Short communicationMitochondrial targeting functional peptides as potential devices for the mitochondrial delivery of a DF-MITO-Porter
Introduction
To date, a number of investigators have reported that genetic defects in mitochondrial DNA are associated with mitochondrial diseases and a variety of human disorders, including neurodegenerative diseases, diabetes mellitus and cancer (Chan, 2006, Schapira, 2012, Taylor and Turnbull, 2005). An efficient mitochondrial targeting system and therapeutic molecules are required to accomplish mitochondrial gene therapy. The use of an endogenous mitochondrial targeting signal (MTS), cell penetrating peptides (CPPs), vesicles, and mitochondrial import proteins has been reported to be useful in delivering various cargoes, including low-molecular weight molecules, nucleic acids and proteins (Adhya et al., 2011, D'Souza et al., 2003, Flierl et al., 2003, Koczor et al., 2009, Mahata et al., 2006, Zhang et al., 2011). Nevertheless, a nanoparticle with a diameter that exceeds 100 nm has not been yet reported to accumulate in mitochondria efficiently compared to low-molecular weight molecules.
In a previous study, we reported on the development of a Dual Function (DF)-MITO-Porter, an innovative nanocarrier for achieving mitochondrial delivery, which has the ability to pass through the endosomal and mitochondrial membranes via step-wise membrane fusion (Yamada and Harashima, 2012, Yamada et al., 2011). In addition, we constructed the DF-MITO-Porter with oligodeoxynucleotide (oligo DNA) encapsulated, and estimated the intracellular multiple processes (Yamada et al., 2012). The rate-limiting step for mitochondrial delivery was concluded to be the mitochondrial targeting process from the cytosol. Thus, to achieve mitochondrial therapy, it was clear that the carrier needed to be improved if it was to be useful as an optimal mitochondrial targeting system. The modified octaarginine (R8) has the ability to enhance cellular uptake and mitochondrial binding (Khalil et al., 2006, Yamada et al., 2008), however we cannot exclude the possibility that it might interact with proteins in the cytosol since R8 is not selective for mitochondria.
Mitochondrial targeting peptides have been studied, in attempts to deliver certain molecular compounds to mitochondria. The R8 peptide, which mimics the trans-activating transcriptional activator (TAT), was used to modify the previously constructed MITO-Porter, and was found to function as a useful moiety for cellular uptake and mitochondrial targeting (Futaki et al., 2001, Khalil et al., 2006, Yamada et al., 2008, Yamada et al., 2011). We also used the 31 amino acid sequence of the MTS derived from an endogenous protein (rat succinyl CoA synthetase) for delivering liposomes to mitochondria (Yamada and Harashima, 2013). In constructing the MTS-MITO-Porter, aggregation was found to be a problem, and we failed to observe mitochondrial targeting activity of MTS-MITO-Porter in living cells. However, inserting polyethylene glycol (PEG) as a linker between the MTS and lipids enabled the preparation of an alternate type of MTS-MITO-Porter (Kawamura et al., 2013). Although the constructed DF-MTS-MITO-Porter showed an enhanced cellular uptake, intracellular trafficking was not greatly improved. We hypothesized that the limited trafficking of the carriers was due to the tendency of the long sequence of MTS to undergo aggregation within cells.
Based on these results, we focused on mitochondrial targeting functional peptides with shorter sequences (Yamada and Harashima, 2008). Szeto and Schiller (2011) reported on the development of a Szeto–Schiller (SS) peptide, a cell permeable antioxidant, in which aromatic residues alternated with basic amino acids. This peptide was reported to be predominantly concentrated in mitochondria within cells (Zhao et al., 2004). Wipf et al. (2005) reported on a conjugate of 4-amino-TEMPO and gramicidin S (GS) segment (XJB-5-131), a mitochondria targeted electron and ROS scavenger (Fink et al., 2007). Mitochondrial penetrating peptides (MPPs) contain repeating sequences of positively charged arginine residues and lipophilic cyclohexylalanine and are designed to exhibit efficient cellular uptake and to accumulate in the mitochondrial matrix (Horton et al., 2008, Horton et al., 2012). The circular peptide (CP) is known to associate with prohibitin in adipose tissue (Hossen et al., 2010, Kolonin et al., 2004), and would be a useful moiety for mitochondrial targeting because the mitochondrial membrane contains prohibitin. We report herein on an investigation of whether the mitochondrial targeting activity of nanocarriers can be enhanced in a similar manner to R8 by modifying a mitochondrial targeting peptide.
