3.2.6. X-ray Diffraction

The results of the XRD analyses of AX, SA, F3 (blank film), and GF3 (GS-loaded film) are presented in Figure 4. SA is semi-crystalline and exhibits characteristic peaks at around 2*θ* 13°, 56°, 20.6°, 20.1°, 29° and 36.4° [45]. The purpose of performing XRD analyses was to investigate the effect of combining film components on the semi-crystalline nature of the SA in AXSA films. The XRD pattern of SA had peaks between 2θ 13° to 38°, whereas AX did not show any sharp peaks attributed to its amorphous nature. The intensities of the semi-crystalline peaks of the SA were reduced in the AXSA films (F3 and F4). This reduction in the peak intensities may originate from the hydrogen bonding interaction of the SA with other amorphous components of the films (AX, Gly, and GS). Previously, Ramakrishnan et al. 2021 suggested a similar decrease in the SA crystallinity due to hydrogen bonding for SA/gum kondagogu blended films [43]. 3.2.6. X-ray DiffractionThe results of the XRD analyses of AX, SA, F3 (blank film), and GF3 (GS-loaded film) are presented in Figure 4. SA is semi-crystalline and exhibits characteristic peaks at around 2*θ* 13◦ , 56◦ , 20.6◦ , 20.1◦ , 29◦ and 36.4◦ [45]. The purpose of performing XRD analyses was to investigate the effect of combining film components on the semi-crystalline nature of the SA in AXSA films. The XRD pattern of SA had peaks between 2*θ* 13◦ to 38◦ , whereas AX did not show any sharp peaks attributed to its amorphous nature. The intensities of the semi-crystalline peaks of the SA were reduced in the AXSA films (F3 and F4). This reduction in the peak intensities may originate from the hydrogen bonding interaction of the SA with other amorphous components of the films (AX, Gly, and GS). Previously, Ramakrishnan et al. 2021 suggested a similar decrease in the SA crystallinity due to hydrogen bonding for SA/gum kondagogu blended films [43].

**Figure 4.** XRD, DSC, TGA and DTG analyses of the AXSA films. **Figure 4.** XRD, DSC, TGA and DTG analyses of the AXSA films.

#### 3.2.7. Thermogravimetric Analyses (TGA) 3.2.7. Thermogravimetric Analyses (TGA)

The thermal degradation and stability of the AXSA films were investigated by TGA. The TG and DTG curves of the blank (F2, F3 and F3) and GS-loaded (GF2, GF3 and GF4) films are presented in Figure 3, while Figure S2 shows the results of the TGA of film components (AX, SA, Gly, and GS). In the first step of thermal degradation, all tested films and pure components (except Gly) exhibited weight loss up to ~120 °C, which was attributable to the loss of free water molecules and water interacting with hydroxyl and carboxylic groups [27]. Glycerol underwent characteristic single-step degradation at temperature range (*Trange*) ~125–300 °C (weight loss (∆W) = 99.3%) with maximum weight loss temperature (*Tmax*) at 256.2 °C. The TG and DTG curves show that AX exhibited degradation at *Trange* < 120 °C (∆W = 5.1%), 260‒320 °C (∆W = 15.8%, *Tmax* = 291), 325‒395 °C (∆W = 28.9%, *Tmax* = 354 °C), and > 395 °C (∆W = 21.7%) attributed to the loss of water, AX backbone and complete pyrolysis [32]. On the other hand, SA showed thermal decomposition at *Trange* < 105 °C (∆W = 5.7%), 265‒415 °C (∆W = 71.3%, *Tmax* = 330 °C), and > 415 °C (∆W = 12.5%) due to water loss, fragmentation of polysaccharide backbone of SA and complete degradation [27]. Similarly, GS underwent thermal degradation at *Trange* < 120 °C (∆W = 13.7%), 245‒295 °C (∆W = 13.4%, *Tmax <sup>=</sup>* 274 °C), 295‒370 °C (∆W = 25.9%, *Tmax* = 330 °C), and >370 °C (∆W = 35.5%), which shows that thermal decomposition of GS starts at The thermal degradation and stability of the AXSA films were investigated by TGA. The TG and DTG curves of the blank (F2, F3 and F3) and GS-loaded (GF2, GF3 and GF4) films are presented in Figure 3, while Figure S2 shows the results of the TGA of film components (AX, SA, Gly, and GS). In the first step of thermal degradation, all tested films and pure components (except Gly) exhibited weight loss up to ~120 ◦C, which was attributable to the loss of free water molecules and water interacting with hydroxyl and carboxylic groups [27]. Glycerol underwent characteristic single-step degradation at temperature range (*Trange*) ~125–300 ◦C (weight loss (∆W) = 99.3%) with maximum weight loss temperature (*Tmax*) at 256.2 ◦C. The TG and DTG curves show that AX exhibited degradation at *Trange* < 120 ◦C (∆W = 5.1%), 260–320 ◦C (∆W = 15.8%, *Tmax* = 291), 325–395 ◦C (∆W = 28.9%, *Tmax* = 354 ◦C), and > 395 ◦C (∆W = 21.7%) attributed to the loss of water, AX backbone and complete pyrolysis [32]. On the other hand, SA showed thermal decomposition at *Trange* < 105 ◦C (∆W = 5.7%), 265–415 ◦C (∆W = 71.3%, *Tmax* = 330 ◦C), and >415 ◦C (∆W = 12.5%) due to water loss, fragmentation of polysaccharide backbone of SA and complete degradation [27]. Similarly, GS underwent thermal degradation at *Trange* < 120 ◦C (∆W = 13.7%), 245–295 ◦C (∆W = 13.4%, *Tmax* = 274 ◦C), 295–370 ◦C (∆W = 25.9%, *Tmax* = 330 ◦C), and >370 ◦C (∆W = 35.5%), which shows that thermal decomposition of GS starts at 245 ◦C [46].

