PEG-PLGA nanoparticles: investigation of BBB translocation according to PEG chain length using a primary endothelial cell permeability model

摘要

The Blood-Brain Barrier (BBB) acts as a physical and metabolic barrier between the Central Nervous System (CNS) and the systemic circulation. This complex cell assembly results in a protective, quasi-impermeable barrier for most therapeutic molecules. Nanotechnologies have been used with various success rates to overcome this problem. In particular, nanoparticles (NP) from the FDA-approved poly(0, L-lactide-co-glycolide) (PLGA), grafted with poly(ethylene glycol) (PEG), have been widely investigated. This alternative confers them reduced loxicity, increased circulation time and BBB penetration. However, no systematic studies have been initiated so far to explore the role of PEG chain length on endocytosls and transcytosis of NP across the BBB. This study aimed therefore to compare the translocation rate of PEG-PLGA nanoparticles across a primary endothelial cell model, according to PEG chain length. PEGylated diblock polymers were synthesized as previously reported by DCC coupling reaction between PLGA chains (24 kDa) and mPEG chains of different lengths (1,2,5 and 10 kDa). Polymers were characterized by GPC, ~1H-NMR and FTIR. In parallel, PLGA chains were covalently labelled with the flurorescent moiety (FLU) anthracene-9-carboxylic acid and verified by ~1H-NMR. PEG-PLGA-FLU NPs were prepared by co-nanoprecipitation of PEG-PLGA and PLGA-FLU and subsequently characterized for their size, polydispersity and charge (DLS/ELS) and surface chemical composition (NMR of NP souspension). Endothelial cells were isolated from adult mice brains, then selected by puromycine (2 days) and cultivated in a home-designed conditioning medium (5 days.) Confluent cells were plated in 24-well Transwell plates. 24-h later, cells were treated with an equivalent amoount of the various PEG-PLGA-FLU NPs; FLU-PLGA NPs served as controls. Transcytosis was quantified by fluorescent detection in both compartments. Endocytosis was monitored by confocal microscopy. A diblock polymer library was created and characterized. Expected molecular weights and PEG content were found, with an overall grafting efficiency ≥ 90%. PEG length affects NP size and surface properties. Thus, nanoprecipitation parameters were optimized to obtain a near-constant NP diameter regardless of the PEG chain lengths to eliminate the confounding effect of NP size on BBB passage. An in vitro BBB permeability assay was designed from primary endothelial cells. Expression of P-gp, GLUT-1, OATP and CAT transporters was used to validate the biorelevancy of transport mechanisms. TEER and Lucifer yellow allowed to assess the model tightness. The effect of PEG length on endothelial cell translocation has been being investigated.