Sugarcane Bagasse-Derived Cellulose Nanocrystal/Polyvinyl Alcohol/Gum Tragacanth Composite Film Incorporated with Betel Leaf Extract as a Versatile Biomaterial for Wound Dressing

In this study, nanocomposite film was fabricated using cellulose nanocrystals (CNCs) as nanofiller in a polymer matrix of polyvinyl alcohol (PVA) and gum tragacanth (GT) via solution casting. CNCs were extracted from sugarcane bagasse using a steam explosion technique followed by acid hydrolysis. Initial analysis of CNCs by transmission electron microscopy (TEM) showed nanosized particles of 104 nm in length and 7 nm in width. Physical and chemical characteristics of neat PVA, PVA/GT, and PVA/GT/CNC films with varying concentrations of CNCs (from 2% to 10%) were analyzed by the scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectrometry, mechanical test, and swelling test. The SEM analysis showed cluster formation of CNCs in the polymer matrix at high concentration. The developed films were transparent. FTIR spectrometry analysis confirmed the chemical functional groups of the various components in the film. The presence of GT and CNCs in the polymer matrix improved the characteristics of films as evident in the prolonged stability for 7 days and increased mechanical properties. The highest elastic modulus of 1526.11 ± 31.86 MPa and tensile strength of 80.39 MPa were recorded in PVA/GT/CNC2 film. The swelling ability, however, decreased from 260% to 230%. Cytotoxicity analysis of the PVA/GT/CNC film showed that it is nontoxic to mouse fibroblast cells L929 with 95% cell viability. Films loaded with betel leaf extract exhibited excellent antibacterial activities against Staphylococcus aureus DMST 8840 and Pseudomonas aeruginosa TISTR 781 with 28.20 ± 0.84 mm and 23.60 ± 0.55 mm inhibition zones, respectively. These results demonstrate that PVA/GT/CNC loaded with the betel leaf extract could act as promising and versatile wound dressings to protect the wound surface from infection and dehydration.


