Development of a Novel Film Based on Bacterial Nanocellulose Reinforced Gelatin/Guar Gum Containing Honey for Wound Healing Applications

Bacterial nanocellulose (BNC) is a type of 3-dimensionally structured polymer gel produced by Acetobacter that has recently attracted increased interest in wound healing concerns. To produce an effective antibacterial wound dressing, researchers investigated the manufacturing and structural features of honey-infused BNC reinforced gelatin/aldehyde-modied Guar gum �lms (H/BNC/Ge/AD-GG). Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), mechanical characteristics, water solubility, and degradability were all used to assess the produced �lms. In addition, the in�uence of honey addition on the produced �lms' various properties has been examined. Antibacterial activity, better degradation capability, improved mechanical qualities, and excellent cell adhesion and proliferation by NIH-3T3 �broblast cells were among the outcomes. The cytotoxicity assay in vitro revealed good cytocompatibility. As a result of the �ndings, the produced H/BNC/Ge/AD-GG �lms appear to have a high potential for antibacterial wound dressing applications.


Introduction
Wound healing is a complex and di cult process that has been studied in various ways ( Bacterial nanocellulose (BNC) is a suitable biological material that meets all the requirements of modern wound dressings (Bielecki et al. 2016). Conventional cellulose dressings are 100 times thicker than BNC bers, which makes BNC bers a completely biocompatible hydropolymer that is also mechanically stable. BNC bers also act as an obstacle against microbial contamination and are therefore useful in making high-performance wound dressings (Moritz et al. 2014;dos Santos et al. 2018). Wound dressings based on BNC have also been shown to induce gas exchange, moisten the wound environment, and absorb secretions (Fu et al. 2013). Suprasorb Ò X + PHMB is a commercial BNC product containing polyhexanide marketed as antiseptic wound dressing for different wounds with different amounts of exudation (Kingley et al. 2009;Dissemond et al. 2010). Guar gum (GG), on the other hand, a biopolymer derived from the seeds of Cyamopsis tetragonoloba has no toxic effect (Ghosh Auddy et al. 2013). Due to its high biodegradability and biocompatibility, this natural material is applied in many biological processes (Thakur et al. 2009), especially as a bioabsorbable material for wound dressing (Bajpai and Raj 2021). In order to expand the use of GG in various elds, its chemical properties can be easily modi ed by surface functionalization (Das et  In view of the above, in this study we aimed to fabricate an antibacterial lm as an effective wound dressing based on honey, BNC reinforced gelatin and aldehyde-modi ed GG. Finally, we developed an antibacterial lm with good antibacterial activity, excellent biocompatibility, increased degradability, and improved mechanical properties, thus succeeding in providing a new wound dressing (H/BNC/Ge/AD-GG) with improved properties.

Synthesis of aldehyde-modi ed Guar gum (AD-GG)
AD-GG was synthesized according to the previous study (1). Firstly, GG solution (0.25 wt%) was prepared by dissolving 0.25 gram GG in 100 ml deionized water at room temperature under stirring. After that, GG solution was oxidized by the addition of 3 mL sodium periodate (0.1 g/mL) solution and the reaction was continued at this condition in the dark place. After 3 hours, the reaction was nished by the addition of 150 μL ethylene glycol to eliminate the excess periodate. Finally, the obtained AD-GG solution was dialyzed with deionized water for 3 days using dialysis membrane (MWCO: 12-14000).

Preparation of bacterial nanocellulose (BNC) reinforced gelatin-AD-GG lms (BNC/Ge/AD-GG)
The Ge powder was dissolved in deionized water at 50°c under stirring to obtain 4% w/v solution. Also, ethylene glycol, as a plasticizer was added to the Ge solution (40% dry weight of the Ge). BNC water dispersions was prepared at 50% 25%, and 12.5% concentration from dilutions of the original source of BNC suspension in deionized water under sonication. Then, BNC suspensions with the predetermined amounts (0.5 ml from the original source and each prepared dispersion of BNC suspension) were separately added to the Ge solution and stirred at 40 °C for 15 min. Finally, the lms were prepared by blending the BNC reinforced Ge and AD-GG solutions with the ratio of 1:2 respectively (with total volume of 15 ml) and were poured into petri dishes to dry at the room temperature for 48 hours. Table 1 shows the formulations of the prepared different sample lms.  The samples chemical structures were determined using FTIR spectroscopy (Bruker-Tensor 27) at the wavenumber range of 400-4000 cm -1 .

