2024 Volume 47 Issue 8 Pages 1415-1421
The efficacy of mesenchymal stem cell (MSC) transplantation has been reported for various diseases. We previously developed a drug delivery system targeting mitochondria (MITO-Porter) by using a microfluidic device to encapsulate Coenzyme Q10 (CoQ10) on a large scale. The current study aimed to confirm if treatment with CoQ10 encapsulated by MITO-Porter enhanced mitochondrial functions in MSCs, with the potential to improve MSC transplantation therapy. We used highly purified human bone marrow-derived MSCs, described as rapidly expanding clones (RECs), and attempted to control and increase the amount of CoQ10 encapsulated in the MITO-Porter using microfluidic device system. We treated these RECs with CoQ10 encapsulated MITO-Porter, and evaluated its cellular uptake, co-localization with mitochondria, changes in mitochondrial respiratory capacity, and cellular toxicity. There was no significant change in mitochondrial respiratory capacity following treatment with the previous CoQ10 encapsulated MITO-Porter; however, mitochondrial respiratory capacity in RECs was significantly increased by treatment with CoQ10-rich MITO-Porter. Utilization of a microfluidic device enabled the amount of CoQ10 encapsulated in MITO-Porter to be controlled, and treatment with CoQ10-rich MITO-Porter successfully activated mitochondrial functions in MSCs. The MITO-Porter system thus provides a promising tool to improve MSC cell transplantation therapy.
Mesenchymal stem cells (MSCs) are multipotent cells that are present in adult tissues and have the potential to differentiate into a various cell lineages, such as bone, cartilage, adipose tissue, tendon, and muscle.1,2) MSCs also have anti-inflammatory properties by interacting with immune cells via cytokine release.3,4) The efficacy of autologous and allogenic transplantation of MSCs has been reported in various diseases.5,6) Furthermore, the immunosuppressive characteristics of MSCs have been clinically applied and are commercially available for treating graft-versus-host disease.
We previously developed a drug delivery system targeting mitochondria (MITO-Porter), which enables the transport of diverse molecules to mitochondria through membrane fusion.7–10) By treating cardiac progenitor cells (CPCs) with MITO-Porter encapsulating resveratrol, as a mitochondrial functional molecule prepared by ethanol dilution method, we successfully created mitochondrial-activated CPCs, described as MITO cells.11) The preventive and therapeutic effects of MITO cell transplantation via intramyocardial injection were also demonstrated in mouse models of doxorubicin-cardiomyopathy and myocardial ischemia–reperfusion, respectively.11,12) Notably, however, the large-scale production of resveratrol encapsulated MITO-Porter by ethanol dilution method has proved challenging, potentially hindering its clinical application.
To overcome this issue, we prepared MITO-Porter using a microfluidic device system, which can form lipid nanoparticles within a microchannel formed on a substrate13); however, resveratrol encapsulated MITO-Porter could not be prepared efficiently using a microfluidic device because of its lipophilic nature and high membrane permeability. Conversely, the poorly water-soluble mitochondrial functional molecule Coenzyme Q10 (CoQ10) could be encapsulated within MITO-Porter (referred to as MP-(CoQ10)) using a microfluidic device,14,15) and could be prepared on a large scale. Furthermore, we successfully enhanced the mitochondrial respiratory capacity of human CPCs by treating them with MP-(CoQ10), and demonstrated its therapeutic efficacy in a myocardial ischemia–reperfusion mouse model via intravenous administration.16)
The human heart consumes large amounts of ATP to maintain its vital functions, and >90% of the energy production in the myocardium is attributed to oxidative metabolism in the mitochondria.17–19) In contrast, MSCs have traditionally been believed to rely primarily on glycolysis for their energy metabolism,20) and enhancing mitochondrial functions in MSCs may require the delivery of more CoQ10 to the mitochondria compared with CPCs.
This study aimed to confirm if treatment with CoQ10 encapsulated MITO-Porter enhanced mitochondrial functions in MSCs, with the potential to improve MSC transplantation therapy (Fig. 1). We used commercially available, highly purified, and clonogenic human bone marrow-derived MSCs, described as rapidly expanding clones (RECs).21–23) We treated these RECs with CoQ10 encapsulated MITO-Porter, and evaluated its cellular uptake, co-localization with mitochondria, changes in mitochondrial respiratory capacity, and cellular toxicity. Furthermore, we aimed to control and increase the amount of CoQ10 encapsulated in MITO-Porter through the utilization of a microfluidic device system (Fig. 1).