Introduction
Natural and synthetic polymers have been investigated over the years as biomaterials for biomedical applications [1,2]. To blend natural polymers with synthetic ones is another unique way of preparing versatile polymeric materials for biomedical applications [3]. Te synthetic polymers have good mechanical properties and thermal stability; however, they can inhibit cell growth due to residue of initiators and other compounds. Natural polymers on the other hand are usually biodegradable, biocompatible, and bioactive in nature [3][4][5]. Polyvinyl alcohol (PVA) is a linear synthetic polymer which is nontoxic, biocompatible, thermostable, and water-soluble with flm forming properties [6,7]. PVA has been used as the main matrix with outstanding physical and chemical properties in biomedical applications [8,9]. However, the scope of PVA application is limited by its instability, insufcient elasticity, and lack of cell-specifc bioactivities [9,10]. To tackle these problems, several attempts have been made by incorporating substances such as chitosan [11,12], dextran [13], polyion liquid [14], and nanocellulose [15,16]. PVA-based composite flms containing bacterial cellulose and epsilon polylysine [16], dextran aldehyde hydrogel [13], and silk fbroin-PVA composite flm coated with chitosan-ZnO nanoparticles [17] were reported for wound dressings. Tese dressings demonstrated good absorbability and tissue regeneration with low cytotoxicity and inhibited bacterial infections with enhanced mechanical properties. However, these materials lacked essential features such as good swelling properties and mediated angiogenesis.
Gum tragacanth (GT) comprises a complex mixture of highly branched heterogeneous hydrophilic polysaccharides mainly D-galacturonic acid and other sugar units with numerous functional groups that provide a suitable medium for cell growth and mediated angiogenesis [18][19][20]. GT is one of the most abundant and renewable natural raw materials. GT is readily accessible, relatively afordable, nontoxic, biocompatible, environmentally friendly, and widely used in biomedical science [21]. As a natural polysaccharide, GT has been applied in various biotechnological industries [19]. GT has been incorporated into PVA for application in drug delivery [22,23], scafold for skin substitutes [24], and wound dressing [25][26][27]. High degradation ability, stability against microbial, and heat in the living systems have triggered several studies on GT for wound dressings [21]. A bio-based wound dressing made with gelatin, gum Arabic, and polyurethane was also reported for wound care application [28]. In a related study, PVA and Iranian gum tragacanth (IGT) biocomposite with nanoclay was reported to have superior degradation and higher chemical and mechanical stability for wound dressing [29]. In addition, GT also serves as a suitable stabilizing agent for biosynthesis of nanoparticles [30]. Te major limitation of GT in biomedical application has to do with its rapid degradation, which afects the mechanical and biological properties. Interestingly, this drawback can be overcome using nanofllers such as cellulose nanocrystals.
Large amount of waste is unfortunately generated from sugarcane processing industries, which composed mainly of bagasse, molasses, flter cake, and leaves [31][32][33]. Te bagasse and leaves are rich in cellulose fbers for nanocellulose production. Cellulose nanocrystals (CNCs) are potential reinforced nanofllers with needle-like shapes ranging from 100 nm to 250 nm in length and from 5 nm to 70 nm in width, high aspect ratio, and high crystallinity [34]. Dispersion of CNCs in synthetic polymers will provide good mechanical properties due to their unique crystalline domains with inherent stability that contributes stifness and elasticity to the material [35]. CNCs have zero or low toxicity and can stimulate long-term cell proliferation [36]. Hence, CNCs have been applied in various aspects of medical felds [37]. Te addition of CNC into carboxymethyl cellulose-glycerol (CMC-G) matrix was reported to improve the physiochemical properties and stability of the flm [38]. A study on bioactive wound dressing using hyaluronic acid-(HA-) based nanofbers with PVA revealed that incorporating CNCs into PVA/HA signifcantly improved swelling and mechanical properties [39]. However, it lacks antimicrobial properties. Antimicrobial activity is an important factor for considering biomaterials for wound dressings. Tis prevents contamination or bacterial infection while improving the healing process. For that matter, plantbased bioactive compounds are widely used as an antibacterial agent in wound dressings [40,41]. An alternative nanocomposite flm for traditional wound dressing incorporated with black pepper and ginger essential oils was reported to signifcantly inhibit the growth of bacteria [42]. In a related study, papain immobilized in bacterial cellulose as a polymer template was reported for wound dressing with strong antibacterial action [43]. Plant extractive from betel leaf (Piper betle L.) was used in this study. Betel leaf is a traditional herbal medicine commonly found in Southeast Asia and East African countries that contains phenolics, favonoids, alcohols, alkaloids, terpenes, fatty acids, and organic acids [44,45]. Tese compounds were reported to exhibit several biological properties including antibacterial, antifungal, anti-infammatory, and antioxidant activities [45][46][47][48].
Diferent treatment strategies and dressings with special properties are required to regenerate damaged tissues in wound management. Wound dressing material should fully cover the afected area, create conducive environment to prevent contamination, and have the ability to absorb exudates on the wound surface. In addition, it must degrade rapidly, be easy to change without causing pain or trauma at the wound bed, be nontoxic, and be cost efective [49,50]. Film dressings are mostly preferred for superfcial injuries to protect skin prone to abrasion or external contamination [50]. Te transparency of flm allows daily observation without its removal and prevents damage to the wound bed [49]. Terefore, combination of diferent polymers impregnated with the plant extract could act as novel flm with unique properties for biomedical consideration in wound dressings.
Herein, PVA, GT, and CNCs were used to produce a versatile flm for wound dressing. Te physicochemical characteristics of the composite flms which are essential for wound dressing were evaluated. Biological properties of PVA/GT/CNC were examined for the flm's ability to support cell viability. Te ability of the flm to prevent bacterial infection was examined by loading the crude betel leaf extract as an antimicrobial agent. A preprint has previously been published [51].

