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

Recent advances in the clinical extracellular vesicles (EVs) 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, in µarray format, for profiling EVs from distinct cellular conditions, employing a multimodal approach that combines surface plasmon resonance imaging (SPRi) and in situ atomic force microscopy (AFM) to decipher EVs' biochemical and biophysical properties. SPRi experiments showed notable EV capture differences on ligands such as Anti-CD36, Anti-CD81, and Anti-ApoA between treated and control conditions, likely due to B[a]P exposure. A complementary AFM study and statistical analyses revealed size differences between EVs from treated and control samples, with ligands like Annexin-V, Anti-CD36, 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. The original combination of SPRi and AFM reveals valuable data on the phenotypical and morphological heterogeneities of EV subsets linked to cytotoxic stresses and highlights the potential of EVs as specific toxicological markers.