Reassembled yeast vacuoles preserve endothelial viability and barrier integrity in an in vitro blood–brain barrier model

Background: The blood–brain barrier (BBB) is a highly selective interface that protects the central nervous system but is vulnerable to chemical- and nanomaterial-induced dysfunction. Therefore, candidate nanocarriers should be evaluated not only for delivery performance, but also for cellular safety and barrier compatibility under physiologically relevant conditions. Objectives: This study aimed to assess the cellular safety and barrier integrity associated with reassembled yeast vacuoles (ReV) compared with normal vacuoles (NorV) in an astrocyte-supported in vitro BBB model, while examining the mechanism by which these carriers influence daunorubicin (DNR) handling under barrier conditions. Methods: In vitro BBB models were established using human brain microvascular endothelial cells (hCMEC/D3) as a monolayer and an astrocyte-supported Transwell co-culture model with human astrocytes seeded on the underside of the membrane. DNR was tested as a free drug or as loaded into ReV or NorV. Endothelial drug-associated fluorescence and transport-related signals were evaluated by fluorescence-based permeability testing and quantitative image analysis. Barrier integrity was monitored using transendothelial electrical resistance (TEER), and endothelial viability was assessed using an MTT assay. Results: TEER remained stable following treatment, indicating no measurable barrier disruption under the tested conditions. MTT analysis showed no significant reduction in hCMEC/D3 viability within the tested dose range. In the monolayer BBB model, the intracellular DNR-associated fluorescence was comparable among the formulations. In contrast, in the astrocyte-supported BBB model, DNR@ReV produced a markedly higher endothelial DNR-associated fluorescence than free DNR or DNR@NorV. Conclusion: Reassembled yeast vacuoles support DNR handling in an astrocyte-supported in vitro BBB model while maintaining endothelial viability and barrier integrity, providing a toxicology-relevant platform for screening yeast-derived nanocarriers for central nervous system-oriented. Graphic abstract: (Figure presented.)