PLGA-Encapsulated Cross-Linked GALC Aggregates as a Hierarchical Nanostructured Platform for Enzyme Stabilization and Delivery.

Enzyme aggregation and polymer encapsulation are complementary strategies used to overcome the intrinsic instability and limited bioavailability of hydrophilic enzymes in medical uses. In this work, we report a systematic study of polymer-encapsulated cross-linked enzyme aggregates (pe-CLEAs) as a hierarchical nanostructured platform for the stabilization and delivery of galactosylceramidase (GALC), a lysosomal enzyme used in the enzyme replacement therapy for Krabbe disease. GALC-CLEAs were produced via cross-linking and controlled precipitation, yielding stable aggregates with small hydrodynamic diameters (40-66 nm) and narrow polydispersity index (PDI < 0.1). Scaling up the synthesis by 50-fold markedly enhanced total enzymatic output, reaching 1225.4 ± 213.2 U·mL-1. GALC-CLEAs were subsequently encapsulated within PLGA nanoparticles. Systematic screening of enzyme-to-polymer ratios revealed an optimal formulation at 75 μg GALC per mg PLGA, displaying a maximum specific activity of 6.0 ± 1.3 U·mg-1. Colloidal stability and retention assays showed that pe-CLEAs preserved full catalytic activity and retained enzyme confinement over 30 days at 4°C. At 37°C, pe-CLEAs exhibited time-dependent size dynamics while retaining substantial activity. In vitro evaluations in primary fibroblasts derived from Twitcher mice (a Krabbe disease model) demonstrated complete cytocompatibility up to 5.0 mg·mL-1 and robust intracellular recovery of GALC activity across tested doses (0.5-5.0 mg·mL-1), outperforming equivalent doses of native enzyme. This work provides a comprehensive physicochemical and functional characterization of pe-CLEAs, elucidating how hierarchical aggregation and polymer encapsulation can synergistically preserve stability and bioactivity. The design principles outlined here support the rational development of polymer-enzyme nanocarriers for enzyme replacement therapy and related biomedical applications.