Abstract
Various types of mitochondrial dysfunctions have been implicated in a variety of human diseases. This suggests that mitochondria would be promising therapeutic drug targets and mitochondrial therapy would be expected to be useful for the treatment of various diseases. We have already reported the development of a MITO-Porter, a liposome-based nano-carrier that delivers its cargo to mitochondria via a membrane-fusion mechanism. In our strategy for delivering cargos to mitochondria using a MITO-Porter, the carriers must fuse with the organelle membrane. Here we report on methodology for screening various types of lipid envelopes that have the potential for fusing with a mitochondrial membrane. The method involves monitoring the cancellation of fluorescence resonance energy transfer (FRET) and evaluating membrane fusion between the carriers and mitochondria in living cells by FRET analysis using a spectral imaging fluorescent microscopy system.
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Flierl A, Jackson C, Cottrell B, Murdock D, Seibel P, Wallace DC (2003) Targeted delivery of DNA to the mitochondrial compartment via import sequence-conjugated peptide nucleic acid. Mol Ther 7:550–557
Vestweber D, Schatz G (1989) DNA-protein conjugates can enter mitochondria via the protein import pathway. Nature 338:170–172
Schatz G (1987) 17th Sir Hans Krebs lecture. Signals guiding proteins to their correct locations in mitochondria. Eur J Biochem 165:1–6
Seibel M, Bachmann C, Schmiedel J, Wilken N, Wilde F, Reichmann H, Isaya G, Seibel P, Pfeiler D (1999) Processing of artificial peptide-DNA-conjugates by the mitochondrial intermediate peptidase (MIP). Biol Chem 380:961–967
Endo T, Nakayama Y, Nakai M (1995) Avidin fusion protein as a tool to generate a stable translocation intermediate spanning the mitochondrial membranes. J Biochem 118:753–759
Wiedemann N, Frazier AE, Pfanner N (2004) The protein import machinery of mitochondria. J Biol Chem 279:14473–14476
Yamada Y, Akita H, Kamiya H, Kogure K, Yamamoto T, Shinohara Y, Yamashita K, Kobayashi H, Kikuchi H, Harashima H (2008) MITO-Porter: a liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion. Biochim Biophys Acta 1778:423–432
Yamada Y, Harashima H (2008) Mitochondrial drug delivery systems for macromolecule and their therapeutic application to mitochondrial diseases. Adv Drug Deliv Rev 60:1439–1462
Kogure K, Akita H, Yamada Y, Harashima H (2008) Multifunctional envelope-type nano device (MEND) as a non-viral gene delivery system. Adv Drug Deliv Rev 60:559–571
Khalil IA, Kogure K, Futaki S, Harashima H (2006) High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression. J Biol Chem 281:3544–3551
Del Gaizo V, Payne RM (2003) A novel TAT-mitochondrial signal sequence fusion protein is processed, stays in mitochondria, and crosses the placenta. Mol Ther 7:720–730
Khalil IA, Kogure K, Futaki S, Hama S, Akita H, Ueno M, Kishida H, Kudoh M, Mishina Y, Kataoka K et al (2007) Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery. Gene Ther 14:682–689
Struck DK, Hoekstra D, Pagano RE (1981) Use of resonance energy transfer to monitor membrane fusion. Biochemistry 20:4093–4099
Maier O, Oberle V, Hoekstra D (2002) Fluorescent lipid probes: some properties and applications (a review). Chem Phys Lipids 116:3–18
Akita H, Kudo A, Minoura A, Yamaguti M, Khalil IA, Moriguchi R, Masuda T, Danev R, Nagayama K, Kogure K et al (2009) Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process. Biomaterials 30:2940–2949
Yamada Y, Akita H, Harashima H (2012) Multifunctional envelope-type nano device (MEND) for organelle targeting via a stepwise membrane fusion process. Methods Enzymol 509:301–326
Haraguchi T, Shimi T, Koujin T, Hashiguchi N, Hiraoka Y (2002) Spectral imaging fluorescence microscopy. Genes Cells 7:881–887
Futaki S, Ohashi W, Suzuki T, Niwa M, Tanaka S, Ueda K, Harashima H, Sugiura Y (2001) Stearylated arginine-rich peptides: a new class of transfection systems. Bioconjug Chem 12:1005–1011
Shinohara Y, Almofti MR, Yamamoto T, Ishida T, Kita F, Kanzaki H, Ohnishi M, Yamashita K, Shimizu S, Terada H (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur J Biochem 269:5224–5230
Shinohara Y, Sagawa I, Ichihara J, Yamamoto K, Terao K, Terada H (1997) Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation. Biochim Biophys Acta 1319:319–330
Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887
Yamada Y, Harashima H (2012) Targeting mitochondria: innovation from mitochondrial drug delivery system (DDS) to mitochondrial medicine. Yakugaku Zasshi 132:1111–1118
Acknowledgements
This work was supported, in part, by the Grant-in-Aid for Young Scientists (A) and Grant-in-Aid for Challenging Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japanese Government (MEXT). We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.
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Yamada, Y., Harashima, H. (2014). A Method for Screening Mitochondrial Fusogenic Envelopes for Use in Mitochondrial Drug Delivery. In: Jain, K. (eds) Drug Delivery System. Methods in Molecular Biology, vol 1141. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0363-4_2
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DOI: https://doi.org/10.1007/978-1-4939-0363-4_2
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