MSC EVs Deliver miRNAs into Cardiomyocytes via EIPA-Sensitive and Clathrin-Mediated Endocytosis.

Background/Objectives: Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in cardiovascular disease, particularly through the transfer of regulatory microRNAs. However, direct evidence of cytosolic microRNA delivery by mesenchymal stem cell (MSC)-derived EVs into cardiomyocytes remains limited. Many prior studies have inferred transfer without fully excluding endogenous microRNA induction or surface-bound artifacts. The objective of this study was to determine whether MSC-derived EV microRNA undergoes functional cytoplasmic delivery into cardiomyocytes and to identify the endocytic pathways involved. Methods: To eliminate background from endogenous microRNA expression, MSC-derived EVs were loaded with the exogenous microRNA cel-miR-39-3p. H9C2 cardiomyocytes were treated with increasing vesicle doses, and intracellular microRNA levels were quantified using real-time polymerase chain reactions. Cytosolic accessibility was assessed indirectly using a cholesterol-modified antagomir targeting cel-miR-39-3p. Pharmacologic inhibitors of macropinocytosis (5-(N-ethyl-N-isopropyl) amiloride (EIPA)) and clathrin-mediated endocytosis (chlorpromazine and ES9-17) were applied to delineate uptake pathways, with transferrin assays confirming clathrin inhibition. Results: EV treatment produced a dose-dependent increase in intracellular cel-miR-39-3p levels. Antagomir administration reduced intracellular microRNA levels by 89%, providing indirect functional evidence that transferred cel-miR-39-3p reached an antagomir-accessible cytosolic compartment. At the highest preparation dose, EIPA and ES9-17 reduced cell-associated cel-miR-39-3p by 71% and 36%, respectively, consistent with contributions from EIPA-sensitive and clathrin-associated uptake processes. Notably, chlorpromazine increased microRNA accumulation despite blocking transferrin uptake, suggesting compensatory or membrane curvature-dependent effects. Conclusions: These findings provide strong functional evidence that exogenous cel-miR-39-3p associated with MSC EV preparations reaches an antagomir-accessible cytoplasmic compartment in H9C2 cardiomyocytes and implicate EIPA-sensitive uptake consistent with macropinocytosis and clathrin-associated uptake. Additional genetic, localization, and pulse-chase studies are required to establish the precise intracellular trafficking mechanisms. This work strengthens the mechanistic foundation for EV-based therapies and informs the rational development of cell-free strategies for cardiovascular disease.