Phosphorodiamidate Morpholino Oligomers-Loaded Nanobubbles for Ultrasound-Mediated Delivery to the Myocardium in Muscular Dystrophy

Microbubbles (MBs) and nanobubbles (NBs) can oscillate and collapse in response to ultrasound exposure, resulting in contrast and delivery effects. Therefore, the retention of the entrapped gas is an important condition in bubble formulations, especially for MBs and NBs with lipid shells, and the stability of the lipid membrane is considered to be affected. We previously developed NBs, which are polyethylene glycol-modified liposomes entrapping an ultrasound contrast gas that can serve as nucleic acid carriers and ultrasound contrast agents. In particular, NBs containing cationic lipids were useful as systemic delivery tools that can load genes and nucleic acids on their surfaces. However, the gas retention of NBs containing cationic lipids were low, leaving room for improvement as ultrasound contrast agents. In this study, we attempted to prepare NBs containing anionic lipids to improve their stability in vivo, and found that they lasted longer in contrast time than previous NBs. In order to utilize anionic NBs, we evaluated their usefulness as systemic delivery tools for cationic-peptide-conjugated phosphorodiamidate morpholino oligomers (PMO). PMO has attracted attention as a therapeutic agent for Duchenne muscular dystrophy (DMD); however, its charge neutrality makes its delivery into muscle fibers challenging, especially more difficult to apply PMO to myocardial damage. We examined the systemic delivery of PMO to the heart using a combination of anionic NBs and ultrasound. Furthermore, we evaluated the usability of octaarginine (R8), a cationic cell-penetrating peptide (CPP), in loading PMO onto the surface of NBs and verified the potential of PMO-loaded NBs as a therapy for cardiac dysfunction in muscular dystrophy.