*3.4. Rheological Evaluation of the HA-FS-NTF Hydrogel*

The naturally available polysaccharide hyaluronic acid has been explored for incorporation in several pharmaceutical preparations because of its unique viscoelastic properties and effective augmentation of soft tissues. However, the degree of cross-linking and preparation method, along with the molecular weight of the natural polymer, have been shown to have a direct relationship on the rheological properties of the final product [32]. Therefore, the impact of our method of preparation and the cross-linking with Gantrez® S-97 on the rheological property of the formulated hydrogel of hyaluronic acid was evaluated in the present study through an analysis of different parameters by the Brookfield viscometer.

It is clear from Figure 6a that there was an obvious increase in the rate of shear with the increase in the shear stress of the hyaluronic acid hydrogel. Further, incorporation of the FS-NTF within the formulated hydrogel was found to be associated with an increase in the shear rate of the developed formulation (Figure 6b). The ηmin for the FS-NTF-loaded hyaluronic acid hydrogel was recorded as 132,318 cp, a number much higher than that for the ηmin for the blank hyaluronic acid hydrogel (110,016 cp). Therefore, application of this formulation within the oral cavity might promote in increasing shear rate during talking, eating, or smiling [33]. This increasing shear rate might promote longer retention of the formulation at the site of application for the effective healing of the ulcers. *Pharmaceutics* **2021**, *13*, x 17 of 23

(**b**)

**Figure 6.** Rheogram of the (**a**) blank hyaluronic acid hydrogel and (**b**) HA-FS-NTF. **Figure 6.** Rheogram of the (**a**) blank hyaluronic acid hydrogel and (**b**) HA-FS-NTF.

ior toward a more non-Newtonian flow [34]. Calculations for the values of the Farrow's constant (N) were performed by plotting the logarithm of the shear rate (G) versus the logarithm of the shear stress (F), where the slope indicates the value of N (Figure 7). A value of less than 1 indicates dilatancy, and a value greater than 1 indicates pseudoplastic flow [34]. The flow behavior of the formulated hydrogels is presented in Table 8. All the hydrogel formulations had thixotropic behavior with pseudoplastic flow, and that is a desirable property for pharmaceutical gels for oral use. This special flow characteristic of hyaluronic acid hydrogel could be due to the breakdown of the structural configuration through the interruption of the existing intermolecular interactions between polymeric chains upon application of shear. There is further reoccurrence of polymerization between the molecules due to the force of the van der Waals interaction between the molecules upon removal of such shear [35,36]. The present study outcomes indicate that the optimal formulation of the HA-FS-NTF could facilitate the mean residence time within the oral cavity. This attribute is mostly due to the greater affinity of hyaluronic acid for the oral

membrane due to its viscosity.

Values calculated by the Farrow's constant (N) indicate a deviation in the fluid of the Newtonian flow behavior, where an increased value indicates a change in the flow behavior toward a more non-Newtonian flow [34]. Calculations for the values of the Farrow's constant (N) were performed by plotting the logarithm of the shear rate (G) versus the logarithm of the shear stress (F), where the slope indicates the value of N (Figure 7). A value of less than 1 indicates dilatancy, and a value greater than 1 indicates pseudoplastic flow [34]. The flow behavior of the formulated hydrogels is presented in Table 8. All the hydrogel formulations had thixotropic behavior with pseudoplastic flow, and that is a desirable property for pharmaceutical gels for oral use. This special flow characteristic of hyaluronic acid hydrogel could be due to the breakdown of the structural configuration through the interruption of the existing intermolecular interactions between polymeric chains upon application of shear. There is further reoccurrence of polymerization between the molecules due to the force of the van der Waals interaction between the molecules upon removal of such shear [35,36]. The present study outcomes indicate that the optimal formulation of the HA-FS-NTF could facilitate the mean residence time within the oral cavity. This attribute is mostly due to the greater affinity of hyaluronic acid for the oral membrane due to its viscosity.

**Figure 7.** Representative plots of the logarithm of the rate of shear (G) versus the logarithm of the shearing stress (F) for (**A**) blank hyaluronic acid hydrogel and (**B**) HA-FS-NTF.


**Table 8.** Rheological parameters of blank hyaluronic acid hydrogel and HA-FS-NTF.

\* Data are expressed as mean ± SD (*n* = 3).

The results in Table 8 show that the viscosity of the developed formulations had different values at a minimum rate of shear (ηmin) and maximum rate of shear (ηmax); the values of the ηmax were less than those for the ηmin for both the formulations. Therefore, it could be assumed from our previous result that there might be structural breakdown of the polymeric chains during shear. Figure 8 shows the relation between the viscosity and shear rate of different gel bases at various concentrations.

