Microneedle-Delivered PDA@Exo for Multifaceted Osteoarthritis Treatment via PI3K-Akt-mTOR Pathway

Abstract

Osteoarthritis (OA) is marked by cartilage deterioration, subchondral bone changes, and an inflammatory microenvironment. The study introduces the Microneedle-Delivered Polydopamine-Exosome (PDA@Exo MN), a therapeutic that not only preserves cartilage and promotes bone regeneration but also improves localized drug delivery through enhanced penetration capabilities. PDA@Exo MN shows strong reactive oxygen species (ROS) scavenging abilities and high biocompatibility, fostering osteogenesis and balancing anabolic and catabolic processes in cartilage. It directs macrophage polarization from M0 to the anti-inflammatory M2 phenotype. RNA sequencing of treated chondrocytes demonstrates restored cellular function and activated antioxidant responses, with modulated inflammatory pathways. The PI3K-AKT-mTOR pathway's activation, essential for PDA@Exo's effects, is confirmed via bioinformatics and Western blot. In vivo assessments robustly validate that PDA@Exo MN prevents cartilage degradation and OA progression, supported by histological assessments and micro-CT analysis, highlighting its disease-modifying impact. The excellent biocompatibility of PDA@Exo MN, verified through histological (H&E) and blood tests showing no organ damage, underscores its safety and efficacy for OA therapy, making it a novel and multifunctional nanomedical approach in orthopedics, characterized by organ-friendliness and biosecurity.

Keywords: macrophage polarization; microneedle drug delivery; osteoarthritis; polydopamine‐exosome complex; reactive oxygen species scavenging.

Figure 1

Synthesis and characterization of PDA@Exo MN. a) Diagrammatic representation of the synthesis pathway for PDA@Exo MN. b) Transmission electron microscopy (TEM) images depicting PDA, Exo, and PDA@Exo. Scale bars: 100 µm. c) Hydrodynamic diameters of Exo, PDA and PDA@Exo determined by Dynamic Light Scattering (DLS). d) Western blot analysis of exosome markers (Alix, CD9, TSG101) and endoplasmic reticulum (ER) markers (calnexin). e) Zeta potential measurements of PDA, Exo, and PDA@Exo. f) UV spectra of PDA, Exo, and PDA@Exo. g) Evaluation of the size stability of PDA@Exo.

Figure 2

Microstructural Analysis and Mechanical Validation of Microneedles. a) SEM Micrographs of MN and PDA@Exo MN. b) Mechanical Characterization of Microneedles: MN versus PDA@Exo MN. c) Photomicrograph of a fluorescent MN. d) 3D reconstruction image of Laser Confocal Microscopy Image of a Microneedle. e) Sequential evaluation of skin recovery at various time points post‐microneedle insertion. Scale bars: 1 cm. f) Immunofluorescent and histological staining of MN penetration in rat skin. Scale bars: 50 and 200 µm, respectively.

Figure 3

Protective effect of PDA@Exo MN on chondrocytes against ROS in vitro. a) Live/Dead staining of chondrocytes treated with Control, PDA, Exo, and PDA@Exo under H₂O₂ stimulation, showing nuclei in blue (33258), live cells in green (AM) and dead cells in red (PI). Scale bars: 20 µm. b) Ratio of live/dead cells measured by MFI in chondrocytes post‐treatment with Control, PDA, Exo, and PDA@Exo under H₂O₂ stimulation. c) ROS detection in chondrocytes treated with Control, PDA, Exo, and PDA@Exo under H₂O₂ stimulation, where green fluorescence (DCF) indicates ROS levels, with DAPI staining for nuclei. Scale bars: 50 µm. d) Quantitative analysis of intracellular ROS levels as indicated by mean fluorescence intensity (MFI) in chondrocytes treated with Control, PDA, Exo, and PDA@Exo following H₂O₂ stimulation. e) ROS detection using FITC in chondrocytes treated with Control, PDA, Exo, and PDA@Exo under H₂O₂ stimulation. f) MFI in chondrocytes treated with Control, PDA, Exo, and PDA@Exo following H₂O₂ stimulation. g) Relative SOD activity restored by PDA@Exo in H₂O₂‐induced chondrocytes. h) Cellular MDA content of chondrocytes treated with Control, PDA, Exo, and PDA@Exo under H₂O₂ stimulation All data are shown as the mean ± standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

PDA@Exo restore the cellular function of chondrocytes under oxidative stress and M2 polarization. a) JC‐1 staining of chondrocytes treated with Control, PDA, Exo, and PDA@Exo under H₂O₂ stimulation to monitor mitochondrial membrane potential. Scale bars: 50 µm. b) Western blot analysis of the expression of apoptosis markers in chondrocytes treated with Control, PDA, Exo, and PDA@Exo under IL‐1β stimulation. c) Western blot analysis of the expression of MMP9, MMP13, SOX9, COL1A2, and RUNX2 in chondrocytes treated with Control, PDA, Exo, and PDA@Exo under IL‐1β stimulation. d) Alizarin red S staining of BMSCs treated with Control, PDA, Exo, and PDA@Exo. e) Western blot analysis of the expression of osteogenesis‐related genes (RUNX2, OPN, and BMP2) in BMSCs treated with Control, PDA, Exo, and PDA@Exo. f) Immunofluorescence staining of the RAW 264.7 cells after being cocultured with control, PDA, Exos and PDA@Exo under LPS stimulation, iNOS (green); CD206 (red); Blue: nuclei. Scale bars: 50 µm. g) Western Blot analysis of RAW 264.7 cells after being cocultured with control, PDA, Exos and PDA@Exo under LPS stimulation. (Scale bars: 50 µm) All data are shown as the mean ± standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Changes in transcriptome profile of PDA@Exo ‐treated OA chondrocytes.a) The volcano plot of differentially expressed genes (DEGs) and b) Venn that are essential for chondrocyte bio‐function and provide cellular protection against inflammation induced by IL‐1β are identified and highlighted. c) Heatmap representing the gene expression profiles of OA chondrocytes stimulated with IL‐1β and OA chondrocytes treated with IL‐1β + PDA@Exo. d–f) The results of Gene Ontology (GO) enrichment analysis in both up‐regulated and down‐regulated genes. g) Enrichment analysis of the KEGG pathways for the differentially expressed genes of interest. h) The expression of p‐PI3K, PI3K, p‐Akt, Akt, p‐mTOR, and mTOR protein in chondrocyte treated with control, PDA, Exos and PDA@Exo under IL‐1β stimulation.

Figure 6

PDA@Exo attenuated OA progression in vivo. a) Representative images of H&E, TB, and SO‐FG staining in rat knee joints, and b) the corresponding OARSI scores after different treatments 8 weeks post‐surgery. Scale bars: 500 µm c) Representative 3D reconstruction images of differently treated rat knees at week 8 (Scale bars: 2 µm) and coronal micro‐CT images of the rat medial tibial plateau in different groups. (Scale bars: 1 µm); d–f) Quantitative analysis of BV/TV and increased Tb. Sp conducted by micro‐CT in 8 weeks post‐surgery. All data are shown as the mean ± standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001.

Scheme 1

Schematic depicting PDA@Exo MN‐mediated modulation of cartilage degradation inhibition, osteogenesis enhancement, and macrophage polarization via the PI3K‐AKT‐mTOR signaling pathway.