Entrapment Efficiency Percentage (EE%)

1

The EE% of the developed and optimized FS-NTF was determined using the indirect method [18], where the EE% was stated as the percentage of the total amount of drugs incorporated within the final formulation. To perform this experiment, the FS-NTF was freeze dried (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) first to remove the traces of the aqueous phase in the formulation. Later, it was

dispersed with methanol and mixed cautiously. The mixture was centrifuged (Centurion, West Sussex, UK), and the supernatant was collected. The residue was washed again with the same solvent to recover the traces of the free fluconazole present in the developed formulation. The supernatant containing the drug was measured to determine the concentration of free fluconazole in the formulation using a UV-visible spectrophotometer (6705 UV/Vis spectrophotometer; JENWAY, Cole-Parmer, Stone, Staffordshire, UK) at 261.6 nm. The correlation coefficient was 0.9997, and the molar absorptivity equaled 0.735 <sup>×</sup> <sup>10</sup><sup>3</sup> L/mol/cm [19].

Finally, the EE% of the formulation was determined using Equation (2).

$$EE\% = \frac{\mathcal{C}\_{total} - \mathcal{C}\_{free}}{\mathcal{C}\_{total}} \times 100\tag{2}$$

where *Ctotal* and *Cfree* are the total quantity of drug added in each formulation according to the experimental design mentioned in Table 1 (25, 50, or 75 mg) and the free drug available outside the formed nanotransfersome vesicles, respectively.