Section snippets
Materials
1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE), sphingomyelin (SM) and rhodamine-DOPE were purchased from Avanti Polar lipids (Alabaster, AL, USA). Phosphatidic acid (PA) and cholesteryl hemisuccinate (CHEMS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Stearyl R8 (STR-R8) and cholesteryl-GALA (cholesteryl-WEAALAEALAEALAEHLAEALAEALEALAA-NH2) were obtained from Kurabo Industries Ltd. (Osaka, Japan). STR-SS (stearyl-Dmt-D-Arg-FK-NH2, Dmt = 2, 6-dimethyltyrosine), STR-S2
Construction of liposomes modified with mitochondrial targeting peptides and an evaluation of mitochondrial binding activity
Mitochondrial targeting peptides, R8, SS, GS, MPP and CP were used to prepare the mitochondrial targeting carriers. We also synthesized an S2 peptide by conjugating two SS peptide sequences, assuming that the mitochondrial targeting ability of the peptide would be enhanced. The liposomes [DOPE/SM (9:2, molar ratio)] were constructed by adding the stearyl peptides at levels of 10 or 15% of the total lipid using the lipid hydration method. MPP6, a sequence of six amino acid residues of MPP, was
Conclusion
A number of researchers have reported that synthesized mitochondrial targeting peptides can associate with mitochondria in living cells by electrostatic interactions or mitochondrial affinity (Fink et al., 2007, Horton et al., 2008, Szeto and Schiller, 2011). However, the use of these peptides did not show a similar behavior when they were used as modifiers of nanoparticles. An evaluation of mitochondrial targeting activity using a homogenate demonstrated that mitochondrial targeting activity
Acknowledgments
This work was supported, in part by, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, Japan (NIBIO), a Grant-in-Aid for Young Scientists (A) and a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japanese Government (MEXT). We also thank Milton Feather for his helpful advice in writing the manuscript.
References (31)
- et al.
Mitochondrial gene therapy: the tortuous path from bench to bedside
Mitochondrion
(2011) - et al.
Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process
Biomaterials
(2009) - et al.
Reprint of: Nanoparticles for ex vivo siRNA delivery to dendritic cells for cancer vaccines: programmed endosomal escape and dissociation
J. Control. Release
(2011) Mitochondria: dynamic organelles in disease, aging, and development
Cell
(2006)- et al.
DQAsome-mediated delivery of plasmid DNA toward mitochondria in living cells
J. Control. Release
(2003) - et al.
Octaarginine- and octalysine-modified nanoparticles have different modes of endosomal escape
J. Biol. Chem.
(2008) - et al.
Hemigramicidin-TEMPO conjugates: novel mitochondria-targeted anti-oxidants
Biochem. Pharmacol.
(2007) - et al.
Targeted delivery of DNA to the mitochondrial compartment via import sequence-conjugated peptide nucleic acid
Mol. Ther.
(2003) - et al.
Mitochondria-penetrating peptides
Chem. Biol.
(2008) - et al.
Ligand-based targeted delivery of a peptide modified nanocarrier to endothelial cells in adipose tissue
J. Control. Release
(2010)
High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression
J. Biol. Chem.
Mitochondrial disease
Lancet
MITO-Porter: a liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion
Biochim. Biophys. Acta
Mitochondrial drug delivery systems for macromolecule and their therapeutic application to mitochondrial diseases
Adv. Drug Deliv. Rev.
Dual function MITO-Porter, a nano carrier integrating both efficient cytoplasmic delivery and mitochondrial macromolecule delivery
Mol. Ther.
Cited by (44)
Mitochondria targeted fluorogenic theranostic agents for cancer therapy
2022, Coordination Chemistry ReviewsCitation Excerpt :Other mitochondria targeting agents used as delivery agents include Szeto-Schiller (SS) peptide [32]. The mitochondria penetrating peptide (MPP) chain exhibits cyclohexylamine as highly hydrophobic residues and alternatively positive charged arginine or lysine residues [33,34]. Collectively, these factors facilitate the peptide tagged agents to cross the plasma membrane and localize into mitochondria efficiently.
Live-cell imaging of the nucleolus and mapping mitochondrial viscosity with a dual function fluorescent probe
2021, Organic and Biomolecular ChemistryExperimental aspects of surface-enhanced Raman scattering for biological applications
2021, Principles and Clinical Diagnostic Applications of Surface-Enhanced Raman SpectroscopyNanotechnology, mitochondria, and cancer: Mitochondria-targeting particles for biomedical applications
2021, Mitochondrial Dysfunction and Nanotherapeutics: Aging, Diseases, and Nanotechnology-Related Strategies in Mitochondrial MedicineTargeted mitochondrial delivery of antisense RNA-containing nanoparticles by a MITO-Porter for safe and efficient mitochondrial gene silencing
2019, MitochondrionCitation Excerpt :The envelopes of MITO-Porter used for the mitochondrial transfection of ASO contain R8 and GALA as cellular uptake and endosomal escape devices, respectively. Based on our previous studies, carriers modified with R8 would be expected to exhibit cytotoxicity at a lipid concentration of more than about 0.1 mM (Kawamura et al., 2013). It is known that GALA improves the endosomal escape of the nanoparticles (Furukawa et al., 2015; Kakudo et al., 2004) but also has a strong toxicity.
- 1
These authors contributed equally as first author.