Research ArticlePharmaceutics, Drug Delivery and Pharmaceutical TechnologyThe Use of a Microfluidic Device to Encapsulate a Poorly Water-Soluble Drug CoQ10 in Lipid Nanoparticles and an Attempt to Regulate Intracellular Trafficking to Reach Mitochondria
Introduction
It was recently reported that 40% of drugs that are currently on the market, as well as ∼90% of new candidates for drugs, are poorly water soluble.1 This suggests that a need exists to develop drug formulations that will permit the expanded use of such drugs. Most of these poorly water-soluble compounds are classified as Class IV drugs in the biopharmaceutics classification system (BCS). Drugs categorized in BCS Class IV are typically sparingly soluble in water and have low gastrointestinal permeability, and it is generally difficult to incorporate them in optimal drug formulations. Coenzyme Q10 (CoQ10) is a representative drug and is classified as a BCS Class IV drug.2
To date, CoQ10 has been developed as an oral therapeutic drug such as Neuquinon® and is used for the prevention and treatment of ischemic diseases. There are many commercially available supplements that contain CoQ10. However, the current CoQ10 drugs are not effective for the treatment of ischemic diseases that require a rapid response because an injectable form of CoQ10 has never been developed, largely because of its poor water solubility. Thus, an injectable form of CoQ10 is strongly required. In addition, cell death via ischemia injury in tissues is accompanied by the production of excess levels of reactive oxygen species and decreased energy production. Therefore, a strategy that permits drugs such as CoQ10 to mitochondria where the production of reactive oxygen species and ATP occur could lead to an effective therapy for the treatment of ischemic injury therapy.3 CoQ10 has strong antioxidant effects and functions as an electron carrier in the oxidative phosphorylation pathway to produce ATP in the inner membranes of mitochondria.4 However, CoQ10 and conventional CoQ10 drugs cannot be delivered to mitochondria by intracellular trafficking. Therefore, regulating the intracellular trafficking of CoQ10 to efficiently deliver it to mitochondria would lead to substantial increase in the therapeutic effect of the drug.
We previously developed a liposome (an “MITO-Porter”) that permits various types of molecules to be delivered to mitochondria via membrane fusion.5, 6, 7 In addition, we succeeded in packaging CoQ10 in such an MITO-Porter (CoQ10-MITO-Porter) and confirmed that the CoQ10-MITO-Porter was delivered to mitochondria showing excellent pharmaceutical effects in vivo.8, 9 Earlier research showed that there are a variety of methods for encapsulating CoQ10 in liposomes.10, 11, 12, 13 We previously reported that a CoQ10-MITO-Porter with the highest loading rate of CoQ10 (ratio of drug to lipid) was obtained, when the particles were prepared by the ethanol dilution method.9 However, this method has disadvantages, in that it is not possible to use it to prepare liposomes on a large scale.
Lipid nanoparticles (LNPs) containing liposomes can be used to encapsulate various types of drugs including conventional small drugs and large molecules such as nucleic acids which, on accumulating at the target site, can have an increased therapeutic effect with a reduction in side effects.14, 15 Moreover, if such a technique could be used to prepare liposomes that encapsulate poorly water-soluble molecules such as CoQ10, this would contribute to an improved dispersibility for such molecules.16, 17 However, there are a limited number of cases where liposome formulations have been put to a practical use. The causes for this include difficulties associated with scaling up the preparation method from the laboratory level to an industrial level, the stability and quality of the formulation, the difficulties associated with such a system being accepted for use. It should be noted that changing a manufacturing process can result in changes in the physicochemical properties of nanoparticle formulations, including liposomal drugs, and that these changes can have a significant influence on the stability of formulations and their therapeutic effect.
