Research Article
Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Validation of a Strategy for Cancer Therapy: Delivering Aminoglycoside Drugs to Mitochondria in HeLa Cells

https://doi.org/10.1002/jps.24686Get rights and content

Abstract

Mitochondria in human cancer cells have been implicated in cancer cell proliferation, invasion, metastasis, and even drug-resistance mechanisms, making them a potential target organelle for the treatment of human malignancies. Gentamicin (GM), an aminoglycoside drug (AG), is a small molecule that functions as an antibiotic and has ototoxic and nephrotoxic characteristics. Thus, the delivery of GM to mitochondria in cancer cells would be an innovative anticancer therapeutic strategy. In this study, we attempted mitochondrial delivery of GM in HeLa cells derived from a human cervical cancer. For the mitochondrial delivery, we used MITO-Porter, a liposomal nanocarrier for mitochondrial delivery via membrane fusion. We first encapsulated GM in the aqueous phase of the carrier to construct GM–MITO-Porter. Flow cytometry analysis and fluorescent microscopy observations permitted us to confirm that the GM–MITO-Porter was efficiently taken up by HeLa cells and accumulated in mitochondria, whereas naked GM was not taken up by the cells. Moreover, cell viability assays using HeLa cells showed that the GM–MITO-Porter induced strong cytotoxic effects related to mitochondrial disorder. This finding is the first report of the mitochondrial delivery of an AG to cancer cells for cancer therapeutic strategy.

Introduction

Waksman discovered streptomycin from Streptomyces spp. in 1944,1 and such aminoglycoside drugs (AGs) are now clinically used in treating infections caused by gram-negative bacteria with a broad antibiotic spectrum all over the world. Some AGs, for example, gentamicin (GM), are preferred for use because of their low cost and higher safety by therapeutic drug monitoring; although AGs can cause irreversible hearing loss and renal toxicity caused by mitochondrial damage, the detailed mechanisms responsible for this remain unclear.2 Mitochondria in the epidermal cells in the organ of Corti or kidneys are vulnerable to AGs,3 thus the patients with mitochondrial disease have a higher risk for AGs.4 Actually unexpected adverse effects of AGs have been reported among patients with undiagnosed mitochondrial disease.5

On the contrary, mitochondria in human cancer cells are closely related to cancer cell proliferation, invasion, metastasis, and even drug-resistance mechanisms, because of the power exerted by cancer cells for the production of usable energy.6, 7, 8 Thus, we hypothesized that the cellular uptake of AGs could kill cancer cells via mitochondrial toxicity, which would lead to an effective strategy for treating cancer in a different way from conventional anticancer agents. It is well known that AGs are efficiently internalized into ear hair cells3 and kidney distal tubule cells9 via nonselective cation channels such as the transient receptor potential vanilloid 4 (TRPV4)10; the studies regarding cellular uptake of AGs by cancer cells have not been examined. To date, our knowledge of the antitumor effect of AG remains limited, probably because the internalization of AG into cancer cells without specific channels would be difficult. To overcome the first barrier to kill cancer cells via the mitochondrial toxicity of AG, the efficient cellular uptake of AG is required.

We previously developed MITO-Porter, a nanocarrier to deliver nucleic acids, proteins, or certain types of small molecules to mitochondria via membrane fusion.11, 12, 13, 14 To date, we showed that the MITO-Porter was efficiently taken up by HeLa cells derived from a human cervical cancer, and bio-functional cargoes that regulate mitochondrial function were delivered to mitochondria. Here, we validated that the mitochondrial delivery of GM (a model AG) using the MITO-Porter resulted in the killing of HeLa cells via mitochondrial pharmacodynamics (Fig. 1). Octaarginine (R8)15, 16 modified on the surface of the MITO-Porter plays important roles in cellular uptake and mitochondrial targeting. It is expected that GM would be delivered to the mitochondrial interior, thus permitting GM to exert a pharmacological effect.

We first prepared a MITO-Porter encapsulating GM conjugated with 7-nitrobenz-2-oxa-1, 3-diazole (NBD) (a fluorescent dye), GM–MITO-Porter, and evaluated its physicochemical properties including diameters and ζ potentials. We then performed flow cytometry to assess the cellular uptake of the GM–MITO-Porter, and observed the intracellular trafficking of the carrier. We also quantified the mitochondrial targeting rate based on obtained images. Moreover, a comparison of cell toxicity between the GM–MITO-Porter and naked NBD–GM was performed by a cell viability assay. Finally, we validated the correlation between the mitochondrial delivery of NBD–GM and cell toxicity.

Section snippets

Materials

Gentamicin sulfate was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) from Tokyo Chemical Industry Company, Ltd. (Tokyo, Japan). The GM sulfate was conjugated with NBD-F to synthesize NBD–GM, which was purified as previously reported,17 and then freeze-dried (see Supplementary Materials for the details). In this experiment, NBD–GM was used as a naked GM and packaged in the MITO-Porter. 1,2-Dioleoyl-sn-glycero-3-phosphatidyl

Construction of the GM–MITO-Porter

We used NBD–GM for the encapsulation in the MITO-Porter, where the mitochondria-fusogenic lipid composition was used.11, 12 After the synthesis and purification of the NBD–GM, we performed a competitive enzyme immunoassay to evaluate the antibacterial activity of NBD–GM using a MaxSignal GM ELISA Test Kit (Bio Scientific Corporation, Austin, Texas). The findings showed that the activity of the NBD–GM was 52 ± 19% of the antibacterial activity of the unconjugated GM (n = 3).

Using a reverse-phase

Discussion

In previous study, it has been reported that AGs have a high affinity for ribosomal RNA and permeability transition in mitochondria, resulting in apoptosis via the inhibition of mitochondrial transcription or exaggerated oxidative stress, which are consistent with the molecular mechanisms for the ototoxicity and nephrotoxicity induced by AGs.24, 25, 26 AGs have been clinically available as antibiotics for many years, and, unfortunately, can exert some irreversible adverse effects, especially in

Conclusion

In order to investigate the intracellular dynamics and cellular toxicity of AGs using cancer HeLa cells, we constructed a GM–MITO-Porter, where NBD–GM (a model AG) with low cellular internalization, was encapsulated into the MITO-Porter. Flow cytometry analysis and fluorescent microscopy observations permitted us to confirm that the GM–MITO-Porter achieved mitochondrial delivery of NBD–GM in cancer HeLa cells. Moreover, we showed that GM–MITO-Porter induced a strong cytotoxicity, suggesting

Acknowledgments

This work was supported, in part, by a grant-in-aid for Scientific Research (B) [grant no. 26282131 (to Y.Y.)] from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), and Kobayashi Foundation for Cancer Research (to Y.Y.). We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.

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  • Cited by (0)

    Jiro, Abe and Yuma Yamada contributed equally to this study.

    This article contains supplementary material available from the authors upon request or via the Internet at http://dx.doi.org/10.1002/jps.24686.

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