245 °C [46]. The F2 and GF2 films exhibited similar thermal degradation events at Trange <120 °C (∆W up to 8%), 125‒245 °C (∆W up to 19.8%), 250‒430 °C (∆W up to 47.1%), and >430 °C (∆W up to 18.8%). Similarly, F3 and GF3 films show similar degradation events at Trange <120 °C (∆W up to 7.2%), 140‒310 °C (∆W up to 36.6%), 310‒410 °C (∆W up to 28.8%), and >410 °C (∆W up to 20.6%). These results suggest that AXSA films undergo The F2 and GF2 films exhibited similar thermal degradation events at Trange <120 ◦C (∆W up to 8%), 125–245 ◦C (∆W up to 19.8%), 250–430 ◦C (∆W up to 47.1%), and >430 ◦C (∆W up to 18.8%). Similarly, F3 and GF3 films show similar degradation events at Trange <120 ◦C (∆W up to 7.2%), 140–310 ◦C (∆W up to 36.6%), 310–410 ◦C (∆W up to 28.8%), and >410 ◦C (∆W up to 20.6%). These results suggest that AXSA films undergo four main events of thermal degradation corresponding to the degradation events of the individual

components of these films. However, the effect of the composition of the AXSA films was observed in the TGA of the films. In F3 and GF3 films, the second degradation event was broader than F2 and GF2 due to the higher AX contents in the F3 and GF3 films, resulting in the merging of the degradation step for Gly and the first step for AX. Similarly, the second degradation step was slightly broader in GS-loaded films than in the blank films due to the merging of degradations steps for Gly and the first degradation step of GS in GS-loaded films. The TG curves of F4 and GF4 also exhibited similar four-step thermal degradation behavior. It was observed that due to the plasticizer's effect, the onset temperature for polysaccharide (AX and SA) degradation shifted to a lower temperature in films vs. pure polysaccharides. These shifts in the TG and DTG curves for the AXSA films indicate the miscibility of the components [47]. These findings for the thermogravimetric analysis of the AXSA films are consistent with previous studies on AX and SA-based films [27,32,33].

