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. 2023 Aug 4:30:169-183.
doi: 10.1016/j.bioactmat.2023.06.012. eCollection 2023 Dec.

Dual-engineered cartilage-targeting extracellular vesicles derived from mesenchymal stem cells enhance osteoarthritis treatment via miR-223/NLRP3/pyroptosis axis: Toward a precision therapy

Weixuan Liu  1   2 , Anqi Liu  3   4 , Xujun Li  5 , Ziyang Sun  2   6 , Zhenghua Sun  2 , Yaru Liu  2 , Gang Wang  2 , Dan Huang  2 , Hao Xiong  2   6 , Shiyang Yu  2   6 , Xintao Zhang  1 , Cunyi Fan  2   6
Affiliations

Dual-engineered cartilage-targeting extracellular vesicles derived from mesenchymal stem cells enhance osteoarthritis treatment via miR-223/NLRP3/pyroptosis axis: Toward a precision therapy

Weixuan Liu et al. Bioact Mater. .

Abstract

Osteoarthritis (OA) is the most common disabling joint disease with no effective disease modifying drugs. Extracellular vesicles released by several types of mesenchymal stem cells could promote cartilage repair and ameliorate OA pathology in animal models, representing a novel therapeutic strategy. In this study, we demonstrated that extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hUC-EVs) could maintain chondrocyte homeostasis and alleviate OA, and further revealed a novel molecular mechanism of this therapeutic effect. miR-223, which could directly bind with the 3'UTR of NLRP3 mRNA, was found to be a key miRNA for hUC-EVs to exert beneficial effects on inflammation inhibiting and cartilage protecting. For enhancing the effect on mitigating osteoarthritis, exogenous miR-223 was loaded into hUC-EVs by electroporation, and a collagen II-targeting peptide (WYRGRL) was modified onto the surface of hUC-EVs by genetic engineering to achieve a more targeted and efficient RNA delivery to the cartilage. The dual-engineered EVs showed a maximal effect on inhibiting the NLRP3 inflammasome activation and chondrocyte pyroptosis, and offered excellent results for the treatment of OA. This study provides a novel theoretical basis and a promising therapeutic strategy for the application of engineered extracellular vesicles in OA treatment.

Keywords: Cartilage-targeting; Extracellular vesicles; NLRP3 inflammasome; Osteoarthritis; Pyroptosis.

