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 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 selleck inhibitor 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 selleck kinase inhibitor were separated to estimate the entrapment efficiency (%). The pellets of the particles in glycerol were suspended in 1 ml of distilled water to 4SC-202 mouse 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 Selleck Baf-A1 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.