RESUMEN
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.
Asunto(s)
Portadores de Fármacos/química , Enzimas/administración & dosificación , Nanopartículas/química , Polímeros/uso terapéutico , Células Cultivadas , Fibroblastos/metabolismo , Galactosilceramidasa/administración & dosificación , Humanos , Métodos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/uso terapéutico , Solubilidad , Tioléster Hidrolasas/administración & dosificación , alfa-Glucosidasas/administración & dosificación , beta-Glucosidasa/administración & dosificaciónRESUMEN
Photodynamic therapy (PDT) is an approach to treating cancer and involves light-induced activation of a photosensitizer that triggers the formation of reactive oxygen species (ROS) in targeted cells and subsequent cell death. Examples of photosensitizers are porphyrins, including the natural compound chlorophyll. These molecules can be delivered alone or co-formulated with an agent, such as quantum dots (QDs), that is able to excite them through a fluorescence resonance energy transfer (FRET)-based mechanism. We encapsulated a chlorophyllin copper complex and CdSe/ZnS core-shell QDs into biodegradable nanoparticles (NPs) composed of poly(lactide-co-glycolide) (PLGA), that allow modification with specific targeting ligands. When excited at 365â nm, FRET occurs between co-encapsulated QDs and chlorophyllin to result in the formation of ROS. This chlorophyllin-QD coformulation allows generation of ROS both in an aqueous environment and in cells, thus confirming the potential of this formulation in PDT.
Asunto(s)
Clorofilidas/farmacología , Fibroblastos/efectos de los fármacos , Nanopartículas/química , Fármacos Fotosensibilizantes/farmacología , Poliglactina 910/química , Puntos Cuánticos/química , Rayos Ultravioleta , Animales , Clorofilidas/química , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Ratones , Células 3T3 NIH , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Terapia de Reemplazo Enzimático/métodos , Galactosilceramidasa/administración & dosificación , Leucodistrofia de Células Globoides/terapia , Nanopartículas/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Galactosilceramidasa/deficiencia , Células HEK293 , Antígenos HLA/metabolismo , Humanos , Leucodistrofia de Células Globoides/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ribonucleasa Pancreática/metabolismo , Valina-ARNt Ligasa/metabolismoRESUMEN
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.