Optimizing Angiopep‐2 Density on Polymeric Nanoparticles for Enhanced Blood–Brain Barrier Penetration and Glioblastoma Targeting: Insights From In Vitro and In Vivo Experiments

Abstract The blood–brain barrier (BBB) poses a formidable challenge to drug delivery to the brain. One promising approach involves receptor‐mediated transcytosis via Angiopep‐2 peptide (Ang‐2)‐conjugated nanoparticles. However, the influence of Ang‐2 density on BBB penetration remains poorly understood. We developed a versatile polymeric nanoparticle system with tunable Ang‐2 surface density and systematically examined its influence on BBB penetration through various in vitro assays and an in vivo study. The results reveal a nuanced relationship between ligand density and BBB penetration across experimental setups. In 2D cell culture, Ang‐2 density positively correlates with nanoparticle association in human cerebral microvascular endothelial cells (hCMEC/D3) with a distinctive inflection point. Conversely, in the Transwell model, higher Ang‐2 density negatively correlate with BBB penetration, while the BBB‐glioblastoma (GBM)‐on‐a‐chip shows the opposite trend. Disparities may be due to differences in avidity under static versus dynamic conditions, modulating nanoparticle interactions due to fluidic forces. In vivo studies align with the microfluidic model. Loading doxorubicin into the optimized nanoparticles achieves controlled pH‐responsive release and enhanced anticancer effects against U87 GBM cells in 2D cell cultures and a 3D BBB‐GBM‐on‐a‐chip. These results underscore the importance of optimizing Ang‐2 density for BBB penetration and emphasize the utility of dynamic models for the preclinical assessment of brain‐targeting nanoparticles.