PLGA-Encapsulated Cross-Linked GALC Aggregates as a Hierarchical Nanostructured Platform for Enzyme Stabilization and Delivery.
- 1Istituto Nanoscienze - CNR, NEST-SNS Piazza San Silvestro 12 56126 Pisa Italy.
- 2Interdisciplinary Research Center "Health Science" Scuola Superiore Sant'Anna Piazza Martiri Della Libertà, 33 56127 Pisa Italy sssup.it.
Abstract
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.
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BibTeX
@article{colagiorgio2026plgaencapsulated,
title = {{PLGA-Encapsulated Cross-Linked GALC Aggregates as a Hierarchical Nanostructured Platform for Enzyme Stabilization and Delivery.}},
author = {Laura Colagiorgio and Mariacristina Gagliardi and Ambra Del Grosso and Sara Carpi and Miriam De Sarlo and Marco Cecchini},
journal = {International journal of biomaterials},
year = {2026},
month = oct,
doi = {10.1155/ijbm/8346433},
url = {https://europepmc.org/article/MED/42898837},
note = {CC BY},
}
RIS
TY - JOUR
TI - PLGA-Encapsulated Cross-Linked GALC Aggregates as a Hierarchical Nanostructured Platform for Enzyme Stabilization and Delivery.
AU - Laura Colagiorgio
AU - Mariacristina Gagliardi
AU - Ambra Del Grosso
AU - Sara Carpi
AU - Miriam De Sarlo
AU - Marco Cecchini
PY - 2026
DA - 2026/10/18
JO - International journal of biomaterials
DO - 10.1155/ijbm/8346433
UR - https://europepmc.org/article/MED/42898837
AB - 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.
SN - 1687-8787
SN - 1687-8795
ER -