thrombin conjugated iron oxide nanoparticles enhanced incisional wound healing [77]. Moreover, FeO NPs were found to promote the macrophage autophagy and inflammatory response in cell and animal models [78]. In addition, curcumin-loaded chitosan-based polymeric micelles considerably improve the in vivo wound healing and anti-diabetic effect [79].
PD-FeO NPs cellular internalization was evaluated in HEK-293 cells by Prussian blue staining counterstained with safranin ( Figure 12A-F). For the comparative analysis, HEK293 cells were treated by CS, PVA, CS/PVA, PD-FeO NPs, and CS/PVA-PD-FeO NPs (0.01%). The cellular iron content was only found in PD-FeO NPs and CS/PVA-PD-FeO NPs treated cells ( Figure 12E,F), while other treatments and control did not observe any iron content ( Figure 12A-D). An early study reports that iron oxide and doxorubicinloaded chitosan nanoparticles successfully internalized in glioblastoma cells, where iron oxide nanoparticles internalized depending on the concentration observed by Prussian blue staining [37]. The cellular iron oxide nanoparticle uptake significantly enhanced the neurite outgrowth in PC12 cells [76].   (Figure 13(Aiv)), followed by CS, PD-FeO NPs, and control ( Figure  13(Ai-iii)). The biocompatible chitosan and PVA provide sustained drug release and metal nanoparticle delivery thereby enhanced cell proliferation and augmented angiogenesis, leading to wound contraction and reduction in cytotoxicity. For example, thrombin conjugated iron oxide nanoparticles enhanced incisional wound healing [77]. Moreover, FeO NPs were found to promote the macrophage autophagy and inflammatory response in cell and animal models [78]. In addition, curcumin-loaded chitosan-based polymeric micelles considerably improve the in vivo wound healing and anti-diabetic effect [79].
Acetic acid was procured from the Daejung chemicals & metals co., Ltd., Siheung-si, South Korea. The manufacturer's information of all the fluorescent staining and culture medium used in the present study is mentioned in our earlier paper. Pinus densiflora leaves were collected from Kangwon National University campus, Chuncheon, Republic of Korea.

Synthesis of FeO NPs
Roughly 10 g of fresh leaves of PD were thoroughly washed with distilled water and boiled with 100 mL of distilled water at 80 • C for 20 min. The aqueous PD leaves extract was collected using Whatman filter paper (No. 1) for the synthesis of FeO NPs. Further, the different ratios (1:9, 2:8, 3:7, 4:7, and 5:5) of FeCl 3 (5 mM) and aqueous PD leaves extract were mixed. Subsequently, NaOH (1 M) was added to the mixture and kept under magnetic stirring for 30 min at 80 • C. The formation of an intense block-colored solution indicated the synthesis of FeO NPs. This was collected by centrifugation at 15,000 rpm, then washed with distilled water, dried at 80 • C, and stored in an airtight container.

Characterization
The synthesized PD-FeO NPs were initially analyzed using a UV-Visible spectrophotometer (SpectraMax ® Plus 384 Microplate Reader from Molecular Devices). The particle size of PD-FeO NPs was observed under a field emission transmission electron microscope (FE-TEM, JEOL-JSM 1200EX Japan). This was followed by particle size distribution, and zeta potential was determined through a particle size Analyzer (Malvern PANalytical Netherland). The presence of phytochemicals, such as total phenol and flavonoids content in PD-FeO NPs, was determined according to the earlier reports [80]. The functional characteristics of PD-FeO NPs, CS, CS/PVA, and CS/PVA-PD-FeO NPs were characterized through FT-IR spectrophotometer (PerkinElmer Paragon 500 USA), and X-ray diffraction analysis (XRD, X'pert-pro MPD-PANalytical Netherland). The surface morphology of CS, CS/PVA, CS/PVA-PD-FeO NPs sponges was observed under an ultra-high-resolution scanning electron microscope (UHR-SEM, Hitachi Japan). The porosity of CS, CS/PVA, CS/PVA-PD-FeO NPs sponges was evaluated by the liquid displacement method, and water sorption capacity was measured according to the earlier report [18].

