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Polymeric nanoparticles (NPs) represent one of the most promising tools in nanomedicine and have been extensively studied for the delivery of water-insoluble drugs. However, the efficient loading of therapeutic enzymes and proteins in polymer-based nanostructures remains an open challenge. Here, we report a synthesis method for a new enzyme delivery system based on cross-linked enzyme aggregates (CLEAs) encapsulation into poly(lactide- co-glycolide) (PLGA) NPs. We tested the encapsulation strategy on four enzymes currently investigated for enzyme replacement therapy: palmitoyl protein thioesterase 1 (PPT1; defective in NCL1 disease), galactosylceramidase (GALC; defective in globoid cell leukodystrophy), alpha glucosidase (aGLU; defective in Pompe disease), and beta glucosidase (bGLU; defective in Gaucher's disease). We demonstrated that our system allows encapsulation of enzymes with excellent activity retention (usually around 60%), thus leading to functional and targeted nanostructures suitable for enzyme delivery. We then demonstrated that CLEA NPs efficiently deliver PPT1 in cultured cells, with almost complete enzyme release occurring in 48 h. Finally, we demonstrated that enzymatic activity is fully recovered in primary NCL1 fibroblasts upon treatment with PPT1 CLEA NPs.

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