Nanocellulose Extraction from Sugarcane Bagasse (SCB).
Te SCB was dried at 55°C for 24 h and then treated by steam explosion (Nitto Koatsu, Japan) at 190°C with a pressure of 13 MPa for 15 min [52]. Te exploded sample without sugarrich liquid fraction was bleached by treating with 1.4% w/w sodium chlorite (NaClO 2 ) solution, which was adjusted to pH of 4.0 with glacial acetic acid (CH 3 COOH) at 70°C. Bleaching chemicals were added every hour until the sample turned white. Te bleached sample was then fltered and washed several times until the pH was neutral [53,54]. Te dried bleached cellulose fbers were dispersed in 64% w/v H 2 SO 4 at a solid-to-liquid ratio of 1 : 20 with constant stirring at 500 rpm for 75 min at 45°C. Te hydrolysis reaction was stopped using cold deionized (DI) water and centrifugation was repeated at 15,000 rpm for 15 min at 4°C. Te supernatant was removed and replaced by clean DI water, followed by dialysis to obtain CNCs. Te CNC was sonicated (Bransonic Model 2201R-MT, USA) and kept at 4°C for further use.

Preparation of Composite
Films. An aqueous solution of PVA (10% w/v) was prepared under continuous stirring at 80°C and, 10% w/w of GT based on a specifc amount of PVA was added to create PVA/GT solution [10]. Te CNC composite flm was fabricated by adding CNC suspension with varying concentrations (2% to 10%) to PVA/GT solution, denoted as PVA/GT/CNC2 to PVA/GT/CNC10. Te mixture was stirred for 30 min to obtain a homogenous state before pouring into Petri dishes and dried at 37°C for 48 h. Te dried flms were soaked in crude betel leaf extract prepared in propylene glycol (PG) at diferent concentrations (2%, 3%, and 4% w/v) for 24 h and dried at 37°C as an antimicrobial agent.

Transmission Electron Microscopy (TEM).
Dimension of CNC particles was examined by TEM (Hitachi Model HT7700, Japan). Briefy, 0.01% w/v CNC suspension was deposited on a carbon-coated copper grid, poststained with 2% w/v uranyl acetate solution and dried for 8 min. Te sample was analyzed with an accelerating voltage of 100 kV [55]. CNC dimensions were determined using the ImageJ program.

Optical and Transparency
Measurement. Film transparency was determined using a Genesys 10 S UV-Vis spectrophotometer (Termo Fisher Scientifc, USA) at a wavelength of 560 nm [56]. Film specimens were cut into rectangular shapes (2 × 40 mm) and placed inside the spectrophotometer cells. An empty spectrophotometer cell was set as a blank. All measurements were conducted in triplicate and the percentage of flm transparency was calculated using the following equation [57]: where T f and T b are the transmittance values of the flm sample and blank cell, respectively.

Scanning Electron Microscopy (SEM).
Surface and cross section of the composite flms were investigated by SEM (Hitachi Model, Joel JSM5600LV, Japan) with an accelerating voltage of 15-20 kV [31]. To observe the cross section, flm specimens were freeze-cracked following immersion in liquid nitrogen. Each piece was deposited on a cylindrical holder and coated with a thin gold layer (5-10 nm thickness) before observation.

Fourier Transform Infrared (FTIR) Spectrometry Analysis.
Te PVA/GT/CNC composite flms were cut into 10 × 10 mm squares at random locations. Infrared spectra of each sample were recorded using an FTIR (Bruker Tensor 27 Spectrometer, USA) in the range of 4000 − 500 cm −1 with a resolution of 4 cm −1 in attenuated total refection (ATR) mode.

Swelling Ratio and Stability.
Swelling ratio and stability of the composite flms were evaluated based on the amount of water absorbed and percentage of weight loss in 7 days, respectively. Briefy, dried flm specimens (15 × 15 mm) were immersed in DI water at ambient temperature. For each turn of measurement [58], swelling ratio was assessed by taking the weight of swollen samples. To examine the stability of composite flms, the swollen samples were taken and dried again to determine the diference in dried weight before and after immersion. All samples were weighed by an analytical balance in triplicate. Te results were calculated using the following equations [13]: where M 0 and M 1 represent sample weight before and after immersion, respectively.

