*3.1. Formulation of Fluconazole-Loaded Nanotransfersome*

The edge activators in the nanotransfersome formulations played a definitive role. They make the formed vesicles ultraflexible so that they are capable of noninvasively penetrating the skin by virtue of their high level of self-optimizing deformability. An elastic nanotransfersomal carrier is described as a lipid droplet of such deformability that it permits easy penetration through pores much smaller than the droplet size, and, also, it is a highly adaptable and stress-responsive complex aggregate. The vesicles are elastic and very deformable; they consist of lecithin in combination with an edge-active surfactant such as Tween. An edge activator is usually a single-chain surfactant that causes the destabilization of the lipid bilayer of the vesicle and increases the vesicle elasticity or fluidity by lowering its interfacial tension. Amongst the various edge activators used widely by different researchers, Spans, Tweens, potassium glycyrrhizinate, sodium deoxycholate, and sodium cholate are widely incorporated for the development of stable and effective formulations [17]. The current approach of formulating fluconazole-loaded nanotransfersomes was achieved by incorporating Tween 20. Tween 20 is a nonionic polyoxyethylene sorbitol ester widely used in pharmaceutical preparations due to its biocompatible characteristics. The development of the nanotransfersomes progressed using this nonionic surfactant [26].

We also incorporated sesame oil in the nanotransfersome formulation. Sesame oil is traditionally used as a pulling agent for dentures; it prevents malodor, bleeding from the gums, tooth decay, and dryness of the lips and throat [27]. Further studies revealed a concentration-dependent antifungal role of sesame oil, specifically against *C. albicans* [10]. Therefore, a total of 19 formulations were prepared according to the Design of Experiment model to formulate an optimized formulation for the next stage of experiment.

*3.2. Optimization of the Fluconazole-Sesame Oil Nanotransfersome: Box–Behnken Statistical Design* 3.2.1. Optimization of the Globule Size of the Fluconazole-Sesame Oil Nanotransfersome Formulations

The globule size of a nanotransfersome formulation is one important parameter in formulation optimization because it can affect the solubility and drug permeation through the skin [28]. Therefore, we analyzed the interactions of the three independent variables in our formulation to determine the globule size of the FS-NTF. During the optimization process, the quadratic model was found to be the best fit. Table 3 shows the statistical outcome of the interactions between the amount of lecithin, fluconazole, and sesame oil in the formulation and the globule size of the FS-NTF. The statistical significance of the different model terms is represented by the *p*-value (<0.05). The model's F-value of 321.4 represents the significance of the model. A lack-of-fit F-value of 4.88 and a respective *p*-value (>0.05) indicated that the lack of fit was not significant relative to the pure error, and this was desirable because we wanted the model to be fit. Close agreement was observed between the actual and predicted globule sizes of the formulations, as shown in Table 1, and also in the predicted and adjusted R<sup>2</sup> values, with a difference of less than 0.2 for the dependent variable of globule size.

**Table 3.** Statistical outcome (ANOVA) of the interaction of three independent variables on globule size of FS-NTF.


The model terms A, B, C, AB, and A2 were significant; they represent the significant effects of the amounts of lecithin, fluconazole, and sesame oil on the globule size of the formulations. A desirable signal-to-noise ratio is higher than 4, and the suggested model has a value of 55.071, indicating an adequate signal. Therefore, this model could be used to navigate the design space.

The generated quadratic equation on the interactions of the three independent variables with the globule size of the formulations is shown in Equation (5).

Y1 = +219.67 + 51.40\*A + 37.95\*B <sup>−</sup> 6.07\*C <sup>−</sup> 29.56\*A<sup>2</sup> + 11.05\*A\*B + 1.30\*A\*C + 1.02\*B<sup>2</sup> + 0.0489\*B\*C <sup>−</sup> 1.63\*C<sup>2</sup> (5)

> Model terms A and B have positive coefficient values of +51.40 and +37.95, respectively, whereas model term C has a negative coefficient value (−6.07) (Equation (5)). The *p*-value for all the model terms was less than 0.05 (Table 3); this indicates a significant increase in the particle size with increasing amounts of lecithin and fluconazole and a decreasing amount of sesame oil in the formulation. The higher value of coefficient of model term A indicates a higher effect on the particle size with changes in the amount of lecithin, followed by the amounts of fluconazole and sesame oil in the formulations. Lecithin is a phospholipid that is responsible for the formation of the concentric lipid bilayers of the formed vesicles. This lipid bilayer is the medium in which the lipophilic drug is located.

