*2.3. Optimization of the Gr-PV-NEs*

The desirability function was the basis of the Design-Expert software for obtaining the optimal NE formulation. The main objective of the optimization process was to find a formulation with the smallest droplet size and mean burn wound diameter. The software chose the solution with the desirability value closest to 1. To guarantee the model's validity and adequacy, the elected formulation was fabricated, depicted, and eventually compared with the response values expected by the software.

#### *2.4. Characterization of the Optimized Formulation*

#### 2.4.1. Determination of Entrapemnt Efficiency

Percentage drug entrapment efficiency (EE%) was determined for the optimum Gr-PV-NE formulation (containing 40 mg PV) using an indirect method. Sample was centrifuged at 15,000 rpm for 30 min using cooling centrifuge (SIGMA 3–30K, Steinheim Germany). After centrifuge, 1 mL of supernatant transparent layer was diluted with 10 mL distilled water. The PV amount in the sample was determined by a reported high-performance liquid chromatography (HPLC) method. In short, the withdrawn samples were diluted with the mobile phase, which composed of 10 mM ammonium acetate, methanol, and triethylamine in a ratio of 40:60:0.17 *v/v/v*. Ten microliters of the prepared samples were injected with a flow rate of 1.0 mL min−1. The PV detection wavelength and elution time were set to be 239 nm and 2.15 min, respectively. Results were taken in triplicate and the average was taken into consideration and EE% was calculated using the following equation.

$$\text{EE\%} = \text{[TD} - \text{FD]} / \text{TD} \times 100\tag{1}$$

where TD is the total added drug amount, FD is the free unentrapped drug
