Therefore, by adding TPP, a competition
would occur between ionotropic LXH254 cross-linking by a polyanion and neutralization through deprotonation of CS. Ionotropic cross-linking is an important property which is broadly used in ionotropic gelation processes. The mild effect of CS on the activity of ASNase II and the higher entrapment efficiency indicated adding TPP into the protein-CS solution as the selected way for nanoparticle preparation in the next steps. Optimization of CS and TPP concentrations CSNPs were prepared by certain amounts of CS (containing 1 mg ASNase II) and TPP. Increasing TPP volume or decrease in CS/TPP ratio led to increased turbidity, indicating a shift in HM781-36B ic50 the size variation of the particles to larger dimensions. Optimization of the CS/TPP ratio revealed that
when this ratio declined to 0.2/0.06, 0.3/0.08, and 0.4/0.11, high turbidity appeared from the increased aggregation of the nanoparticles. Thus, the CS/TPP ratios of 0.2/0.06, 0.3/0.08, and 0.4/0.11 Selleckchem Evofosfamide (Table 1) were discarded because of aggregation which was confirmed microscopically [14, 30]. Nanoparticle aggregation occurs under circumstances such as the rise in pH of suspension , inadequate speed of homogenization, or high level of cross-linker . López et al.  suggested that since the pK α value of the chitosan is close to the neutral pH, particles spontaneously aggregate in slightly basic pH, where they become completely uncharged. The final pH of the prepared ASNase II-loaded CSNP suspensions was between 6.2 and 6.3 in all CS/TPP ratios, which many was lower than the pK α of chitosan. Moreover, increase in TPP concentration
might be a more important agent for particle aggregation via cross-linking, as was observed through a raise in TPP volume. Aggregation might be prevented by using a high-speed homogenizer or by sonication during CSNP preparation, but such approaches would lead to inactivation of ASNase and thus could not be used. Table 3 shows that the average size of the particles increased with a lower CS/TPP ratio (PDI < 0.4) and was positively associated with ASNase II entrapment efficiency. Entrapment efficiency was the highest (70%) when the concentration of CS/TPP was 0.4/0.095. These results might be due to an increased number of interacting units at higher polymer concentrations and to cross-linker levels that lead to the observed increase in particle size and entrapment efficiency [32, 33]. Table 3 The size, polydispersity index (PDI < 5 and unimodal size distribution), and entrapment efficiency of nanoparticles CS (% w/ v)/TPP (% w/ v) Size (nm) PDI EE (%) 0.2/0.04 138 ± 7 0.35 59.1 0.3/0.06 180 ± 8 0.35 60.2 0.4/0.08 224 ± 10 0.44 62.7 0.2/0.05 187 ± 9 0.43 64.0 0.3/0.075 209 ± 11 0.47 67.3 0.4/0.095 247 ± 10 0.4 70.