For this reason, the increase in the lecithin concentration within the formulated liposomes leads to an increased number of bilayers of this multilamellar vesicle, and this leads to an increase in the size of the formed vesicles. A similar effect is reflected in the perturbation plot, where positive slopes are evident with model terms A and B and there is a flattened negative curve with model term C. The curve with model term A is stiffer than the curve with model term B, and this is per the respective coefficient value in Equation (2). Similar effects on the interaction of the amounts of lecithin and fluconazole are reflected by color changes in the contour plot (see Figure 2a) and by changes in the color and slope of the three-dimensional surface plot (see Figure 2b).

**Figure 2.** (**a**) Contour plot showing the interactions of lecithin and fluconazole with the globule size of the FS-NTF. (**b**) 3D surface plot showing the interactions of lecithin and fluconazole with the globule size of the FS-NTF.

3.2.2. Box–Behnken Statistical Designs: Optimization of the Entrapment Efficiency of the Fluconazole-Sesame Oil Nanotransfersome Formulations

The EE% of the nanoformulation is one of the hurdles in developing novel deliveries, where nanoformulations with a higher EE% are desirable [29]. Therefore, we analyzed the interactions of the three independent variables in our formulation to achieve a higher percentage of fluconazole to be successfully entrapped in the FS-NTF. During the optimization process, the quadratic model was found to be the best fit for a response such as the EE%. The statistical outcome of the interactions of the amounts of lecithin, fluconazole, and sesame oil in the formulation on the EE% of the FS-NTF is shown in Table 4. The model *F*-value of 386.29 represents the significance of the model. An F-value of 0.76 for the lack of fit and the respective *p*-value (>0.05) indicated that the lack of fit was not significant relative to the pure error; this was desirable as it indicated the model was fit. Close agreement was observed between the predicted and actual EE% of the formulations, as shown in Table 1, and the difference between the predicted and actual R<sup>2</sup> values was found to be less than 0.2.

In Table 4, the *p*-value indicates a significant effect of the amounts of lecithin, fluconazole, and sesame oil on the EE% of the formulations. When the coefficient value of model terms A, B, and C in the quadratic equation (Equation (6)) indicated that a desirable signal-to-noise ratio was more than 4, we found that the suggested model had a value of 60.699, indicating an adequate signal. Therefore, this model could be used to navigate the design space.


**Table 4.** Statistical outcome (ANOVA) of the interactions of three independent variables on the EE% of the FS-NTF.

The generated quadratic equation on the interactions of the three independent variables on the EE% of the formulations is shown in Equation (6).

Y2 = +75.19 + 3.70\*A <sup>−</sup> 3.10\*B + 1.38\*C <sup>−</sup> 0.9091\*A<sup>2</sup> <sup>−</sup> 0.1381\*A\*B <sup>−</sup> 0.3881\*A\*C <sup>−</sup> 16.57\*B<sup>2</sup> <sup>−</sup> 0.1381\*B\*C <sup>−</sup> 0.2650\*C<sup>2</sup> (6)

> From the equation, it can be seen that the model terms A and C have a positive coefficient value of +3.70 and +1.38, respectively, whereas model term B has a negative coefficient value (−3.10). The p-value for all the model terms was less than 0.05 (Table 4). This indicates a significant increase in the EE% with an increasing amount of lecithin and sesame oil and a decreasing amount of fluconazole in the formulation. A similar effect is reflected in the perturbation plot, where positive slopes are evident with model terms A and C and a positive slope is observed for model term C up to a certain extent and the curve of the model term goes down with an increasing amount of fluconazole. The curve with model term A is stiffer than the curve with model term C; this is per the respective coefficient value in Equation (3), so it can be inferred that the effect of the amount of lecithin is higher than for the amount of sesame oil in the EE% of the formulation. The effect of the interactions between the amounts of lecithin and fluconazole is reflected by the color changes in the contour plot (Figure 3a) and by the changes in color and slope in the three-dimensional surface plot (see Figure 3b). It is interesting to note that for the contour plot and three-dimensional surface plot (see Figure 3), an increasing amount of drug in the nanotransfersome formulation leads to an increase in the EE% up to a certain point; however, a further increase in drug concentration leads to a decrease in the EE% of the formulation. This phenomenon might be explained by the inefficiency of entrapping a higher amount of drug in the nanotransfersome formulation. At each level for the lecithin amount, the increase of fluconazole will increase the EE% until a certain level is reached, but then a further increase in the level of fluconazole will lead to a decrease in the EE%. This occurs as the space available for the incorporation of the added fluconazole becomes insufficient for the excess amount added, consequently leading to a decrease in the EE% at a higher level of fluconazole.

**Figure 3.** (**a**) Contour plot showing the interactions of lecithin and fluconazole with the EE% of the FS-NTF. (**b**) Threedimensional surface plot showing the interactions of lecithin and fluconazole with the EE% of the FS-NTF.
