Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.

Key words: Mitochondria, mitochondrial drug delivery, mitochondrial macromolecule delivery, MITO-Porter, membrane fusion, multifunctional envelope-type nano device (MEND), mitochondrial protein therapy, mitochondrial gene therapy, mitochondrial protein import machinery, mitochondrial DNA. Yamada, Y., et al. AbstractMitochondrial dysfunction has been implicated in a variety of human disorders-the so-called mitochondrial diseases. Therefore, the organelle is a promising therapeutic drug target. In this review, we describe the key role of mitochondria in living cells, a number of mitochondrial drug delivery systems and mitochondria-targeted therapeutic strategies. In particular, we discuss mitochondrial delivery of macromolecules, such as proteins and nucleic acids. The discussion of protein delivery is limited primarily to the mitochondrial import machinery. In the section on mitochondrial gene delivery and therapy, we discuss mitochondrial diseases caused by mutations in mitochondrial DNA, several gene delivery strategies and approaches to mitochondrial gene therapy. This review also summarizes our current efforts regarding liposome-based delivery system including use of a multifunctional envelope-type nano device (MEND) and mitochondrial liposome-based delivery as anti-cancer therapies. Furthermore, we introduce the novel MITO-Porter-a liposome-based mitochondrial delivery system that functions using a membrane-fusion mechanism.Yamada, Y., et al. 3

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Mitochondrial dysfunction is associated with a variety of human diseases including inherited mitochondrial diseases, neurodegenerative disorders, diabetes mellitus, and cancer. Effective medical therapies for mitochondrial diseases will ultimately require an optimal drug delivery system, which will likely be achieved through innovations in the nanotechnology of intracellular trafficking. To achieve efficient mitochondrial drug delivery, two independent processes, i.e., "cytoplasmic delivery through the cell membrane" and "mitochondrial delivery through the mitochondrial membrane" are required. In previous studies, we developed an octaarginine (R8) modified nano carrier for efficient cytoplasmic delivery, showing that R8-modified liposomes were internalized into cells efficiently. On the other hand, we also constructed MITO-Porter for the mitochondrial delivery of macromolecules, a liposome-based carrier that delivers cargos to mitochondria via membrane fusion. Here, we report the development of a dual function MITO-Porter (DF-MITO-Porter), based on the concept of integrating both R8-modified liposomes and MITO-Porter. We show that the DF-MITO-Porter effectively delivers exogenous macro-biomolecules into the mitochondrial matrix, and provide a demonstration of its potential use in therapies aimed at mitochondrial DNA.