#### 3.2.8. Differential Scanning Calorimetry (DSC)

The results of the DSC analyses of the AXSA films (F2, F3, F4 GF2, GF3, and GF4) are depicted in Figure 4, while DSC curves of AX, SA, and GS are given in Figure S4. The DSC curves of the tested samples exhibited endotherms from 60 ◦C up to ~125 ◦C, indicating the removal of the water molecules. SA exhibited prominent exotherm at 297 ◦C, corresponding to the thermal degradation of the SA polysaccharide backbone as discussed above [27]. The DSC thermogram of AX showed characteristic exotherms at 290 and 312 ◦C (due to polysaccharide degradation), while a melting endotherm was found in the DSC of the GS at ~250 ◦C, which are in close agreement with the previous report [32]. On the other hand, two prominent endotherms were found in the DSC curves of the tested AXSA films at ~95 ◦C (water loss) and ~210 ◦C (due to glycerol). However, in DSC GS-loaded GF2 and GF3 films, a third endotherm was present at ~245 and 250 ◦C, respectively, attributed to the melting of GS. The second endotherm was broader in the DSC curve of the GF4, which might result from merging the endotherms due to glycerol and melting of GS. The exothermic peaks due to degradation of SA and AX were merged in the AXSA films and are present at ~310 ◦C. The DSC curves of the AXSA films did not show any significant shift in the DSC peaks of the components of the film. These findings of DSC analysis are consistent with previous studies of AX- and SA-based films and suggest that components of the AXSA films were highly miscible, and their stability was not affected during the casting process [27,32].

#### 3.2.9. Expansion Profile

Water uptake (or hydration) is a vital characteristic of polymeric materials designed for wound dressing application. After application to the wounds, the dressings should absorb water (exudate) and expand in size while maintaining their integrity and shape [48]. Hydration also plays a key role in the dissolution of the drugs entrapped in the films [38]. Therefore, the expansion profiles of the AXSA films were determined using the gelatin model [32], and the results are shown in Figure 5a,b.

The expansion profile of AXSA films shows that films hydrated (uptake water) and expanded rapidly during the first hour of contact with solidified gelatin. After that, the expansion of the AXSA films slowed down. Among the blank AXSA films, F2 exhibited the highest expansion, followed by F3 and F4, respectively. Similarly, the expansion of GF2 films was highest among the GS-loaded AXSA films. Moreover, all AXSA films maintained their shape after 24 h. Higher expansion of the F2 films can be attributed to greater SA content in these films. The carboxylic group of SA converts to carboxylate ions in water, resulting in electrostatic repulsion among the polymeric chains, leading to the film expansion [49]. Therefore, higher expansion was observed in films containing more SA. As discussed above, an increase in the AX concentration in the films results in hydrogen bonding between the polymers, leading to the formation of a denser network and a decrease in free carboxylate ions available for interaction with water [50]. These factors lead to a further reduction in the expansion of the F3 and F4 films. The expansion behavior of AXSA films was in close

agreement with previously reported simvastatin-loaded SA-pectin films [29]. However, the expansion percentage of the AXSA films was less than that previously reported for pure AX and AX-GL films, which can be explained by the hydrogen bonding interaction in the AXSA films [32,33]. Moreover, the AXSA films were intact (maintained their shape) throughout the expansion study compared to previously reported AX and AX-GL films, which started degrading after 5 to 6 in the expansion study [32,33]. Nevertheless, the expansion profile of AXSA films suggests that they hold the capacity to absorb the wound exudate while maintaining their shape and are suitable for wound dressing application [30]. tatin-loaded SA-pectin films [29]. However, the expansion percentage of the AXSA films was less than that previously reported for pure AX and AX-GL films, which can be explained by the hydrogen bonding interaction in the AXSA films [32,33]. Moreover, the AXSA films were intact (maintained their shape) throughout the expansion study compared to previously reported AX and AX-GL films, which started degrading after 5 to 6 in the expansion study [32,33]. Nevertheless, the expansion profile of AXSA films suggests that they hold the capacity to absorb the wound exudate while maintaining their shape and are suitable for wound dressing application [30].

drogen bonding between the polymers, leading to the formation of a denser network and a decrease in free carboxylate ions available for interaction with water [50]. These factors lead to a further reduction in the expansion of the F3 and F4 films. The expansion behavior of AXSA films was in close agreement with previously reported simvas-

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 13 of 18

**Figure 5.** Expansion profiles of blank (**a**) and GS-loaded (**b**) AXSA films; and (**c**) drug release profile (mean ± SD, *n =* 3). **Figure 5.** Expansion profiles of blank (**a**) and GS-loaded (**b**) AXSA films; and (**c**) drug release profile (mean ± SD, *n* = 3).