Thermogravimetric analysis (TGA)
To assess the thermal stability and decomposition temperature of the samples, TGA was carried out by TGA instrument (LINSEIS SPA PT 1600 device, Germany) from 25 °C to 700 °C, under a nitrogen atmosphere ow.

Mechanical behaviors of the fabricated lms
The mechanical behaviors of the fabricated lms were investigated by tensile analyzer (SANTAM Machine, Iran) according to the guidelines established by ASTM. Brie y, the lms were cut into the dumbbell-shaped (with 5.4×3 mm sizes) and placed between two grips; the test speed was set at 10 mm/min.

Morphology of the fabricated lms
The surface morphology of the obtained lms was assessed by scanning electron microscopy (SEM, TESCAN, MIRA3). For this purpose, the lms were sputter-coated with a gold layer and the images were captured. where W i and W f represents the initial dry weight and nal weight of the sample at the predetermined time.

Antibacterial study
The antibacterial capacity of the fabricated lms was evaluated by disk diffusion manner on agar plate against Staphylococcus aureus (S. aureus as gram-positive) and Escherichia coli (E. coli as gramnegative) bacteria. Brie y, the optimal lm and the lm containing honey were cut into the circular pieces with 10 mm diameter, UV light sterilized, placed to Muller Hinton Agar, which was cultured with 10 6 CFU/mL of bacterial suspension, and incubated overnight at 37 ° C. The bacterial growth inhibition was assessed by diameters of the inhibition zones.

Cell viability and proliferation assays
To consider the biocompatibility of the lms, MTT assay was carried out using NIH-3T3 broblast cells (Yavari Marou and Ghorbani 2021). NIH-3T3 broblast cells were cultured in RPMI-1640 medium containing 10% FBS and 1% Pen/Strep at 37 °C with 5% CO 2 . After adequate cellular con uency, the biocompatibility evaluations were ful lled. In brief, the fabricated lms were cut into the small pieces and sterilized under UV light for 1 h. The lms were placed into the cell culture plate and the cell suspension with density of 5000 cells were added to each well. At the predetermined times (1, 3, and 5 days after seeding), the medium was taken out, 3 mg/ mL of MTT solution (in PBS) was added to each well, and incubated for 4 h at 37 °C. The MTT solution was removed and DMSO added to dissolve formazan crystals formed inside cells. Finally, the optical density (OD) of the produced violet formazan crystals was determined at 570 nm using spectrophotometer plate reader (Awareness Technologies Stat Fax 2100 Microplate Reader).

Cell attachment study by SEM
For evaluating the NIH-3T3 broblast cells attachment on the fabricated lms, the fabricated samples were cut into the small pieces and sterilized under UV light. Cells were cultured on each lm according to the previous section. After 3 days of cell seeding, following process was used to x cells on the lms: rstly, the medium was removed, lms containing cells were washed with PBS, xed with glutaraldehyde solution (4%), dehydrated by serial ethanol solutions (50%, 60%, 70%, 80%, 90%, and 100%), and nally cells were visualized by SEM imaging.

Statistical analysis
Values were reported as mean ± SD. Statistical differences (P < 0.05) were detected by One-way variance (ANOVA) test by using GraphPad Prism software.

Morphological characterization of hydrogels
In this study, we fabricated antibacterial lms based on BNC reinforced with Ge/AD-GG containing honey. Fig. 1 shows the SEM images of the BNC/Ge/AD-GG lms without and with 15% of honey. The developed lms have revealed a uniform surface morphology. Moreover, the honey incorporation caused the formation of lms with some thicker diameters which related to the variation in viscosity of polymer solution via the incorporation of honey.

