A Physiological Microfluidic Blood–Brain‐Barrier Model for In Vitro Study of Nanoparticle Trafficking and Accumulation

ABSTRACT Although the blood–brain barrier (BBB) restricts passage of most molecules, various naturally occurring and synthetic nanoparticles (NPs) are nonetheless found within the brain parenchyma. To study the mechanisms underlying this phenomenon, we developed a microfluidic BBB model (mBBB) using human cerebral microvascular endothelial cells (HCMECs) in direct contact with primary human astrocytes and pericytes within a physiologically relevant extracellular matrix. The horizontal architecture enables high‐resolution imaging across the full barrier interface and allows direct assessment of nanoparticle transport and accumulation. This in vitro platform recapitulates key features of the BBB, including selective permeability, junctional protein expression, and receptor‐mediated uptake pathways. Using this system, the trafficking and accumulation of structurally distinct nanoparticles, including liposomes, nanoplastics, and extracellular vesicles (EVs), were compared. Among these, heterologous EVs exhibit the highest transport efficiency. Analysis of nanoparticle properties suggest that ligand presentation and membrane composition, rather than size or stiffness, primarily govern BBB penetration. The mBBB platform provides a high‐throughput, imaging‐based framework to systematically interrogate nanoparticle trafficking across the BBB and offers a translational tool for both drug delivery and neurotoxicity screening.