In Vitro Iron Release
The amount of iron release from the CS/PVA-PD-FeO NPs (0.01%) was determined by ICP-OES (inductively coupled plasm optical emission spectrometer, Agilent 5900) analysis. Firstly, the previously described weight of composite sponges was placed in the 20 mL of phosphate buffer (pH 7.4), in a dissolution bath at 37 • C under shaker (150 rpm). 2 mL of the sample was collected from the dissolution bath at each predetermined time interval (1, 3, 6, 12, and 24 h) and an equal amount buffer solution was replaced to maintain the equal volume. Next, collected samples were diluted to ten-fold for iron quantification, using ICP-OES analysis. The content of iron release was calculated with the known concentration of iron (Correlation coefficient: 0.999) using Agilent Technologies software (Version 7.5.1.11869).

Antioxidant, Antidiabetic and Antibacterial Assays
The antioxidant (DPPH and ABTS + ) radical scavenging ability of PD aqueous extract and PD-FeO NPs was determined according to the earlier reports [81]. The antidiabetic potential of PD aqueous extract and PD-FeO NPs was determined in terms of digestive enzyme (α-glucosidase and α-amylase) inhibition, as described earlier [82]. The antibacterial activity of PD-FeO NPs, and CS/PVA-FeO NPs was evaluated against bacterial pathogens, such as Bacillus cereus (ATCC 14579), Staphylococcus aureus (ATCC 19095), Escherichia coli (ATCC 43888), and Salmonella enterica (ATCC 14028). The minimum inhibitory concentration of PD-FeO NPs and CS/PVA-PD-FeO NPs were determined by the broth dilution method, according to the earlier report [83]. Furthermore, the antibacterial potential of PD-FeO NPs incorporated CS/PVA composite sponges confirmed in terms of zone of inhibition.

High-Resolution Transmission Electron Microscopy (HR-TEM)
The antibacterial effect of CS/PVA-PD-FeO NPs treated each Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria was determined using HR-TEM (JEOL-JSM 1200EX Japan). To prepare bacteria for HR-TEM analysis, control and treated bacterial cells were initially centrifuged (5000 rpm for 10 min), and the pellet was washed with phosphate buffer (pH 7.4). Further, the pellet was fixed with glutaraldehyde (2.5%) for 4 h, and then samples were dehydrated by 30, 50, 80, and 100% ethanol for 2 h at each step [80]. Finally, air-dried samples were observed under HR-TEM.

Flow Cytometry Analysis
The CS/PVA-PD-FeO NPs treated bacterial cells were further evaluated by flow cytometry (FACSCalibur, USA) analysis. For flow cytometry analysis, the bacterial sample and staining (Syto-9 and propidium iodide (PI)) solution were prepared according to the manufacture's protocol [66].

Cell Viability
The cytotoxicity of PD-FeO NPs, CS, CS/PVA-PD-FeO NPs were evaluated in the human embryonic kidney cell line (HEK-293 cells). The HEK-293 cells were obtained from the Korean cell line bank and cultured in DMEM medium supplemented with FBS (10%) and antibiotics (1%) in a 5% CO 2 incubator at 37 • C. For the cell viability assay, HEK 293 cells were cultured in 96 well plates for 24 h. After reaching the 70% of confluence, 10 µL of various concentration (3.9, 7.81, 15.62, 31.25, 62.5, 125, 250, 500, 1000 µg/mL) of respective samples were treated to each well and incubated for 24 h in CO 2 (5%) incubator. After the treatment, 10 µL of WST reagent was added to each well and incubated for 1 h, and then absorbance was measured at 450 nm. The percentage of cell viability was calculated using the following Equation (1). % of cell viability = (Treated cells absorbance @ 450 nm)/(Control cells absorbance @ 450 nm) × 100 Further, cytotoxicity of PD-FeO NPs, CS, CS/PVA-PD-FeO NPs treated HEK 293 cells was evaluated under a fluorescence microscope using various fluorescence staining (AO/EB, PI, RH123, and DCFH-DA).