#### *3.3. Gentamicin Release Profile and Kinetics*

The GS release profile from AXSA (GF2 and GF3) films, investigated by Franz diffusion cell, is presented in Figure 5c. The GF2 and GF3 films were selected for the antibiotic release experiment owing to their better characteristics (EAB%, WVTR and expansion profile) for wound dressing application. The release study results show that GF2 and GF3 released 45% and 41% of the incorporated GS during the first hour of the release study. The GS release became slower after that, and reached equilibrium by 24 h. The maximum cumulative release after 24 h was 83.4% and 81.2% for GF2 and GF4, respectively. This initial release from the AXSA films is less than that of previously reported pure AX films and higher than that of AX-GL films [32,33]. These results suggest that 10 cm (diameter) GF2 and GF3 films will release up to 21 mg GS in 24 h. Previous studies on GS-based wound dressing materials suggest that 0.1% GS is effective for wound infections [14]. Moreover, topical delivery of 0.1% GS is considered safe for avoiding the toxic effects (ototoxicity and nephrotoxicity) associated with systematic GS administration [41,51].

The drug release from the dressing material depends on various factors, such as the solubility of the drug in the release media, relaxation, swelling and erosion of the polymeric network, and diffusion rate of the drug [33,38,52]. Therefore, mathematical models were applied to investigate the GS release kinetics and mechanism of release from the GS-loaded AXSA films. The results are shown in Table 2. The higher regression coefficient "*R <sup>2</sup>*" values indicate the best fitting model. The results (Table 2) indicate that "*R <sup>2</sup>*" values, obtained by fitting zero-order (0.4498 and 0.4243), first-order (0.543 and 0.5856), and Higuchi (0.6804 and 0.7015) equations, were less than those obtained with the Korsmeyer–Peppas equation (0.9788 and 0.9451). These results suggest that the Korsmeyer–Peppas model is the bestfitted model ("*R* <sup>2</sup>" close to 1) for GS release from GF2 and GF3 films. The values of *n* (release exponent) for the Korsmeyer–Peppas model were 0.4603 and 0.4982 for GF2 and GF3 films, respectively. According to Korsmeyer–Peppas, for thin films, *n* values below 0.5 indicate that Fickian diffusion is the mechanism governing drug release [53]. Therefore, the *n* values of GS release from the AXSA films indicate that Fickian diffusion was the predominant mechanism, with the rate of GS release being limited by solvent penetration rate. A similar release mechanism was reported by Pires et al. for extract release from chitosan and alginate membranes intended for wound dressing [54]. These findings for the GS release profile suggest that the AXSA films hold potential for the treatment of infected wounds by delivering an initial high concentration of GS, followed by slower release up to 24 h.


**Table 2.** Mathematical modeling of GS release from AXSA films.

#### *3.4. Antibacterial Effect of the Films*

The results of the antibacterial effect of GS solution, F3, GF2, and GF3 films are depicted in Figure 6a,b. In this experiment, blank AXSA film (F3) was used as a negative control, and filter paper disks dipped in 0.1% (*w*/*w*) GS solution were used as a positive control. The F3 (blank) films formed no clear inhibition zones, suggesting an insignificant antibacterial effect of AXSA films against the tested bacterial strains. However, opaque regions were observed around F2 films due to bacterial growth in the expanded blank AXSA films. In contrast, positive control (GS std.) and GS-loaded AXSA films (GF2 and GF3) exhibited significant antibacterial effects against both Gram-positive (*S. aureus*) and Gram-negative (*E. coli and P. aeruginosa*) bacteria. The values of inhibition zones for GF2 and GF3 were slightly higher than for the positive control. The antibacterial effect of the films was slightly higher against *E. coli* compared to the other two test strains, but this difference

was statistically insignificant. The antibacterial effect of the AXSA films was better than the effect of recently reported chitosan-SA/GS nanofibrous wound dressing [35]. These results indicate GS-loaded AXSA films' potential to protect wounds against infections caused by Gram-positive and Gram-negative bacteria. difference was statistically insignificant. The antibacterial effect of the AXSA films was better than the effect of recently reported chitosan-SA/GS nanofibrous wound dressing [35]. These results indicate GS-loaded AXSA films' potential to protect wounds against infections caused by Gram-positive and Gram-negative bacteria.

control. The F3 (blank) films formed no clear inhibition zones, suggesting an insignificant antibacterial effect of AXSA films against the tested bacterial strains. However, opaque regions were observed around F2 films due to bacterial growth in the expanded blank AXSA films. In contrast, positive control (GS std.) and GS-loaded AXSA films (GF2 and GF3) exhibited significant antibacterial effects against both Gram-positive (*S. aureus*) and Gram-negative (*E. coli and P. aeruginosa*) bacteria. The values of inhibition zones for GF2 and GF3 were slightly higher than for the positive control. The antibacterial effect of the films was slightly higher against *E. coli* compared to the other two test strains, but this

