2.4.2. Zeta Potential Determination

Zeta potentiap (ZP) of optimum Gr-PV-NEs (containing 40 mg PV) was examined via diluting 100 μL of the formulation with 900 μL of double distilled water in a volumetric flask. The diluted samples were thoroughly mixed, and 100 μL of the mixed samples were employed to determine its ZP using a Zeta track analyzer (Microtrac, Inc., Montgomeryville, PA, USA) [38]. Assessment of the sample was carried out in triplicates, and the results were presented as the mean ± standard deviation (SD).

#### 2.4.3. Measurement of Burn Wound Diameter and Interlukin-6 Level

As in previously described evaluations of different Gr-PV-NE formulations, the test in this study was repeated on 15 rats that were divided into 5 groups. The first group (Group A) was treated with the optimum formulation proposed by the design and followed to determine the outcomes of the first stage of the animal test. The second group (Group B) was treated with the optimum formulation that contained no PV. The third group (Group C) was treated with the optimum formulation prepared utilizing oleic acid in lieu of Gr oil. The fourth group (Group D) was treated with Gr-PV mixture. The fifth group (Group E) was treated with normal saline and served as a negative control. The test continued for 14 days. The formulations were applied once a day and the required parameters were evaluated. All tested formulations contained PV amount equal to 40 mg/g of the tested formulation.

For the assessment of interleukin-6 (IL-6), a previously described quantitative sandwich enzyme-linked immunoassay technique (R&D Systems, Inc., Minneapolis, MN, USA) was employed. A microplate was covered with a particular monoclonal antibody that was specified for rat IL-6. Consequently, the animals' IL-6 that was present in the tested sample was engaged by the antibody. Following the elimination of any unbound interleukin, an enzyme-linked polyclonal antibody particular for rat IL-6 was appended, and the original blue color was transformed into a yellow one. Finally, the IL-6 level in the tested samples was evaluated by measuring the intensity of the produced color [36].
