DNA Origami-Based CD44-Targeted Therapy Silences Stat3 Enhances Cartilage Regeneration and Alleviates Osteoarthritis Progression

Abstract

Osteoarthritis (OA) is a widespread musculoskeletal disorder affecting ≈600 million people globally, and small interfering RNA (siRNA) therapy shows potential in targeting OA progression. However, the efficient and targeted delivery of siRNA remains a major challenge due to issues with tissue specificity and degradation in vivo. In this study, A DNA origami-based chondrocyte-targeted delivery system (OCS) is designed for siRNA delivery to OA-affected cartilage. The DNA origami is engineered to load with siRNA targeting signal transducer and activator of transcription 3 (Stat3), a key regulator of inflammation and cartilage degradation, and is functionalized with anti-CD44 aptamers for selective targeting of OA chondrocytes. In vitro, the DNA origami system effectively delivers siRNA to diseased chondrocytes, silencing matrix metalloproteinases expression and reducing inflammation. In OA rat models, it preserves cartilage integrity, promotes regeneration, and mitigates ECM degradation without evident side effects. These findings highlight DNA origami as a promising platform for siRNA-based OA therapy, offering a promising solution to the challenges of targeted and efficient siRNA delivery.

Keywords: CD44 targeting; DNA triangle origami; osteoarthritis; si‐Stat3.

Figure 1

Schematic illustration of the design and construction of a DNA origami‐based chondrocyte‐targeted delivery system (OCS) for in vivo OA treatment. OCS was injected into the knee joint of OA rats, where it specifically targeted chondrocytes with high CD44 expression. This facilitated the selective recruitment of OCS by the diseased chondrocytes, promoting its internalization and enabling the effective release of si‐Stat3. As a result, Stat3 gene silencing was achieved, leading to the downregulation of MMP13 expression, a significant reduction in crtilage degradation biomarkers, and an increase in the expression of anabolic factors. Upper‐right corner: AFM image of OCS. Scale bar: 100 nm. Diagram of osteoarthritis adapted from Liu et al. Signal Transduct Target Ther. 8, 138 (2023), under a CC BY 4.0 license.

Figure 2

The cellular uptake, antioxidant properties, and cytoprotective effects of OCS. A) Schematic illustration and representative confocal images of OCS cellular uptake in chondrocytes under normal and inflammatory conditions (induced by IL‐1β). The cytoskeleton is labeled with phalloidin (red), OCS is labeled with FITC (green), and the nuclei were counterstained with DAPI (blue). Scale bar: 20 µm. B) Schematic and representative confocal images of ROS expression levels in OA chondrocytes after 24 h of incubation with si‐Stat3, origami, and OCS. Ctrl group: healthy chondrocytes with no IL‐1β induction or treatment; IL‐1β group, IL‐1β‐induced chondrocytes. C) Relative fluorescence intensity analysis of ROS expression levels in chondrocytes after different treatments. Data were presented as means ± SD (n = 3). D) Flow cytometry analysis of ROS levels in chondrocytes after different treatments. E) Cell viability of chondrocytes after 24 h of different treatments assessed by the CCK‐8 assay. Data were presented as means ± SD (n = 6). Statistical comparisons were performed using one‐way ANOVA. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, ns indicates no statistical significance. Diagram of osteoarthritis (A) adapted from Liu et al. Signal Transduct Target Ther. 8, 138 (2023), under a CC BY 4.0 license.

Figure 3

OCS modulates catabolic and anabolic proteins in OA chondrocytes. A) Schematic illustration of the cellular OA model establishment in rat chondrocytes by IL‐1β induction, followed by treatment with si‐Stat3, Origami, and OCS. Ctrl group: healthy chondrocytes with no IL‐1β induction or treatment; IL‐1β group, IL‐1β‐induced chondrocytes. B) Western blot analysis of Stat3, MMP3, MMP13, COL‐II, and SOX9 protein expression in chondrocytes after different treatments. C–G) Quantitative analysis of the western blot results. Data were presented as means ± SD (n = 3). Statistical comparisons were performed using one‐way ANOVA. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, ns indicates no statistical significance. H–J) Representative Immunofluorescence images of the protein expression of COL‐II, SOX9, and ACAN in chondrocytes after different treatments. Scale bar: 20 µm.

Figure 4

In vivo distribution and biosafety of OCS in rats. A) Representative in vivo fluorescence images of the knee joints in Sham and OA rats at various time points following intraarticular injection with FITC‐OCS (n = 3 per group). B) Quantitative analysis of fluorescence radiation in (A). C–E) Blood biochemical indicators of renal function, BUN: blood urea nitrogen, CREA: creatinine, UA: uric acid. Data were presented as means ± SD (n = 3). F–K) Liver function test indexes of rats. ATL: alanine transaminase, AST: aspartate transaminase, ALB: albumin, DBIL: direct bilirubin, TBIL: total bilirubin, γ‐GT: gamma‐glutamyl transferase. Data were presented as means ± SD (n = 3). L) Representative H&E staining images of the major organs from both OA and Sham groups of rats. Scale bar: 100 µm.

Figure 5

Therapeutic effects of OCS on articular cartilage in a rat OA model. A) A schematic diagram illustrating the experimental design. An OA model was established using the ACLT and pMMx surgical technique. After four weeks, intra‐articular injections of normal saline, si‐Stat3, origami, or OCS were administered weekly, totaling four injections. At the ninth week, cartilage recovery was evaluated using micro‐CT imaging and histological staining. Sham group: only the skin and muscles were separated, leaving the joint structures intact. OA group: treated with normal saline. B) Representative 3D micro‐CT images of the knee and 2D images in the coronal (COR), sagittal (SAG), and axial planes (AX). C,D) Quantitative analysis of tibial subchondral bone parameters: bone mineral density (BMD) and volume/tissue volume (BV/TV). Data were presented as means ± SD (n = 3). Statistical comparisons were performed using one‐way ANOVA. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, ns indicates no statistical significance. E–G) Histological analyses of the knee joint, including H&E, Masson, and Safranin O staining. Photographs of the right are higher magnification of the black rectangular areas. Scale bars: 200 µm.

Figure 6

Immunohistochemical (IHC) staining of knee joints from rats in different treatment groups of rats after four weeks of treatment. A) Representative IHC staining images of CoL‐II, SOX9, and ACAN in rat cartilage sections under different treatment conditions. Scale bar: 500 µm. B) The CoL II‐positive (CoL II⁺) area is calculated from(A). C) The SOX9‐positive (SOX9⁺) area is calculated from (A). D) The ACAN‐positive (ACAN⁺) area is calculated from (A). Data were presented as means ± SD (n = 3). Statistical comparisons were performed using one‐way ANOVA. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, ns indicates no statistical significance. E) Representative IHC staining images of Stat3 in rat cartilage sections under different treatment conditions. Scale bar: 100 µm.