In Vitro Glucose Uptake
Cellular glucose uptake efficiency of PD-FeO NPs, CS, and CS/PVA-PD-FeO NPs was determined in insulin-resistant hepatocellular carcinoma (IR-HepG2) cells according to the earlier report [84,85].

In Vitro Wound Healing Activity
The effect of PD-FeO NPs (0.01%), CS (0.5%), and CS/PVA-PD-FeO NPs (0.01%) treatments on wound healing activity was evaluated by in vitro scratch assay [86]. HEK-Antibiotics 2021, 10, 524 16 of 20 293 cells were cultured in six-well plates in a CO 2 incubator at 37 • C, and we allowed cells to reach the proper monolayer confluence. Then, the artificial wound was created by scarping the cell monolayer in a straight line, using a pipette tip (200 µL), and cell debris was washed with PBS. The wounded cell monolayer was treated with each sample incorporated DMEM medium, while the control cells received the DMEM medium alone and were incubated in a CO 2 incubator. This was followed by the wound healing being observed under a light microscope at each predetermined time interval (0 h, 6 h, 12 h, 24 h, and 48 h). The wound area was measured by ImageJ software. Further, the percentage of wound healing was calculated by the following Equation (2). % of wound healing = (wound area on the initial day − wound area on the final day)/(wound area on the initial day) × 100 (2) 3.6.5. PD-FeO NPs Cellular Internalization The cellular internalization of FeO NPs was observed by Prussian blue staining assay according to an earlier report [87], with some modifications. In brief, CS, CS/PVA, PD-FeO NPs (0.01%) and CS/PVA-PD-FeO NPs (0.01%) treated HEK-293 cells were incubated for 24 h at a 5% CO 2 incubator. After incubation, cells were washed with PBS twice, to remove the excess amount of FeO NPs. Then cells were stained with a 1:1 ratio of K 4 Fe(CN) 6 (10%) and HCl (20%) for 20 min in the incubator. Further cells were counterstained with safranin and washed with 0.02% of acetic acid three times and then observed under a light microscope at 10× magnification.

Statistical Analysis
All of the experimental data were presented as mean ± standard deviation. The oneway analysis of variance (ANOVA) was used for the statistical analysis. The p-value < 0.05 was considered statistically significant.

Conclusions
The results reveal that PD aqueous extracts have a higher amount of total phenol and flavonoid resulting in significant antioxidant and antidiabetic activity. The PD aqueous extract-mediated iron oxide NPs exhibited good biological activity. Further, the bioactive potential of iron oxide NPs (0.01%) loaded CS/PVA nanocomposite sponge displayed highly porous properties, and potential water sorption properties. The sustained and lower iron release (%) was observed from nanocomposite sponges. CS/PVA-PD-FeO NPs (0.01%) exhibited a substantial antibacterial activity against tested bacterial pathogens. Further, CS/PVA-PD-FeO NPs (0.01%) augmenting cell proliferation in HEK-293 cells observed in in vitro wound healing scratch assay. The plant-mediated synthesized iron oxide nanoparticles, chitosan, and poly (vinyl alcohol) consisted of nanocomposite sponges (CS/PVA-PD-FeO NPs) which supported gaseous exchange, wound exudated absorption, and microbial inhibition in the diabetic wound. Hence, we conclude that the existence of antioxidant, antidiabetic, and antibacterial properties of CS/PVA-PD-FeO NPs would aid diabetic wound healing. However, further in vivo studies are required to evaluate the molecular mechanism of diabetic wound healing of developed sponges.