Mechanical Properties.
Tensile strength and elongation at break of the composite flms were evaluated using a universal testing machine (AGS-J 1 kN, Japan) according to the ASTM D882-02 standard method. Briefy, the flms were cut into rectangular shapes (10 × 50 mm) and kept at 25°C with relative humidity of 50% ± 2% until reaching constant weight. Te flms were tightly fxed in the grips with 30 mm initial space and pulled apart by a 1 kN load cell. Te experiment was conducted in triplicate. Te tensile strength International Journal of Biomaterials 3 and elongation at break were calculated from the following equations, respectively [57]: where F, T, and X are the maximum force, flm thickness, and width of flm, respectively.
where D 1 is the distance of rupture and D 0 is the initial distance between grips.
2.10. Cytotoxicity. Cytotoxicity test was conducted using the colorimetric assay which is based on the ability of the cells (cellular oxidoreductase enzymes) to reduce the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its insoluble formazan, following the ISO 10993-5 standard [59]. Mouse fbroblast cells L929 (NCTC clone 929 : CCL1 from the American Type Culture Collection (ATCC), strain L) were cultured in minimum essential medium (MEM) with an appropriate cell density of 10 5 cells/ ml and incubated for 24 ± 2 h. Te flms were sterilized by UV irritation for 15 min, prepared at 3 cm 2 /ml, and extracted for 24 ± 2 h at 37 ± 1°C. Te extracts were further incubated for 24 ± 2 h before being stained with MTT assay for 2 h. A Termanox (Nunc) coverslip was used as the negative control, while polyurethane flm containing 0.1% zinc diethyldithiocarbamate served as the positive control. After culturing with fbroblast cells for 24 h, tested samples were compared to the negative and positive controls. A microplate reader was used to determine cell viability at absorbance of 570 nm using equation (6) [25]. Cell viability above 70% from equation (6) was considered noncytotoxic.
where OD sample and OD blank are measurements of optical density of the test sample and blank sample, respectively.
2.11. Antimicrobial Ability. Antimicrobial property of the flms loaded with the crude betel leaf extract was studied against Staphylococcus aureus DMST 8840 and Pseudomonas aeruginosa TISTR 781 using the agar difusion method. Before the experiment, S. aureus and P. aeruginosa suspensions of 10 6 CFU/ml were evenly spread over nutrient agar plates. Films loaded with the betel leaf extract were placed on an inoculated agar surface, with paper discs loaded with erythromycin used as the positive control and PG as the negative control. Te culture plates were incubated at 37°C for 18 h, and the inhibition zone was measured at the end of the incubation time using digital Vernier caliper [60].

Statistical Analysis.
Te statistical analysis of data was done by one-way analysis of variance (ANOVA) at a confdence level of 0.05 using the Minitab program. All data were expressed as mean ± standard deviation. A p value <0.05 was considered statistically signifcant.

