External magnetic-field driven delivery of exosomes modulates immune and metabolic changes of dystrophic muscle II

Exosomes are nanosized extracellular vesicles which have been recently demonstrated as promising agents for tissue repair/regeneration by inducing and guiding appropriate immune responses in dystrophic pathologies. Unfortunately, the accurate manipulation of exosomes by controlling their biodistribution still poses significant challenges. Here we overcome this limitation by developing an externally controlled delivery system for primed ANXA1 myo-exosomes (Exomyo). Effective nanocarriers are realized by immobilizing the Exomyo onto ferromagnetic nanotubes (NT-MAG) to achieve a controlled delivery and localization of Exomyo into skeletal muscles by an applied external magnetic field. Quantitative muscle-level analyses revealed that macrophages dominate the uptake of Exomyo from NT-MAG in vivo, to synergistically promote beneficial muscle responses in a murine animal model of Duchenne Muscular Dystrophy (DMD) thanks to the successful localization of therapeutic Exomyo upon systemic injection. These findings provide valuable insights into the development of exosome-based therapies for muscle diseases and in general highlight the formulation of effective functional nanocarriers aimed at optimizing exosome biodistribution. To characterize the molecular traits induced by repetitive magnetic delivery of anti-inflammatory exosomes Exomyo, we performed two main experiments. The first experiment involved three groups of mice that received intravenous magnetic NT-MAG-CFSE-ExoMyo infusion once (NT-MAG-Exomyo I), twice, one a week (NT-MAG-Exomyo II) or three times, once a week (NT-MAG-Exomyo III). In the second experiment, NT-MAG-Exomyo III was compared to a group receiving intravenous non-magnetic NT-CFSE-ExoMyo delivery three times, once a week (NT-ExoMyo III). A control group of mdx mice, receiving intravenous administration of saline for three weeks (vehicle), was included in each of the main experiments. After 30 days from the first injection, mice were sacrificed, and hind limb muscle tissues prepared for RNA sequencing analyses of single muscle fibers (MS) and freshly FACS-isolated circulating macrophages that infiltrate muscles and entrapped CFSE positive exosomes (Ly6ChiCFSEhi; iMAC).