Research Article
Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Mitochondrial Delivery of Doxorubicin Using MITO-Porter Kills Drug-Resistant Renal Cancer Cells via Mitochondrial Toxicity

https://doi.org/10.1016/j.xphs.2017.04.058Get rights and content

Abstract

Most anticancer drugs are intended to function in the nuclei of cancer cells. If an anticancer drug could be delivered to mitochondria, the source of cellular energy, this organelle would be destroyed, resulting in the arrest of the energy supply and the killing of the cancer cells. To achieve such an innovative strategy, a mitochondrial drug delivery system targeted to cancer cells will be required. We recently reported on the development of a MITO-Porter, a liposome for mitochondrial delivery. In this study, we validated the utility of such a cancer therapeutic strategy by delivering anticancer drugs directly to mitochondria. We succeeded in packaging doxorubicin (DOX) as a model cargo in MITO-Porter to produce a DOX-MITO-Porter. We evaluated cellular toxicity of OS-RC-2 cell, a type of DOX-resistant cancer cell, after delivering DOX to mitochondria using the MITO-Porter system. Cell viability was decreased by the DOX-MITO-Porter treatment, while cell viability was not decreased in the case of naked DOX and a conventional DOX liposomal formulation. We also found a relationship between cellular toxicity and mitochondrial toxicity. The use of a MITO-Porter system for mitochondrial delivery of a toxic agent represents a possible therapeutic strategy for treating drug-resistant cancers.

Introduction

The fact that numerous reports have appeared regarding multidrug-resistant cancer cells1 suggests that the use of conventional chemotherapy is not a viable option for treating clinically untreatable cancers. Most anticancer drugs are intended to function in the nuclei and ultimately kill the cancer cells. Mitochondria have been implicated in the proliferation of cancer cells, invasion, metastasis, and even drug resistance mechanisms,2 thus making them a potential target organelle for cancer therapy. To achieve such an innovative therapeutic strategy, a system for delivering a drug to mitochondria2, 3 of cancer cells is required.

To date, several groups have reported that the mitochondrial delivery of doxorubicin (DOX), an anthracycline antitumor drug that damages the nuclear genome, was achieved via the direct chemical conjugation of the cargo with mitochondrial targeting ligands.4, 5 Han et al.5 reported on the mitochondrial delivery of DOX via a modified type of lipophilic triphenylphosphine (TPP). TPP, originally designed by Murphy et al.6 is a mitochondriotropic ligand that is taken up by the mitochondrial membrane potential because of its high lipophilicity and stable cationic charge. Han et al. showed that TPP modified DOX was delivered to mitochondria of a DOX-resistant human breast cancer cell line (MDA-MB-435/DOX) more effectively than naked DOX. These reports indicate that the mitochondrial delivery of DOX represents a potentially useful therapeutic strategy for targeting DOX-resistant cancer cells.

Promising reports such as those described above promote the development of mitochondrial targeting DOX for cancer therapy and related research. However, these mitochondrial targeting drugs are not guaranteed to selectively target the tumor. Thus, side effects such as cardiotoxicity in the case of DOX are a real concern. Doxil is a liposomal formulation encapsulating DOX and accumulates at increased levels in cancer cells via the enhanced permeability retention effect,7 permitting its therapeutic effect to be enhanced.8 Thus, this liposome (LP) can regulate biodistribution and reduce access to heart tissue, avoiding side effects such as cardiotoxicity. It has attracted attention as the world’s first liposomal formulation for use in cancer therapy, although there are concerns regarding differences in its therapeutic effect in animal experiments versus clinical trials involving humans.9, 10

To date, several other liposomal formulations for cancer therapy have been reported,10, 11 while reports of mitochondrial targeting liposomal carriers for cancer therapy are few in number and no reports of DOX-resistant tumor-bearing model animals in in vivo experiments. Concerning liposomal carriers for mitochondrial delivery, it has been reported that DQAsomes and a TPP-modified liposome (TPP-LP), developed by Weissig et al.,12, 13 and the MITO-Porter, developed in our laboratory,14, 15 are viable vesicles. The MITO-Porter is an LP-based mitochondrial delivery system that functions via membrane fusion.14, 16 To date, we have shown that the MITO-Porter can be used to deliver a variety of therapeutic cargoes, including an antiapoptosis chemical and an antioxidant chemical, to mitochondria of human cells.17, 18, 19 As a result, the mitochondrial delivery of therapeutic cargoes have the potential for functioning as a mitochondrial therapeutic strategy in in vitro experiments. Moreover, we evaluated the antioxidant effect conferred by the mitochondrial delivery of Coenzyme CoQ10 (CoQ10) using an ischemic/reperfusion injury model mouse in in vivo experiments.20 As a result, we confirmed that the systemic injection of the CoQ10-MITO-Porter resulted in a significant therapeutic effect compared with naked CoQ10 and other carriers. The results indicate that the MITO-Porter represents a potentially useful carrier for mitochondrial medicine.

