Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Validation of the Mitochondrial Delivery of Vitamin B1 to Enhance ATP Production Using SH-SY5Y Cells, a Model Neuroblast

https://doi.org/10.1016/j.xphs.2021.08.033Get rights and content

Abstract

Large amounts of ATP are produced in mitochondria especially in the brain and heart, where energy consumption is high compared with other organs. Thus, a decrease in ATP production in such organs could be a cause of many diseases such as neurodegenerative diseases and heart disease. Based on thus assumption, increasing intracellular ATP production in such organs could be a therapeutic strategy. In this study, we report on the delivery of vitamin B1, a coenzyme that activates the tricarboxylic acid (TCA) cycle, to the inside of mitochondria. Since the TCA cycle is responsible for ATP production, we hypothesized delivering vitamin B1 to mitochondria would enhance ATP production. To accomplish this, we used a mitochondrial targeted liposome a “MITO-Porter” as the carrier. Using SH-SY5Y cells, a model neuroblast, cellular uptake and intracellular localization were analyzed using flow cytometry and confocal laser scanning microscopy. The optimized MITO-Porter containing encapsulated vitamin B1 (MITO-Porter (VB1)) was efficiently accumulated in mitochondria of SH-SY5Y cells. Further studies confirmed that the level of ATP production after the MITO-Porter (VB1) treatment was significantly increased as compared to a control group that was treated with naked vitamin B1. This study provides the potential for an innovative therapeutic strategy in which the TCA cycle is activated, thus enhancing ATP production.

Introduction

Mitochondria produce ATP through oxidative phosphorylation, and the resulting ATP is used as an energy for various purposes such as synthesis of biological substances, active transport and muscle movement. Thus, a decrease in ATP production could be one of causes of many diseases. Based on this possibility, it would be is expected that a strategy for increasing intracellular ATP production could be a therapeutic strategy for such diseases. The main action site of vitamin B1 is the tricarboxylic acid (TCA) cycle in the mitochondria. It is involved with pyruvate dehydrogenase complex (PDHC) and 2-oxoglutarate dehydrogenase complex (OGDHC) and activates the TCA cycle to promote ATP production.1

Vitamin B1 is transported into the mitochondria by a transport protein on the mitochondrial membrane (SLC25A19).2 However, when the vitamin B1 that is taken up by the cell via active transport reaches a certain concertation in the cellular component, the remaining vitamin B1 is taken up by passive diffusion. Therefore, a drug delivery system (DDS) for the cellular uptake of vitamin B1 is needed to increase the cellular concentration of vitamin B1, if a therapeutic effect greater than that for normal cellular uptake is to be achieved. Furthermore, the delivery of vitamin B1 to mitochondria, the location where vitamin B1 promotes ATP production, would be expected to further enhance the therapeutic effect of this strategy.

We previously developed a MITO-Porter3, 4, 5, 6, 7, 8 a liposomal DDS for delivering cargoes to mitochondria, and reported that such a MITO-Porter system successfully delivered various cargoes including low-molecular-weight compounds (e.g., anti-cancer drugs,9 a porphyrin-type chemical,10 coenzyme Q1011) and macromolecules such as nucleic acids12, 13, 14, 15 to mitochondria via mitochondrial membrane fusion. We assumed that the MITO-Porter system could accelerate the cellular uptake of vitamin B1 and would eventually reach the mitochondria. As a result, ATP production would increase via activation of the TCA cycle by vitamin B1 (Fig. 1). The purpose of this study was to validate that the delivery of vitamin B1 to mitochondria via the MITO-Porter system would enhance the production of ATP.

In this study, we first optimized the process used to prepare a MITO-Porter that contained encapsuled vitamin B1 (MITO-Porter (VB1)). The cellular uptake and intracellular localization of the MITO-Porter (VB1) were then evaluated using flow cytometry and confocal laser scanning microscopy. SH-SY5Y cells, model neuroblastoma cells, were used as typical cells in these experiments. The amount of ATP production after treating the cells with the MITO-Porter (VB1) was then quantitatively evaluated.

Section snippets

Materials

1,2-dioleoyl-sn‑glycero-3-phosphoethanolamine (DOPE) was obtained from the NOF Corporation (Tokyo, Japan). Sphingomyelin (SM) and DOPE-N-(7-nitro-2–1,3-benzoxadiazole-4-yl) (NBD-DOPE) were purchased from Avanti Polar lipids (Alabaster, AL). Stearylated R8 (STR-R8)16 was obtained from KURABO Industries (Osaka, Japan). Cholesteryl RP aptamer (Chol-RP, cholesteryl 5′-CUCCCUGAGCUUCAGG-3′)17, 18, 19 was purchased from Greiner bio-one (Tokyo, Japan). Thiamine pyrophosphate (vitamin B1) and fetal

Packaging vitamin B1 in lipid envelopes of the MITO-Porter using the REV method

The encapsulation of vitamin B1 in the MITO-Porter was initially investigated. The lipid composition used was DOPE and SM (9:2, molar ratio), and the liposomes were prepared at 5.5 mM total lipid using the REV method by varying the concentration of vitamin B1. The physicochemical properties of the prepared samples are shown in Table 1. The size of the particles decreased with decreasing initial concentration of vitamin B1. We also conducted PDI measurements as a measure of particle homogeneity

Discussion

In this study, the REV method was used to encapsulate vitamin B1 into the envelopes of the MITO-Porter. The diameters of the MITO-Porter (VB1) were 100 – 150 nm, and the encapsulation efficiency was in excess of 25% (Tables 1, 2 and Fig. 2). As shown in Table 1, the diameters of the MITO-Porter (VB1) increased with increasing initial concentration of vitamin B1. We discuss possible explanations for this issue. In the REV method, the lipid-containing organic solvent and aqueous vitamin B1

Supporting information

SI includes Materials and methods regarding Characterization of the prepared carriers, Evaluation of cellular uptake by flow cytometry analysis and Intracellular observation of the carriers by confocal laser scanning microscopy, Evaluation of cell viability, Supplementary Figures (Figure S1-S5) and Supplementary Table (Table S1).

Acknowledgments

This work was supported, in part by, a Grant-in-Aid for Scientific Research (B) [Grant No. 20H04523 to Y.Y.] and Scientific Research (C) [Grant No. 17K11063 to K.I., Y.Y.] from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), Tokyo Biochemical Research Foundation and Mochida Memorial foundation for Medical and Pharmaceutical research. We also wish to thank Dr. Milton Feather for his helpful advice in writing the manuscript.

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