Charge-driven lipid nanoparticle encapsulation of enzymes for enhanced ERT with reduced immunogenicity.

Efficient intracellular delivery of therapeutic enzymes remains a central limitation of protein-based therapies. In lysosomal storage disorders such as Pompe disease, classical enzyme replacement therapy (ERT) is particularly constrained by inefficient delivery to skeletal muscle, limited cellular uptake, and immunogenicity. Here, we introduce a lipid nanoparticle (LNP) platform designed for charge-mediated enzyme encapsulation, enabling high enzyme loading (up to 100 enzymes per LNP) while preserving catalytic activity. Developed enzyme-loaded LNPs achieve efficient delivery to muscular tissues and significantly increase cellular uptake compared with free enzyme administration. High particle-level enzyme loading allows each internalization event to deliver a large enzymatic payload, reducing the number of uptake events required per cell and resulting in up to 15-fold higher intracellular enzyme activity in muscle cells without cytotoxicity. Structurally, enzymes are internally sequestered within multilamellar vesicular structures rather than exposed on the particle surface, overcoming limitations of earlier low-yield or adsorption-based approaches and achieving encapsulation efficiencies of up to 60%. Upon repeated dosing in mice, the encapsulated enzymes exhibit markedly reduced immunogenicity, with more than a 5-fold decrease in anti-drug antibody formation and elimination of antibody-mediated infusion-associated reactions. Together, these results demonstrate that encapsulation-driven enhancement of tissue delivery, cellular uptake, and intracellular enzyme availability directly addresses key limitations of classical ERT. The scalable and biocompatible LNP platform, therefore, provides a generalizable framework for improving enzyme therapeutics for lysosomal storage disorders.