SH-SY5Y NEUROBLASTOMA-DERIVED SMALL EXTRACELLULAR VESICLES AT THE BLOOD-BRAIN BARRIER <EM>IN VITRO</EM>: UPTAKE PATTERNS AND PIPELINE FOR INTERACTION ASSESSMENT

Small extracellular vesicles (sEVs) represent promising therapeutic delivery vehicles for crossing the blood-brain barrier (BBB), yet their specific uptake mechanisms and cellular interactions remain poorly understood. This study investigates the behaviour of selected neuroblastoma-derived sEVs at the BBB interface using a human in vitro model. sEVs were isolated and characterised from four neural cell lines for size, marker expression and morphology. Uptake efficiency was screened in hCMEC/D3 brain endothelial cells to select optimal candidates. Internalisation mechanisms were elucidated by pharmacological inhibition studies, and intracellular localisation was assessed via confocal microscopy. Uptake and permeation were analysed under normoxic and oxygen/glucose deprivation (OGD) conditions. BioID2-engineered SH-SY5Y cells were developed to produce biotinylated sEVs for protein interaction mapping. Following comprehensive characterisation, SH-SY5Y sEVs demonstrated superior internalisation from the brain-facing direction (4219±1854 a.u.). Inhibition studies identified caveolae-mediated endocytosis and macropinocytosis as primary uptake pathways. Confocal microscopy revealed lysosomal accumulation over 24 hours. Despite efficient cellular uptake without compromising barrier integrity, sEVs failed to cross the BBB under normoxic conditions. OGD reduced sEV uptake, but permeation remained undetectable even with severely disrupted barrier conditions. BioID2-sEVs achieved successful self-biotinylation, establishing a pipeline for future sEV-cell interaction studies despite current background limitations. This work defines specific internalisation mechanisms for neuronal sEVs at the BBB and demonstrates a BioID2-sEV pipeline for molecular interaction studies. The absence of sEV transcytosis despite efficient uptake suggests lysosomal degradation as the predominant fate, limiting therapeutic delivery potential under current conditions studied to a better understanding of CNS delivery strategies.