(A) The amount of CoQ10 encapsulated in MITO-Porter was controlled using a microfluidic device. By initially changing the concentration of CoQ10 suspension, we successfully prepared MITO-Porter with approximately two-fold (MP-(2× CoQ10)) or three-fold (MP-(3× CoQ10)) the amount of CoQ10 compared with the previous MP-(CoQ10). (B) Treatment with CoQ10 encapsulated MITO-Porter increased the mitochondrial respiratory capacity of rapidly expanding clones (RECs).
Commercially available RECs were obtained at passage 5 and cultured with Dulbecco’s modified Eagle’s medium (DMEM) with low-glucose-containing 20% fetal bovine serum, 1% 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, 1% penicillin streptomycin, and 0.1% basic fibroblast growth factor at 37 °C and 5% CO2, following the manufacturer’s guidelines (Wako Pure Chemical Corporation, Osaka, Japan). The medium was refreshed two or three times per week, and RECs from 7–11 passage processes in total were used in the experiments.
Preparation of MITO-PorterWe prepared MITO-Porter by using a microfluidic device system, as reported previously.14) We prepared MITO-Porter with approximately two-fold (MP-(2× CoQ10)) or three-fold (MP-(3× CoQ10)) the amount of encapsulated CoQ10 compared with the previous MP-(CoQ10) by initially changing the concentration of the CoQ10 suspension.
The particle properties of MITO-Porter were evaluated using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.). The estimated lipid content of the MITO-Porter was measured by determining the fluorescence intensities of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-labeled liposomes using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific Life Sciences, Waltham, MA, U.S.A.). The CoQ10 content in the MITO-Porter was assessed using HPLC using an Arc™ HPLC system, as reported previously.14)
Intracellular Dynamics of MITO-PorterCellular absorption of MITO-Porter was evaluated by quantifying the fluorescence intensity of DiI-labeled MITO-Porter internalized by RECs using flow cytometry (Fig. 2). Confocal laser scanning microscopy (CLSM) was used to analyze the intracellular localization of DiI-labeled MITO-Porter (green fluorescence) and mitochondria labeled with MitoTracker Deep Red (red fluorescence) in RECs (Fig. 3).
(A) Histogram showing fluorescence intensities of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) taken up by RECs after treatment with MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), or MP-(3× CoQ10) at a final lipid concentration of 20 µM compared with non-treatment (NT) group. (B) Cellular internalization expressed as mean fluorescence intensity. Data expressed as mean ± standard deviation (S.D.) (n = 3). Significant difference (* p < 0.05, ** p < 0.01) calculated by one-way ANOVA followed by Student–Newman–Keuls test.
Co-localization was visualized between mitochondria (red-fluorescence, A–E) and MITO-Porter (green-fluorescence, F–J), evidenced by yellow signals in the merged image (K–O). Scale bars, 50 µm.
The Seahorse XFp Analyzer (Agilent Technologies, Santa Clara, CA, U.S.A.), an extracellular flux analyzer, was used to assess the mitochondrial oxygen consumption rate (OCR). Basal and maximal OCR were assessed by the stepwise addition of oligomycin (a Complex V inhibitor), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (a mitochondrial oxidative phosphorylation uncoupler that maximizes mitochondrial respiratory capacity), followed by rotenone (a Complex I inhibitor) along with antimycin A (a Complex III inhibitor) (Supplementary Fig. S1).
Further methodological details and materials are summarized in Supplementary Materials.
In this study, we initially tried to enhance mitochondrial function in RECs by treating with MP-(CoQ10) for 24 h at a final lipid concentration of 50–60 µM as described previously, which successfully activated mitochondrial function in CPCs.16) However, this resulted in no significant changes in mitochondrial respiratory capacity evaluated by extracellular flux analyzer (Supplementary Fig. S2). These results suggested that it was more difficult to improve mitochondrial functions in RECs compared with CPCs, and that the previous MP-(CoQ10) might not improve mitochondrial function.
To enhance mitochondrial respiratory capacity in RECs, we therefore prepared MITO-Porter containing more CoQ10 using a microfluidic device system (Fig. 1). By changing the concentration of CoQ10 suspension, we successfully prepared MP-(2× CoQ10) and MP-(3× CoQ10). We also prepared MITO-Porter without CoQ10 (referred to as MP-(Empty)) as a negative control.
