Engineering\na 3D Biomimetic Peptides Functionalized-Polyethylene\nGlycol Hydrogel Model Cocultured with Endothelial Cells and Astrocytes:\nEnhancing In Vitro Blood–Brain Barrier Biomimicry

The blood–brain barrier (BBB) poses a significant\nchallenge\nfor drug delivery and is linked to various neurovascular disorders.\nIn vitro BBB models provide a tool to investigate drug permeation\nacross the BBB and the barrier’s response to external injury\nevents. Yet, existing models lack fidelity in replicating the BBB’s\ncomplexity, hindering a comprehensive understanding of its functions.\nThis study introduces a three-dimensional (3D) model using polyethylene\nglycol (PEG) hydrogels modified with biomimetic peptides that represent\nrecognition sequences of key proteins in the brain. Hydrogels were\nfunctionalized with recognition sequences for laminin (IKVAV) and\nfibronectin peptides (RGD) and chemically cross-linked with matrix\nmetalloprotease-sensitive peptides (MMPs) to mimic the extracellular\nmatrix of the BBB. Astrocytes and endothelial cells were seeded within\nand on the surface of the hydrogels, respectively. The barrier integrity\nwas assessed through different tests including transendothelial electrical\nresistance (TEER), the permeability of sodium fluorescence (Na–F),\nthe permeability of Evan’s blue bound to albumin (EBA), and\nthe expression of zonula occluden-1 (ZO-1) in seeded endothelial cells.\nHydrogels with a combination of RGD and IKVAV peptides displayed superior\nperformance, exhibiting significantly higher TEER values (55.33 ±\n1.47 Ω·cm<sup>2</sup>) at day 5 compared to other 2D controls\nincluding HAECs-monoculture and HAECs-cocultured with NHAs seeded\non well inserts and 3D controls including RGD hydrogel and RGD-IKVAV\nmonoculture with HAECs and RGD hydrogel cocultured with HAECs and\nNHAs. The designed 3D system resulted in the lowest Evan’s\nblue permeability at 120 min (0.215 ± 0.055 μg/mL) compared\nto controls. ZO-1 expression was significantly higher and formed a\nrelatively larger network in the functionalized hydrogel cocultured\nwith astrocytes and endothelial cells compared to the controls. Thus,\nthe designed 3D model effectively recapitulates the main BBB structure\nand function in vitro and is expected to contribute to a deeper understanding\nof pathological CNS angiogenesis and the development of effective\nCNS medications.