2.4.4. Ex-Vivo Permeation Study

The test was performed according to a previously described procedure [40]. A freshly excised section of abdominal rat skin was obtained from a male Wistar rat and utilized to determine the permeation profile of PV from various formulations (i.e., Gr-PV-NE optimum formulation, PV-NE prepared with oleic acid instead of Gr oil, Gr-NE prepared without PV, physical mixture of PV and Gr oil, PV aqueous dispersion, and plain NE prepared without PV or Gr oil). All formulations except for the plain nanoemulsion contained PV amount equal to 40 mg/g of the tested formulation. The institutional animal ethics committee approved the experimental protocol. In brief, the abdominal skin hair was guardedly clipped and removed without causing any skin damage. Then, rats were slaughtered, and abdominal skin was separated from the subsidiary connective tissues and cleaned with a Ringer solution before experimentation. Automated Franz diffusion cells (MicroettePlus; Hanson Research, Chatsworth, CA, USA) having a permeation area of 1.76 cm2 were employed for determining the ex-vivo skin permeation of the PV from the previously stated formulations. The skin (2.5 × 2.5 cm2) was inserted between the donor and receptor chambers of the cells with the stratum corneum layer oriented toward the donor compartment. The receptor milieu was phosphate buffer saline (PBS, pH 7.4) adjusted at 37 ± 0.5 ◦C in a sufficient volume to meet the sink condition for ex-vivo permeation studies. One milliliter of the various tested formulations was applied to the stratum corneum layer of the skin through the donor chamber and covered with Parafilm to diminish evaporation. At time intervals of 1, 2, 4, 6, 8, 10, 12, and 24 h, 0.5 mL aliquots were extracted from the receptor compartment. Immediately, a fresh receptor milieu previously heated to 37 ± 0.5 ◦C replaced the withdrawn samples. The PV amount in the gathered samples was determined by a reported high-performance liquid chromatography (HPLC) method previously described in Section 2.4.1.

#### 2.4.5. Antibacterial Activity Evaluation

The antimicrobial action of the previously stated formulations (Section 2.4.2) was examined applying a disc diffusion method [41]. The well-known Gram-positive bacterium *S. aureus*, ordinarily present in infected burns, was utilized as a test bacterium. Following the procedure endorsed by the Clinical and Laboratory Standards Institute, an *S. aureus* suspension was made to the 0.5 McFarland turbidity standard and mounted on Mueller– Hinton agar plates. Filter-paper discs (with a diameter of 10 mm) were immersed in the tested samples and then put in the center of the agar plate and incubated for 24 h at 37 ◦C. When inhibitory concentrations were reached, a clear so-called inhibition zone containing no colonies could be seen around the discs. Lastly, the inhibition zones' diameters were measured for each examined formulation.

#### 2.4.6. Statistical Analysis

Compiled IL-6 serum level data were tested using the one-way ANOVA followed by the post-hoc Tukey honest significant difference (HSD) test for multiple comparisons, and the level of significance was set at a *p*-value of less than 0.05 using SPSS software (version 22, Chicago, IL, USA). The obtained data were tested for normality using the Kolmogorov–Smirnov (K-S) test.

#### **3. Results and Discussion**

#### *3.1. Assessment of Gr-PV-NE Droplet Size*

Regarding the topical administration of active agents, the physicochemical specifications of nanoemulsions are fundamental factors that must be determined in the fabrication process [42].

The droplet size is an essential key parameter and can identify an emulsion as being a microemulsion or a nanoemulsion. The present investigation revealed that the developed formulations had a droplet size of between 61 ± 1.5 and 138 ± 3.1 nm (see Table 2), with an acceptable polydispersity index ranging from 0.09 to 0.40, confirming the acceptable homogeneity and favorable size distribution of the developed formulations.