We previously discussed the non-Newtonian behavior of the hyaluronic acid hydrogel. Figure 8 demonstrates that there is an inverse relationship between the viscosity of hydrogel and the applied shear rate. Therefore, the pseudoplastic flow of the formulated hydrogel is confirmed from the obtained results. Our results are in agreement with the available information in the literature on the relationship between shear rate and viscosity [36].

**Figure 8.** Representative plots of the rate of shear (G) versus the viscosity (η) for the (**A**) blank hyaluronic acid hydrogel and (**B**) HA-FS-NTF. *Pharmaceutics* **2021**, *13*, x 19 of 23

#### *3.5. In Vitro Release Profile of the HA-FS-NTF* The results of the in vitro release study of the formulated HA-FS-NTF containing the

The results of the in vitro release study of the formulated HA-FS-NTF containing the 0.5% marketed formulation of fluconazole and the HA suspension containing the 0.5% fluconazole using the Type I USP dissolution apparatus are presented in Figure 9. From the results, it could be clearly seen that the release of fluconazole from the HA-FS-NTF was faster and completed within the time frame of 3 h, when compared with the other two formulations. The release of drug from the marketed gel and aqueous suspension was slow, at approximately 43% and 12%, respectively. There was a significantly higher rate of release (<0.05) for our formulated FS-NTF-loaded hydrogel (~85%). From Figure 9, it can be demonstrated that the initial release rate of the drug from the HA-FS-NTF was retarded due to incomplete gel formation, but the release of fluconazole was gradual following complete hydration of the gel and remained at a steady state thereafter owing to pseudoplastic flow. Swelling of the polymer due to hydration led to a change in the physical and chemical parameters of the hydrogel configuration, to tune the porous structure uniformly [37]. The decreased release of fluconazole from the suspension and even from the gel might be due to a larger particle size or a less porous configuration, whereas the development of the nanotransfersome delivery facilitated the release of the drug from the semipermeable membrane owing to the smaller vesicle size and enhanced solubility. Our results are in agreement with those of the existing literature, where the release of an entrapped hydrophobic agent (resveratrol) was found to be improved when delivered via this nanotransfersome method [26]. 0.5% marketed formulation of fluconazole and the HA suspension containing the 0.5% fluconazole using the Type I USP dissolution apparatus are presented in Figure 9. From the results, it could be clearly seen that the release of fluconazole from the HA-FS-NTF was faster and completed within the time frame of 3 h, when compared with the other two formulations. The release of drug from the marketed gel and aqueous suspension was slow, at approximately 43% and 12%, respectively. There was a significantly higher rate of release (<0.05) for our formulated FS-NTF-loaded hydrogel (~85%). From Figure 9, it can be demonstrated that the initial release rate of the drug from the HA-FS-NTF was retarded due to incomplete gel formation, but the release of fluconazole was gradual following complete hydration of the gel and remained at a steady state thereafter owing to pseudoplastic flow. Swelling of the polymer due to hydration led to a change in the physical and chemical parameters of the hydrogel configuration, to tune the porous structure uniformly [37]. The decreased release of fluconazole from the suspension and even from the gel might be due to a larger particle size or a less porous configuration, whereas the development of the nanotransfersome delivery facilitated the release of the drug from the semipermeable membrane owing to the smaller vesicle size and enhanced solubility. Our results are in agreement with those of the existing literature, where the release of an entrapped hydrophobic agent (resveratrol) was found to be improved when delivered via this nanotransfersome method [26].

**Figure 9.** In vitro release profile of fluconazole from aqueous suspension, marketed gel, and HA-FS-NTF formulation. Data are expressed as mean ± SD (*n* = 3). and aqueous suspension (Table 9). **Figure 9.** In vitro release profile of fluconazole from aqueous suspension, marketed gel, and HA-FS-NTF formulation. Data are expressed as mean ± SD (*n* = 3).

*3.6. Permeation Parameters of Fluconazole from Optimized HA-FS-NTF by Ex Vivo Permeation* 

period from the 0.5% HA-FS-NTF optimized hydrogel, marketed 0.5% fluconazole gel, and aqueous 0.5% fluconazole suspension, respectively. With respect to the examined formulations, the optimized HA-FS-NTF hydrogel had maximum fluconazole permeation across the oral mucosa, and this is statistically significant (*p* < 0.05) when compared with the other two formulations. Our ex vivo permeation results of the tested formulations could be correlated with the in vitro release profile of the respective formulations, where the highest in vitro release from the HA-FS-NTF is reflected by the highest ex vivo perme-

HA-FS-NTF were found to be superior when compared with those of the marketed gel

The cumulative permeation of fluconazole through the sheep buccal mucosa was

(Table 9) after 3 h of an experimental

). The D, EF, Pc, and Jss of the optimized

found to be 400 ± 57, 294 ± 34, and 122 ± 18 μg/cm<sup>2</sup>

ability from the same formulation (400 μg/cm<sup>2</sup>

*Studies* 
