Engineering exosomes derived from subcutaneous fat MSCs specially promote cartilage repair as miR-199a-3p delivery vehicles in Osteoarthritis

Abstract

Osteoarthritis (OA) is a degenerative joint disease involving cartilage. Exosomes derived from Mesenchymal stem cells (MSCs) therapy improves articular cartilage repair, but subcutaneous fat (SC) stromal cells derived exosomes (MSCs^SC-Exos), especially engineering MSCs^SC-Exos for drug delivery have been rarely reported in OA therapy. This objective of this study was to clarify the underlying mechanism of MSCs^SC-Exos on cartilage repair and therapy of engineering MSCs^SC-Exos for drug delivery in OA. MSCs^SC-Exos could ameliorate the pathological severity degree of cartilage via miR-199a-3p, a novel molecular highly enriched in MSCs^SC-Exos, which could mediate the mTOR-autophagy pathway in OA rat model. Intra-articular injection of antagomiR-199a-3p dramatically attenuated the protective effect of MSCs^SC-Exos-mediated on articular cartilage in vivo. Furthermore, to achieve the superior therapeutic effects of MSCs^SC-Exos on injured cartilage, engineering exosomes derived from MSCs^SC as the chondrocyte-targeting miR-199a-3p delivery vehicles were investigated in vitro and in vivo. The chondrocyte-binding peptide (CAP) binding MSCs^SC-Exos could particularly deliver miR-199a-3p into the chondrocytes in vitro and into deep articular tissues in vivo, then exert the excellent protective effect on injured cartilage in DMM-induced OA mice. As it is feasible to obtain human subcutaneous fat from healthy donors by liposuction operation in clinic, meanwhile engineering MSCs^SC-Exos to realize targeted delivery of miR-199a-3p into chondrocytes exerted excellent therapeutic effects in OA animal model in vivo. Through combining MSCs^SC-Exos therapy and miRNA therapy via an engineering approach, we develop an efficient MSCs^SC-Exos-based strategy for OA therapy and promote the application of targeted-MSCs^SC-Exos for drug delivery in the future.

Keywords: Autophagy; Cartilage-targeted; Engineering exosomes; Osteoarthritis; miR-199a-3p.

Fig. 1

Identification of MSCs^SC and MSCs^SC-Exos. A Flow cytometry analysis of MSCs^SC markers in P4-ADSCs, the solid red curves represent measured surface markers and the solid blue curves represent isotype controls. B Schematic illustration of MSCs^SC-Exos isolation using differential centrifugation. C Transmission electron microscopy (TEM) analysis for the morphology of MSCs^SC-Exos (Scale bar: 100 nm). D Nanoparticle tracking analysis (NTA) for measuring the size distribution and concentration of MSCs^SC-Exos. E Western blot analysis of protein markers CD9, CD81, Alix, Tsg101, and Calnexin in MSCs^SC-Exos and MSCs^SC

Fig. 2

MSCs^SC-Exos treatment repairs the damaged articular cartilage in OA rat model. A Representative image of DiO-labelled MSCs^SC-Exos were uptaken by the damaged cartilage. White dotted lines indicate articular surface and the green dots indicate DiO-labelled MSCs^SC-Exos. Scale bar: 100 μm. B Schematic diagram of MSCs^SC-Exos treatment strategy in OA rats. C Safranin O/fast green staining of cartilage morphology in each group rats, scale bar: 500 μm (up) and 100 μm (down). D The OARSI scoring system quantify the severity of cartilage destruction. n = 6 for each group. E and F The immunofluorescence analysis of COL2A1 and MMP-13 in articular cartilage of OA rat model. n = 6 for each group. Scale bar: 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ns not significant

Fig. 3

The autophagy level was up-regulated in OA rats treated with MSCs^SC-Exos. A RNA-Seq analysis was conducted in total cartilage mRNA of OA rats, which was extracted from control group (PBS-treated, n = 2) and MSCs^SC-Exos group (Exos^SC-treated, n = 2). Volcanic map of different expression genes (DEGs) between control group and Exos^SC group. Red spots represent up-regulated genes and blue spots represent down-regulated genes. B KEGG enrichment for control group and Exos^SC group. C GSEA enrichment analysis of control group and Exos^SC group. D The immunofluorescence analysis of LC3B in articular cartilage of OA rat model. n = 6 for each group. scale bar: 100 μm. E Representative image of DiO-labelled Exos^SC absorbed by rat chondrocytes. Scale bar: 20 μm. F and G The immunofluorescence analysis of COL2A1 and MMP-13 in chondrocytes of OA model. n = 4 for each group. Scale bar: 10 μm. H Rat chondrocytes were infected with RFP-GFP-LC3 adenovirus and effects of Exos^SC on RFP-GFP- LC3 puncta. The numbers of RFP- and GFP-LC3 dots per cell were counted. n = 4 for each group. Scale bar: 10 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ns not significant

Fig. 4

MSCs^SC-Exos up regulated the autophagy process via inhibiting the mTOR expression. A Heatmap showing the hierarchical cluster of differential expression genes in OA cartilage treated with MSCs^SC-Exos or PBS. B Relative expression levels of the autophagy-related genes in primary chondrocytes treated with IL-1β and MSCs^SC-Exos by qRT-PCR. C, D The immunofluorescence analysis of mTOR in articular cartilage of OA rat model. n = 6 for each group. Scale bar: 100 μm. E Protein levels of mTOR and P-p70s6 were examined in chondrocytes treated with IL-1β and MSCs^SC-Exos by western blotting. F Protein level of mTOR was examined by western blotting in chondrocytes treated with IL-1β, MSCs^SC-Exos and MHY1485. G and H The immunofluorescence analysis of COL2A1 and MMP-13 in chondrocytes of OA chondrocytes treated with MHY1485. n = 4 for each group. Scale bar: 10 μm. I Rat chondrocytes were infected with RFP-GFP-LC3 adenovirus and effects of MHY1485 on RFP-GFP- LC3 puncta. The numbers of RFP- and GFP-LC3 dots per cell were counted. n = 4 for each group. Scale bar: 10 μm. J Protein levels of P-p70s6 were examined in articular cartilage in OA rat model by western blotting. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ns not significant