The average particle size, polydispersity index (PDI), a parameter indicating the distribution of particle sizes, and ζ-potential are shown in Table 1. The particle sizes of MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), and MP-(3× CoQ10) were 41 ± 2.7, 51 ± 7.2, 57 ± 4.4, and 58 ± 6.2 nm, respectively. As the amount of encapsulated CoQ10 increased, there was a tendency for the particle size to also increase. The MITO-Porter acquired positive charge due to the modification of the particle surface by Stearylated R8 (STR-R8). The PDI and ζ-potential values of MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), and MP-(3× CoQ10) were approximately 0.180–0.220 and 18–20 mV, respectively.
| Initial amount of CoQ10 for preparation (mM) | Size (nm) | PDI | ζ-Potential (mV) | Lipid recovery rate (%) | CoQ10 recovery rate (%) | Drug:lipid ratio (w:w) | |
|---|---|---|---|---|---|---|---|
| MP-(Empty) | — | 41 ± 2.7 | 0.182 ± 0.043 | 20 ± 1.4 | 79 ± 3.3 | — | — |
| MP-(CoQ10) | 0.7 | 51 ± 7.2 | 0.219 ± 0.013 | 20 ± 1.8 | 86 ± 7.1 | 75 ± 4.0 | 0.16 ± 0.01 |
| MP-(2× CoQ10) | 1.3 | 57 ± 4.4 | 0.202 ± 0.025 | 19 ± 0.6 | 78 ± 11.5 | 77 ± 1.3 | 0.37 ± 0.06 |
| MP-(3× CoQ10) | 2.2 | 58 ± 6.2 | 0.179 ± 0.021 | 18 ± 1.8 | 85 ± 9.2 | 80 ± 2.9 | 0.57 ± 0.07 |
MITO-Porter was constructed using an ethanol suspension containing 4.2 mM lipids (DOPE:SM:DMG-PEG2000:STR-R8 [9 : 2 : 0.33 : 1.1, molar ratio]). Average particle size, polydispersity index (PDI), ζ-potential, lipid and CoQ10 recovery rates, and drug:lipid ratio (w:w) represent properties of the constructed MITO-Porter. Data expressed as mean ± standard deviation (S.D.) (n = 4). DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; SM, sphingomyelin; DMG-PEG2000, 1,2-dimyristoyl-sn-glycerol-methoxy polyethylene glycol 2000; STR-R8, stearylated R8.
The recovery rates of the lipid and CoQ10 are presented in Table 1. The lipid recovery rates of MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), and MP-(3× CoQ10) were approximately 75–85% and the CoQ10 recovery rates of MP-(CoQ10), MP-(2× CoQ10), and MP-(3× CoQ10) were approximately 75–80%. The drug-to-lipid ratios (weight:weight) of MP-(CoQ10), MP-(2× CoQ10), and MP-(3× CoQ10) were 0.16 ± 0.01, 0.37 ± 0.06, and 0.57 ± 0.07, respectively. These results demonstrated that it was possible to control the amount of CoQ10 encapsulated in MITO-Porter by using a microfluidic device system, which might have positive implications for the modification of mitochondrial function in RECs.
Cellular Uptake Analysis by Flow CytometryThe internalization of MITO-Porter into cells was assessed by flow cytometry analysis. The fluorescence intensity resulting from the uptake of the MITO-Porter by RECs were measured after treating cells with MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), or MP-(3× CoQ10) labeled with the fluorescent lipid indicator DiI for 24 h at a final lipid concentration of 20 µM (Fig. 2).
Cellular uptakes of MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), and MP-(3× CoQ10) were significantly increased compared with the non-treatment (NT) group. Conversely, encapsulation of CoQ10 significantly reduced the intracellular uptake of MITO-Porter, and it further decreased with increasing content of encapsulated CoQ10. These results indicate that CoQ10 encapsulated MITO-Porter may be internalized by RECs, and its uptake is partially influenced by the presence of CoQ10.
Intracellular Observation by CLSMWe assessed the intracellular localization and dynamics of DiI-labeled MITO-Porter by detecting green fluorescence using CLSM. RECs, including mitochondria stained with red fluorescence using MitoTracker Deep Red, were treated with MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), or MP-(3× CoQ10) for 24 h at a final lipid concentration of 20 µM (Fig. 3).
The cellular internalizations of the different MITO-Porter preparations were visualized by the presence of green dots in RECs, which showed that all the carriers had the capability for cellular uptake. Additionally, co-localization was observed between red-fluorescent mitochondria and green-fluorescent MITO-Porter, demonstrated by the occurrence of yellow signals, indicating that a portion of the MITO-Porter was taken up by the mitochondria.