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Figures

Image 1
Graphical abstract
Scheme 1
Scheme 1
A schematic overview of the study.
Fig. 1
Fig. 1
Identification and characterization of hUCMSCs and hUC-EVs (A) hUCMSCs displayed a representative spindle-like morphology. Scale bar: 100 μm. (B) Osteogenesis, adipogenesis, and chondrogenesis were all capable of being induced in hUCMSCs. Alizarin Red S staining, scale bar: 100 μm. Oil Red O staining, scale bar: 100 μm. Alcian Blue staining, scale bar: 25 μm. (C) Flow cytometric analysis of mesenchymal positive markers (CD73, CD90) and negative markers (CD34, CD45). Isotype controls are represented by blue histograms, and detected markers are represented by red histograms. (D) Morphology of hUC-EVs observed by transmission electron microscopy (TEM). Scale bar: 100 nm. (E) Particle size distribution of hUC-EVs measured by nanoparticle tracking analysis (NTA). (F) Exosome markers (CD81, Alix, and Tsg101) and the endoplasmic reticulum protein Calnexin measured by western blotting. These experiments were repeated three times independently, and representative results are shown.
Fig. 2
Fig. 2
HUC-EVs promote cell survival and anabolism of IL-1β-induced chondrocytes. (A) Schematic of in vitro experiments. (B) Cellular uptake of DiO-labelled hUC-EVs by chondrocytes observed using confocal micrograph, scale bar: 10 μm. (C) Cell viability of chondrocytes measured using CCK-8 assays. (D–E) Protein levels of Collagen II, SOX9, and MMP13 in chondrocytes detected by western blotting. The color blocks correspond to the groups. (F–H) Collagen II and Aggrecan expressions in chondrocytes measured by immunofluorescence, scale bar: 50 μm. MFI: mean fluorescence intensity. ***P < 0.001. These experiments were repeated three times independently, and representative results are shown.
Fig. 3
Fig. 3
HUC-EVs alleviate the inflammation and cartilage degeneration in OA rat model (A) Schematic diagram of animal experiments. (B) ELISA analysis of pro-inflammatory factors (MMP-2, PGE2, IL-1β, and TNF-α) in OA models. (C) qRT-PCR analysis of anabolic factors (Collagen II, SOX9), and catabolic factor (MMP13) in cartilage samples. (D) H&E, Safranin O/Fast Green, and immunohistochemical staining of knee joint sections. Scale bar: 100 μm. Cartilage degradation evaluated by (E) the OARSI scoring system and (F) Mankin's score. (G) Semi-quantitative analysis of Collagen II expression in immunohistochemical staining. n = 3 for each group. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the PBS group.
Fig. 4
Fig. 4
miR-223 mediates the inhibitory effect of hUC-EVs on NLRP3 (A) Volcano plot illustrating miR expression of OA rat knee joint cartilage treated with or without hUC-EVs. Red dots, up-regulated miRs, blue dots, down-regulated miRs, gray dots, miRs with no significant difference. (B) Heat map of miRNA sequencing (miRNA-seq) analysis. Red arrow: miR-223. (Red: high expression, blue: low expression, n = 3 per group) (C) qRT-PCR analysis of miR-223 (n = 3 per group). (D) Binding seq of NLRP3 and miR-223. (E) Dual-luciferase reporter gene assay validating the interaction between NLRP3 and miR-223. (F) Protein levels of NLRP3 examined by western blotting. **P < 0.01. These experiments were repeated three times independently, and representative results are shown.
Fig. 5
Fig. 5
CTP- and Mir-engineering of hUC-EVs (A) Schematic diagram of EVs engineering (CTP modifying and miR-223 loading). (B) Schematic of the plasmid constructs. SP, signal peptide; TM, transmembrane domain; CT, C terminus. (C) Overexpression of EGFP-lamp2b or CTP-EGFP-lamp2b in hUCMSCs observed using fluorescence imaging. Scale bar = 50 μm. (D) Cartilage-targeting ability of CTP-modified EVs in rat knees observed using fluorescence imaging. Scale bar = 100 μm. These experiments were repeated three times independently, and representative results are shown.
Fig. 6
Fig. 6
CTP- and Mir-engineering both amplify the effect of hUC-EVs on promoting survival and improving anabolism of chondrocytes via miR-223/NLRP3/pyroptosis axis (A) Schematic and (B) grouping of in vitro experiments, the color blocks correspond to the groups throughout this figure. (C) Cell viability of chondrocytes measured by CCK-8 assays. (D) LDH activity analysis of chondrocyte supernatant by its assay kit. (E–F) Protein expression levels of Collagen II, SOX9, MMP13, NLRP3 inflammasome-related molecules (NLRP3, cleaved caspase-1, and GSDMD-N) in chondrocytes measured by western blotting and the grayscale scanning analysis. (G–L) Detection of Collagen II, Aggrecan, MMP13, NLRP3, and GSDMD-N expressions in chondrocytes by immunofluorescence. Scale bar: 50 μm. MFI: mean fluorescence intensity. *P < 0.05, **P < 0.01, ***P < 0.001 compared with IL-1β group. #P < 0.05 compared with Mir-EVs group. &P < 0.05 compared with CTP-EVs group. %P < 0.05 compared with CTP/Mir-EVs group. These experiments were repeated three times independently, and representative results are shown.
Fig. 7
Fig. 7
Dual-engineered CTP/Mir-EVs further promote cartilage repair, inhibit inflammation and ameliorate the severity of OA. (A) Immunofluorescence analysis of NLRP3 and GSDMD-N expressions in sections of knee joints. Scale bar: 50 μm. (B) ELISA analysis of pro-inflammatory factors in OA models, including IL-1β, MMP-2, TNF-α, and PGE2. (C) H&E, Safranin O/Fast Green, and IHC staining (Collagen II) of knee joint sections. Scale bar: 100 μm. Cartilage degradation evaluated by (D) the OARSI scoring system and (E) Mankin's score. (F) Semi-quantitative analysis of Collagen II expression in IHC staining. n = 3 for each group. *P < 0.05, ***P < 0.001 compared with PBS group. #P < 0.05 compared with Mir-EVs group. &P < 0.05 compared with CTP-EVs group.
Fig. 8
Fig. 8
Micro-CT demonstrating engineered CTP/Mir-EVs significantly delay the progression of knee OA. (A) Sectional micro-CT images showing the knee joint in an anterior-posterior and lateral view. (B) Three-dimensional reconstruction of the knee joint. (C) The relative joint space widths measured from anterior-posterior images. (D) The relative joint space widths measured from lateral images. (E) The relative bone volume fraction (BV/TV) measured from three-dimensional reconstruction. n = 3 for each group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with PBS group. #P < 0.05 compared with hUC-EVs group.
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