Fig. 5

miR-199a-3p transferred by MSCs^SC-Exos decrease the expression of mTOR in OA model. A Heatmap showing the hierarchical cluster of differential miRNA expression in MSCs^SC-Exos. B The relative expression of miR-199a-3p on OA cartilage with MSCs^SC-Exos treatment was detected by RT-PCR. C The relative expression of miR-199a-3p on chondrocytes with MSCs^SC-Exos treatment was detected by qRT-PCR. D The effects of MSCs^SC-Exos and antagomir-199a-3p on the luciferase activity of the IL-1β-induced chondrocytes. E Schematic representation of a predicted binding site of miR-199a-3p in the 3ʹUTR of mTOR mRNA, and the mutant mTOR 3ʹUTR. F The luciferase activity was determined using the dual-luciferase reporter system. G Protein levels of mTOR and P-p70s6 were examined by western blotting. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ns not significant

Fig. 6

Antagomir-199a-3p abrogates MSCs^SC-Exos therapeutic effect on damaged cartilage in OA model. A Schematic diagram of antagomiR-199a-3p and MSCs^SC-Exos treatment strategy in OA rats. B Safranin O/fast green staining of cartilage morphology in each group rats, scale bar: 500 μm (up) and 100 μm (down). C The OARSI scoring system quantify the severity of cartilage destruction. n = 6 for each group. D and E The immunofluorescence analysis of COL2A1 and MMP-13 in articular cartilage in OA rat model. n = 6 for each group. Scale bar: 100 μm. F and G The immunofluorescence analysis of mTOR and LC3B in articular cartilage in OA rat model. n = 6 for each group. Scale bar: 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ns not significant

Fig. 7

A Schematic diagram of the plasmid constructs containing Lamp2b, CAP-EGFP-Lamp2b and CAP-Lamp2b, and the engineering exosomes derived from MSCs^SC. B Transmission electron microscopy (TEM) analysis for the morphology of CAP-MSCs^SC-Exos (Scale bar: 100 nm). C Nanoparticle tracking analysis (NTA) for measuring the size distribution and concentration of CAP-MSCs^SC-Exos. D Western blot analysis of protein markers CD9, CD81, Alix, Tsg101, and Calnexin in whole cell lysates (WCLs) and purified exosomes including CAP-MSCs^SC-Exos and MSCs^SC-Exos. E Schematic illustration of rat chondrocytes and synovial cells co-culture assays. F Efficient delivery of miR-199a-3p into chondrocytes by CAP-EGFP-MSCs^SC-Exos. Scale bar: 10 μm. G Specific delivery to chondrocytes instead of synovial cells by CAP-EGFP-MSCs^SC-Exos. Scale bar: 10 μm. H The relative expression of miR-199a-3p in chondrocytes treated with different MSCs^SC-Exos delivery systems was detected by RT-PCR. I The immunofluorescence analysis of COL2A1 and MMP-13 in chondrocytes of OA model treated with different MSCs^SC-Exos delivery systems. n = 6 for each group. Scale bar: 10 μm. J Rat chondrocytes were infected with RFP-GFP-LC3 adenovirus and effects of different MSCs^SC-Exos delivery systems on RFP-GFP- LC3 puncta. The numbers of RFP- and GFP-LC3 dots per cell were counted. n = 6 for each group. Scale bar: 10 μm. K Protein level of mTOR in chondrocytes of OA model treated with different MSCs^SC-Exos delivery systems was examined by western blotting. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ns not significant

Fig. 8

A Schematic illustration of the animal experimental procedure. MSCs^SC-Exos or CAP-MSCs^SC-Exos loading with FAM-miR-199a-3p were intra-articular injected into DMM-induced mice, and the joint samples were collected after 48 h. B Fluorescent images of cartilage tissues treated with MSCs^SC-Exos or CAP-MSCs^SC-Exos loading with FAM-miR-199a-3p. scale bar: 5 μm. C Schematic diagram of different MSCs^SC-Exos delivery systems treatment strategy in DMM-induced OA mice. D The relative expression of miR-199a-3p in cartilage tissues treated with different MSCs^SC-Exos delivery systems was detected by RT-PCR in DMM-induced OA mice. E Protein level of mTOR in cartilage tissues treated with different MSCs^SC-Exos delivery systems was examined by western blotting in DMM-induced OA mice. F Safranin O/fast green staining of cartilage morphology in each group mice, scale bar: 500 μm (up) and 100 μm (down).G The OARSI scoring system quantify the severity of cartilage destruction. n = 6 for each group. H and I The immunohistochemistry analysis of COL2A1 and MMP13 in articular cartilage of OA mice model. n = 6 for each group. Scale bar: 50 μm. J Transmission electron microscopy (TEM) image of autophagic vesicles in cartilage of OA mice. The white arrow indicates the cell bilayer membrane structure of autophagic vesicles. Scale bar: 0.5 μm. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ns not significant