2.2.6. Ex Vivo Skin Permeation Studies

An ex vivo permeability study was performed using sheep buccal mucous membrane by the Franz diffusion cell method (MicroettePlus®, Teledyne Hanson, Chatsworth, CA, USA). The freshly excised buccal mucous membrane was obtained from the slaughterhouse and kept in phosphate buffered saline. A prepared buccal mucous membrane of 2 × 2 cm was carefully arranged between the acceptor and donor compartments of the diffusion cell area (1.75 cm<sup>2</sup> ). The permeation characteristics were evaluated for the developed HA-FS-NTF containing the 0.5% marketed formulation of fluconazole and the suspension containing the 0.5% fluconazole. Eight milliliters of phosphate buffered saline (pH 6.8) prepared from disodium hydrogen phosphate (Na2HPO<sup>4</sup> anhydrous) = 14.4 g sodium dihydrogen phosphate (KH2PO<sup>4</sup> anhydrous) = 2.4 g sodium chloride (NaCl) = 80.0 g, and potassium chloride (KCl) = 2.0 g, was kept in the receptor chamber, which was maintained at 32 ± 2 ◦C with the help of a warm water jacket and stirred at 410 to 430 rpm using the magnetic bead. The samples were autosampled at predetermined time intervals and quantified in parallel using high-performance liquid chromatography (HPLC) [22]. Diffusion of the fluconazole from the donor compartment to the receptor compartment through the mounted mucous membrane was determined by plotting the cumulative release pattern of the drug per unit of time and area. We calculated the diffusion coefficient (D), enhancement factor (EF), permeability coefficient (Pc), and steady-state flux (Jss) to determine whether the developed formulation was superior. Finally, the percentage of fluconazole permeated and the total amount of fluconazole diffused across the receptor chamber was calculated using Equation (4):

$$\text{Percent percentage} = \frac{F\_P}{F\_T} \times 100\tag{4}$$

where *Fp* and *F<sup>T</sup>* are the permeated amount of fluconazole in the receptor chamber and the loaded amount of fluconazole in the donor chamber, respectively.
