Injectable photocrosslinking spherical hydrogel-encapsulated targeting peptide-modified engineered exosomes for osteoarthritis therapy

Abstract

Osteoarthritis (OA) is a common degenerative joint disease urgently needing effective treatments. Bone marrow mesenchymal stromal cell-derived exosomes (Exo) are considered good drug carriers whereas they have limitations such as fast clearance and low retention. This study aimed to overcome the limitations of Exo in drug delivery using multiple strategies. Novel photocrosslinking spherical gelatin methacryloyl hydrogel (GelMA)-encapsulated cartilage affinity WYRGRL (W) peptide-modified engineered Exo were developed for OA treatment and the performance of the engineered Exo (W-Exo@GelMA) loaded with a small inhibitor LRRK2-IN-1 (W-Exo-L@GelMA) was investigated in vitro and in vivo. The W-Exo-L@GelMA showed an effective targeting effect on chondrocytes and a pronounced action on suppressing catabolism and promoting anabolism in vitro. Moreover, W-Exo-L@GelMA remarkably inhibited OA-related inflammation and immune gene expression, rescuing the IL-1β-induced transcriptomic responses. With enhanced retention in the joint, W-Exo-L@GelMA demonstrated superior anti-OA activity and cartilage repair ability in the OA murine model. The therapeutic effect was validated in the cultured human OA cartilage. In conclusion, photocrosslinking spherical hydrogel-encapsulated targeting peptide-modified engineered Exo exhibit notable potential in OA therapy. Engineering Exo by a series of strategies enhanced the targeting ability and retention and cartilage-targeting and Exo-mediated drug delivery may offer a novel strategy for OA treatment.Clinical trial registration: Not applciable.

Keywords: Exosome; Hydrogel; Nanomedicine; Osteoarthritis; Surface modification.

Fig. 1

LRRK2-IN-1 suppresses the IL-1β-induced inflammation and catabolism and induces anabolism without causing the inhibition of chondrocyte viability. A Schematic diagram of cell treatment and experimental procedures. B Cell viability assessed by CCK8 assay. No obvious inhibition of chondrocyte proliferation was observed when treated with 0.5, 1.0, 2.5, and 5.0 µM LRRK2-IN-1 for 24 h. Data represent mean ± SD; N = 6/group; one-way ANOVA; ns, not significant. C Western blot analyses of the protein levels of anabolic, catabolic, inflammatory factors in the IL-1β-induced chondrocytes treated with 0.5, 1.0, 2.5, and 5.0 µM LRRK2-IN-1 for 24 h. LRRK2-IN-1 suppressed MMP3, MMP13, iNOS, and COX2 and induced COL2 and SOX9 in a dose-dependent manner. D Quantitative analyses of the western blot results. Data represent mean ± SD; N = 3/group; *P < 0.05; **P < 0.01 by one-way ANOVA. E Immunofluorescence of iNOS, MMP13, and aggrecan expression in the IL-1β-induced chondrocytes treated with 5.0 µM LRRK2-IN-1 for 24 h. Scar bar: 400 μm

Fig. 2

Characterization of W-Exo-L@GelMA. A The protocol of W-Exo-L@GelMA preparation. L, LRRK2-IN-1; EXO, exosome; W, cartilage affinity WYRGRL (W) peptide; UV, ultraviolet. B Western blot analyses showing that MSC exosomes could express characteristic marker proteins, such as TSG101, CD81, and CD9. C Scanning electron microscopy showing that the exosomes were round-shaped and the diameter of target peptide-modified exosomes increased compared with that of unmodified exosomes. D Diameter measurement of MSC exosomes. The diameter measurement showed the diameter of the exosomes after the modification of the targeted peptide increased compared with that of the unmodified exosomes. E Surface potential analysis of MSC exosomes. The surface potential of exosomes was increased after peptide modification. F Optical and fluorescent images of W-Exo-L@Gel. The exosomes containing Dil fluorescence were successfully encapsulated by GelMA microspheres with a size of about 100–200 μm. G Scanning electron microscopy showing the morphology and size of GelMA microspheres. H Infrared spectrum analysis of W-Exo-L@GelMA. The amide I (1646 cm^–1), amide II (1534 cm^–1), amide III (1316 cm^–1), and amide A (3270 cm^–1) and amide B (3064 cm^–1), are peaks of infrared characterization of amide. 1285 cm⁻¹ and 1420 cm⁻¹ belong to C-N. I The drug release curve of Exo-L@GelMA in vitro. The drug release time of Exo-L@GelMA was prolonged by 1 week. Data represent mean ± SD; N = 3/group; *P < 0.05; **P < 0.01 by Student’s t-test