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**Figure 6.** (**a**) Antibacterial effect of F3 (1), gentamicin sulphate (GS) (2), GF2 (3), GF3 (4); (**b**) inhibition zones and (**c**) cell viability of arabinoxylan (AX)- sodium alginate (SA) films (mean ± SD, *n* = 3). Asterisk (\*) indicate statistically significant difference (*p* < 0.05) as compared to blank film (F3). **Figure 6.** (**a**) Antibacterial effect of F3 (1), gentamicin sulphate (GS) (2), GF2 (3), GF3 (4); (**b**) inhibition zones and (**c**) cell viability of arabinoxylan (AX)-sodium alginate (SA) films (mean ± SD, *n* = 3). Asterisk (\*) indicate statistically significant difference (*p* < 0.05) as compared to blank film (F3).

#### *3.5. Indirect Cell Viability Assay 3.5. Indirect Cell Viability Assay*

The indirect cell viability assay results of AXSA films against MRC-5 are depicted in Figure 6c. The results display that the viability of the cells treated with the extract of F2 and GF2 films exhibited no significant difference from that of untreated (control) cells. Cell viability assays are typically performed to predict films' safety for application on living tissues [7]. The extracts of F2 and GF2 films demonstrated no cytotoxic effects on normal cells and did not decrease cell viability. The cell viability (%) of the tested AXSA films was higher than the previously reported AX films indicating that the biocompatibility of the films increased by combining AX with SA in film formulation [33]. Therefore, The indirect cell viability assay results of AXSA films against MRC-5 are depicted in Figure 6c. The results display that the viability of the cells treated with the extract of F2 and GF2 films exhibited no significant difference from that of untreated (control) cells. Cell viability assays are typically performed to predict films' safety for application on living tissues [7]. The extracts of F2 and GF2 films demonstrated no cytotoxic effects on normal cells and did not decrease cell viability. The cell viability (%) of the tested AXSA films was higher than the previously reported AX films indicating that the biocompatibility of the films increased by combining AX with SA in film formulation [33]. Therefore, it can be suggested that AXSA films are safe for in vivo applications.

#### it can be suggested that AXSA films are safe for in vivo applications. **4. Conclusions**

The findings of this study demonstrate that the optimized AXSA films possess various desirable characteristics for antibacterial wound dressings. FTIR, XRD, and TGA analyses suggested that AX, SA, Gly, and GS were finely blended and formed hydrogen bonds. The prepared AXSA films were smooth, flexible, and transparent, which can aid in inspecting wounds without removing the dressing. The skin-like mechanical strength and flexibility of AXSA films make them suitable for their application on wounds in problematic body areas like joints. The moderate water uptake (expansion) and transmission (WVTR) of these films could be helpful for protecting moderately exudating wounds from dehydration and to maintain a moist environment for healing. The AXSA films released more than 80% of the loaded GS in 24 h with the initial rapid release (in the first hour), which can be used to control infections. The inhibition zones formed by the GS-loaded AXSA films in Gram-positive and Gram-negative bacteria were similar to those of GS solution, suggesting that these films can be used to control bacterial growth in infected wounds. The AXSA films also demonstrated excellent cytocompatibility in MTT assay, suggesting their safety for in vivo applications. Overall, we demonstrated that the AXSA films are a potential candidate for antibacterial wound dressing applications.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23052899/s1.

**Author Contributions:** Conceptualization, A.I.A., N.K.A. and N.A.; methodology, N.A.; formal analysis, A.I.A., M.M.A. and N.A.; investigation, A.I.A., M.M.A., A.M.B., M.U.M., M.S.B.S., Z.A.A. and N.A.; resources, A.I.A., A.V.D., Z.S.A. and S.S.A.; data curation, A.I.A.; writing—original draft preparation, A.I.A., M.M.A. and N.A.; writing—review and editing, A.M.B. and A.V.D.; supervision, A.I.A. and N.A.; project administration, A.I.A.; funding acquisition, A.I.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Deanship of Scientific Research at Jouf University through grant number 40/267. The APC was funded by Jouf University, Saudi Arabia.

**Data Availability Statement:** Data sharing is not applicable to this article.

**Acknowledgments:** The authors extend their appreciation to the Central laboratory at Jouf University for technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

