*4.3. Water Contact Angle*

The ability of a liquid to contact the solid surface is known as wetting. When the liquid and solid surfaces are brought together, they result from intermolecular interactions. A force balance between adhesive and cohesive forces determines the degree of wetting. Wettability is essential in the bonding or adherence of two different materials. Wettability describes the material hydrophilicity and hydrophobicity caused by the surface forces that control wetting. A liquid drop spreads across the surface due to adhesive forces between the liquid and the solid. Due to cohesive forces within the liquid, the drop balls up and avoid contact with the surface [24]. We have observed the wetting behavior of the hydrogels at different times (zero and ten seconds) and with an increased amount of GO. As time and GO amount increase, the hydrogel's wetting behavior shifts from hydrophobicity to hydrophilicity. BSG-4 and BSG-1 had water contact angles of 103.8◦ and 65.4◦ at zero seconds, respectively. However, after ten seconds, the water contact angles for BSG-4 and BSG-1 were 63.40◦ and 96.10◦, respectively. The wetting of BSG-2 = 75.50◦ and BSG-3 = 83.40◦ was observed at zero seconds as shown in Figure 3. The wetting analysis clearly shows that the hydrophilicity to hydrophobicity ratio has shifted. That is due to the increasing amount of GO, as it contains several oxygen-based functional groups that caused hydrogen bonding and other weak interactions [25]. A more hydrophilic nature for BSG-4 was observed with increased hydrophilicity by an increasing GO amount and prolonged contact time. Hence, BSG-4 will be helpful to absorb more wound exudate and keep the burn and chronic wound hydrated.

**Figure 3.** The wetting analysis of the hydrogel was conducted via water contact angle to determine the hydrophilicity and hydrophobicity nature of the hydrogels. The wetting behavior was investigated at different time intervals and 4(**a**–**d**) wetting behavior at 0 s while 4(**e**–**h**) wetting behavior of hydrogels at 10 s.

#### *4.4. Swelling*

Swelling is a significant feature of hydrogels for burn and chronic wound healing and drug delivery. The impacts of pH on the swelling behavior of hydrogels were studied at various pH levels, as shown in Figure 4a,b. All hydrogels showed minimal swelling in acidic and basic pH, with amazing swelling at pH 7. It reveals that hydrogels were extremely sensitive to changes in pH. The hydrophilic groups on the bacterial cellulose, PVA, and GO structures caused swelling due to H-bonding. The water penetrates the void space inside the hydrogels in neutral media. Functional groups in BC, such as carboxyl groups, were protonated to produce more electrostatic repulsion forces [26,27]. The available oxygen-based functional facilitate hydrogen bonding. However, in acidic media, the –OH functional groups have protonated those decreases in swelling, dissociating at higher pH. The hydrogels also have different swelling due to a variable amount of GO. It may act as a crosslinker as it has such a closely packed hydrogel structure due to the enhanced weak

intra-hydrogel bonding and electrostatic interactions [28,29]. BSG-1 has maximum, and BSG-4 has minimum swelling in aqueous and PBS media. At pH 7, electrostatic repulsion causes hydrogen bonding and void spaces in hydrogels, resulting in maximum swelling. As a result, the tested hydrogels cause the hydrogel to swell dramatically.

**Figure 4.** The physicochemical analysis of the hydrogels was conducted to study to analyze the behavior of the hydrogel with biofluid during application. Swelling in PBS (**a**) and aqueous (**b**) media, (**c**) biodegradation in PBS buffer solution (**d**), and stress–strain curve of hydrogels.

