Gelated microvesicle-mediated delivery of mesenchymal stem cell mitochondria for the treatment of myocardial infarction

Abstract

Mitochondrial dysfunction is closely linked to cardiomyocyte injury following myocardial infarction (MI). While mitochondrial transplantation is a promising therapeutic strategy, challenges remain in maintaining mitochondrial structural integrity, enhancing delivery efficiency, and increasing the mitochondrial supply. Herein, we developed a gelated microvesicle-based mitochondria delivery system (Mito@Microgels) for transplanting mesenchymal stem cell mitochondria, addressing the aforementioned issues. Further decoration of phosphatidylserine on the surface of Mito@Microgels boosted cellular uptake efficiency by cardiomyocytes. These Mito@Microgels effectively deliver active mitochondria to cardiomyocytes, improving the mitochondrial network architecture and function and consequently reducing the cellular injury induced by oxidative stress. Moreover, Mito@Microgels attenuated the inflammatory phenotype of macrophages, helping resolve excessive local inflammation. In vivo animal studies using a rat MI model further validated the therapeutic efficacy of the Mito@Microgels, as evidenced by improved myocardial function, prevention of infarcted left ventricular wall thinning, and increased cardiomyocyte survival. Our study introduces an efficient mitochondrial delivery strategy with significant potential for cardiac repair post-MI and other mitochondria-related diseases.

Keywords: gelated microvesicle; mitochondrial delivery; myocardial infarction; myocardiocyte rescue.

Fig. 1.

Schematic illustration of the preparation of Mito@Microgels and their role in mitigating myocardial injury post-MI.

Fig. 2.

Preparation and characterization of Mito@Microgels. (A) Schematic illustration showing the preparation of Mito@Microgels. (B) Representative fluorescence images showing microgels retain structural integrity in PBS upon Triton X-100 treatment. The GelMA hydrogel was stained with FITC (green), and liposome membranes were stained with DiI (red). (C) The particle sizes of microgels of various sizes analyzed using flow cytometry (FC). (D) The zeta potential of microgels of various sizes analyzed via dynamic light scattering. (E) Representative fluorescence images showing microgels of various sizes being taken up by H9C2 cells. The microgels were fluorescently labeled with DiI (red), while F-actin was stained with phalloidin (green), and the nuclei were counterstained with DAPI (blue). (F) The uptake efficiency of microgels of various sizes analyzed using FC. (G) Representative fluorescence images showing the successful loading of mitochondria within the Mito@Microgels. The microgels were fluorescence-stained with FITC (green), and the mitochondria were stained with TMRM (red). (H) The mitochondrial loading rates of Mito@Microgels of various sizes analyzed using FC. (I) Representative transmission electron microscopy image showing the successful loading of intact mitochondria within 1 μm Mito@Microgels. The mitochondrial loaded in Mito@Microgels was marked with white arrows. (All data are presented as the mean ± SD. *P < 0.05, **P < 0.01.)

Fig. 3.

Alleviation of oxidative-induced cellular injury in NRCMs by Mito@Microgels. (A) Representative fluorescence images using an HIS-SIM microscope showing the delivery of MSC mitochondria to NRCMs by Mito@Microgels. The mitochondria of NRCMs were fluorescently labeled with PK Mito Red (red), the mitochondria within the Mito-Microgels were labeled with Mito-tracker Deep Red FM (cyan), and the autophagosomes were stained with an Autophagy Staining Assay Kit with MDC (green), with nuclei counterstained with Hoechst 33342 (blue). ① Mitochondria from Mito@Microgels colocalized and fused with NRCM mitochondria, ② mitochondria from Mito@Microgels colocalized with autophagosomes, ③ mitochondria from Mito@Microgels partially integrated into NRCM mitochondria network. (B) Representative fluorescence images showing the architecture of the mitochondrial network in NRCMs via Mito-Tracker Green staining. (C–E) The mitochondrial architecture analyzed via the ImageJ plug-in MINA2.0. (F) Representative fluorescence images showing the MMP of NRCMs via TMRM staining. (G) The average fluorescence intensity of TMRM in individual cells analyzed via ImageJ. (H) Intracellular ATP production of NRCMs measured by chemiluminescence. (I) The ADP/ATP ratio of NRCMs measured by chemiluminescence. (J) Representative fluorescence images showing the intracellular ROS levels in NRCMs detected by the ROS probe DCFH-DA. (K) The average fluorescence intensity of DCFH-DA in individual cells analyzed via ImageJ. (L) Representative immunofluorescence images showing the expression of Cx43 (green) and α-Actinin (red) in NRCMs. (M and N) The positive area ratio of Cx43 and α-Actinin analyzed via ImageJ. (O and P) Spontaneous calcium transients (O) and extracted frequency signals (P) in NRCMs. (All data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 vs. H₂O₂.)