Evaluation of the Mitochondrial Respiratory Capacity in RECs by Extracellular Flux AnalysisMitochondrial respiratory capacity was assessed in RECs by assessing the mitochondrial OCR by extracellular flux analysis. We first conducted an experiment to verify the optimal quantity of MITO-Porter-encapsulated CoQ10 to enhance the mitochondrial respiratory capacity of RECs (Supplementary Fig. S3). The OCR was measured 24 h after treatment with MP-(CoQ10), MP-(2× CoQ10), or MP-(3× CoQ10) at a final lipid concentration of 20 µM. Treatment with MP-(2× CoQ10) resulted in the maximal OCR in RECs.
We then measured the OCR in non-treated RECs (NT group), RECs treated with MP-(Empty), and RECs treated with MP-(2× CoQ10) for 24 h with a final lipid concentration of 20 µM (Fig. 4A). There were no significant changes observed in basal OCR among any of the groups (Fig. 4B); however, a significant increase in the maximal OCR was observed in RECs treated with MP-(2× CoQ10) compared with the NT group and RECs treated with MP-(Empty) (Fig. 4C). These findings indicated that MP-(2× CoQ10) had the greatest effect on peak mitochondrial respiratory capacity in RECs, but had no effect on baseline capacity.
Oxygen consumption rate (OCR) was evaluated following treatment with phosphate-buffered saline (non-treatment (NT) group), MP-(Empty), or MP-(2× CoQ10) with a final lipid concentration of 20 µM (A). The OCRs relative to basal respiration (B) and maximal respiration (C) were calculated by normalizing to the OCR in the NT group. Maximal OCR was significantly increased in RECs treated with MP-(2× CoQ10) compared with the NT group and RECs treated with MP-(Empty). Data expressed as mean ± S.D. (n = 12). Significant difference (* p < 0.05) calculated by one-way ANOVA followed by Student–Newman–Keuls test.
We assessed the cytotoxicity of MITO-Porter by cell viability assessment employing the WST-1 reagent. The viabilities of RECs treated with MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), or MP-(3× CoQ10) for 24 h were compared with that of the NT (Fig. 5).
The cytotoxicities of MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), and MP-(3× CoQ10) were evaluated by cell viability testing using WST-1 reagent and measured by a Varioskan LUX multimode microplate reader. There was no significant difference in cell viability among the MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), and MP-(3× CoQ10) groups within a final lipid concentration of 10–40 µM. Data expressed as mean ± S.D. (n = 3). Significant difference calculated by one-way ANOVA followed by Student–Newman–Keuls test.
The cell viabilities (%) of the respective MITO-Porter groups were 108 ± 10.4, 95 ± 10.4, 103 ± 5.3, and 102 ± 5.2, respectively, at a final lipid concentration of 10 µM, 102 ± 13.4, 101 ± 7.6, 97 ± 2.2, and 104 ± 8.2, respectively, at a final lipid concentration of 20 µM, and 103 ± 8.2, 94 ± 11.0, 93 ± 4.0, and 91 ± 6.5, respectively, at a final lipid concentration of 40 µM. Cell viability (%) in the NT group was 100 ± 4.6. There was no significant difference in cell viability among the NT, MP-(Empty), MP-(CoQ10), MP-(2× CoQ10), and MP-(3× CoQ10) groups within a final lipid concentration 10–40 µM. These results suggest that CoQ10 encapsulated MITO-Porter did not affect the viability of RECs, even as the amount of encapsulated CoQ10 increased.
In this study, we used a microfluidic device system to formulate MITO-Porter encapsulating larger amounts of CoQ10 than previously used because the previous MP-(CoQ10) failed to enhance mitochondrial respiratory capacity in RECs. By changing the concentration of CoQ10 suspension before preparing the MITO-Porter, we successfully prepared CoQ10-rich MITO-Porter, which increased REC’s mitochondrial respiratory capacity.
We previously reported that treatment with MP-(CoQ10) increased mitochondrial respiratory capacity in human CPCs and rat skeletal muscles16,24); in contrast, however, the maximal OCR in RECs did not increase significantly following similar treatment with MP-(CoQ10) containing CoQ10 with a drug:lipid ratio of 0.16 ± 0.01,16) while treatment with MP-(2× CoQ10) containing CoQ10 with a drug : lipid ratio of 0.37 ± 0.06 significantly increased the maximal OCR in RECs. Furthermore, the increase in maximal OCR in RECs treated with MP-(2× CoQ10) was smaller than that in human CPCs treated with MP-(CoQ10).16) These results indicate that enhancing mitochondrial respiratory capacity in MSCs, as in this study, represents a challenging task. In fact, 40–50% of the energy production in RECs is attributed to glycolysis (Supplementary Fig. S4), which may constitute a higher percentage than in CPCs.17–19) The results of this study indicate that microfluidic device technology has the potential to control mitochondrial function by varying the amount of CoQ10 encapsulated in MITO-Porter, possibly in any cell type.
