2.2.2. Preparation of Fluconazole-Sesame Oil Nanotransfersomes (FS-NTF)

Development of the drug-loaded NTF was approached by the thin-layer evaporation technique using the optimized ratios of the components suggested by the statistical design [17]. In brief, accurately measured quantities of Tween 20®, as an edge activator, were incorporated with the phospholipid to form the vesicular structure of the nanotransfersome. The Tween® and phospholipid were placed in a round-bottom flask, along with the sesame oil and fluconazole, using the solvent mixture of chloroform:methanol at a ratio of 1:1 (*v*/*v*). The organic solvents in the round-bottom flask were evaporated at reduced pressure using the rotary vacuum evaporator (BUCHI Rotavapor R-205, Marshall Scientific, Hampton, NH, USA) at 45 ◦C, and the rotation of the flask was maintained at 80 rpm. Later, following the removal of traces of organic solvents in the mixture, a thin film of the components was formed within the inner wall of the round-bottom flask. Lastly, phosphate buffered saline (pH 7.4) was used to hydrate the thin layer in order to form the lipid vesicles at room temperature. The multilamellar vesicles were formed upon hydration, and they were further sonicated using a probe sonicator (Branson, Danbury, CT, USA) for 15 min to form our desired drug-loaded unilamellar vesicles (FS-NTF). Finally, the formulation was collected after it had passed through polycarbonate membranes of the desired size and stored at 4 ◦C for further characterization and experiments. Figure 1


**Figure 1.** Schematic representation of steps of preparation of fluconazole-sesame oil nanotransfersomes (FS-NTF).