#### *4.5. Biodegradation*

Biodegradation is a characteristic of drug delivery hydrogels that are likely related to drug release. In vitro degradation of hydrogels was carried out under in vitro conditions in PBS buffer solution. The hydrogels exhibited different biodegradation due to different GO amounts, as shown in Figure 4c. The hydrogel BSG-1 has maximum weight loss, and BSG-4 has the most minor. It may be due to the increasing amount of GO that acts as a crosslinker to hold the polymeric chain tightly. Biodegrading also occurs due to alkyl linkage and glycosidic bonds in BC, facilitating drug release [30]. The different biodegradation also confirms the successful hydrogel fabrication, and the difference in degradation ratio was due to varying amounts of GO. The increased GO amount provides a closely packed structure with increased crosslinking density. The available alkyl linkage and glycosidic bonds of BC breakdown cause biodegradation [31]. Hence, controlled swelling facilitates controlled drug release and other therapeutic agents essential for wound healing.

#### *4.6. Mechanical Testing*

The mechanical behavior of hydrogels was studied using a stress–strain curve. The mechanical properties of hydrogels provide structural integrity and substantial strength to resist swelling and degradation. The mechanical properties of hydrogels can be controlled by optimizing crosslinker and filler (GO) amounts. It is worth mentioning that increasing the GO amount improved the tensile strength and elastic modulus from BSG-1 to BSG-4, as shown in Figure 4d. Hence, the mechanical strength confirms that our hydrogels have

been successfully fabricated with sufficient strength to resist swelling after absorbing wound exudate.

#### *4.7. In Vitro Drug Release*

Stimulated release, degradation-controlled release, solvent-controlled release, and diffusion-controlled release are the four mechanisms of drugs released from the polymer network of hydrogels. Solvent-controlled mechanisms include osmotic pressure-controlled and swelling-controlled mechanisms. The swelling of hydrogels is an essential factor in their structural design. It is strongly associated with the controlled release of drugs from hydrogels. The extracellular matrix of hydrogel expands on swelling and makes the drug available on the surface via diffusion pathways. We have taken BSG-4 to load curcumin, which is a natural antibacterial and anticancer drug, due to its optimum physicochemical characterizations. In vitro drug release of curcumin-BSG-4 loaded was determined via the Franz diffusion method under different pH-level (6.4, 7.4, and 8.4) in PBS media, and the drug release profile has been shown in Figure 5. The hydrogels are pH-responsive and offer other drug release mechanisms under different pH levels. The drug release mechanism was found in 7.4 > 6.4 > 8.4 pH, and it is strongly related to swelling and biodegradation of hydrogel, as shown in Figure 6a–c. The pore size of hydrogels increases during swelling due to the diffusion process and the drug is released [32]. Hence, maximum drug release was found at 7.4 pH, and the residual drug could not be determined as the hydrogel films broke into tiny pieces. The drug release can be prolonged by increasing the crosslinker amount. The increasing crosslinker amount can control swelling and degradation, essential factors for the drug delivery system [33]. The drug release at different pH is strongly linked to the hydrogels' swelling behavior that facilitates drug release on swelling under various other mechanisms. From the drug release profile, a smooth drug release was observed ≈ 91.38% at pH 7.4 after 33 h as seen in Figure 6b. The drug contains hydrogel broken down into pieces and it is not easy to determine drug release from small pieces. The controlled drug release time can be increased or decreased purely depending on crosslinking factors, which optimizes the swelling and degradation of the hydrogels that are necessary parameters for controlled drug release. Hence, these hydrogels could be desired dressing materials with pH-sensitive behavior that could be helpful in controlled drug delivery for burn and chronic wound healing.

**Figure 5.** The drug release of curcumin was studied at 37 ◦C under different pH levels to determine the release analysis behavior: (**a**) Curcumin release at pH 6.4; (**b**) Curcumin release at pH 7.4 and; (**c**) Curcumin release at pH 6.4.

**Figure 6.** The hydrogel sample BSG-3 was taken to on a random basis to determine curcumin release kinetics at different pH levels (6.4 (**a**–**f**), 7.4 (**g**–**l**), and 8.4 (**m**–**r**)) against different mathematical models (Zero order, First order, Higuchi, Korsmeyer-Peppas, Hixson & Bakers-Lonsdale).