The increase in maximal OCR in RECs was higher with MP-(2× CoQ10) than MP-(3× CoQ10). The reason for this is unclear; however, it is possible that the delivery of CoQ10 may have exceeded the processing capacity of the RECs, resulting in the mitochondrial respiratory capacity reaching a plateau. Alternatively, although the cell viability remained unchanged according to WST-1 assay, excessive delivery of CoQ10 may be toxic to the mitochondria. A third possibility is that excessive encapsulation of CoQ10 in MITO-Porter may lead to structural changes in its composition, affecting CoQ10 release, resulting in decreased effect. Indeed, we previously reported that an increase in the molecules encapsulated in lipid nanoparticles may result in changes in their internal structure, potentially influencing their effects.25,26)
Flow cytometry analysis showed that the cellular internalization of MITO-Porter declined with the presence of encapsulated CoQ10 in RECs. A prior investigation indicated that the binding energy between the nanoparticle and the membrane increased as the nanoparticle’s minimum radius decreased.27,28) The increase in particle size due to the encapsulation of CoQ10 might have influenced the intracellular uptake in RECs. It has been reported that the stiffness of the particles influences intracellular uptake, and that lipid nanoparticles containing internal molecules become stiffer than empty ones.29) In this study, encapsulating CoQ10 in the MITO-Porter may have altered the particle stiffness, potentially affecting intracellular uptake in RECs.
Hypoxia-inducible factor-1α (HIF-1α), a promotor of glycolytic gene expression, was highly expressed in undifferentiated bone-marrow derived MSCs, while expression levels of the HIF-1α gene and glycolytic enzyme decreased during osteogenic or adipogenic differentiation in bone-marrow derived MSCs.20,30,31) The differentiation of MSCs is thus assumed to be linked to decreased glycolysis and increased mitochondrial function.20) Enhancement of mitochondrial function by treatment with CoQ10 encapsulated MITO-Porter may thus affect MSC differentiation, which may have a positive impact on MSC cell transplantation therapy. It has been reported that hypoxic condition, which is the physiological environment of MSCs, may promote MSC proliferation, survival, and migration.32,33) On the other hand, the transplantation site may not be a hypoxic environment, as represented by myocardium.18) In our previous study, we demonstrated that treatment with CoQ10 encapsulated MITO-Porter to CPCs can improve their antioxidant capacity. Therefore, even if the increase in mitochondrial function resulting from treatment of MSCs with CoQ10 encapsulated MITO-Porter to MSCs was minimal or absent, replenishing cells with the antioxidant CoQ10 may still aid cell survival, especially when the cellular transplantation destination is not hypoxic environment. We need further study regarding this in vitro and in vivo.
In conclusion, utilization of a microfluidic device enabled the amount of CoQ10 encapsulated in MITO-Porter to be controlled, and treatment with CoQ10-rich MITO-Porter successfully activated mitochondrial respiratory capacity in MSCs. This technology leveraging the MITO-Porter system offers a promising tool to control mitochondrial function and improve MSC cell transplantation therapy.
This work was supported, in part, by a Grant-in-Aid for Scientific Research (A) (Grant number 23H00541 to Y.Y.) from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), JST FOREST (Grant number JPMJFR203X to Y.Y.) and JST SPRING (Grant number JPMJSP2119 to Y.M. and M.S.) from the Japan Science and Technology Agency (JST), a Grant from the Takeda Science Foundation (to Y.Y.), and AMED under Grant Number JP24ym0126801 (to Y.Y.). We are grateful to Dr. Manabu Tokeshi and Dr. Masatoshi Maeki for providing the microfluidic device (iLiNP device).
Conceptualization, Y.Y.; methodology, Y.M., M.S., M.H. and J.A.; validation, Y.M. and Y.Y.; formal analysis, Y.M. and Y.Y.; investigation, Y.M.; writing original draft presentation, Y.M.; writing review and editing, Y.Y.; supervision, A.T. and Y.Y.; project administration, Y.Y.; funding acquisition, Y.M., M.S. and Y.Y. All authors have read and agreed to the published version of the manuscript.
The authors declare no conflict of interest.
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