Brain-targeted enzyme loaded nanoparticles: a breach through the blood brain barrier for enzyme replacement therapy in Krabbe disease

Lysosomal storage disorders (LSDs) result from an enzyme deficiency within lysosomes. The systemic administration of the missing enzyme, however, is not effective in the case of LSDs with central nervous system (CNS)-involvement. Here, an enzyme delivery system based on the encapsulation of cross-linked enzyme aggregates (CLEAs) into poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) functionalized with brain targeting peptides (Ang2, g7 or Tf2) is demonstrated for Krabbe disease, a neurodegenerative LSD caused by galactosylceramidase (GALC) deficiency. We first synthesize and characterize Ang2-, g7- and Tf2-targeted GALC CLEA NPs. We study NP cell trafficking and capability to reinstate enzymatic activity in vitro. Then, we successfully test our formulations in the Twitcher mouse. We report enzymatic activity measurements in the nervous system and in accumulation districts upon intraperitoneal injections, demonstrating activity recovery in the brain up to the unaffected mice level. Together, these results open new therapeutic perspectives for all LSDs with major CNS-involvement

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

Nanocarriers for Protein Delivery to the Cytosol: Assessing the Endosomal Escape of Poly(Lactide-co-Glycolide)-Poly(Ethylene Imine) Nanoparticles

Therapeutic proteins and enzymes are a group of interesting candidates for the treatment of numerous diseases, but they often require a carrier to avoid degradation and rapid clearance in vivo. To this end, organic nanoparticles (NPs) represent an excellent choice due to their biocompatibility, and cross-linked enzyme aggregates (CLEAs)-loaded poly (lactide-co-glycolide) (PLGA) NPs have recently attracted attention as versatile tools for targeted enzyme delivery. However, PLGA NPs are taken up by cells via endocytosis and are typically trafficked into lysosomes, while many therapeutic proteins and enzymes should reach the cellular cytosol to perform their activity. Here, we designed a CLEAs-based system implemented with a cationic endosomal escape agent (poly(ethylene imine), PEI) to extend the use of CLEA NPs also to cytosolic enzymes. We demonstrated that our system can deliver protein payloads at cytoplasm level by two different mechanisms: Endosomal escape and direct translocation. Finally, we applied this system to the cytoplasmic delivery of a therapeutically relevant enzyme (superoxide dismutase, SOD) in vitro

Endocytosis of Nanomedicines: The Case of Glycopeptide Engineered PLGA Nanoparticles

The success of nanomedicine as a new strategy for drug delivery and targeting prompted the interest in developing approaches toward basic and clinical neuroscience. Despite enormous advances on brain research, central nervous system (CNS) disorders remain the world’s leading cause of disability, in part due to the inability of the majority of drugs to reach the brain parenchyma. Many attempts to use nanomedicines as CNS drug delivery systems (DDS) were made; among the various non-invasive approaches, nanoparticulate carriers and, particularly, polymeric nanoparticles (NPs) seem to be the most interesting strategies. In particular, the ability of poly-lactide-co-glycolide NPs (PLGA-NPs) specifically engineered with a glycopeptide (g7), conferring to NPs’ ability to cross the blood brain barrier (BBB) in rodents at a concentration of up to 10% of the injected dose, was demonstrated in previous studies using different routes of administrations. Most of the evidence on NP uptake mechanisms reported in the literature about intracellular pathways and processes of cell entry is based on in vitro studies. Therefore, beside the particular attention devoted to increasing the knowledge of the rate of in vivo BBB crossing of nanocarriers, the subsequent exocytosis in the brain compartments, their fate and trafficking in the brain surely represent major topics in this field

Cross linked enzyme aggregates as versatile tool for enzyme delivery: application to polymeric nanoparticle.

ABSTRACT: 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

Cross-Linked Enzyme Aggregates as Versatile Tool for Enzyme Delivery: Application to Polymeric Nanoparticles

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-<i>co</i>-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