Impact of the protein corona in RGD-functionalized nanoliposomes delivering enzymatic replacement therapy for Fabry disease.

Abstract

The protein corona (PC) formed upon nanoparticle exposure to blood critically defines their biological identity and in vivo fate, yet its impact on targeted liposomal enzyme replacement therapies (ERTs) remains incompletely understood. Here, we systematically investigated how RGD functionalization (including varying ligand densities) and α-galactosidase A (GLA) loading influence PC composition, cellular uptake, and biodistribution using a panel of liposomal formulations with and without targeting ligands and therapeutic cargo. Quantitative proteomics revealed that both RGD functionalization and GLA loading reshape the PC by enriching key opsonins (e.g., C3 and IgG subclasses) together with dysopsonins (such as apolipoproteins and albumin), generating distinct and balanced immune signatures. Notably, RGD promoted complement recruitment, indicating increased immune recognition potential. In vitro, RGD functionalization significantly enhanced endothelial uptake even in the presence of a pre-formed PC, demonstrating that targeting functionality is modulated but not abolished by corona formation. In vivo, GLA-loaded liposomes increased enzymatic activity in plasma and tissues compared to free GLA, and higher RGD density enhanced delivery to liver, spleen, kidney, and heart, albeit with reduced plasma persistence. All GLA-loaded formulations showed brain-associated enzymatic activity in perfused brain tissue. Additional capillary-depletion analysis indicated that, at 30 min after administration, the measurable signal was predominantly associated with the cerebrovascular fraction, that could be potentially linked to coronas enriched in ApoE, transferrin, and transthyretin. Together, these findings support a hybrid model in which the protein corona modulates, rather than masks, targeting ligands through a balanced opsonin/dysopsonin profile. This work provides mechanistic insight into how nanoparticle surface properties and biological identity interplay, offering general design principles for optimizing targeted nanomedicines beyond liposomal ERT.