Nanovesicles derived from iron oxide nanoparticles-incorporated mesenchymal stem cells for cardiac repair

Abstract

Because of poor engraftment and safety concerns regarding mesenchymal stem cell (MSC) therapy, MSC-derived exosomes have emerged as an alternative cell-free therapy for myocardial infarction (MI). However, the diffusion of exosomes out of the infarcted heart following injection and the low productivity limit the potential of clinical applications. Here, we developed exosome-mimetic extracellular nanovesicles (NVs) derived from iron oxide nanoparticles (IONPs)-incorporated MSCs (IONP-MSCs). The retention of injected IONP-MSC-derived NVs (IONP-NVs) within the infarcted heart was markedly augmented by magnetic guidance. Furthermore, IONPs significantly increased the levels of therapeutic molecules in IONP-MSCs and IONP-NVs, which can reduce the concern of low exosome productivity. The injection of IONP-NVs into the infarcted heart and magnetic guidance induced an early shift from the inflammation phase to the reparative phase, reduced apoptosis and fibrosis, and enhanced angiogenesis and cardiac function recovery. This approach can enhance the therapeutic potency of an MSC-derived NV therapy.

Fig. 1. Cellular uptake of IONPs and IONP-triggered cellular modification.

(A) Schematic illustration and TEM image of IONPs. PEG, polyethylene glycol. (B) Fluorescent images of IONP (red) uptake by MSCs 24 hours after IONP treatment [green, cell membrane; and blue, 4′,6-diamidino-2-phenylindole (DAPI)]. Scale bars, 100 μm. (C) The cytotoxicity of IONPs 2, 4, 7, 10, and 14 days after IONP treatment to MSCs, as evaluated by CCK assay (n = 4 per group; NS, no significant difference). (D) Relative mRNA expression levels of various cardiac repair–favorable genes in MSC and IONP-MSC, as evaluated by qRT-PCR (n = 4 per group). (E) Schematic illustration of the IONP-mediated intracellular signaling cascades in MSCs and Western blot analysis for the intracellular signaling cascade molecules and associated therapeutic molecules (n = 3 per group). *P < 0.05 by two-tailed t test. All values are means ± SD.

Fig. 2. Preparation and characterization of MSC-derived NVs.

(A) (i) TEM images of N-NVs and IONP-NVs and (ii) confocal images of IONP-NVs. White arrows indicate IONPs within IONP-NVs. In the fluorescent confocal images, NV lipid layers and IONPs were labeled with DiO (green) and RITC (red), respectively. Scale bars, 1 μm (ii). (B) Representative size distribution of N-NVs and IONP-NVs derived from MSCs and IONP-MSCs, respectively, as evaluated by DLS. (C) Relative mRNA levels in N-NVs and IONP-NVs, as evaluated by qRT-PCR (n = 4 per group). (D) Representative images of Western blots of therapeutic molecules and the marker of EV, CD9 (n = 3 per group). (E) Fold changes in levels of cardiac repair–related microRNA (miRNA) in IONP-NVs compared to N-NVs. All columns represent log₂-fold changes. *P < 0.05 by two-tailed t test. All values are means ± SD.

Fig. 3. Therapeutic effects of IONP-NVs in vitro.

(A) Relative cell viability as evaluated by the CCK assay after treatment (n = 4 per group). (B) Antifibrotic effects of NVs and MSCs. Relative mRNA expression levels of cardiac myofibroblast–related genes in CFs after treatment, as evaluated by qRT-PCR (n = 4 per group). (C) Macrophage (MΦ) polarization after treatment. Relative mRNA expression levels of the markers of inflammatory M1 macrophages (Il1b, Il6, and Tnfa) and reparative M2 macrophages (Arg1, Il10, and Vegf) in lipopolysaccharide (LPS)–treated macrophages (M1 MΦ) after treatment, as evaluated by qRT-PCR (n = 4 per group). All data were normalized to normoxia or M0 MΦ data. (D and E) Capillary tube formation (n = 6 per group) and cell migration (n = 5 per group) of HUVECs after treatment. Red lines indicate borders of the cell-free area. Scale bars, 200 and 500 μm, respectively. NT indicates no treatment. *P < 0.05, using one-way analysis of variance (ANOVA), followed by post hoc Bonferroni test. All values are means ± SD.

