Macromolecular Crowding Effect on Chitosan-Hyaluronic Acid Complexation and the Activity of Encapsulated Catalase

The associative phase separation of charged biomacromolecules plays a key role in many biophysical events that take place in crowded intracellular environments. Such natural polyelectrolyte complexation and phase separation often occur at nonstoichiometric charge ratios with the incorporation of bioactive proteins, which is not studied as extensively as those complexations at stoichiometric ratios. In this work, we investigated how the addition of a crowding agent (polyethylene glycol, PEG) affected the complexation between chitosan (CS) and hyaluronic acid (HA), especially at nonstoichiometric ratios, and the encapsulation of enzyme (catalase, CAT) by the colloidal complexes. The crowded environment promoted colloidal phase separation at low charge ratios, forming complexes with increased colloidal and dissolution stability, which resulted in a smaller size and polydispersity (PDI). The binding isotherms revealed that the addition of PEG greatly enhanced the ion-pairing strength (with increased ion-pairing equilibrium constant K_a from 4.92 × 10⁴ without PEG to 1.08 × 10⁶ with 200 g/L PEG) and switched the coacervation from endothermic to exothermic, which explained the promoted complexation and phase separation. At the stoichiometric charge ratio, the enhanced CS-HA interaction in crowded media generated a more solid-like coacervate phase with a denser network, slower chain relaxation, and higher modulus. Moreover, both crowding and complex encapsulation enhanced the activity and catalytic efficiency of CAT, represented by a 2-fold increase in catalytic efficiency (K_cat/K_m) under 100 g/L PEG crowding and CS-HA complex encapsulation. This is likely due to the lower polarity in the microenvironment surrounding the enzyme molecules. By a systematic investigation of both nonstoichiometric and stoichiometric charge ratios under macromolecular crowding, this work provided new insights into the complexation between natural polyelectrolytes in a scenario closer to an intracellular environment.