Pharmaceutics, Drug Delivery and Pharmaceutical TechnologyValidation of the Mitochondrial Delivery of Vitamin B1 to Enhance ATP Production Using SH-SY5Y Cells, a Model Neuroblast
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.
References (24)
- et al.
MITO-Porter: a liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion
Biochim. Biophys. Acta
(2008) - et al.
Evolution of drug delivery system from viewpoint of controlled intracellular trafficking and selective tissue targeting toward future nanomedicine
J Controlled Rel
(2020) - et al.
Mitochondrial delivery of an anticancer drug via systemic administration using a mitochondrial delivery system that inhibits the growth of drug-resistant cancer engrafted on mice
J Pharm Sci-Us
(2020) - et al.
The use of a microfluidic device to encapsulate a poorly water-soluble drug CoQ(10) in lipid nanoparticles and an attempt to regulate intracellular trafficking to reach mitochondria
J Pharm Sci-Us
(2019) - et al.
Development of a nanoparticle that releases nucleic acids in response to a mitochondrial environment
Mitochondrion
(2020) - et al.
Validation of gene therapy for mutant mitochondria by delivering mitochondrial rna using a MITO-Porter
Mol Ther-Nucl Acids
(2020) - et al.
The use of a MITO-Porter to deliver exogenous therapeutic RNA to a mitochondrial disease's cell with a A1555G mutation in the mitochondrial 12S rRNA gene results in an increase in mitochondrial respiratory activity
Mitochondrion
(2020) - et al.
A dual-ligand liposomal system composed of a cell-penetrating peptide and a mitochondrial RNA aptamer synergistically facilitates cellular uptake and mitochondrial targeting
J Pharm Sci
(2016) - et al.
PNPASE regulates RNA import into mitochondria
Cell
(2010) - et al.
Targeted mitochondrial delivery of antisense RNA-containing nanoparticles by a MITO-Porter for safe and efficient mitochondrial gene silencing
Mitochondrion
(2019)
A mitochondrial delivery system using liposome-based nanocarriers that target myoblast cells
Mitochondrion
Elevated lactate secondary to gastrointestinal beriberi
J Gen Intern Med
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