*3.7. In Vitro Drug Release Studies*

Drug release studies were carried out through dialysis bag diffusion technique over 48 h, as previous described by Silva et al. and Abouhussein et al. [9,81]. Simulated nasal electrolyte solution (SNES) and phosphate-buffered solution (pH 6.4) were used as release media. SNES was prepared by dissolving 12.9 mg of potassium chloride, 745 mg of sodium chloride, and 3.6 mg of calcium chloride in 1000 mL of ultrapure water. Phosphate-buffered solution pH 6.4 was prepared according to the European Pharmacopoeia (Ph. Eur.) [9,31,48]. The release profile of rivastigmine from the NLC was evaluated for the optimized formulations produced by HPH and ultrasound technique. Briefly, 2.5 mL of NLC with 1.2 mg/mL of rivastigmine were filled in a dialysis bag (cellulose membrane with molecular weight cut-offs of 300 kDa, 12–15 cm long, Spectra/Por® Biotech, US), clamped, and immersed in a glass vial containing 250 mL of release medium at 37 ± 0.5 ◦C, then stirred at 50 rpm. At predetermined time intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 36, and 48 h), 1.0 mL of sample was collected and the release medium was replaced with the same volume of fresh medium to guarantee sink conditions. Collected samples were passed through a syringe filter (0.21 µm) and diluted in 1 mL of acetonitrile, being the amount of rivastigmine measured by HPLC in a Thermo Scientific™ Dionex™ UltiMate™ instrument, with a detection wavelength of 237 nm using an analytical reverse-phase C<sup>18</sup> column (100 mm × 4.6 mm, 5 µm) from Thermo Scientific Acclaim™ (Portugal). The results were reported as the mean ± SD of three replicates (*n* = 3). The cumulative rivastigmine released was calculated and expressed as a percentage of the theoretical maximum drug content.