Fig. 4. Retention of IONP-NVs in the infarcted myocardium.

Fluorescent dye–labeled IONP-MSCs and IONP-NVs were injected into the rat infarcted myocardium. In some groups, magnetic guidance (M) was applied above the hearts for 24 hours after injection. (A) Representative ex vivo fluorescent imaging of infarcted hearts and the quantitative data 24 hours after the injection of VivoTrack 680–labeled MSCs, IONP-MSCs, N-NVs, and IONP-NVs (n = 3 animals per group). VivoTrack 680 labeled the membranes of cells and NVs. *P < 0.05, using one-way ANOVA, followed by post hoc Bonferroni test. (B) Representative magnetic resonance images and the quantification of IONP within heart 24 hours after the injection of IONP-NVs (n = 3 animals per group). The yellow line indicates IONP retention area. *P < 0.05, using two-tailed t test. All values are means ± SD. ROI, region of interest.

Fig. 5. Attenuation of cell apoptosis and inflammation by IONP-NV injection in vivo.

(A) Representative images of TUNEL-positive (green) apoptotic cells and immunostaining for cardiac troponin T (cTnT) (CM marker) and the quantitative data 1 day after injection (n = 5 animals per group). Scale bars, 100 μm. (B) Representative images of immunohistochemical staining for Cx43 in the peri-infarct zone and the quantitative data 1 day after injection (n = 5 animals per group). Scale bars, 100 μm. (C) Relative mRNA expression levels of M2 macrophage–specific markers (Cd206, Arg1, and Il10) and M1 macrophage–specific markers (Nos2, Il1b, and Il6) in the infarcted myocardium, as evaluated by qRT-PCR (n = 5 animals per group). (D) Immunohistochemical staining for CD68 (macrophage marker), CD206 (M2 macrophage marker), and nitric oxide synthase 2 (NOS2) (M1 macrophage marker); and the quantitative data in the peri-infarct zone 1 day after injection (n = 5 animals per group). Scale bars, 50 μm. *P < 0.05, using one-way ANOVA, followed by post hoc Bonferroni test. All values are means ± SD.

Fig. 6. Enhanced blood vessel density, the improvement of left ventricular remodeling, and the promotion of cardiac function recovery.

(A) Representative images of functional blood vessels visualized by perfusion of isolectin B4 and immunostaining for cTnT (CM marker) in the infarct zone and border zone 4 weeks after injection and the quantitative data (n = 5 animals per group). Scale bars, 50 μm. (B) Representative Masson’s trichrome–stained infarcted hearts four weeks after injection (blue, scar tissue; red, viable myocardium). (C) The fibrosis area, scar size, viable myocardium area, and thickness of the left ventricular (LV) wall 4 weeks after injection (n = 5 animals per group). *P < 0.05, using one-way ANOVA, followed by post hoc Bonferroni test. (D) Representative M-mode images 1 and 4 weeks after treatment. Yellow arrows between wall motions indicate left ventricular dimension. Distance between yellow arrows and arrowheads in top and bottom area indicates left ventricular wall thickness and left ventricular posterior wall thickness, respectively. (E and F) Ejection fraction and fractional shortening at various time points (n = 5 animals per group). *P < 0.05 versus NT, #P < 0.05 versus IONP, †P < 0.05 versus N-NV, and ‡P < 0.05 versus IONP-NV, using two-way ANOVA, followed by post hoc Bonferroni test. (G and H) Left ventricular end diastolic dimension (LVIDd) and left ventricular end systolic dimension (LVIDs) at various time points (n = 5 animals per group). (I and J) Posterior wall thickness and septal wall thickness at various time points (n = 5 animals per group). *P < 0.05, using two-way ANOVA, followed by post hoc Bonferroni test. The group names in (E to J) are referred to Fig. 6 (C and D). All values are means ± SD.