A microfluidic human blood-brain barrier model reveals neurovascular toxicity and barrier disruption induced by E-cigarette additives

The increasing popularity of e-cigarettes raises concerns about their neurotoxic potential. Exposure to flavoring additives may compromise the blood-brain barrier (BBB); however, their effects and underlying mechanisms remain poorly understood, largely due to the lack of physiologically relevant in in vitro models. Here, we developed a dynamic microfluidic BBB co-culture model (CPAC-Co) incorporating human brain endothelial cells and astrocytes under physiological shear stress. CPAC-Co model showed superior barrier integrity, efflux activity, and drug-permeability prediction over conventional models. Using this validated platform, we evaluated seven widely used e-cigarette additives (ethanol, menthol, WS-23, lactic acid, benzoic acid, ethyl maltol, and methylcyclopentenolone). Ethanol, ethyl maltol, and methylcyclopentenolone markedly increased nicotine permeability and reduced transepithelial electrical resistance, indicating BBB disruption. These changes were accompanied by endothelial Apoptosis, downregulation of tight-junction genes, and activation of oxidative/nitrosative stress and inflammatory responses. Transcriptomics further revealed upregulation of inflammation- and stress-related pathways, including NF-κB and NOD-like Receptor signaling. Our study not only establishes CPAC-Co as a robust, physiologically relevant model for neurovascular toxicity screening but also provides experimental evidence for the safety evaluation of e-cigarette additives, underscoring the need to integrate their central nervous system potential risks into regulatory policies.