Ultra-Long-Term Anti-Inflammatory Polyphenol Capsule to Remodel the Microenvironment for Accelerating Osteoarthritis Healing by Single Dosage

Abstract

Osteoarthritis (OA) is a common chronic inflammatory disease that leads to disability and death. Existing therapeutic agents often require frequent use, which can lead to drug resistance and long-term side effects. Polyphenols have anti-inflammatory and antioxidant potential. However, they are limited by their short half-life and low bioavailability. This work presents a novel pure polyphenol capsule for sustained release of polyphenols, which is self-assembled via hydrophobic and hydrogen bonds. The capsule enhances cellular uptake, scavenges reactive oxygen and nitrogen species, reduces inflammatory markers, and remodels the OA microenvironment by inhibiting the p38 MAPK pathway. The capsule overcomes the limitations of short half-life and low bioavailability of polyphenols and achieves single-dose cure in mouse and dog OA models, providing an optimal therapeutic window for OA repair. Taking advantage of simple manufacturing, convenient administration, and pure polyphenol composition, these capsules show great potential for clinical treatment of osteoarthritis and chronic inflammatory diseases.

Keywords: antioxidant; capsules; osteoarthritis; polyphenol; single‐dose cure.

Scheme 1

Schematic illustration of PC capsules in the treatment of ROS‐related diseases. a) PC capsules with broad‐spectrum and long‐term ROS scavenging ability are synthesized by a simple and green method. b) Due to the inhibition of the MAPK signaling pathway in vivo, PC capsules exhibit therapeutic effects against osteoarthritis.

Figure 1

Preparation and characterization of PC capsules. a) SEM image of PC@CaCO₃. b) SEM image of PC capsules. c) TEM image of PC capsule. d) Size distribution of PC@CaCO₃ and capsules. e) EDS analysis of PC@CaCO₃ and capsules. f) CLSM image of PC capsule. g) Stability of the capsules in 1) pure water, 2) PBS, 3) 0.9% NaCl, and 4) DMEM. h) In vitro accumulative release profiles of PC from PC capsules at pH 7.4 and 5.5. All the values are expressed as mean ± SD, n = 3.

Figure 2

ROS and RNS scavenging ability of PC capsules. a) total antioxidant capacity, b) H₂O₂, c) O₂ ⁻, d) DPPH, and e) ABTS radical scavenging ability of PC capsules. Storage in an oxidant environment for 0 h, one month, and six months, ROS scavenging capacity of f) VC solution, g) PC solution, and h) PC capsule was measured. ROS scavenging capacity included total antioxidant capacity, H₂O₂, O₂ ⁻, and DPPH radical scavenging ability. The yellow column represents the effect after 0 h of placement, the blue column represents the effect after one month of placement and the green column represents the effect after six months of placement. p values: ns p > 0.5, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, all the values are expressed as mean ± SD, n = 3.

Figure 3

Mechanism of capsule formation. a) XPS data of PC and capsule where capsules were prepared by removing the template by HCl and EDTA, respectively. b) Spectra of Ca²⁺ of PC and capsule where capsules were prepared by removing the template by HCl and EDTA, respectively. c) Fourier transform infrared (FTIR) spectra of PC and capsules. d) The 3D structural formula of PC. e) The representative snapshot (0, 40, and 440 ns) from the all‐atom molecule dynamics simulation trajectory showed the aggregation of high‐density (100 mM L⁻¹) PCD molecules. The PCD molecules were shown in licorice. f) The hydrogen bonds (blue dashed line) and π − π interaction (yellow dashed line) among PCD molecules. This structure was obtained from the snapshot of 440 ns in the all‐atom molecule dynamics simulation trajectory showed the aggregation of 100 mM PCD molecules. g) The average hydrogen bond number formed between PCD molecules evolution with time for low‐density PCD (8.5 mM L⁻¹, blue line) and high‐density PCD (100 mM L⁻¹, red line). h) The percentage of PC capsules remaining after 8 h of incubation with 100 mM of NaCl, urea, or Tween 20, highlighting the dominant interactions of PC. i) SEM images of capsules after 8 h of incubation with 100 mM of NaCl, urea, or Tween 20. j) The solvent accessible area of low‐density (8.5 mM L⁻¹) PCD molecules and high‐density (100 mM L⁻¹) PCD molecules. All the values are expressed as mean ± SD, n = 3.

Figure 4

ROS‐responsiveness of PC capsules in cellular. Cell viability of a) RAW 264.7 cells and b) ATDC5 cells after incubating with PC solution and capsules for 24 h. Cellular uptake of capsules in c) RAW 264.7 cells and d) ATDC5 cells. Cell viability induced by H₂O₂ of e) RAW 264.7 cells and f) ADTC5 cells. g) Apoptotic chondrocytes were detected using Annexin V‐FITC/PI staining and flow cytometry. h) TUNEL fluorescent images of ADTC5 cells stimulated with IL‐1β and capsules. i) The apoptosis rates of flow cytometry. j) The survival rate of ADTC5 induced by TUNEL fluorescent images. k) Quantification of total fluorescence intensity of DCFH‐DA in RAW 264.7 cells. (P values: ns p > 0.5, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, all the values are expressed as mean ± SD, n = 3).

Figure 5

The assessment of therapeutic efficiency in vivo on osteoarthritis. a) Schematic representation of the establishment and treatment of osteoarthritis mice. b) H&E staining of mouse models of osteoarthritis. c) qPCR data in mouse model of osteoarthritis for IL‐6, TNF‐α and MMP‐13. d) Representative immunohistochemical images of PBS, PC and capsule‐treated osteoarthritis sections stained with IL‐6, TNF‐α and MnSOD. e) Schematic representation of the surgical and therapeutic protocols used in a dog model of ACLT‐induced dog OA. f) Macroscopic appearance of the femoral condyle and tibial plateau cartilage and their g) scores after 12 weeks of treatment. (Red dashed line: articular cartilage damage). Representative images of SO&FG, H&E and PR staining in h) dog femoral condyle and i) tibial plateau cartilage after 12 weeks of treatment. Macroscopic cartilage scores based on the OARSI scoring system and for two major weight‐bearing surfaces, including the j) medial femoral plateau and k) medial tibial condyle. (p values: ^*** p < 0.001, ^**** p < 0.0001, all the values are expressed as mean ± SD, n = 3).

Figure 6

PC capsules inhibited MAPK expression in osteoarthritis. a) Volcano plot displaying DEGs between capsule‐ and blank control‐treated osteoarthritic mice. b) Heatmap of DEGs in the osteoarthritis mouse model. c) GSEA analysis of the MAPK gene set in the osteoarthritic mouse model. ADTC5 cells were exposed to IL‐1β then treated with PC capsules. d) Phosphorylation of proteins involved in MAPK signaling (JNK, p38) as determined by western blotting. e) Expression of p38, p‐P38, total caspase3, cleaved caspase3, and Bcl‐2. f) p38, p‐P38, iNOS, and COX‐2 was assessed by western blotting. g) Quantification of (d). h) Quantitation of (e). i) western blotting band quantitation of (f). (p values: ^** p < 0.01, ^*** p < 0.001, all values are expressed as mean ± SD, n = 3).

Figure 7

Schematic illustration of the potential mechanism by which intracellular PC capsules exert anti‐inflammatory effects by inhibiting apoptosis. Capsules could regulate the MAPK‐p38 signaling pathway in the Osteoarthritis of mice.