A quadratic model of polynomial analysis was employed to scrutinize the collected data on droplet size. The chosen design showed the employed model's competence in exploring the influence of the amount of Gr oil (A), amount of PV (B), and HLB of the surfactant mixture (C) on the developed emulsions' droplet size. The advocated mathematical model had an adjusted R<sup>2</sup> value of 0.9729 and predicted R2 value of 0.9445, which were closely related, as shown in Table 3. The ANOVA analysis of the data provided the following equation.

Globule size = +78.26 <sup>+</sup> 14.87A <sup>+</sup> 1.29B <sup>+</sup> 0.1909C <sup>+</sup> 3.04AB <sup>−</sup> 0.1560AC <sup>−</sup> 1.51BC <sup>+</sup> 0.5087A2 <sup>−</sup> 2.68B2+35.64C2 (2)

As perceived, the amount of Gr oil (factor A) had a significantly agonistic action on the droplet size at a *p*-value of less than 0.0001; thus, any increase in the amount of Gr oil would eventually increase the droplet size. Increasing the amount of Gr oil might have provided more space in which the PV could be housed within the nanoemulsion, giving droplets a larger diameter. Further, the increase in the amount of Gr oil would be accompanied with a corresponding decrease in the amount of cosurfactant used and its capacity to downsize a nanoemulsion's droplets, leading to the formation of larger droplets, as similar findings had previously reported [36]. On the other hand, the amount of PV and HLB value of the surfactant mixture exerted an insignificant effect on the droplet size.

Based on the above equation and Figure 1, which show the effect of the studied factors on the droplet size, an interesting result was detected: There was a significant impact exerted by the quadratic term of factor C (i.e., C2) on the droplet size of the developed formulations. As shown in Figure 1A, such an observation implies that the peripheral levels of factor C had a positive effect on the droplet size, whereas the middle level exerted a negative effect, yielding droplets with a smaller diameter. Such phenomena could be due to the fact that each lipid material must have a particular HLB value to lower the nanoemulsion droplet size, as reported by other researchers [43,44]. Therefore, it might be deduced that the best HLB value for the Gr oil used in the formulations should be the middle value among those used in the proposed design.

**Table 3.** Regression analysis results for Y1 and Y2 responses.


**Figure 1.** Main effect diagram (**A**), contour plot (**B**), and three-dimensional (3D) surface plot (**C**) showing the effects of different independent variables on the droplet size (Y1) of different Gr-PV-NE formulations.

#### *3.2. Assessment of Wound Healing*

The healing of open wounds is a complicated dynamic chain of events that encompasses some sequential and overlapping processes, including hemostasis, inflammation, epithelialization, cell proliferation, revascularization, and collagen development [45]. In the present investigation, the healing impact of the fabricated formulations on burn wounds was determined by measuring the wound diameter. The diameters fluctuated between 2.5 ± 0.30 and 10 ± 1.2 mm (see Table 2) and followed a quadratic model of polynomial analysis. The chosen experimental design employed the model's adequacy to observe the impact of independent variables on the diameter of burn wounds (Y2). The statistical model had an adjusted R<sup>2</sup> value of 0.9817, which was in line with an expected R2 value of 0.9642, as shown in Table 3. The ANOVA analysis of the collected data yielded the following equation.

$$\begin{array}{l}\text{Mean burn would diameter} = +6.03 - 1.64\text{A} - 1.51\text{B} + 0.057\text{C} + 0.5102\text{AB} + 0.1786\text{AC} + 0.1548\text{BC} - \\ \text{ } & 0.3194\text{A}^2 - 0.2792\text{B}^2 + 0.41108\text{C}^2 \end{array} \tag{3}$$

It was noticed that there was an inverse relationship between the amounts of Gr oil and PV and response Y2 values at a *p*-value of less than 0.0001. The potential of the PV to decrease the burn wound size might be related to its ability, as a member of the statin class of drugs, to promote the output of the VEGF at the injury site; VEGF is a key element for developing new blood vessels [46]. Further, PV is also thought to inhibit mevalonate and farnesyl pyrophosphate production, leading to enhanced epithelialization and renovation of tissues of wounded skin [47]. Additionally, the wound healing action of Gr oil (factor A) might be due to its strong antibacterial activity against the Gram-negative bacterial strains that are mostly responsible for wound infections and that are resistant to treatments, and similar findings were reported in the literature [45]. The influence of the studied factors on the mean burn wound diameter is shown in Figure 2.