The objective of this study is to validate the utility of a cancer therapeutic strategy by delivering anticancer drugs directly to mitochondria with the goal of killing anticancer drug-resistant cancer cells. In this study, we prepared a DOX-MITO-Porter, which packages DOX as a cargo, and evaluated cellular toxicity using OS-RC-2 cells, a DOX-resistant renal cancer cell. The schematic image indicates that an anticancer therapeutic effect is observed as the result of the mitochondrial delivery of DOX in cancer cells using the MITO-Porter system (Fig. 1). Under an ideal scenario, the DOX-MITO-Porter is internalized by the cells, and then escapes from endosomes. In the cytosol, the carrier delivers DOX to mitochondria via membrane fusion, resulting in the killing of DOX-resistant OS-RC-2 cells via mitochondrial toxicity. Naked DOX is internalized by cells in concentration-dependent manner, and then enters the nucleus, resulting in intercalation into nuclear DNA and the disruption of topoisomerase-II-mediated DNA repair to kill the cells. In the case of Doxil, naked DOX is released from Doxil that accumulates in tumor tissue, and the naked DOX is then internalized to cells, killing them in a manner similar to that described above. The mechanism of DOX resistance involves complex mechanisms including P-glycoproteins as efflux pumps of anticancer drugs and the regulation of topoisomerase-II (target molecule of DOX) expression in the nucleus.21 In such a situation, naked DOX, which acts in the nucleus, would not be able to kill drug-resistant cells. Thus, the mitochondrial delivery of DOX using a MITO-Porter system would lead to an innovative strategy for cancer therapy by killing DOX-resistant cancer cells via mitochondrial toxicity.

In this study, we prepared a DOX-MITO-Porter using a pH loading method, in which DOX is contained in the aqueous phase of the MITO-Porter, and investigated the physicochemical properties of the preparation, including diameters, ζ-potentials, and DOX encapsulation efficiencies. We also prepared a DOX-PEG-LP, which is a polyethylene glycol (PEG) modified LP encapsulating DOX as a Doxil analog control. We compared cell toxicity among the DOX-MITO-Porter, naked DOX, and DOX-PEG-LP using a cell viability assay. Moreover, we observed the intracellular localization of the DOX using confocal laser scanning microscopy (CLSM), when samples were treated with OS-RC-2 cells. Finally, we evaluated the efficiency of adenosine triphosphate (ATP) production and mitochondrial membrane potentials after the cells were treated with the preparations, and validated the relationship between mitochondrial toxicity via the mitochondrial delivery of DOX and cell death.

Section snippets

Materials

Cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and sphingomyelin (SM) were purchased from Avanti Polar lipids (Alabaster, AL). Egg yolk phosphatidylcholine (EPC) and N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG 2000) were obtained from the NOF Corporation (Tokyo, Japan). Stearylated octaarginine (STR-R8)22 was obtained from Kurabo Industries Ltd. (Osaka, Japan). DOX hydrochloride and Chol E were obtained

Preparation of the DOX-MITO-Porter Using the pH Loading Method

In this experiment, we prepared the DOX-MITO-Porter using the pH loading method, as shown in Figure 2. The construction of the DOX-MITO-Porter requires the following steps: (1) LP preparation by the hydration method, (2) controlling particle diameter to 100-200 nm by extrusion, (3) exchange of external buffer to prepare LPs with a transmembrane pH gradient, while the interior is buffered acidic whereas the exterior is buffered neutral, (4) encapsulation of DOX via a transmembrane pH gradient,

Discussion

In this study, we validated the utility of cancer therapeutic strategy by the mitochondrial delivery of anticancer drugs for killing anticancer drug-resistant cancer cells. To achieve this, we packaged DOX (a model anticancer drug) into a MITO-Porter, an LP for mitochondrial delivery, and investigated the anticancer therapeutic effect conferred by mitochondrial delivery of DOX in OS-RC-2 cells, DOX-resistant cells. Intracellular observations by CLSM confirmed that the MITO-Porter system

Conclusion

We report on the validation that the cellular uptake of DOX kills cancer cells via mitochondrial toxicity, which has the potential to lead to an innovative strategy for killing DOX-resistant cancer cells. We succeeded in packaging DOX in a MITO-Porter by a pH loading method to prepare a DOX-MITO-Porter. Evaluation of cell viability using OS-RC-2 cells (DOX-resistant cancer cell) showed that the DOX-MITO-Porter significantly decreased cell viability compared with naked DOX and a conventional DOX

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (B) (grant 26282131 to Y.Y.) and a Grant-in-Aid for Challenging Exploratory Research (grant 25560219 to Y.Y.) from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), and the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from Japan Agency for Medical Research and

References (26)

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Conflicts of interest: The authors declare no conflict of interest associated with this manuscript.

This article contains supplementary material available from the authors by request or via the Internet at http://dx.doi.org/10.1016/j.xphs.2017.04.058.

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