Results and Discussion
3.1. Characterization of Nanocellulose. SCB (Figure 1(a)) consists of cellulosic and noncellulosic components. A series of treatment steps are involved in removing the noncellulosic components (hemicellulose and lignin) to promote defbrillation. Removal of major noncellulosic components increased the cellulose content (Figure 1(b)). Te bleached fber was subsequently subjected to acid hydrolysis that caused disordering in the glycosidic linkages and breakdown of the fbrous structures. Tese resulted in the reduction of fber size to produce CNCs. Te crystalline CNCs were presented as a stable colloidal suspension (Figure 1(c)). Te surface of CNCs was linked to negatively charged particles of sulfate half ester groups derived from the acid hydrolysis process [35]. TEM analysis was carried out to determine particle size and distribution of CNCs. Results showed needle-like shapes of individual and aggregated particles (Figure 1(d)). Dimensions of the CNCs were approximately 104 nm in length and 7 nm in width, similar to sizes reported earlier [61,62]. Te stable colloidal property coupled with nanosize of particles make the CNCs an ideal candidate in composite flm preparation.  [63] and aggregation of CNCs as the concentration increased [64]. Optical properties also relate to the rearrangement in the internal structure of PVA molecules during the drying process [63,65]. Although the opaqueness of flms increased at higher CNC concentration (Table 1), visual observation showed they are all colorless and transparent ( Figure 2). Te flm is ideal for wound dressings, as it will allow daily observation without its removal and prevents damage to the wound bed [49]. Optically transparent wound dressings ofer an opportunity to monitor the wound healing progress without having to replace the dressing. In addition, the direct naked-eye observation is the most appropriate and efective way for detecting wound infection during the healing process [66,67]. Films with the lowest and highest concentrations of CNCs were, therefore, selected for SEM analysis. (Figure 3) shows the surface and cross section of neat PVA, PVA/GT, and PVA/GT/ CNC flms. Te neat PVA flm had a uniform texture with a smooth planer surface and cross section (Figures 3(a) and 3(e)). However, the addition of GT changed the flm surface to slightly rough, characterized by the presence of white dots on the surface and cracks in the cross section (Figures 3(b) and 3(f )). Mostafavi et al. [57] reported that the chemical structure of GT could organize into a more open and porous network [47]. Similarly, the flm containing GT showed reduced homogeneous quality [65,68]. Dispersion of CNCs in the PVA/GT flms (Figure 3(c)) resulted in the disappearance of some white dots on the surface structure, suggesting that CNC particles flled in the polymeric matrix. Increasing the CNC content led to assemblage and cluster formation of CNCs in the flm (Figure 3(d)). Tis result agreed with a previous study by Jahan et al. [69]. Te CNC particles were observed on the fractured shapes in the cross section (Figures 3(g) and 3(h)). Te nanocellulose reinforced polymer network formed aggregates with a wide range of sizes and shapes in random directions. However, higher concentrations of nanocellulose induced brittle fracture due to the aggregation at localized points. Figure 4 shows the FTIR analysis of chemical functional groups of GT powder and PVA, PVA/ GT, and PVA/GT/CNC flms. Te FTIR spectra for GT powder with an absorption band at 2149 cm −1 corresponded to various carbonyl groups in the gum while peaks of carbonyl stretching in aldehydes, ketones, and carboxylic acids were presented at 1750 cm −1 [70]. Te bands at 1635 cm −1 and 1442 cm −1 were attributed to asymmetrical and symmetrical stretching of carboxylate groups, respectively, while peaks at 1242 cm −1 and 1020 cm −1 displayed C-O stretching vibration in polyols and alcoholic groups, respectively [22,24,25]. Te band observed in all samples at 3285 cm −1 was characteristic of O-H stretching groups from intra-and intermolecular hydrogen bonds [15,69] while a wider band of O-H stretching in the GT structure observed at 3420 cm −1 was caused by OH and COOH groups [25,57].

International Journal of Biomaterials
Asymmetrical and symmetrical stretching vibrations of methylene groups were presented at 2939 cm −1 and 2908 cm −1 , respectively [24], while the peak at 1086 cm −1 was assigned to C-O stretching [57,69]. Vinyl C-H in plane bending of PVA was confrmed at 1419 cm −1 . Furthermore, the absorption band centered at 842 cm −1 represented C�C bending [71,72]. No signifcant changes were observed in the chemical structure of PVA after the incorporation of GT and CNCs. Notably, addition of CNCs at high concentration contributed C�C stretching at 1655 cm −1 [73].   Figure 5 show the swelling ability of the various flms. At the initial stage, the large number of free hydroxyl groups in the PVA flm absorbed water molecules to reach a maximum state of approximately 260% before reducing at 1.5 h. PVA/GT flms increased gradually and reached a steady rate of 250% due to the presence of hydrogen bonds in hydroxyl and carboxyl functional groups [70]. Te porous structure of GT helped in trapping water molecules [20,68,74]. After 6 h, swelling ability slightly decreased to 230% for 7 days due to degradations in the polymer matrix [75]. Tere were no obvious diferences in PVA/GT/CNC2 and PVA/GT flms. Addition of small amounts of CNCs increased water absorption in the polymer matrix. However, as CNC concentration increased, extra swelling capacity was restrained [58,69,76] due to the reinforcing efects of CNCs. Tis reduction illustrated the hydrogen bonding between CNCs and polymers. Tus, water molecules could not freely pass through the polymer [76,77]. Te PVA/GT/CNC2 flm provided swelling behavior that could create an environment suitable for wound healing [14,17,78,79]. Swelling of wound dressing is an important factor that relates to wound exudate absorption and prevention of infection. Tis is as a result of physical and chemical changes in material structure that help water molecules to difuse internally, leading to an increase in free volume [69,78,80].

