Design of a versatile nanoparticle-based delivery system for intracellular and in vivo-targeted protein replacement therapy

Protein replacement therapy is to-date one of the main clinical approaches to treat a wide number of diseases that are correlated to a protein dysfunction or absence. However, the systemic administration of proteins rarely translates into a successful therapy, due to the fragile nature of these molecules and their inability to reach specific targets in vivo. Pharmaceutical technology strategies such as the encapsulation into a nanovector could overcome the limits of protein administration, since nanovectors can protect the payload from degradation and rapid clearance, and they can be functionalized to reach specific targets. In this thesis, a nanoparticle (NP) protein delivery system based on the biodegradable polymer poly(lactideco- glycolide) (PLGA) was designed and tailored to the delivery of active therapeutic proteins towards specific intracellular targets (i.e. the lysosome and the cytosol) and in vivo districts, with a special attention to overcome the blood brain barrier and deliver the therapeutics to the central nervous system. The synthesis approach is based on the modification of proteins into cross-linked enzyme aggregates (CLEAs) and their encapsulation into PLGA with a straightforward and simple nanoprecipitation method. This formulation strategy allowed excellent encapsulation efficiencies and catalytic activity retention, taking significant steps forward compared to encapsulation methods described so far in literature for PLGA NPs. The obtained CLEA NP system was first applied to the delivery of lysosomal enzymes involved in the treatment of lysosomal storage disorders, namely, Infantile Neuronal Ceroid Lipofuscinosis and Krabbe Disease, and their efficacy as enzyme replacement therapy agents was demonstrated both in vitro and in vivo. Indeed, brain-targeted versions of CLEA NPs were demonstrated to promote the complete enzymatic activity recovery in cell models of both diseases and to deliver the therapeutic payload to the cell lysosome, that represents the intracellular target of LSDs. Moreover, upon systemic administration CLEA NPs are able to cross the blood brain barrier and restore the missing enzymatic activity in the brains of mouse models of LSDs. Finally, the application of CLEA NPs was extended to allow delivery even of cytosolic proteins, thus significantly expanding the therapeutic applicability of this platform, by incorporation of an endosomal escape agent in the formulation. Such modified CLEA NPs were demonstrated to evade the endo-lysosomal route upon cell uptake and deliver therapeutic enzymes also to the cytosol thanks to two distinct mechanisms, namely, direct translocation through the cell membrane and endocytosis followed by endosomal disrupture. The new formulation successfully allows the delivery of a therapeutic enzyme to the cytosol and subsequent enzymatic activity increase in vitro, adding strength to the versatility of this system as protein delivery platform for virtually any protein-related disorder.