The promising results obtained in cell transfection of pBAE polyplexes with BB4 encouraged us to also explore its transmigration potential across the blood-brain barrier (BBB). Since TfR1 is also highly expressed in the brain endothelium, BB4 might also drive the transport of pBAE-CRHR-BB4-Zw/GFP nanoparticles across the brain endothelial cells . Brain shuttle peptides have previously been shown to transport large cargoes across the BBB . Although previously showed that BB4 is an efficient brain shuttle peptide for protein cargoes, it had not yet been applied for the transport of nanoparticles. To measure the BBB permeability of BB4-modified pBAE-CRHR-Zw, we first set up a well validated human cell-based in vitro BBB model reported by . This model is formed by a tight monolayer of CD34+ endothelial-like cells from human umbilical cord seeded on Transwell® inserts. Upon co-culture with pericytes, these cells display a BBB phenotype including tight junctions and BBB receptors, including similar levels of TfR1 than human endothelium in vivo . Lucifer yellow was used as an internal standard to ensure the barrier integrity in all wells.