Optimization of CS and TPP concentrations To optimize the CS/TPP ratio based on particle size and the entrapment efficiency, various CS concentrations (0.2%, 0.3%, and 0.4% (w/v)) were prepared from the stock solution. The concentrated TPP Selleckchem 3-deazaneplanocin A solution (0.5% (w/v)) was used in order not to dilute the CS/ASNase II mixture

more than necessary. From this stock solution, different volumes of TPP solution (Table 1) were added dropwise (10 μl per 10 s interval) to 1 ml of each CS concentration (containing 1 mg lyophilized ASNase II) with stirring (about 800 rpm), with particular care taken to avoid foam formation. In addition to the applied volumes of TPP, Table 1 shows the final concentrations of the added TPP (% w/v). All procedures were carried out at room BYL719 cell line temperature (25°C). After 10 min of stirring, the particles were collected by centrifugation at 25,000 × g, 25°C for 30 min in 50-μl glycerol bed. The supernatants AZD5153 order were separated to estimate the entrapment efficiency (%). The pellets of the particles in glycerol were suspended in 1 ml of distilled water to determine the average sizes (nm). Table 1 Chitosan concentrations,

TPP volumes from TPP stock solution (0.5%  w / v ), and final TPP concentrations in final prepared nanoparticle suspensions CS (% w/ v) TPP (ml) TPP (% w/ v) 0.2 0.1 0.04 0.12 0.05 0.14 0.06 0.3 0.15 0.06 0.18 0.07 0.21 0.08 0.4 0.2 0.08 0.24 0.095 0.28 0.11 Optimization of protein loading The stable and suitable CS/TPP ratio from the previous step was selected in order to investigate the optimal entrapment efficiency and loading capacity of CSNPs, loaded with five different Janus kinase (JAK) amounts of protein

(1, 2, 3, 4, and 5 mg). Nanoparticles were prepared according to the procedure given above by adding a certain amount of lyophilized ASNase II in 1 ml of optimal CS solution. After centrifugation, the supernatants were separated to estimate the entrapment efficiency. The pellets of the particles in glycerol were suspended in 1 ml of DDW and dispersed by sonication. The size (nm), zeta potential (mV), protein content (mg), entrapment efficiency (%), and loading capacity (%) of the particles were determined. Entrapment efficiency estimation In order to determine the entrapment efficiency of the nanoparticles, it was necessary to detect by the Lowry method [21] the amount of free enzyme in the clear supernatant.