Research Article
Pharmaceutics, Drug Delivery and Pharmaceutical Technology
A Dual-Ligand Liposomal System Composed of a Cell-Penetrating Peptide and a Mitochondrial RNA Aptamer Synergistically Facilitates Cellular Uptake and Mitochondrial Targeting

https://doi.org/10.1016/j.xphs.2016.03.002Get rights and content

Abstract

It has been reported that the use of mitochondrial RNA aptamers including RNase P (RP) results in the selective mitochondrial delivery of endogenous and exogenous RNAs. The issue of whether these aptamers would be useful ligands for the mitochondrial targeting of a nanoparticle has not been demonstrated to date because nanocarriers modified with these RNA aptamers are insufficiently internalized by cells. We report here on the development of a dual-ligand liposomal system composed of octaarginine (R8), a device that enhances cellular uptake, and an RP aptamer for mitochondrial targeting to permit a nanocarrier to be efficiently delivered to mitochondria. Surprisingly, the cellular uptake of the R8-modified nanocarrier was facilitated by modification with an RP aptamer. The optimal composition of a nanocarrier needed for efficient cellular uptake and mitochondrial targeting was determined. In a confocal laser scanning microscopy analysis, the dual-ligand–modified nanocarrier was found to result in effective mitochondrial targeting through an ATP-dependent pathway and was much more effective than a single-ligand R8-modified nanocarrier. This is the first report of the regulation of intracellular trafficking by a mitochondrial RNA aptamer–modified nanocarrier system.

Introduction

If it was possible to develop an active targeting system that could target a specific organelle, this would open a new field of research directed toward therapy for various diseases. Mitochondria are promising targets for delivering therapeutic molecules.1 Mitochondrial dysfunctions are implicated in a variety of human diseases, including neurodegenerative disorders, ischemia reperfusion injury, cancer, and inherited mitochondrial diseases.2, 3, 4, 5 Accordingly, it would be expected that delivering therapeutic molecules to mitochondria in diseased cells be a strategy for the treatment of mitochondrial dysfunctions, resulting in the suppression of mitochondria-related diseases.

The mitochondrial targeting signal peptide, which is necessary for targeting nuclear-encoded protein to mitochondria, is useful as a specific ligand for mitochondria.6, 7 Previous reports showed that the conjugation or direct modification of mitochondrial targeting signal peptide permitted a macromolecule, such as a protein, DNA, and a liposomal nanocarrier to be delivered to mitochondria.8, 9, 10, 11, 12 On the other hand, some researchers have reported the use of a mitochondrial RNA aptamer for mitochondrial delivery. The mitochondrial import of mitochondrial ribozyme, RNase P (RP), and mitochondrial RNase P (MRP) were reported to be mediated by polynucleotide phosphorylase.13 Wang et al.13, 14 showed that allotropically encoded mitochondrial mRNAs and transfer RNA (tRNA) were imported by RP and MRP aptamers into mitochondria. Adhya et al.15 reported that the combination of a mitochondrial tRNA import signal (D-arm) and a RNA import complex, which were found in Leishmania tropica, induced the transport of tRNA and antisense RNA into mitochondria in living cells. However, the direct modification of a mitochondrial RNA aptamer on a nanocarrier has not been a subject of extensive investigation.

The present study focused on enhancing mitochondrial targeting by modification of a liposomal-based nanocarrier with a mitochondrial RNA aptamer. The mitochondrial RNA aptamers used in this study included RP and MRP, in which mitochondrial delivery occurs through polynucleotide phosphorylase,13 and the D-arm with a high affinity with the tubulin antisense–binding protein located on the mitochondrial outer membrane16 (Table 1). These RNA aptamers themselves have mitochondrial targeting activity, but modifying the carrier with a single aptamer would not be sufficient to allow the particle to be internalized by a cell. We recently reported on the development of a mitochondrial delivery system, a MITO-Porter in which the surface is modified with octaarginine (R8). The R8 functions as both a cellular uptake device through macropinocytosis and as a mitochondrial targeting peptide through electrostatic interactions with negatively charged mitochondria.17, 18, 19 We also developed a dual-ligand system in which the nanocarrier is modified with a specific ligand and R8.20, 21 These systems have a synergistic effect on both selectivity and cellular uptake.

