Small extracellular vesicles (sEVs), through their natural ability to interact with biological membranes and exploit endogenous processing pathways to convey biological information, are quintessential for the delivery of therapeutically relevant compounds, such as microRNAs (miRNAs) and proteins. Here, we used a fluorescently‐labelled miRNA to quantify the efficiency of different methods to modulate the cargo of sEVs. Our results showed that, compared with electroporation, heat shock, permeation by a detergent‐based compound (saponin) or cholesterol‐modification of the miRNA, Exo‐Fect was the most efficient method with > 50% transfection efficiency. Furthermore, qRT‐PCR data showed that, compared with native sEVs, Exo‐Fect modulation led to a > 1000‐fold upregulation of the miRNA of interest. Importantly, this upregulation was observed for sEVs isolated from multiple sources. The modulated sEVs were able to delivery miR‐155‐5p into a reporter cell line, confirming the successful delivery of the miRNA to the target cell and, more importantly, its functionality. Finally, we showed that the membrane of Exo‐Fect‐loaded sEVs was altered compared with native sEVs and that enhanced the internalization of Exo‐Fect‐loaded sEVs within the target cells and decreased the interaction of those modulated sEVs with lysosomes.

Introduction: Pulmonary Hypertension (PH) is characterized by increased right atrial (RA) stretch and pressure. The major genetic predisposing risk factor for PH involves mutations in the bone morphogenetic protein (BMP) receptor 2 (BMPR2), for which BMP9 and BMP10 are ligands. Although BMP9 is mostly produced by hepatocytes, BMP10 is predominantly produced by adult RA cardiomyocytes. Therefore, its role is of interest in PH despite an elusive BMP10 release mechanism. Hypothesis: Increased RA wall stress in PH may be a trigger for BMP10 secretion. Methods: We first investigated BMP10 gene and protein expressions, as well as BMP activity, in RA tissue samples from control (N=5) and PH-patients (N=4). We also quantified BMP10, BMP9, IL-6 and active TGF-β plasma levels in controls (N=16) and PH-patients (N=48) by ELISA. We studied BMP10 and BMP9 activities, using a BMP-inducible reporter assay. Last, we established correlations between BMP10 activity and PH clinical parameters. Results: BMP10 mRNA, protein and activity (pSMAD1/5/8 and ID3) were increased in RA tissue of PH-patients compared to controls (Figure1 A-E). BMP10, BMP9 and IL-6 plasma levels were also augmented (Figure1 F-H); while TGF-β plasma levels, and BMP10 and BMP9 activities between PH-patients and controls were preserved. Finally, we divided the PH cohort on median BMP10-activity (0.33 AU). Higher BMP10 activity in PH-patients was associated with diminished RA compliance, reduced right ventricular function, lower stroke volume and elevated NT-proBNP (Figure1 I). Conclusions: Although BMP10 plasma levels were increased, BMP10 activity was preserved in PH-patients, probably because of high inflammatory cytokines, such as IL-6. High BMP10 activity was associated with increased RA pressure and worse disease severity in PH-patients.