Stability.
Stability of the wound dressing was assessed as the percentage weight loss of composite flm over a period of 7 days to indicate its prolonged use. Te result in Figure 6 shows that the PVA flm exhibited the highest rate of weight loss of 9.8% on the frst day, which increased to 17.2% on the 7 th day due to solvation and fragmentation of the flm [80].
Tere was signifcant diference between the neat PVA and the composite flms. Te addition of GT improved the flm stability by maintaining weight loss below 12% for 7 days. Bassorin fragments in GT are insoluble in water; these probably reduced the flm's solubility [81]. Te solubility was further reduced when CNCs were added. Tis could be explained as due to the formation of a strong matrix of hydrogen bonds through the three-dimensional structure of CNCs and the polymeric matrix that reduced free hydroxyl groups and restricted water penetration [58,77].

Mechanical Properties.
Mechanical properties of the flms are summarized in Table 2. Wound dressing should be strong, fexible, and elastic for efcient treatment; thus, the flm was evaluated in terms of tensile strength, elongation at break, and elastic modulus. Te neat PVA flm exhibited a tensile strength of 54.63 MPa, which reduced slightly to 49.26 MPa when GT was added. Tere was no signifcant diference (p < 0.05) between neat PVA and PVA/GT. Tis result agreed with Ojagh et al. [82] who found that the addition of GT had no signifcant efect on mechanical properties. Highest tensile strength was recorded in PVA/ GT/CNC2 (80.39 MPa), while PVA/GT/CNC6 gave the lowest strength of 45.05 ± 3.39 MPa. Te addition of CNCs increased the strength of the material by entrapping inside the matrix. Tis allowed strong hydrogen bond formation between the nanocellulose and PVA matrix, thus impacting mechanical integrity [61,83]. However, high concentration of CNCs led to agglomeration of particles, increased rigidity, and poor distribution in polymer matrices, which afected the formation of hydrogen bonds among polymer chains and inhibited reinforcing properties [15,71]. Elastic modulus of PVA and PVA/GT was 1223.08 ± 182.08 MPa and 1062.51 ± 101.65 MPa, respectively. Highest elastic modulus of 1526.11 ± 31.86 MPa was recorded in the PVA/GT/CNC2 flm, which decreased to 1260.45 ± 76.94 MPa in PVA/GT/ CNC10. Te high value was attributed to the crystalline nature of CNCs that resulted in better alignment and enhanced elastic modulus [69,83]. Tere was no signifcant diference in elastic modulus of neat PVA and composite flms loaded with CNCs, which implies the nanoparticles has no negative efect on the elastic nature of PVA. Neat PVA had elongation at break of 48.52% and this slightly decreased to 44.48% in PVA/GT. However, elongation at break of PVA/GT/CNC flms decreased drastically with increasing concentration of CNCs. Te elongation at break signifcantly decreased to 8.11% in PVA/GT/CNC2 and dropped further to 3.92% in the PVA/GT/CNC10 flm due to the rigid nature of CNCs. Since CNCs are nondeformable, strong interaction between CNCs and the polymer matrix did not allow elongation in the composite materials [84]. Te tensile strength of wound dressings should be adequate for application and storage to ensure that it is not easily damaged by handling [85,86]. Terefore, PVA/GT/CNC composite materials in the range of 45-80 MPa showed good mechanical properties compared to previous studies [86][87][88] for wound dressing. Te stress-strain curves of PVA, PVA/ GT, and PVA/GT/CNC with diferent concentrations of CNC are presented in Figure 7.
3.2.7. Cytotoxicity. Te cytotoxicity of PVA, PVA/GT, and PVA/GT/CNC10 flms was evaluated using the MTT assay. Te sample with the highest CNC concentration (PVA/GT/ CNC10) was tested to confrm nontoxicity and safety of the flm for cell growth. Te result in Figure 8 shows all the test samples except that the positive controls (which is expected) are nontoxic to fbroblast cells with reference to ISO 10993-5 standard with acceptable limit of 70% cell viability [59,89]. Cell viability of PVA and PVA/GT was 93% and 84%, respectively. Te decrease in cell viability of the PVA/GT flm may be due to the diversity in chemical composition that slightly infuenced the biological properties of the flm [20]. Te PVA/GT/CNC10 flm, which contains high concentration of CNCs, was found to be nontoxic to fbroblast cells with 95% cell viability. Te value is closer to that of the negative control of 99% cell viability. Incorporation of CNCs in the polymeric matrix and reaction between CNCs and PVA/GT improved cell viability in the flms. Te flm satisfed noncytotoxic requirement of cell viability above 70% in agreement with previous reports [24,89,90].  International Journal of Biomaterials 3.2.8. Antibacterial Activity. Figure 9 shows clear zones around discs, which increased with increasing concentration of the betel leaf extract. Disc difusion result revealed that flms loaded with the betel leaf extract at diferent concentrations of 2%, 3%, and 4%, exhibited excellent antibacterial activity against both gram-negative (P. aeruginosa) and gram-positive (S. aureus) bacteria. Tere was high signifcant diference in inhibition zone between the positive control (erythromycin) and the flms loaded with extracts ( Table 3). As expected, there was no inhibition zone in the negative control. Te flm loaded with 4% extract (PVA/GT/ CNC2_4%) recorded the highest inhibition zone of   International Journal of Biomaterials 9 23.60 ± 0.55 mm and 28.20 ± 0.84 mm for P. aeruginosa (Figure 9(a)) and S. aureus (Figure 9(b)), respectively. In all, S. aureus is more susceptible than P. aeruginosa which may be as a result of the diferences in their cell wall structure [91,92]. Te cell wall of S. aureus has only peptidoglycan layer, while that of P. aeruginosa is made of three layers such as the inner or cytoplasmic membrane, peptidoglycan layer, and outer membrane [91,93,94]. Te betel leaf extract contained an essential oil and phenolic compounds which are reported to inhibit bacterial cell growth [95][96][97]. Te presence of oxygenated terpenoids including alcohols and phenolic terpenes [96] as well as hydroxyl groups in hydrophobic fatty acids and fatty acid ester components caused destabilization of the cytoplasmic membrane, disrupted proton and electron fow, and decreased adenosine triphosphate (ATP) synthesis, leading to cell death [44,98].

