Detection Of Peptides Capable Of Crossing TheBlood-Brain Barrier (Bbb) And Being IntegratedInto Drug Delivery Systems Using PhysiologicallyRelevant Human Bbb Models

The human blood-brain barrier (BBB), consisting of brain microvascular endothelialcells, astrocytes, and pericytes, is a critical protective layer, isolating the brain fromthe bloodstream. It prevents the entry of harmful agents and foreign molecules,including many therapeutics, into the brain. Consequently, treating neurologicaldiseases—the second leading cause of mortality and the leading cause of morbidityglobally—presents significant challenges. To overcome these challenges and addressthe urgent need for shuttles capable of delivering drugs into the brain, this studytakes a unique approach for selecting peptides that can cross the BBB and reach thebrain, utilizing a physiologically relevant, human cell-based BBB-on-a-chip modelthat recapitulates the in vivo blood flow conditions. We developed a two-channel microfluidicsystem where the induced pluripotent stem cell-derived brain endothelialcells were seeded in one channel to mimic the brain capillaries, and primary humanastrocytes and pericytes were seeded in the other, separated by a porous membrane,to obtain brain compartment. The barrier integrity of the model was validated usingpermeability assays. Initially, wild-type M13 bacteriophages displaying no peptideswere perfused through the blood channel to measure their ability to cross the BBB.Subsequently, M13 bacteriophages displaying ∼109 different peptides were perfusedthrough the blood channel, and the peptide-displaying phages that cross the BBBwere quantified. Additionally, we performed a negative selection on human primarylung, kidney, and gut endothelial cells before biopanning of bacteriophages on BBBon-a-chip models, for brain specificity. The peptides on the crossing-phages weresequenced and results were compared.