FTIR
The spectra of honey, Ge, pure GG, and AD-GG are revealed in Fig. 2a, b, c, and d, respectively. The pure honey spectrum appeared peaks at 3360 cm -1 and 2935 cm -1 , which were related to the O-H and C-H stretching of carboxylic acid groups, respectively. The detected bonds at 1650 cm -1 and 920 cm -1 were found to be related to the C=O bonds stretching and C-H bending of carbohydrate groups, respectively. The pure Ge FT-IR spectrum indicates the typical speci c amide bands of proteins. The characterization absorption bond at 3400 cm -1 is represented to the O-H and N-H stretching vibration. The C-H bonds stretching vibration was revealed at 2930 cm -1 . The pure GG spectrum appeared absorption bonds at 3350 cm -1 and 2910 cm -1 , which were represented to the O-H and C-H stretching, respectively. The absorption peak at 1124 cm -1 was found to be assigned to the C-O bonds stretching. The aldehyde agents formation through the oxidation process of GG and its conversion to AD-GG led to the two absorption peaks reveals at ~1740 cm -1 and 889 cm -1 in FTIR spectrum. Besides, the FTIR spectra of pure BNC, Ge/AD-GG, BNC/Ge/AD-GG, and H/BNC/Ge/AD-GG were revealed in Fig. 3a, b, c, and d, respectively.
For pure BNC, a broad characteristic bond at 3400 cm -1 and 2940 cm -1 were attributed to O-H and aliphatic C-H stretching vibration, respectively. Another intense peak located at 1600 cm -1 was related to C-O-C stretching vibration. To verify the covalent interaction among the NH 2 of Ge and the aldehyde agents of AD-GG, the spectrum of optimum Ge/AD-GG hydrogel (Ge/AD-GG 1:2) demonstrated an absorption bond at 1682 cm -1 , which can be related to the newly created amide groups . For the FTIR spectra of BNC/Ge/AD-GG and H/BNC/Ge/AD-GG lms, all characteristic peaks of BNC remained to be present, moreover, some additional peaks appeared in the spectrum from the characteristic groups of Ge, honey, and AD-GG. Moreover, the revealed some shifted at the wavenumber and intensity of absorption peaks are due to the impact of honey interactions with BNC/Ge/AD-GG lms.

TGA analysis
The TGA thermograms of the pure Ge, GG, Ge/AD-GG, BNC/Ge/AD-GG, and H/BNC/Ge/AD-GG were speci ed in Fig. 4. For all samples, as can realize from these thermograms, the rst stage of weight loss (25-150 •C) was assigned to the loss of moisture and free water content adsorbed in the prepared lms. The second and main weight loss happened at around range of 250-350 •C which is generally related to the polymer structure thermal decomposition as well as the degradation of the honey components followed by honey contents carbonization. The covalent cross-links presence in the structure of Ge/AD-GG hydrogel between dialdehyde groups of AD-GG and amine groups of Ge has led to thermal durability.

Mechanical property
The tensile stress behavior of developed lms with BNC and with varying ratios of Honey 5%, 10%, and 15% are shown in Fig. 5. The good strength of developed lms along with the presence of DAGG, is owing to the cross-linking between the amine groups of Ge and dialdehyde groups DAGG via a covalent bond. The prepared lms with BNC incorporation showed fewer mechanical properties (Fig. 5A). Generally, BC is regarded as lacking elastic properties. As a result, the honey incorporation in developed lms led to improved mechanical strength properties (Fig. 5B). Owing to the creation of more interpolymer bonds and enhanced physical cross-linking density via the honey addition, the hydrogel mechanical strength was improved noticeably ).

Swelling studies
The swelling pro le of the prepared BNC/Ge/AD-GG lms without (control) and with different honey concentrations of 5%, 10%, and 15% are revealed in Fig. 6. It was detected that although the prepared neat BNC/Ge/AD-GG lms were revealed a high water absorption capacity. But, as detected in Fig.6, adding and increasing the honey concentration within the BNC/Ge/AD-GG lms reduced its swelling capability. Based on the literature review, honey is recognized for its great water uptake capability and high water solubility (Mohd Zohdi et al. 2012). Such high water solubility results in increasing the degradation degrees of lms and so losing their compacted porous structure that can maintain water. So, this nally affects considerably reduction in swelling capacity. This was con rmed in the very low swelling abilities of the BNC/Ge/AD-GG lms with 15% of honey after 5 h.

