When the model was ready, we labelled the DNA with Hoechst 33258, a fluorescent dye that binds AT sequences in the miner groove of double stranded DNA. DNA-labelled polyplexes were formed at three ratios of pBAE-CRHR-Zw-BB4 to pBAE-CRHR-Zw, including 1:9, 1:3, 1:1, and a control without BB4. The Lucifer yellow internal standard indicated the formulations did not alter BBB permeability (Table ). Remarkably, polyplexes with 25% of pBAE-CRHR-Zw-BB4 resulted in a significant increase of transport (+ 25%) across the tight endothelial monolayer with respect to the control pBAE-CRHR-Zw without BB4 (Figure , Table ). Lower or higher percentages of pBAE-CRHR-Zw-BB4 show lower permeability because BBB transport is highly sensitive to the surface modification of the nanocarriers. While too little peptide may provide inefficient engagement with the brain endothelium, excess functionalization may cluster the receptors and/or prevent adequate intracellular sorting, decreasing transport efficiency .