A ROS-responsive hydrogel encapsulated with matrix metalloproteinase-13 siRNA nanocarriers to attenuate osteoarthritis progression

Abstract

RNA interference (RNAi) and oxidative stress inhibition therapeutic strategies have been extensively utilized in the treatment of osteoarthritis (OA), the most prevalent degenerative joint disease. However, the synergistic effects of these approaches on attenuating OA progression remain largely unexplored. In this study, matrix metalloproteinase-13 siRNA (siMMP-13) was incorporated onto polyethylenimine (PEI)-polyethylene glycol (PEG) modified Fe₃O₄ nanoparticles, forming a nucleic acid nanocarrier termed si-Fe NPs. Subsequently, a poly(vinyl alcohol) (PVA) crosslinked phenylboronic acid (PBA)-modified hyaluronic acid (HA) hydrogel (HPP) was used to encapsulate the si-Fe NPs, resulting in a bifunctional hydrogel (si-Fe-HPP) with reactive oxygen species (ROS)-responsive and RNAi therapeutic properties. Studies in vitro demonstrated that si-Fe-HPP exhibited excellent biocompatibility, anti-inflammatory effects and prolonged stable retention time in knee joint. Intra-articular injection of si-Fe-HPP significantly attenuated cartilage degradation in mice with destabilization of the medial meniscus (DMM)-induced OA. The si-Fe-HPP treatment not only notably alleviated synovitis, osteophyte formation and subchondral bone sclerosis, but also markedly improved physical activity and reduced pain in DMM-induced OA mice. This study reveals that si-Fe-HPP, with its ROS-responsive and RNAi abilities, can significantly protect chondrocytes and attenuate OA progression, providing novel insights and directions for the development of therapeutic materials for OA treatment.

Keywords: Metalloproteinase-13 siRNA nanocarrier; Osteoarthritis; ROS-responsive Hydrogel.

Fig. 1

Schematic diagram of preparation process and action mechanism of si-Fe-HPP hydrogel to attenuate osteoarthritis progression

Fig. 2

Preparation, characterization, and biological properties of Fe₃O₄-PEI-PEG NPs and si-Fe NPs. a Schematic diagram of preparation of Fe₃O₄-PEI-PEG NPs and si-Fe NPs. b–c TEM image of b Fe₃O₄-PEI-PEG NPs and c si-Fe NPs. Scale bar: 50 μm. d The average hydrodynamic particle size distribution of Fe₃O₄-PEI-PEG NPs and si-Fe NPs. e Zeta potentials of si-Fe NPs at various weight ratios of siRNA:Fe. f Agarose gel retardation assay of si-Fe NPs at various weight ratios of siRNA:Fe. g The protective effect of Fe₃O₄-PEI-PEG NPs on siRNA against RNase digestion. h Cell viabilities of C28/I2 cell line detected by MTT with various concentrations of si-Fe NPs. i Fluorescence image of C28/I2 cell line incubated with naked FAM-siRNA or FAM si-Fe NPs for 12 h. scale bar: 50 μm. j MMP-13 gene silencing efficacy of si-Fe NPs on C28/I2 cell line

Fig. 3

Preparation, characterization, and biological properties of HPP hydrogel. a Schematic diagram of preparation of HPP hydrogel. b The formation process of HPP hydrogel. c SEM image of HPP hydrogel. Scale bar: 200 μm. d–f Rheological characterization of HPP hydrogel. d Strain sweep test; e Frequency sweep test; f Time sweep test. g Degradation of HPP hydrogel in PBS and different concentrations of H₂O₂. h UV–vis spectroscopy of NaI/H₂O₂ solution with or without HPP hydrogel co-incubation. i–j Cell viabilities of C28/I2 cell line detected by MTT assays i with various concentrations of HPP hydrogel and j with various concentrations of H₂O₂ with or without HPP Hydrogel. k–l Cell viabilities (k) of C28/I2 cell line detected by Live/Dead staining at 1 mM H₂O₂ with or without HPP Hydrogel, followed by quantitative analysis of the results (l). Scale bar: 200 μm. m Residence time of the HPP hydrogel in knee joint of normal and model mice

Fig. 4

Preparation, characterization, and biological properties of si-Fe-HPP hydrogel. a Schematic diagram of preparation of si-Fe-HPP hydrogel. b Formation of si-Fe-HPP hydrogel. c SEM image of Si-Fe-HPP hydrogel. Scale bar: 10 μm. d Injectable properties of si-Fe-HPP hydrogel. e Rheological characterization of HPP hydrogel and si-Fe-HPP hydrogel. f Release behavior of si-Fe NPs in si-Fe-HPP hydrogel at various concentrations of H₂O₂. g–h Cell viabilities of C28/I2 cell line detected by MTT method at various concentrations of si-Fe-HPP hydrogel. i–l Evaluation of anti-inflammatory effect of si-Fe-HPP hydrogel on inflammation-induced C28/I2 cell line

Fig. 5

si-Fe-HPP attenuated OA progression in vivo. a Schematic diagram of the flow of animal experiment. b–f S.O. staining and Alcian blue staining (b) and quantification of Osteoarthritis Research Society International (OARSI) grading (c), cartilage thickness (d), chondrocyte number (e) and relative cartilage area (f). Scale bar: 50 μm. g–j Representative images of immunohistochemical staining of MMP-13 + (g) and Col II (i) in the cartilage of knee joints, as well as the relative quantification of MMP-13 + (h) and Col II (j). Scale bar: 50 μm. Data are expressed as mean ± SD

Fig. 6

si-Fe-HPP alleviated synovitis and subchondral bone sclerosis in OA mice. a Schematic diagram of HE staining and Micro-CT detection. b–c Body weight (b) and knee joint diameter (c) of mice at the endpoint of the animal experiment. d–e Representative images of H&E-stained mouse knee joint sections (d) and quantitative scores of synovitis (e). Scale bars: 50 μm. f Micro-CT analysis and 3D reconstructed images of mouse knee joints and sagittal views of the medial joint compartment showing changes in femoral and tibial surfaces and SBP thickness, respectively. Scale bars: 1 mm. g–m Quantification of the number of osteophytes (g), BV (h), SBP thickness (i), subchondral BMD (j), and the ratio of BV/TV (k), Tb.N (l), and Tb.Th (m) of the knee joint. n Schematic representation of the working mechanism of si-Fe-HPP in the knee joint of OA mice. Data are expressed as mean ± SD

Fig. 7

si-Fe-HPP improved the behavioral performance of mice. a A representative trajectory plot of the spontaneous activity of mice in the open field test. b–e the quantitative analyses of the relative activity (b), the duration of the activity (c), the distance traveled (d) and the average speed (e) of mice within the 180-s experimental period. f Gait analysis of mice. Dotted blue line, stride length; dotted red line, step length; dotted green line, front/aft footprint. Red footprint, forepaw; blue footprint, hindpaw. g–i Quantification of mouse footprints, including relative stride length (g), relative step length (h), and relative fore/rear footprint length (i). j Measurement of mechanical sensitivity of mice by the von Frey test. Data are expressed as mean ± SD

Fig. 8

Satisfactory biocompatibility and safety of si-Fe-HPP in vivo. a Representative images of H&E staining of heart, liver, spleen, lung, kidney excised from mice treated with si-Fe-HPP hydrogel treatment and without treatment. Scale bars, 200 μm. b–g The levels of ALT (b), AST (c), ALB (d), CHO (e), BUN (f), and LDH (g) in serums from mice treated with si-Fe-HPP hydrogel and no treatment. The two dashed lines in figures represent the range of normal values for each serum biochemical index. Data are expressed as mean ± SD