Conclusions
Te PVA/GT/CNC composite flm was successfully prepared as wound dressing material. Te flms exhibited higher swelling ratio and mechanical properties with good transparency, which are ideal for wound dressing. Tese properties could help to absorb exudates with easy daily observation and minimize trauma to the wound bed.
Incorporation of CNCs from 2% to 10% improved the physicochemical properties of the flm. Te PVA/GT/CNC2 showed excellent properties among the CNC-based composite flms. Apart from the physicochemical enhancements, the cytocompatibility was also improved. Te flm loaded with the betel leaf extract exhibited excellent antibacterial activity against P. aeruginosa and S. aureus strains. Terefore, the prepared nanocomposite flm with good features will provide the optimum conditions for wound healing, especially in cutaneous wound dressing application. Te visual inspection property of PVA/GT/CNC coupled with antibacterial activity from the betel leaf extract and other excellent physiochemical characteristics of the flm are suitable for wound healing application. Tis dressing can protect the wound surface from infection and dehydration, which deprives wound tissues of oxygen and the nutrients necessary for healing.

Data Availability
Te data that support the fndings of this study are available upon reasonable request from the corresponding author.

Disclosure
A preprint of this article has previously been published. For values with the same letter, the diference is not statistically signifcant, while diferent letters mean statistical signifcance (p ≤ 0.05).

Conflicts of Interest
Te authors declare that they have no conficts of interest.