#### *4.8. Drug Release Kinetics*

Drug release provides a deep understanding of the mass transport mechanisms involved. A therapeutic system must provide a specific drug release profile to mathematically calculate the resulting drug release kinetics. Various mathematical models were used to develop simple and complex drug delivery systems and predict overall release behavior. They make it possible to measure an extensive range of vital parameters and apply a model fitting to the experimental release data of curcumin from BSG-4 as presented in Figure 6. Understanding how to use these equations is crucial to grasping the various factors influencing drug release velocity. Their dissolution behaviors affect the efficacy of a patient's therapeutic regimen [34,35]. These mathematical models have different values of the regression coefficient (*R*2) at different pH levels. All the different parameters have been summarized in Table 1, these values provide drug release behavior through the polymeric matrix. Among all the factors, *R*<sup>2</sup> is the most prominent factor that describes the drug release behavior from different therapeutic systems. The release of curcumin follows various mechanisms at different pH levels that can be described based on the maximum of *R*2. We have calculated the value of *R*<sup>2</sup> against all fitting models. It was observed that at pH 6.4, curcumin follows the Hixson model with a maximum *R*<sup>2</sup> value (0.99054). The Hixson model describes the release of the curcumin dissolution approach. The release of curcumin was observed to follow zero-order at 7.4 pH by *R*<sup>2</sup> value (098106). The zero-order describes the quick release of therapeutic agents that is very important to control the attack of the pathogens. However, the curcumin release behavior was observed at pH 8.4 to follow zero-order due to the maximum *R*<sup>2</sup> value (0.9782). The release of curcumin from the composite hydrogel at pH 8.4 is found to be as effective as it is at pH 7.4. However, the mathematical fitting models have been summarized in Table 1.

**Table 1.** The drug release kinetics summarizes against different models to determine the best-fitted model at different pH levels (6.4, 7.4, and 8.4).



**Table 1.** *Cont.*

#### *4.9. Antimicrobial Activities*

Chronic and burn wounds, and many surgical procedures are all susceptible to bacterial infection. It can lead to severe disease, muscle tissue death, septicemia, and even death. Antimicrobial agents that are effective in treating bacteria are desperately needed. The prepared hydrogels were evaluated against severe wound infection-causing microbes (*S. aureus*, *E. coli*, and *P. aeruginosa*) via the disc diffusion method. The prepared hydrogels were applied to the bacterial lawns in Petri dishes, and these plates were kept in incubation for 12 h. Antibacterial activities were determined via measuring zone inhibition, as shown in Figure 7. These hydrogels have different zone inhibitions against treated bacterial strains. The hydrogel BSG-1 performed minimum, and BSG-4 exhibited maximum antibacterial activity. The maximum antibacterial activities of BSG-4 may be due to the maximum GO amount that ruptures the bacterial membrane with sharp edges of GO [9,36]. On the other hand, the bacterial cell membrane is composed of lipopolysaccharides and phospholipids. The polymeric part of the hydrogel may interact with the bacterial membrane to surround and penetrate several available functional groups to hinder bacterial growth [37,38]. The covalently crosslinked hydrogels via TEOS have inter/intra-hydrogen bonding and weak interactions. BSG-4 exhibited maximum antimicrobial activities, which can be polymeric, and GO components of hydrogels that may communicate with DNA to stop bacterial replication. Hence, the prepared hydrogel could be a potential biomaterial with promising antibacterial activities, which is helpful to treat burns and chronic wounds.

**Figure 7.** The antibacterial activities of composite hydrogels against different severe skin infections causing Gram-positive and Gram-negative pathogens. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 and n = 3.

