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.

Diffuse intrinsic pontine glioma (DIPG) is a chemo-resistant, incurable pediatric tumor of the central nervous system (CNS). The blood−brain barrier (BBB) remains intact in the course of the disease, preventing drugs from entering the brain and resulting in therapeutic failure. The topoisomerase I inhibitor SN-38 shows strong anticancer activity in a patient-derived DIPG cell line in vitro, though a low CNS bioavailability and anti-DIPG efficacy in vivo. In this work, we produced SN-38-loaded polymeric nanoparticles of an amphiphilic chitosan (CS)-g-poly(methyl methacrylate)-poly(acrylic acid) copolymer that were surface-modified with a peptide shuttle that improves transport across the BBB. Drug-loaded nanoparticles displayed a size of ∼200 nm (intensity distribution) and a ζ-potential of +16 mV. The cytocompatibility and endocytosis assayed in DIPG cells (both attached and in suspension) indicated that the nanoparticles are compatible and mainly internalized by clathrin-mediated endocytosis and that the anticancer activity of SN-38 is preserved after nanoencapsulation. In addition, a tandem permeability/anticancer activity study utilizing a coculture model of BBB endothelial cells and DIPG cell spheroids demonstrated that the modified nanoparticles cross a BBB endothelial cell monolayer to a higher extent than the unmodified counterparts and are taken up by DIPG cells. After 72 h of exposure, both SN-38-loaded nanoparticles killed ∼84-88% of the DIPG cells in suspension, indicating that they reach a concentration above the inhibitory concentration 50 of the drug. Finally, the brain accumulation of the drug-loaded nanoparticles upon intravenous injection to Hsd:ICR mice was preliminarily characterized by light sheet fluorescence microscopy. As opposed to unmodified SN-38-loaded nanoparticles, the modified counterparts bind the brain blood vessels and accumulate in the cerebral parenchyma to a large extent. These results confirm the potential of this nanotechnology platform to deliver anticancer agents to the brain in DIPG and other brain tumors with fully conserved BBB. KEYWORDS: diffuse intrinsic pontine glioma (DIPG), amphiphilic polymeric nanoparticles, peptide shuttles, targeting of the central nervous system (CNS)