Molecular Permeability Behavior and Catalytic Effectiveness in Zwitterionic Polymer Hydrogel-Enzyme Hybrid Nanocarrier

Enzyme-encapsulated nanogels serve as promising platforms for constructing enzyme nanoreactors in which mass transport plays a crucial role in the enzymatic performance. However, it remains a challenge to investigate the relationship between the permeability of nanoreactors and their catalytic efficiency owing to their small dimensions. The molecular permeability behavior and enzyme activity of two types of nanogels with different hydrophobicities are compared using 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence assays. Partitioning experiments with different fluorescent dyes demonstrate that hydrophobic and electrostatic interactions govern the molecular distributions within the nanogels. Total internal reflection microscopy (TIRF) further demonstrates that a less hydrophobic microenvironment facilitates the faster mass transport of hydrophobic resorufin molecules. This contributes to the higher catalytic activity and greater reaction heterogeneity observed in the single-particle assays. These results underscore the importance of hydrogel molecular permeability in modulating enzyme kinetics and offer valuable insights into the rational design of efficient enzyme nanoreactors.