Differential protein and mRNA cargo loading into engineered large and small extracellular vesicles reveals differences in in vitro and in vivo assays.

Extracellular vesicles (EV) represent an advanced platform for genetic material and protein delivery, particularly when they are loaded through the so-called endogenous loading method. This study investigates the differences between large EV (lEV) and small EV (sEV) obtained from genetically engineered C2C12 myoblasts overexpressing two different model biomolecules. Erythropoietin (EPO) is a secretory protein with anti-inflammatory, angiogenic and hematopoietic effects, while TGL is a chimeric cytosolic protein containing green fluorescent protein (GFP) and luciferase, used for imaging. We compared these EV subtypes in terms of protein and nucleic acid loading, intercellular cargo transfer capacity, and subsequent functional effects both in vitro and in vivo. Our results demonstrated that lEV exhibited higher protein and mRNA cargo content than sEV, which also translated into increased intercellular cargo transfer capacity, even when dosing according to the constitutive sEV and lEV secretion ratio (10,1). Indeed, we showed that, although receptor cells successfully internalized both EV subtypes, cells treated with lEV displayed stronger intracellular luciferase signals and higher EPO protein secretion compared to those treated with sEV. In terms of functional effects, both EV subtypes exerted anti-inflammatory and antioxidant effects in lipopolysaccharide-activated macrophages, as well as angiogenic effects in human umbilical vein endothelial cells. Finally, in vivo studies evidenced that subcutaneously administered lEV led to a more significant increase in hematocrit levels and red blood cell counts than sEV. Taken together, these findings suggest that the protein and mRNA cargo differ between endogenously loaded EV subtypes, and that this variation in cargo loading leads to differences in their functional outcomes. Therefore, the choice of EV subtype could be critical for optimizing EV-based delivery strategies for biologic drugs.