NanoBioAnalytical (NBA) Platform to Decipher Extracellular Vesicles Secreted by Microvascular Endothelial Cells Under Benzo[a]pyrene Exposure

Recent advancements in the clinical EV field highlight their potential as biomarkers for diverse diseases and therapeutic applications. This study provides an in-depth characterization of 10K EVs from human microvascular endothelial cells (HMEC-1) exposed to benzo-a-pyrene (B[a]P), a polycyclic aromatic hydrocarbon found in food and smoke. Given EVs’ complexity, with numerous surface and cargo proteins, phenotyping remains challenging. Here, we introduce a multiplex biosensor for profiling EVs from distinct cellular conditions, employing a multimodal approach that integrates surface plasmon resonance imaging (SPRi) and atomic force microscopy (AFM) to examine how cytotoxic environments impact EV biochemical and biophysical properties. SPRi experiments showed notable Evs capture differences ion ligands as Anti-CD36, Anti-CD81, and Anti-ApoA between treated and control conditions, likely due to B[a]P exposure. Complementary AFM scanning and statistical analyses revealed size differences between EVs from treated and control samples, with ligands like Anti-CD81, Anti-ApoA, Annexin-V, Anti-NOX2, and Anti-VEGFR1 emerging as ligands specific to potential cytotoxicity biomarkers. Our findings suggest that B[a]P exposure may increase EV size and alter marker expression, indicating phenotypic shifts in EVs under cytotoxic stress. This study highlights the utility of SPRi and AFM in identifying EV markers linked to cytotoxic responses, providing insights into EV heterogeneity in stress conditions.