Functional Nanopore Engineering for Encapsulation and Activity Modulation of Horseradish Peroxidase

The development of structured hybrid materials with calibrated nanopores and homogeneous functionalization is crucial for enzyme encapsulation with optimal loading efficiency and specific activity. This work showcases the encapsulation of horseradish peroxidase (HRP) via the direct synthesis of functional nanopores with diameters spanning from 4 to 31 nm, by employing polyion complex (PIC) micelles as structure-directing and -functionalizing agents for silica. Micelle size was controlled by reversible addition–fragmentation chain transfer (RAFT) synthesis of double-hydrophilic block copolymers (poly(oligo(ethylene glycol)methyl ether acrylate)-block-poly(acrylic acid), POEGMEAn-b-PAAm): The hydrodynamic diameter of the PIC micelles scales with the copolymer length, and the nanopore size in silica materials is proportional to the micelle core. These well-calibrated nanopores benefit from intrinsic and uniform acrylic acid functionality (close to 1.9 mmolacrylic acid·gSiO2–1), which allows elucidation of their structure–activity relationship in terms of HRP encapsulation and specific activity. The loading capacity was optimal for pore diameters of about 9–17 nm, reaching about 35 mg·gmaterial–1. The lower loadings obtained for smaller and larger pores indicate mass transfer and low specific area limitations, respectively. Nanopores larger than the hydrodynamic diameter of the enzyme are required to maintain the specific activity of HRP. The highest enzyme activity achieved (ca. 1200 U·gmaterial–1) was competitive with the literature and corresponded to materials with a pore diameter of 17 nm. This optimized functional nanopore size exceeds the hydrodynamic diameter of the enzyme, facilitating mass transfer and preserving its specific activity, while also being sufficiently small to maximize the available functional surface area.