Thus, we expected that a dual-ligand liposomal system modified with both R8 and a mitochondrial RNA aptamer would show an enhanced mitochondrial delivery. In this study, we prepared a dual-ligand–modified MITO-Porter composed of R8 with different amounts of mitochondrial RNA aptamers including RP, MRP, and the D-arm. We then evaluated the cellular uptake efficiency of the carriers using flow cytometry. The extent of intracellular trafficking was observed by confocal laser scanning microscopy (CLSM), and the mitochondrial targeting rate and mitochondrial occupancy rate were estimated based on the obtained CLSM images.

Section snippets

Chemicals and Materials

1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin, and DOPE-N-(7-nitro-2-1,3-benzoxadiazole-4-yl) (NBD-DOPE) were purchased from Avanti Polar lipids (Alabaster, AL). Stearylated R822 was obtained from KURABO Industries (Osaka, Japan). Cholesterol covalently linked to the 3′ end of 2′-O-methyl RNAs (Chol-RNA aptamer) containing the RP sequence (Chol-RP), MRP sequence (Chol-MRP), and the D-arm sequence (Chol-D-arm) were obtained from Hokkaido System Science Co., Ltd. (Sapporo,

Construction of Dual-Ligand–Modified MITO-Porter

Chol-RNA aptamers, including RP, MRP, and the D-arm, were used to prepare the dual-ligand–modified MITO-Porter (RP/R8-modified MITO-Porter, MRP/R8-modified MITO-Porter, and D-arm/R8-modified MITO-Porter). Information regarding the RNA aptamers used in this study is listed in Table 1. The physicochemical properties of the prepared carriers are summarized in Table 2. Their diameters were approximately 100-150 nm. Modification of a negatively charged RNA aptamer on the positively charged

Discussion

The present study focused on strategies for enhancing mitochondrial targeting by modification of a liposomal-based nanocarrier with a mitochondrial RNA aptamer. To evaluate the targeting of the nanocarrier to mitochondria in living cells, we developed a dual-ligand system, in which the nanocarrier is modified with a mitochondrial RNA aptamer and R8. We expected that R8, which is reported to function as a cellular uptake device for a liposomal carrier,17, 18, 19 would assist the cellular

Conclusions

The results presented here constitute the first report of the use of a mitochondrial RNA aptamer–modified nanocarrier system to regulate intracellular trafficking, although lipophilic and cationic peptide-based mitochondrial targeting have been reported in previous studies.12, 19, 33, 34, 35 In this study, we determined the optimal dual-ligand system for a nanocarrier for achieving efficient cellular uptake and mitochondrial targeting (RP/R8-modified MITO-Porter). However, more improvements in

Acknowledgments

This work was supported, in part, by a Grant-in-Aid for Scientific Research (B) (grant 26282131 to Y.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology, the Japanese Government (MEXT), the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and the Uehara Memorial Foundation. We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.

References (36)

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    Citation Excerpt :

    We focused on the fact that mitochondria are continuously repeating fusion and fission in living cells and this permits bio macromolecules (nucleic acids, mtDNA, proteins and etc) to be shared between mitochondria, and we proposed the concept of a MITO-Porter, a liposome to achieve mitochondrial delivery of various types of cargoes via a membrane fusion. We successfully identified highly fusible mitochondrial fusogenic lipid compositions for this purpose [9,70–72]. A MITO-Porter, a particle in which the surface is modified with the membrane-permeable peptide (R8) is efficiently taken up by cells.

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The authors Yuma Yamada and Ryo Furukawa contributed equally.

This article contains supplementary material available from the authors by request or via the Internet at http://dx.doi.org/10.1016/j.xphs.2016.03.002.

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