Original research articleA mitochondrial delivery system using liposome-based nanocarriers that target myoblast cells
Introduction
Skeletal muscle mitochondrial dysfunction is an important issue, not only in aging and mitochondrial disease, but also in numerous other types of diseases. In type 2 diabetes, it has been reported that mitochondrial abnormalities associated with skeletal muscle cause a reduced capacity for exercise (Takada et al., 2014; Takada et al., 2013; Takada et al., 2015; Toledo and Goodpaster, 2013). Skeletal muscle mitochondrial dysfunction, skeletal muscle abnormalities, and low exercise tolerance are observed in the case of renal and heart failure (Matsumoto et al., 2018; Nishikawa et al., 2015; Okita et al., 2013; Takada et al., 2016). These dysfunctions affect the quality of life, activity related to daily living, and a poor prognosis (Piepoli et al., 2004). It would be possible to improve this prognosis by treating and improving the skeletal muscle mitochondria that are impaired by various diseases.
Mitochondrial gene therapy and delivering drugs to mitochondria are possible therapeutic approaches to addressing these types of mitochondrial abnormalities. Delivering a substance to mitochondria can be difficult and is limited by factors that include the molecular weight of the material to be delivered, low therapeutic efficiency, and toxicity. Furthermore, a method for directly delivering a substance to mitochondria in such cases has not been reported in skeletal muscle or myoblast cells. In previous studies, we reported on the development of mitochondrial targeted liposome-based nanocarriers which are capable of delivering encapsulated substances to the mitochondria via membrane fusion. Such a “MITO-Porter” is a liposome-based mitochondrial delivery system (Yamada et al., 2008) and has a lipid composition that was optimized for use in HeLa cells, derived from a human cervical carcinoma. The MITO-Porter was modified with octaarginine (R8) peptide, which is reported to stimulate micropinocytosis-mediated uptake by liposomal modification (Khalil et al., 2006). It has been shown that the cellular uptake of MITO-Porter is dependent on the cell type (Ishikawa et al., 2018) and, as a result, it is necessary to validate their efficiency of uptake for each type of cell.
In this study, using skeletal myoblast cells (C2C12 cells), we attempted to develop a liposome-based nanocarrier that is efficiently internalized by cells and is capable of efficiently transporting genes into the mitochondria of these cells. We first investigated the capacity of cellular uptake, and identified a lipid composition that could be used in preparing a carrier that could be efficiently internalized into C2C12 cells. We then evaluated mitochondrial transgene expression in C2C12 cells after delivering a mitochondrial DNA vector (pCMV-mtLuc (CGG)) using liposomes modified with different type functional devices. We also observed the intracellular localization of each type of liposomes by confocal laser scanning microscopy (CLSM).
Section snippets
Materials
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (hydrochloride) (DC-Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldiocatadecylammonium bromide (DDAB), egg yolk phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2,1,3-benzoxadiazol-4-yl) (NBD-DOPE) were purchased from Avanti Polar Lipid (Alabaster, AL, USA). Stearylated R8 (Futaki et al., 2001) was obtained from
Screening for a lipid composition with a high affinity for C2C12 myoblast cells
We evaluated the cellular uptake of liposomes by C2C12 myoblast cells by measuring the fluorescence intensity of cells that had been incubated with liposomes that contained NBD, a fluorescent dye. We compared each of the lipid compositions of the liposomes (Table 1); a MITO-Porter with a high cellular uptake capacity in HeLa cells as a candidate or one without the cell-penetrating R8 (“R8-MITO-Porter” or “DOPE/SM-LP”), the β-MEND with a high cellular uptake efficiency in mouse pancreatic β
Discussion
Mitochondrial targeting liposome-based nanocarriers are known to have different cellular uptake efficiencies depending on the cell type under consideration (Ishikawa et al., 2018). The lipid composition of the β-MEND conferred an enhanced efficiency for cellular uptake by C2C12 myoblast cells than the MITO-Porter (Fig. 1), which are similar to that in MIN6 cells derived from mouse pancreatic β cells. The relationship between cell type and lipid composition regarding cellular uptake efficiency
Conclusions
The findings reported herein show that a β-MEND modified with the KALA peptide is taken up by skeletal muscle cells, and that it has the ability to efficiently transfect genes into mitochondria.
Funding
This work was supported, in part by, Japan Agency for Medical Research and Development (AMED), Japan [grant number 17ek0109192h002 to S.K.], a Grant-in-Aid for Scientific Research (B) [grant numbers 26282131 and 17H02094 to Y.Y.] from the Ministry of Education, Culture, Sports, Science and Technology of Japanese Government (MEXT) and the Noguchi-Shitagau Research Grant (to Y.Y.).
Disclosures
The authors state that there are no disclosures to announce.
Declaration of Competing Interest
None.
Acknowledgements
We wish to thank Miwako Yamane, Yuki Kimura, Noriko Ikeda, and Chihiro Ogawa for their technical assistance. The flow cytometry analysis in this study was supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from Japan AMED. We also wish to thank Dr. Milton Feather for his helpful advice in preparing this manuscript.
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