In vitro blood-brain barrier models for the study of brain shuttle peptide transport

Brain shuttles, such as antibody fragments, bispecific antibodies, peptides, or nanocarriers, are engineered to exploit the blood-brain barrier (BBB) transport mechanisms to deliver therapeutics to the brain. This strategy has emerged as a potential game-changer to overcome the therapeutic challenge of many brain diseases: the hindered passage of molecules across the BBB. Due to special tight junctions, low intracellular vesicles, negatively charged glycocalyx, and highly regulated transport, brain endothelial cells limit central nervous system drug delivery. To facilitate drug passage to the brain, brain shuttles utilize endogenous transport pathways, such as receptor-mediated and adsorptive-mediated transcytosis. In vitro BBB models provide a controlled environment to evaluate the passage of brain shuttles across the BBB. Here, we present the cellular and methodological basis for selecting an appropriate model type for any shuttle study, with relevant barrier tightness and cellular composition. We explain how to apply rigorous experimental controls to assess transport efficiency, receptor specificity, cytotoxicity, and barrier integrity, which helps to identify specific transport phenomena, and exclude artefacts.