Development of Myotropic Extracellular Vesicles for Targeted Delivery of Therapeutics to Skeletal Muscle

Muscular pathologies comprise a family of diseases and disorders resulting in the degeneration of skeletal muscle tissue. Current therapeutics are non-specific to skeletal muscle, which often reduces efficacy and causes harmful off-target effects in patients. Thus, a myotropic (muscle-targeted) drug delivery system is needed to shuttle therapeutics to muscle cells to improve efficacy and limit off-target effects. The objective of this investigation was to develop biological lipid nanoparticles, extracellular vesicles (EVs), with enhanced myotropism. We hypothesized that displaying myotropic transmembrane proteins at the surface of EVs would improve delivery of molecular cargo into muscle cells. To this end, we displayed the myotropic transmembrane proteins MyoMaker (MYMK), MyoMixer (MYMX) and M-Cadherin (M-CAD) in the membranes of EVs from Human Embryonic Kidney (HEK293) cells. Using flow cytometry, we measured the incorporation of each protein candidate into the EVs via endogenous green fluorescent protein (GFP) tags included on the cytosolic c-terminus of each protein. We then measured the delivery of fluorescently-labeled protein by each myotropic EV candidate in mouse (C2C12) myotubes. In addition to using EVs as delivery vehicles for exogenously loaded therapeutics, the native EV cargo may also be of therapeutic relevance. Given this, we examined the endogenous protein cargo of non-engineered HEK293-EVs via mass spectrometry. MYMK displayed the highest degree of incorporation into EVs relative to control HEK293-EVs (p = 0.006). The MYMK-EV formulation displayed the greatest increase in protein cargo delivered into myotubes relative to control HEK293-EVs (~125%, p = 0.0005). Proteomic analysis revealed the top biological processes and molecular functions of HEK293-EV protein cargo to be ribonucleic acid binding, processing, and metabolism, potentially altering translation in recipient cells. The in vitro data in the present study establish the feasibility of using MYMK-EVs as a myotropic delivery system. Future investigations are needed to fully elucidate the cell/tissue tropism and physiological effects of MYMK-EVs in vivo.