**Figure 2.** Main effect diagram (**A**), contour plot (**B**), and 3D surface plot (**C**) showing the effects of different independent variables on the mean burn wound diameter (Y2) obtained after the application of different Gr-PV-NE formulations.

#### *3.3. Optimization and Evaluation of Nanoemulsion Formulations*

Following the completion of the tests described, a nanoemulsion formulation with the most appropriate specifications (i.e., the optimum formulation) was defined. Varying combinations of independent variables were suggested by the experimental design. The optimum formulation had 275 mg of Gr oil, 40 mg of PV, and a surfactant mixture with an HLB of 12 with a desirability value of 0.784. The fabricated optimal Gr-PV-NE had a droplet size of 95 ± 2.4 nm and a mean burn wound diameter of 3 ± 0.3 mm. Such results were in close accordance with the predicted values of the same responses, which were 90 nm for the droplet size and 3.18 mm for the mean burn wound diameter. Figure 3 clarifies the desirability ramp and bar chart for different levels of the studied factors and predicted dependent variables of the optimal formulation. Optimum formulation acquired an EE% of 91.3 ± 2.6% and ZP value of −17.3 ± 1.2 mV indcating the acceptable drug loading and stability of the developed optimal formulation.

**Figure 3.** Desirability ramp and bar chart for optimization. (**A**) The desirability ramp shows the levels of independent variables and predicted values for the responses of the optimum formulation. (**B**) The bar chart shows the desirability values for the combined responses.

3.3.1. Wound Healing Action Assessment

Burn Wound Diameter Evaluation

As can be observed in Figure 4, the optimum Gr-PV-NE (formulation A) had the lowest mean burn wound diameter of 3 ± 0.5 mm, while the group treated with normal saline (formulation E) had the largest wound diameter, 12 ± 1.5 mm, compared with the other tested formulations. It was also noticed that the nanoemulsion containing no PV (formulation B) had a wound diameter (i.e., 6 ± 0.75 mm) greater than that of formulation A but very close to that of formulation C (i.e., 5.5 ± 0.5 mm), which was a nanoemulsion containing oleic acid instead of Gr oil. Such outcomes affirm the synergistic wound healing activity of PV and Gr oil. On the other hand, the Gr-PV mixture (formulation D) had a mean burn wound diameter of 8 ± 1 mm, which was greater than that of the optimum nanoemulsion formulation, indicating the predominant wound healing activity of the nanosized formulation compared with the mixture of Gr oil and PV. Figure 5 also illustrated the wound healing results of rat skin after 14 days of treatment The obtained rsults were found to be significant at asignificance level of 0.05 (*p*-value < 0.05)

**Figure 4.** Mean burn wound diameter for different formulations: optimum Gr-PV-NE (**A**), Gr-NE (**B**), PV-NE (**C**), PV-Gr mixture (**D**), and normal saline (**E**).

**Figure 5.** Mean burn wound diameter for different formulations: optimum Gr-PV-NE (**A**), Gr-NE (**B**), PV-NE (**C**), PV-Gr mixture (**D**), and normal saline (**E**).

#### IL-6 Level Evaluation

IL-6 is an abundant cell protein that helps in modulating immune system responses. The IL-6 level is usually raised by triggers such as injuries, inflammatory conditions, microbial infections, disturbances of the immune system, and malignant tumors; accordingly, IL-6 could be an advantageous marker for detecting inflammation and immune system activation [48].