Fig. 4.

Suppression of the inflammatory macrophage phenotype by Mito@Microgels. (A) Representative fluorescence images using an HIS-SIM microscope showing the delivery of MSC mitochondria to macrophages by Mito@Microgels. The mitochondria of macrophages were fluorescently labeled with PK Mito Red (red), the mitochondria within the Mito-Microgels were labeled with Mito-tracker Deep Red FM (cyan), and the autophagosomes were stained with an Autophagy Staining Assay Kit with MDC (green), with nuclei counterstained with Hoechst 33342 (blue). ① Mitochondria from Mito@Microgels colocalized and fused with macrophage mitochondria, ② mitochondria from Mito@Microgels partially integrated into macrophage mitochondria network, ③ mitochondria from Mito@Microgels colocalized with autophagosomes. (B) The percentages of CD86⁺ and CD206⁺ macrophages analyzed via FC. (C) Representative fluorescence images showing iNOS and ARG1 expression in macrophages via immunofluorescence staining. (D and E) The average fluorescence intensities of iNOS and ARG1 in individual cells analyzed via ImageJ. (All data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. M0, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 vs. M1.)

Fig. 5.

Surface modification of Mito@Microgels with PS enhances their uptake by NRCMs. (A) Schematic illustration showing PS modification on the lipid membrane of Mito@Microgels via hydrophobic insertion. (B) Representative fluorescence images showing PS modification of Mito@Microgels-PS via Annexin V staining. (C) Representative fluorescence images showing that PS modification improved the uptake efficiency by NRCMs. The Mito@Microgels and Mito@Microgels-PS were labeled with DiI (red), F-actin was stained with phalloidin (green), and the nuclei were stained with DAPI (blue). (D) Uptake efficiency of the Mito@Microgels and Mito@Microgels-PS analyzed via FC. (E) Representative fluorescence images showing the retention of Mito@Microgels and Mito@Microgels-PS in myocardial tissue of rats postinjection following MI. (All data are presented as the mean ± SD. *P < 0.05.)

Fig. 6.

Mito@Microgels mitigate myocardial damage in MI model rats. (A) A timeline illustrating the assessment of myocardial damage in MI model rats. (B) Representative echocardiographic M-mode, Vevo Strain, motion heatmap, and selected point motion curve images showing the LV structure and wall motion. (C) Assessment of LVEF and LVFS via Vevo Lab software. (D) Representative images showing the myocardial fibrosis via Masson’s staining. Collagen was stained blue, whereas muscle tissue appeared red. (E) The LV wall thickness in 12 segments analyzed by ImageJ individually. (Sham group, n = 5; MI group, n = 5; Microgel group, n = 5; Mito@Microgel group, n = 5; Mito@Microgel-PS group, n = 5. All data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Sham, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 vs. MI.)

Fig. 7.

Immunofluorescence assessment of tissue sections from the central infarct regions of hearts from MI model rats. (A) Representative fluorescence images showing cardiomyocyte profiles via WGA staining, with cardiomyocyte size measured via ImageJ. (B) Representative fluorescence images showing the expression of Cx43 (green) and α-Actinin (red), with the positive ratio analyzed via ImageJ. (C) Representative fluorescence images showing blood vessels costained with CD 31 (green) and α-SMA (red), with quantitative analysis of vessel density via ImageJ. (D) Representative fluorescence images showing macrophage infiltration. The macrophage subsets were analyzed by labeling them with the universal macrophage marker CD68 (cyan), the M1 macrophage marker CD86 (red), and the M2 macrophage marker CD206 (green). (Sham group, n = 5; MI group, n = 5; Microgel group, n = 5; Mito@Microgel group, n = 5; Mito@Microgel-PS group, n = 5. All data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Sham, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 vs. MI.)

Fig. 8.

Comparison of the DEPs reveals the therapeutic mechanism of the Mito@Microgels in the MI rat model. (A) The first (PC1) and second (PC2) PCA of the protein expression levels showing complete separation among all groups. (B) Heatmap representation showcasing the DEPs involved in myocardial energetics and metabolism regulation. The major fatty acid transporters, β-oxidation enzymes, and enzymes involved in the regulation of glycolysis, gluconeogenesis, the TCA cycle, and mitochondrial energy metabolism are shown. (C) Heatmap representation showcasing the DEPs involved in mitochondrial organization and function. Major mitochondrial dynamics-related proteins, mitochondrial membrane proteins, mitochondrial respiratory chain proteins, and mitochondrial calcium ion homeostasis-regulated proteins are shown. (D) Gene set enrichment analysis (GSEA) of target pathways of MI model rats. (Sham group, n = 3; MI group, n = 3; Microgel group, n = 3; Mito@Microgel group, n = 3; Mito@Microgel-PS group, n = 3.)