#### *4.10. Anticancer Activities*

Figure 8 presents the anticancer activity via cell viability after different time intervals (24, 48, and 72 h) and cell morphology of all samples of composite hydrogels and curcuminloaded composite hydrogels against U87 cell lines. After 72 h of contact time with composite hydrogels and curcumin-BSG-4 composite hydrogels, nearly 79.56 to 87.58 percent of U87 cells were determined to be nonviable, respectively, as exhibited in Figure 8a. Curcumin combined with PVA/BC-*f*-GO was discovered to have improved anticancer properties. The BSG-4 composite hydrogel also had the highest cell nonviability among the composite hydrogels, which could be owing to the increased GO content [20,39]. The curcuminloaded-BSG-4 had the strongest antitumor effect. This could be due to the interaction of the composite hydrogel with the cellular membrane, allowing curcumin to conduct anticancer functions. Cell clustering may also be found for BSG-1 and BSG-2, as shown by the red circle, even after 72 h, whereas some cell adherence can be seen for both samples as presented in Figure 8(b–e). However, the red arrows indicated cell detachment, which might occur as a result of cell death, as well as the cell repute. Curcumin was delivered via the drug-loaded BSG-3 composite hydrogel membrane, resulting in increased anticancer activity [36,40]. As a result of the synergetic impact of the release of curcumin-loaded-BSG-3 composite hydrogels after 72 h, the anticancer activities are at their peak. As a result, the curcumin-loaded-BSG-3 composite hydrogel could be used as a biomaterial for wound care and treatment in cancer patients.

**Figure 8.** The anticancer activities of composite hydrogels against U87 cell line, (**a**) cell viability in terms of anticancer activities, and (**b**–**f**) cell morphology with different indications.

#### **5. Conclusions**

The pH-sensitive composite hydrogels were prepared by blending bacterial nanocellulose with PVA and GO and crosslinked with optimized TEOS. The controlled drug delivery

was determined at various pH levels, and the drug release mechanism was investigated using different mathematical models. In hydrogels, FTIR reveals all of the functional groups of bacterial nanocellulose, GO, and TEOS and well-defined crosslinking and hydrogen bonding. GO-flakes were observed via SEM and increasing GO-flakes were observed due to the increasing GO amount. BSG-4 was more stable than other hydrogels. Due to complex polymerization formation, the biodegradation rate of hydrogels was slowed by increasing the GO content. In addition, increasing the GO amount increased the mechanical and hydrophilicity properties. Swelling in the buffer and non-buffer solutions demonstrates that the prepared hydrogels are pH-sensitive. Therefore, hydrogels are a good fit for controlled drug release due to their responsive behavior, and they could be used to release curcumin in a controlled manner. Furthermore, these newly composite hydrogels have exhibited substantial potential, but BSG-4 and curcumin-loaded-BSG-4 have potential antibacterial and anticancer activities. Hence, these composite hydrogels could be promising biomaterials for chronic wound healing applications.

**Author Contributions:** Conceptualization, M.U.A.K.; Data curation, M.U.A.K. and S.I.A.R.; Formal analysis, M.U.A.K. and W.S.A.-A.; Funding acquisition, M.U.A.K. and S.I.A.R.; Investigation, S.I.A.R. and S.M.A.; Methodology, M.U.A.K., S.I.A.R. and H.H.A.; Project administration, S.I.A.R., H.H.A. and S.M.A.; Resources, M.U.A.K., S.M.A. and S.I.A.R.; Software, M.U.A.K.; Supervision, M.U.A.K., S.I.A.R. and W.S.A.-A.; Validation, M.U.A.K. and S.I.A.R.; Visualization, M.U.A.K. and W.S.A.-A.; Writing—original draft, M.U.A.K.; Writing—review & editing, M.U.A.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is supported through the Annual Funding track by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Project No. AN000593).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data is available in the article.

**Acknowledgments:** We highly acknowledge the countless efforts and expertized of Muhammad Umar Aslam Khan for the completion and publication of the project.

**Conflicts of Interest:** All authors declared no conflict of interest.

#### **References**

