Crossing the blood-brain barrier (BBB) to deliver lipid nanoparticles (LNPs) for central nervous system (CNS) therapies remains a major challenge. Here, we present a compartmentalized, human-derived 3D BBB tissue model incorporating five cell types, integrating a BBB with a protein composite scaffold system, to support parenchymal elements within a single integrated brain tissue system. This tissue model recapitulates key structural and functional features of the native human BBB, including enhanced tight junction formation, neuronal maturation, polarized endothelial morphology, low permeability, and a more homeostatic microenvironment. The silk-collagen composite provided physiologically relevant extracellular matrix stiffness that supported long-term culture stability and parenchymal development. Translational utility for evaluating CNS-penetrating LNPs was demonstrated by showing that LNP transport efficiency and parenchyma penetration capability in vitro correlated with in vivo brain delivery following systemic administration in mice. Furthermore, this in vitro tissue model enables mechanistic investigation of LNP transport via receptor modulation using siRNA knockdown and pharmacological inhibition, revealing scavenger receptor class B type I (SR-B1) and insulin receptor (INSR) as key mediators of receptor-dependent transcytosis. By enabling the integrated assessment of permeability, transport mechanisms, and toxicity within a single human-relevant in vitro tissue platform, this model serves as a tool to bridge the translational gap between LNP design, in vitro screening, and in vivo validation, to support the optimization of CNS drug delivery. Among the tested formulations, LNP1 exhibited superior BBB penetration, neuronal transfection, and low toxicity, highlighting its potential as a promising lead candidate for CNS mRNA therapeutics for neurological diseases.