Fig. 3

W-Exo-L@GelMA exhibits a strong chondrocyte-targeting effect and a pronounced action on promoting anabolism and suppressing catabolism and inflammation without causing the inhibition of chondrocyte viability. A Cell viability assessed by CCK8 assay. No obvious cytotoxicity on chondrocytes was observed when treated with W-Exo-L@GelMA loaded with 0.5, 1.0, 2.5, and 5.0 µM LRRK2-IN-1 for 48 h. Data represent mean ± SD; N = 6/group; one-way ANOVA; ns, not significant. B Immunofluorescence of Dil-labeled exosomes. The uptake of exosomes was observed in the chondrocytes when treated with Exo-L, Exo-L@GelMA or W-Exo-L@GelMA for 48 h. Dil was used for labeling exosomes (red), DAPI to label nuclei (blue), and Phalloidin to label the cytoskeleton (green). Scar bar: 200 μm. C Western blot analyses of the protein levels of anabolic, catabolic, and inflammatory factors in the IL-1β-induced chondrocytes treated with W-Exo-L@GelMA loaded with 0.5, 1.0, 2.5, and 5.0 µM LRRK2-IN-1 for 48 h. W-Exo-L@GelMA promoted COL2 and SOX9 and inhibited iNOS, COX2, MMP3, and MMP13 protein levels in a dose-dependent manner. D Quantitative analysis of the western blot results. Data represent mean ± SD; N = 3/group; *P < 0.05; **P < 0.01 by one-way ANOVA

Fig. 4

W-Exo-L@GelMA suppresses the IL-1β-induced transcriptomic responses related to catabolic effect, inflammation, and immune response. A Principal component analysis of the overall transcriptome of each sample. B Scatter plots of the differentially expressed genes (DEGs) in the comparison between the IL-1β and VEH groups or between the W-Exo-L@GelMA/IL-1β and IL-1β groups. The up- or down-regulated genes are indicated in red or blue color. The top 20 DEGs in the ranked gene list sorted in descending order of expression variance values are labeled by official gene symbols and the rescued genes are labeled in bold. Expression variance is defined by the absolute value of the difference in normalized gene expression (FPKM). C Overlap of the DEGs in each comparison. D The scatter plot of the completely-rescued (Type-1) and partially-rescued (Type-2) genes. E Top 30 IL-1β-induced or suppressed genes notably rescued by W-Exo-L@GelMA. F KEGG pathway analysis of the rescued genes (up-regulated by IL-1β and down-regulated by W-Exo-L@GelMA). G qPCR validations of the key rescued genes. Data represent mean ± SD; N = 3/group. **P < 0.01 by Student’s t-test

Fig. 5

W-Exo-L@GelMA shows a significant joint retention effect and attenuates cartilage lesions in the OA murine model. A Joint retention assessed by in vivo fluorescence method. The hydrogel with Cy5-loaded exosomes or the Cy5-loaded exosomes alone was injected into the knee joint of the 12-week-old C57BL/6 mice. W-Exo-L@GelMA delayed the dissipation of the Cy5 fluorescence signal, indicating that the hydrogel can be retained 14 days after injection. Data represent mean ± SD; N = 3/group. **P < 0.01 by Student’s t-test; the colored groups versus Exo-L@GelMA. #P < 0.05; ##P < 0.01 by Student’s t-test, the colored groups versus W-Exo-L@GelMA; $P < 0.05 by Student’s t-test, W-Exo-L@GelMA versus Exo-L@GelMA. B Safranin O-fast green staining of knee sections. W-Exo-L@GelMA exhibited a stronger ability to attenuate cartilage wear and degeneration induced by destabilization of the medial meniscus (DMM) than L@GelMA, Exo@GelMA, or Exo-L@GelMA. Scar bar: 200 μm. C Immunohistochemistry representative images of aggrecan protein levels. Scar bar: 200 μm. Aggrecan expression was increased in local chondrocytes after the intervention, especially W-Exo-L@GelMA. D Immunohistochemistry representative images of MMP13 protein levels. Scar bar: 200 μm. The expression of MMP13 in local chondrocytes decreased after treatments. E Statistical analysis of OARSI histological scores. Data represent mean ± SD; N = 6/group. *P < 0.05; **P < 0.01 by one-way ANOVA. ##P < 0.01 by one-way ANOVA, the DMM group versus the control group. F Quantitative statistics of immunohistochemistry analyses of aggrecan and MMP13. Data represent mean ± SD; N = 6/group. *P < 0.05; **P < 0.01 by one-way ANOVA. ##P < 0.01 by one-way ANOVA, the DMM group versus the control group

Fig. 6

W-Exo-L@GelMA inhibits osteophyte formation and subchondral bone loss in the OA murine model. A Micro-CT representative images of the bone surface and the coronal plane of the subchondral bone of joints. W-Exo-L@GelMA notably reduced the osteophyte formation of the surface and the loss of subchondral bone. B Quantitative analysis of the parameters of tibial subchondral bone: trabecular number (Tb. N), volume/tissue volume (BV/TV), trabecular thickness (Tb. Th), and trabecular separation (Tb. Sp). Data represent mean ± SD; N = 6/group. *P < 0.05; **P < 0.01 by one-way ANOVA

Fig. 7

W-Exo-L@GelMA exhibits a therapeutic effect on human OA cartilage explants by promoting aggrecan and suppressing MMP13 expression. A Schematic diagram of the procedures of the human OA cartilage explant harvest and treatments. The human OA cartilage explants were harvested from the medial condyle of the femur of the OA patients undergoing total knee arthroplasty. B Representative image of the harvested human OA cartilage explant. C Representative images of Immunohistochemistry analyses of the aggrecan and MMP13 expression levels in the IL-1β- maintained explants treated with or without W-Exo-L@GelMA (5.0 µM) for 72 h. Scar bar: 200 μm. D Quantitative analyses of immunohistochemistry. Data represent mean ± SD; N = 3/group. *P < 0.05 by paired t-test