Mitochondrial delivery of Coenzyme Q10 via systemic administration using a MITO-Porter prevents ischemia/reperfusion injury in the mouse liver
Graphical abstract
Introduction
In recent years, various mitochondrial dysfunctions have been implicated in a variety of diseases [1], [2], [3]. Thus, research and development directed toward mitochondrial medicine would be expected to have great medical benefits to society in general. Thus, researchers dealing with mitochondrial drug delivery systems are encouraged to develop such therapeutic strategies based on the in vivo mitochondrial delivery of therapeutics. Various types of mitochondrial delivery systems have been reported during the past decades [4], [5], [6], [7], [8], but only a limited number of these approaches have the potential for use in mitochondrial therapy. These strategies face many problems including cell internalization, size limitations and the physicochemical properties of the cargo, modification of a functional device and the denaturation of the cargo [5], [9], [10].
We previously reported on the construction of a MITO-Porter, a liposome-based mitochondrial delivery system that functions via membrane fusion [5], [11]. This membrane fusion mechanism-based strategy can deliver a cargo to mitochondria independent of its size and physical properties. To date, we have shown that the MITO-Porter can be used to deliver a variety of therapeutic cargoes, including an anti-apoptosis chemical and an anti-oxidant chemical, to the mitochondria of human cells [12], [13]. As a result, the mitochondrial delivery of therapeutic cargoes has potential for functioning as a mitochondrial therapeutic strategy, indicating that the MITO-Porter represents a potentially useful carrier for use in mitochondrial medicine, based on in vitro experiments.
The purpose of this study was to validate the utility of the mitochondrial therapeutic strategy by targeting in vivo mitochondria as shown in Fig. 1. The schematic image indicates the antioxidant effect conferred by delivering coenzyme Q10 (CoQ10), an anti-oxidant, to liver mitochondria in a mouse ischemia/reperfusion injury (I/R injury) model, using the MITO-Porter. Under an ideal scenario, the MITO-Porter encapsulating CoQ10 reaches the liver tissue via systemic injection, and the carrier is then internalized into hepatocytes via macropinocytosis. In the cytosol, the carrier delivers CoQ10 to mitochondria via membrane fusion, resulting in the creation of a pharmacological effect of CoQ10 in mitochondria. Reactive oxygen species (ROS) are mainly produced in the mitochondrial respiratory chain, and are associated with a variety of diseases including I/R injury, neurodegenerative diseases, tumor metastasis, metabolic syndrome and aging [5], [14], [15], [16], [17], [18]. Thus, a therapeutic strategy for the mitochondrial delivery of antioxidant chemicals could be useful for the treatment of these diseases. To evaluate the anti-oxidant effects resulting from the mitochondrial delivery of CoQ10, we used hepatic I/R injury induced mice that overexpress mitochondrial ROS in the liver. In such a situation, serum alanine aminotransferase (ALT) levels would be increased.
In this study, we prepared the CoQ10-MITO-Porter by the ethanol dilution method, in which CoQ10 is contained in the lipid envelopes of the MITO-Porter, and attempted appropriately adjust the size of the particles. We also investigated the effect of the size of the CoQ10-MITO-Porter on the distribution of the carrier in liver tissue post systemic injection by histological observations using confocal laser scanning microscopy (CLSM). A knowledge of the extent of mitochondrial binding and the fusogenic activities of the CoQ10-MITO-Porter permitted us to determine the optimal composition for the constructing a CoQ10-MITO-Porter with a high mitochondrial fusogenic activity. Histological observations by CLSM and the use of a carrier labeled with a radio isotope (RI) verified that the CoQ10-MITO-Porter was delivered liver mitochondria via systemic injection. Finally, we injected mice with the optimized CoQ10-MITO-Porter via the tail vein, and hepatic I/R injury was then induced, followed by measurements of serum ALT levels, a marker of liver injury.
Section snippets
Materials
1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), egg yolk phosphatidyl choline (EPC), sphingomyelin (SM), 7-nitrobenz-2-oxa-1,3-diazole labeled DOPE (NBD-DOPE) and rhodamine-DOPE were purchased from Avanti Polar lipids (Alabaster, AL, USA). 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol 2000 (DMG-PEG 2000) was obtained from the NOF Corporation (Tokyo, Japan). Stearylated octaarginine (STR-R8) [19] was obtained from Kurabo Industries Ltd (Osaka, Japan). CoQ10 was obtained
Distribution of the MITO-Porter in liver tissue and mitochondrial accumulation
We first investigated the distribution of the MITO-Porter in the liver after systemic injection of the carriers. We prepared the MITO-Porter with highly mitochondrial fusogenic envelopes [5], [9], [11], [21], where, of the total lipids, 10 mol% R8 was modified on the carrier surface, by the hydration method. We also prepared PEG-LP without R8 as a control carrier. The physiochemical properties of these carriers are summarized in Table S1. For histological observations, carriers labeled with DiI
Discussion
We confirmed the utility of a mitochondrial therapeutic strategy that involves targeting in vivo mitochondria using a MITO-Porter, which delivered therapeutic cargoes to mitochondria and showed a mitochondrial therapeutic effect as well as in an in vitro experiment [12], [13]. In the present study, the MITO-Porter was designed to deliver therapeutic cargoes to liver mitochondria of mice via systemic injection for prevention of hepatic I/R injury. Thus, we first investigated the distribution of
Conclusion
We report on the successful delivery of CoQ10 to liver mitochondria of mice via the systemic injection of a CoQ10-MITO-Porter. We confirmed that controlling the size of the carrier is an important factor and that the use of R8 contributed to liver accumulation and the mitochondrial targeting. We also confirmed that the in vivo mitochondrial delivery of CoQ10 via MITO-Porter prevents the development of hepatic I/R injury. Moreover, the findings show that the mitochondrial membrane fusion
Acknowledgment
This work was supported, in part by, a Grant-in-Aid for Young Scientists (A) [Grant No. 23680053 (to Y.Y.)] from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), and A-step feasibility study program in Japan Science and Technology Agency (JST) [Grant No. AS251Z00277Q (to Y.Y.)], Northern Advancement Center for Science & Technology (Noastec Foundation, HokkaidoJapan) [Grant No. T-1-42 (to Y.Y.)], the Mochida Memorial Foundation for Medical and
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These authors contributed equally as first author.