Cetuximab-loaded self-assembled polymeric nanoparticles for the integrin-targeted therapy of glioblastoma

The blood-brain barrier (BBB) restricts the delivery of therapeutic small-molecule and macromolecular drugs to the central nervous system and precludes the therapeutic application of monoclonal antibodies in glioblastoma (GBM). In this work, we investigate integrin-targeted polyelectrolyte complex nanoparticles for the delivery of the anti-epidermal growth factor receptor monoclonal antibody cetuximab (CET) across the BBB in GBM. For this, an amphiphilic chitosan-graft-poly(methyl methacrylate-co-acrylic acid) copolymer is functionalized with cyclo(Arg-Gly-Asp-D-Phe-Val) (PEP) to engage ανβ3 and ανβ5 integrins expressed by the BBB endothelium and overexpressed by the GBM cells and co-assembled with sodium alginate using a T-junction microfluidics device. This double self-assembly method results in optimal CET encapsulation efficiency (∼99%) and the formation of loaded nanoparticles with a diameter of ∼322 nm, with very good structural fidelity and ∼16% w/w payload. Nanoparticles show very good compatibility with the human brain microvascular endothelium cell line hCMEC/D3 and the GBM cell line U87. Moreover, PEP-conjugated nanoparticles display significantly greater apparent permeability (P_app) in hCMEC/D3 monolayers grown on semi-permeable membranes, an in vitro model of the BBB, than the unmodified counterparts; P_app values being consistent with macromolecular systems capable of achieving measurable BBB transport in vivo. Cell uptake studies in both cell types using confocal laser scanning microscopy and imaging flow cytometry reveal an energy-dependent internalization pathway and a significant increase of the targeted nanoparticles with respect to the unmodified ones. Finally, CET-loaded nanoparticles significantly reduce the viability of the U87 cells, a model of GBM in vitro, suggesting its very good antitumoral activity in vitro. Overall results support the potential of these doubly self-assembled polymeric nanoparticles for the encapsulation and targeted delivery of therapeutic macromolecules to the brain.