In Vitro Release Kinetics of Lysozyme-loaded Chitosan Nanoparticles

The dialysis bag (MWCO: 12–14 kDa) was used as the release barrier. The isotonic nanosuspension of LZ–CSNPs and LZ–AqS (control) were subjected to in vitro release study using STF as a release medium. The 6.8 g of NaCl, 2.2 g of NaHCO₃, 1.4 g of KCl, and 0.08 g of CaCl₂.2H₂O were dissolved in 1000 mL purified water to prepare the STF (pH 7.4). An equivalent volume of nanosuspension of LZ–CSNPs and LZ–AqS (containing 1000 µg of LZ) was filled into the pre–activated dialysis bags and the ends of the bags were closed using closures. The dialysis bags were put into beakers containing STF (50 mL each). The set-ups were placed in a shaking (100 rpm) water bath maintained at 35 ± 1 °C. At fixed time intervals, 1 mL of each sample was collected from each beaker, and after sampling equal volume of fresh STF was added to each beaker. The collected samples were centrifuged and 20 µL of the supernatant was injected into the HPLC–UV system to determine the drug concentration. The LZ–AqS was formulated by suspending LZ (10 mg) in 10 mL of 0.25% (w/v) Polysorbate–20 aqueous solution [38 (link),39 ]. The experiment was executed three times for each LZ–formulations. The cumulative amount of drug released (DR%) was calculated by the following expression Equation (3).
$$DR\%=\frac{Conc.\left(\mathsf{\mu }\mathrm{g}/\mathrm{mL}\right)\times DF\times Volume of release medium \left(\mathrm{mL}\right)}{Initial quantity of LZ used for the experiment \left(\mathsf{\mu }\mathrm{g}\right)}\times 100$$
where “DF” is the dilution factor. The release data were fitted to different kinetic models “(Zero–order, First–order, Higuchi–Matrix Square–Root, Hixson–Crowell Cube–Root, and Korsmeyer–Peppas)”. The best–fitted kinetic model for LZ release from CSNPs was categorized based on the highest value of the coefficient of correlation (R²). From the slopes and intercepts of the different release plots, the release exponent (n-value) was calculated [40 (link)]. The n-value would suggest the mechanism of LZ release from CSNPs [13 (link),41 ,42 (link)].