In vitro degradation study
The degradation behavior of the developed lms following the enhancing of honey concentration was studied for 24 and 48 h. As revealed in Fig. 7, via increasing the concentration of honey inside BNC/Ge/AD-GG lms, the improved degradation ability was detected after incubation in PBS at 37 •C for about 48 h. This can be attributed to the great water solubility of honey. Therefore, in the developed BNC/Ge/AD-GG lms containing a higher concentration of honey (15%), the degradation rate improvement was illustrated.

In vitro biocompatibility study
In this study, comparing the pure lms as control, the viability of the 3T3 cells treated with BNC/Ge/AD-GG lms after 1, 3, and 5 days was tested via the WST1 assay. Fig. 8 revealed the cell viability of neat BNC/Ge/AD-GG lms and BNC/Ge/AD-GG with different content of honey (5, 10, and15 wt%) near 100% and con rmed good viability with increasing the weight addition of honey, showing that these obtained lms are nontoxic and have excellent biocompatibility.

Proliferation Test
The potential of prepared lms to support the attachment, growth, and cell proliferation is critical for tissue engineering applications. Fig. 9 revealed the NIH 3T3 broblast cells adhesion and penetration on BNC/Ge/AD-GG lms without and with various content of honey (5%, 10%, and 15 wt%) via SEM technique after culture for overnight. As shown in this Figure, the broblast cells were attached properly on the BNC/Ge/AD-GG lms surface that revealed the ne interaction between the prepared lm and cells.
The cell growth behavior of BNC/Ge/AD-GG lms showed improved cell adhesion with the 15 wt% of honey. So, according to the previous investigations [53], the incorporation of honey into the scaffolds may increase signals and endorsing cellular adhesion and proliferation. These obtained results con rmed that the developed H/BNC/Ge/AD-GG lm is suitable for adhesion, growth, and cell proliferation, and has no cytotoxicity effect on broblast cells.
3.9. Antimicrobial activity Fig. 10 and Table 2 show the inhibition activity against E. coli (Gram-negative) and S. aureus (Grampositive) bacteria for the blank lms and honey incorporation in BNC/Ge/AD-GG lm. The blank lms as the control group represented no inhibition activity against selected bacteria. The highest inhibition zones were detected by BNC/Ge/AD-GG lms with 15% of honey which those values were 13.0 ± 0.1 mm (against S. aureus) and 15.0 ± 1.0 mm (against E. coli). The antibacterial property of honey is represented by the high sugar content, acidity as well as ability for producing hydrogen peroxide. Hence, the honey incorporated BNC/Ge/AD-GG lms revealed inhibition zones against selected bacteria. Related to similar previous reports [49], it could be noted that via increasing the honey concentration, the inhibition zone values were increased against both selected bacteria. Accordingly, the highest inhibition zones were achieved by developed lms containing 15 wt% of honey.

Conclusion
This paper describes a novel method for creating a high-yield antibacterial lm using honey-infused BNC reinforced gelatin/aldehyde-modi ed Guar gum (H/BNC/Ge/AD-GG). Honey is a natural wound healer that has been reintroduced to clinical ulcer treatment due to its potent antibacterial properties. Various methodologies were used to investigate the physicochemical and biological aspects of successful produced lms with and without honey. Honey content had a noticeable effect on the swelling behavior of hydrogel lms. As a result, lms containing 5% honey showed the most swelling, whereas lms containing 15% honey showed the least swelling. The produced lms demonstrated signi cant antibacterial e cacy against gram-positive and gram-negative bacteria, as well as cytocompatibility with human skin broblast cell lines. In addition, increased honey concentration resulted in improved mechanical properties, great cell adhesion, growth, and proliferation. Accordingly, the produced H/BNC/Ge/AD-GG lms appear to have good promise as an antibacterial dressing for wound healing.

Declarations
Ethics approval The study procedure was approved by the Ethical Committee of Tabriz University of Medical Science (Approval ID: IR. TBZMED.VCR.REC.1398.443).