Based on such conditions, a method for preparing LNPs using a microfluidic device has attracted considerable attention as one of the techniques for preparing nanoparticle formations.18, 19, 20, 21 In this study, we evaluated the use of a microfluidic device for preparing LNPs based on the ethanol dilution method. The rapid mixing of ethanol containing lipids and an aqueous solution on a microfluidic device to rapidly dilute the ethanol make it possible to produce LNPs by a simple continuous flow process. The size of LNPs can generally be controlled by adjusting the concentration of lipid phase, the type of aqueous phase being used, the total flow rate, and the flow rate ratio, which corresponds to the ethanol dilution concentration. While the use of a microfluidic device to encapsulate a poorly water-soluble drug belonging to BCS Class IV such as CoQ10 in LNPs has been rarely reported,17 homogenous LNPs containing CoQ10 with diameters of less than 100 nm have never been prepared. It should also be noted that the use of a microfluidic device for preparing LNPs with mitochondrial targeting ability has not been attempted.
Herein, we report on attempts to encapsulate CoQ10 as a model of a poorly water-soluble drug in an MITO-Porter using a microfluidic device (CoQ10-MITO-Porter [μ]) (Fig. 1). In this study, a microfluidic device incorporating a baffle mixer named invasive lipid nanoparticle production (iLiNP) device was used.22 As shown in Figure 1b, the iLiNP device contains the basic structure of 20 baffle mixer structure sets. Using such a device, it is possible to achieve a more precise size control compared with conventional products. The physical properties of the CoQ10-MITO-Porter [μ] including homogeneity, size, and preparation volume were compared with those for a CoQ10-MITO-Porter prepared by the ethanol dilution method (CoQ10-MITO-Porter [ED]). In addition, the intracellular trafficking of the 2 types of CoQ10-MITO-Porters prepared by each method to reach mitochondria was evaluated by a cellular uptake analysis and intracellular observations.
Section snippets
Materials
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), and DOPE-N-(7-nitro-2-1,3-benzoxadiazole-4-yl) (NBD-DOPE) were purchased from Avanti Polar lipids (Alabaster, AL). 1,2-Dimyristoyl-sn-glycerol, methoxy polyethylene glycol 2000 (DMG-PEG 2000) was obtained from the NOF Corporation (Tokyo, Japan). Stearylated R8 (STR-R8) was obtained from Toray Industries, Inc. (Tokyo, Japan). CoQ10 was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals
Effect of Ethanol Concentration on the Particle Size of the CoQ10-MITO-Porter Prepared by Means of the Microfluidic Device
In the preparation where the microfluidic device was used, the particle diameter can be controlled by changing the ethanol concentration by adjusting the flow rate ratio between the lipid phase and the aqueous phase. In these experiments, ethanol concentrations of 10%, 20%, 30%, and 40% were used to evaluate the effect of the particle size (Fig. 2a; Table S1). Particle size increased and PDI decreased with increasing ethanol concentration when the microfluidic device was used [Fig. 2a(a)].
Discussion
Rapid dilution of an organic solvent containing lipids mixed with an aqueous solution allows small-sized particle formation.17, 23 The iLiNP device used in this study contained the basic structure of 20 baffle mixer structure sets. We previously reported that mixing an organic solvent with an aqueous solution at a fully expected mixing speed was sufficient after passing through the 10th baffle mixer at a total flow rate of 500 μL/min,22 which are similar conditions of this study. If an organic
Conclusions
It was possible to prepare a homogeneously distributed, small-sized CoQ10-MITO-Porter [μ] on a large scale by using a microfluidic device. In addition, this was also the optimum method for stably modifying the particle surface with a functional device such as STR-R8. Analyses of intracellular trafficking showed that a CoQ10-MITO-Porter with a small size was efficiently internalized into cells and accumulated in mitochondria. This study promises to be useful, not only a technique for
Acknowledgments
This work was supported, in part, by a Grant-in-Aid for Scientific Research (B) (grant no. 17H02094 to Y.Y.) and a Grant-in-Aid for Challenging Exploratory Research (grant no. 17K20076 to Y.Y.) from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), a grant from JST CREST (grant no. JPMJCR17H1 to M.M. and M.T.), a grant from the Special Education and Research Expenses of the MEXT, and a grant from the Tokyo Biochemical Research Foundation (to
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The authors Mitsue Hibino and Yuma Yamada equally contribute to this study.
This article contains supplementary material available from the authors by request or via the Internet at https://doi.org/10.1016/j.xphs.2019.04.001.