Development of a nanoparticle that releases nucleic acids in response to a mitochondrial environment
Introduction
Mitochondria, with their own genome (mtDNA), have attracted attention from various fields including the life sciences, novel therapeutic strategies, beauty and health (Chan, 2006, Schapira, 2006, Taylor and Turnbull, 2005, Verechshagina et al., 2019, Yamada and Harashima, 2008, Yamada and Harashima, 2017). This organelle is of interest regarding possibility targeting the mitochondrial genome including mtDNA, mitochondrial RNA and mitochondrial micro RNA. Based on our limited knowledge of the unique intracellular space in mitochondria, it appears that this is a major barrier to progress in mitochondrial medicine and science. Nucleic acids that target the nucleus and cytoplasm have been subjects of considerable interest and the results of such studies have been a major factor in clinical applications using antisense therapy and gene repair therapy. Extensive research has focused the development of drug delivery systems (DDS) that can efficiently release nucleic acids into the cytoplasm and nucleus by introducing nucleic acid nanoparticles into these organelles (Bertrand et al., 2011, Mann et al., 2014, Mann et al., 2011, Sato et al., 2016, Takemoto et al., 2014, Yamada et al., 2012a, Yamada et al., 2012c).
In recent years, research on mitochondrial DDS has also come of age (Biswas and Torchilin, 2014, Ma et al., 2016, Milane et al., 2015, Nakamura et al., 2019, Sato et al., 2017, Wen et al., 2016, Zhang et al., 2011) and there is a high probability that it will have an impact in developing methods for delivering nucleic acids to mitochondria. In a previous study, we reported on the development of a MITO-Porter, an innovative nanocarrier that can deliver a cargo to mitochondria via mitochondrial membrane fusion (Nakamura et al., 2019, Yamada et al., 2008, Yamada and Harashima, 2017). To date, we succeeded in mitochondrial delivery of various molecule to regulate mitochondrial function (Abe et al., 2018, Ishikawa et al., 2018, Takano et al., 2017, Yamada et al., 2019a, Yamada et al., 2011, Yamada et al., 2015). As one of reports, we succeeded in delivering antisense RNA (AS RNA) targeting mtDNA-encoded mRNA to mitochondria of HeLa cells using a MITO-Porter, and were able to achieve mitochondrial RNA knockdown (about 50%) and in regulating mitochondrial function (Furukawa et al., 2015) (Fig. S1A). Furthermore, we succeeded in efficiently packaging oligo nucleic acids in the MITO-Porter via a nanoparticle packaging method. The method involves the use of a nanoparticle containing oligo nucleic acids concentrated with stearylated octaarginine (STR-R8), a polycation, via electrostatic interactions, and is then packaged into the lipid envelope (Yamada et al., 2012b).
We expected that the efficient packaging of nanoparticles of nucleic acids in the MITO-Porter would increase the accumulation of nucleic acids in mitochondria, thus enhancing the mitochondrial RNA knockdown effect. Therefore, we attempted to deliver a nanoparticle of AS RNA to mitochondria using the MITO-Porter. In this experiment, an AS RNA nanoparticle was formed with STR-R8 and mitochondrial delivery was achieved using the MITO-Porter. We evaluated the antisense effect using HeLa cells by measuring mitochondrial membrane potential. Unfortunately, the knockdown effect was limited to 50%, similar to a MITO-Porter that contained encapsulated naked AS RNA. We also observed that the polycations that were used to form the nanoparticle were toxic to mitochondria (Fig. S1B). Detailed information regarding this can be found in the Results section 3.1.
Based on the results, we conclude that it is essential to proceed with, not only further developments of mitochondrial DDS but also nucleic acid science research if mitochondrial nucleic acid medicine is to be successful. In other words, unlike nuclear / cytoplasmic targeting, it is essential to form nanoparticles of nucleic acids that can function effectively in the mitochondrial environment and design optimal polycations that can be used to form the nanoparticle. The objective of this study was to develop a nanoparticle that can release nucleic acids in response to the mitochondrial environment, which contains high levels of anionic biomolecules and which the pH is higher than 8.0. To achieve this, we prepared nanoparticles using various poly cationic peptides, and measured their diameters and ζ-potentials. We then evaluated the efficiency of release of nucleic acids from the nanoparticles under conditions that mimic the mitochondrial environment.
Section snippets
Materials
STR-histidine (H) 8, STR-R8 (Futaki et al., 2001), STR-H4R4, STR-H2R2H2R2, STR-H2R4H2 and STR-R2H4R2 were obtained from Greiner Bio-One GmbH (Kremsmuenster, Austria). Native Protamine sulfate from salmon milt, was purchased from CALBIO CHEM (Darmstadt, Germany). Sequence information for these peptides is summarized in Table 1. Darm modified antisense 2′-O-Methyl (2′-OMe) RNA targeting mitochondrial cytochrome c oxidase subunit II (COX II) (Darm ASO [COX2]) (5′-
Design of polycations for forming pH-responsive nanoparticles that release nucleic acids
We expected that efficient packaging of nanoparticles of nucleic acids in the MITO-Porter would increase the degree of accumulation of the nucleic acids in mitochondria, thus enhancing mitochondrial therapeutic effects (RNA knockdown effect). Therefore, we attempted to deliver a nanoparticle of AS RNA to mitochondria using the prepared MITO-Porter. In this experiment, AS RNA nanoparticles were formed with STR-R8 and the actual mitochondrial delivery involved the use of the MITO-Porter. We also
Conclusions
The findings reported in this study show that nucleic acid nanoparticles can, in fact, be prepared with STR-H4R4, STR-H2R2H2R2 and STR-R2H4R2 in the acidic to neutral region (Fig. 3B). Moreover, the release efficiency of nucleic acids from these nanoparticles was superior to the R-based polymers, including Protamine and STR-R8, under in the presence of an anionic compound under alkaline conditions (Fig. 4B). This indicates that STR-H4R4, STR-H2R2H2R2 and STR-R2H4R2 are suitable polymers for
Acknowledgements
This work was supported, in part by, a Grant-in Aid for Challenging Exploratory Research [Grant No. 15595383 (to Y.Y.)] and a Grant-in-Aid for Scientific Research (B) [Grant No. 17923708 (to Y.Y.)] from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT). We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.
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