Aggregation Behavior and Inner Structure of Nanoparticles from Trypsin and Chondroitin Sulfate in a Wide pH Range–Implications for the Design of Biocompatible Nanocarriers

Nanoparticles having a radius of ca. 200 nm are prepared from the proteolytic enzyme trypsin (TRY) and the anionic polysaccharide chondroitin sulfate (CS) by electrostatic complexation at pH 4.3 and subsequent thermal treatment. 3D dynamic light scattering and synchrotron small-angle X-ray scattering are used to assess their size distribution and their inner structure in the pH range up to 11.3. They feature a monomodal size distribution up to pH 8, whereas the distribution becomes very broad above. Their inner structure depends strongly on pH, again with a significant change at pH 9. SAXS reveals hierarchical organization within the NPs. At pH 4.3–8.0, compact TRY-CS complexes at intermediate length scales (∼30 nm), whose size increases by thermal treatment, are present within the NPs. At pH 10.1–11.3 the spherical NPs transform into fractal aggregates. The spherical internal substructure vanishes, and only smaller substructures stay present. At low pH values, where TRY is homogeneously positively charged, these results are attributed to the electrostatic interactions of TRY with CS. At intermediate pH values, where the electrostatic surface potential of TRY is patchy, the TRY-TRY interactions seem to play an important role. The morphological properties of the TRY-CS NPs and their length-scale-dependent response to thermal treatment (20 min at 60 °C) and increase of pH are expected to be crucial for the drug/nutrient loading and release properties and the interactions with other biopolymers, proteins, cells, and tissues. They can also be a useful guide for other polysaccharide–protein biocompatible nanocarriers.