As could be seen in Figure 6, formulation A (i.e., Gr-PV-NE) had the lowest IL-6 serum level of 944 ± 100 U/mL, whereas formulation E (i.e., normal saline) had the highest level of IL-6 (3600 ± 450 U/mL); this indicated the superiority of formulation A in counteracting inflammation compared with the other tested formulations. The superior anti-inflammatory activity of the optimum nanoemulsion formulation could be due to its content of PV and Gr oil [49]. It is well known that statins can depress the output of proinflammatory cytokines [50] via inhibiting HMG-CoA reductase, which could invigorate the mevalonate pathway. As a result, PV might minimize the occurrence of the isoprenylation and geranylgeranylation of proteins, particularly Ras protein prenylation. The suppression of Ras diminishes the efficiency of transcription factor nuclear factor kappa B, which plays a pivotal role in many inflammatory reactions [51].

The anti-inflammatory action of Gr oil might be connected to the prohibition of some intracellular signaling pathways encompassing several inflammatory mediators' actions. Abe et al. [52] revealed the ability of Gr oil to depress the adherence response of human neutrophils in vitro and diminish the induced neutrophil mobilization in the peritoneal cavity following the intraperitoneal administration of the oil. Several studies have explored the components that might be responsible for such bioactivity [53]. It was found that the major components of the oil, namely, geraniol, citronellol, and linalool, were proven to have anti-inflammatory influences [54,55].

**Figure 6.** IL-6 levels for different formulations: optimum Gr-PV-NE (**A**), Gr-NE (**B**), PV-NE (**C**), PV-Gr mixture (**D**), and normal saline (**E**).

Formulations B and C had Il-6 values of 1415 ± 200 U/mL and 1611 ± 150 U/mL, respectively. Although these values indicated a higher effect of formulation C, which contained PV and oleic acid instead of Gr oil in the nanoemulsion, compared with formulation B, which contained only Gr oil in the nanoemulsion, such difference was found to be insignificant using Tukey post test. Moreover, formulation D, which was composed of PV and Gr oil, had an IL-6 value of 3200 ± 400 U/mL, affirming the favorable anti-inflammatory behavior of the optimum nanoemulsion formulation (formulation A).

IL-6 serum levels of the different tested groups were examined for normal distribution characteristics using the K-S test of normality. The observed high *p*-values and low D-values for all of the formulations suggested that there was no considerable difference between the collected data and the data that were normally distributed.

The outcomes of the ANOVAs confirmed that all of the tested formulations (except for formulation D) had much higher IL-6 levels than formulation E, with a *p*-value of less than 0.01; therefore, the noticed variations between formulations could not happen by chance. The Tukey HSD post-hoc test revealed that the IL-6 serum levels of all of the formulations varied significantly from each other (*p*-value < 0.01), except that the comparisons between formulations C and B and D and E were found to be insignificant. Such findings are quite reasonable because formulation B and formulation C each contained one component (i.e., PV in the case of B and Gr oil in the case of C) that had an IL-6–lowering effect.

#### 3.3.2. Ex-Vivo Permeation Study

Upon reviewing the ex-vivo permeation results presented in Table 4 and Figure 7, the following information was detected. First, the nanoemulsion formulation that contained PV and oleic acid instead of Gr oil promoted drug permeation across the skin by more than 5.8 when compared with the PV aqueous dispersion. More importantly, the optimized Gr-PV-NE encouraged skin permeation of PV by 7.6-fold and 2.7-fold in comparison with the PV aqueous dispersion and PV-Gr physical mixture, respectively. Such enhancement might be due to a synergistic effect of a nanoemulsion as a drug delivery system and the effect of Gr oil (which contains components like citronellol and geraniol, which acted as penetration enhancers) on the permeation by PV; similar results were found in the literature [44]. Nanosized emulsions are known to offer a large surface area for drug permeation, in addition to their surfactant and cosurfactant contents, which are thought to fluidize the stratum corneum layer, which is the main barrier to the permeation of drugs through the skin [56]. The obtained rsults were found to be significant at a significance level of 0.05 (*p*-value < 0.05).


**Table 4.** Inhibition zones against *S. aureus* and percentage PV permeated for varying formulations.

**Figure 7.** Ex-vivo permeation profiles of PV from different tested formulations.
