MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion

Abstract

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.

Mitochondrial delivery of Coenzyme Q10 via systemic administration using a MITO-Porter prevents ischemia/reperfusion injury in the mouse liver

“…The fusion activity of the carriers with isolated mitochondrial membranes was assessed by FRET as previously described [9,11,21]. In this experiment, lipid envelopes of the dual-labeled carriers were labeled with both NBD (excitation at 460 nm and emission at 534 nm) and rhodamine (excitation at 550 nm and emission at 590 nm) so that the energy would be transferred from NBD to rhodamine.…”

“…We prepared the MITO-Porter with highly mitochondrial fusogenic envelopes [5,9,11,21], where, of the total lipids, 10 mol% R8 was modified on the carrier surface, by the hydration method. We also prepared PEG-LP without R8 as a control carrier.…”

“…We previously reported on the construction of a MITO-Porter, a liposome-based mitochondrial delivery system that functions via membrane fusion [5,11]. This membrane fusion mechanism-based strategy can deliver a cargo to mitochondria independent of its size and physical properties.…”

Localization of exogenous DNA to mitochondria in skeletal muscle following hydrodynamic limb vein injection

“…To verify the mitochondrial delivery of pDNA, the purity of the isolated mitochondria needs to be verified. In this experiment, the crural muscles were harvested following HLV injection and homogenized, and a mitochondria-enriched fraction was then isolated from the homogenate by differential centrifugation, as described in a previous report [27,28]. Samples of the homogenate and mitochondria-enriched fraction were subjected to western blotting using primary antibodies against the COX IV, Lamin A+C and GAPDH, as mitochondrial, nuclear and cytosolic fraction markers (Fig.…”

Progress and prospects: gene therapy for mitochondrial DNA disease

“…Another liposome-based carrier, MITO-Porter has also been shown to co-localise to mitochondria in intact cells. 15 MITO-Porter loaded with GFP was shown in isolated mitochondria to be delivered to the intermembrane space and to co-localise with mitochondria in cultured cells. These data suggest that such liposomal preparations can fuse with the outer mitochondrial membrane, but will it prove to be able to access the inner membrane that is necessary for the cargo to access the mitochondrial matrix?…”