Amphiphilic Polymeric Nanoparticles Modified with a Retro-Enantio Peptide Shuttle Target the Brain of Mice

The blood–brain barrier (BBB) is a challenge for the treatment of diseases of the central nervous system (CNS) from the systemic circulation. The design of novel strategies to increase drug bioavailability in the CNS is called for. In this work, we synthesized amphiphilic polymeric nanoparticles by the self-assembly of graft copolymers of chitosan (CS, hydrophilic copolymer backbone) hydrophobized in the side-chain with poly­(methyl methacrylate) (PMMA)/poly­(acrylic acid) (PAAc) blocks and surface-decorated with a biologically stable retro-enantio peptide shuttle that improves brain transport. Nanoparticles showed one size population in the 190–210 nm range (intensity distribution) and a relatively small polydispersity index, as measured by dynamic light scattering. The surface charge estimated by the zeta-potential decreased from +35 mV in the unmodified nanoparticles to +14 mV in the modified ones, confirming the exposure of the peptide shuttle at the nanoparticle surface. The cell compatibility and uptake were assayed in hCMEC/D3 cells, a model of BBB endothelium, by a metabolic assay, confocal laser scanning fluorescence microscopy, and imaging flow cytometry in the absence and the presence of endocytosis inhibitors. Results indicated that the peptide shuttle modification improves their cell compatibility and that they are internalized by a clathrin-mediated pathway. In vitro permeability studies conducted in hCMEC/D3 cell monolayers showed that peptide shuttle-modified nanoparticles increase the apparent permeability with respect to the unmodified ones by 3.4 times. Finally, the brain accumulation was investigated upon i.v. administration to Hsd:ICR mice by using fluorescently labeled nanoparticles in an in vivo imaging system and light sheet fluorescence microscopy. Unmodified nanoparticles could be hardly detected in the brain blood vessels and parenchyma. Conversely, nanoparticles modified with the peptide shuttle could be detected after 10 min, with a maximum accumulation at 30 min and a slow concentration decline later on. Calculation of the area under the curve confirmed a 4-fold statistically significant increase in the accumulation of the modified nanoparticles with respect to the unmodified counterparts. These findings demonstrate the promise of this strategy to improve the delivery of nanoencapsulated cargos to the CNS.