Microenvironmental Enzyme-Responsive Methotrexate Modified Quercetin Micelles for the Treatment of Rheumatoid Arthritis

Abstract

Purpose

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease involving synovial inflammation and joint destruction. Although therapeutic drugs for RA have some efficacy, they usually cause severe side effects and are expensive. RA is characterized by synovial hyperplasia, intra-articular hypoxia, upregulated expression of matrix metalloproteinases, and excessive accumulation of reactive oxygen species. The adverse microenvironment further aggravates activated macrophage infiltration. Therefore, controlling the microenvironment of diseased tissues and targeting the activated macrophages have become new therapeutic targets in RA patients.

Methods

Here, microenvironment-targeting micelles (PVGLIG-MTX-Que-Ms) were synthesized using the thin film hydration method. In the inflammatory microenvironment, PVGLIG was cleaved by the highly expressed MMP-2, PEG₅₀₀₀ was eliminated, MTX was exposed, macrophage activation was targeted, and Que enrichment was enhanced. The cytotoxicity, targeting, antioxidant, and anti-inflammatory properties of drug-loaded micelles were tested in vitro. The drug-loaded micelles were used to treat CIA rats. In vivo targeting, expression of serum inflammatory factors, immunohistochemistry of the articular cartilage, and changes in immunofluorescence staining were observed.

Results

The developed micelles had a particle size of (89.62 ±1.33) nm and a zeta potential of (−4.9 ±0.53) mV. The IC₅₀ value of PVGLIG-MTX-Que-Ms (185.90 ±6.98) μmol/L was significantly lower than that of free Que (141.10 ±6.39) μmol/L. The synthesized micelles exhibited slow-release properties, low cytotoxicity, strong targeting abilities, and significant anti-inflammatory effects in vitro. In vivo, the drug-loaded micelles accumulated at the joint site for a long time. PVGLIG-MTX-Que-Ms significantly reduced joint swelling, improved bone destruction, and decreased the expression of serum inflammatory factors in CIA rats.

Conclusion

The smart-targeting micelles PVGLIG-MTX-Que-Ms with strong targeting, anti-inflammatory, cartilage-protective, and other multiple positive effects are a promising new tool for RA treatment.

Keywords:

• matrix metalloproteinases
• quercetin
• macrophages
• methotrexate
• anti-inflammatory

Abbreviations

RA, rheumatoid arthritis; PVGLIG-MTX-Que-Ms, mmp enzymes respond to peptide and methotrexate modified quercetin micelles; CVD, chronic inflammation-induced cardiovascular disease; MMP, matrix metalloproteinases; Que, quercetin; PVGLIG, MMP-responsive peptide; MTX, methotrexate; Ms, micelles; FR-β, folate receptor-β; LPS, lipopolysaccharide; DiR, 1.1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide; SRB, Sulforhodamine B; Cou, coumarin.

Acknowledgments

Yandong Li is the principal corresponding author and Yingli Wang is the secondary correspongding author for this study. This work was supported by the National Natural Science Foundation of China (Grant No. 81903813), the Shanxi Province Natural Science Foundation General Project (No. 202303021211171), the Scientific and Technological Innovation Ability Cultivation Project of Shanxi University of Chinese Medicine (Grant No. 2022PY-TH-12), the Scientific Research Fund of Shanxi Administration of Traditional Chinese Medicine (Grant No. 2022ZYYC087), and the Discipline Construct Funds of Shanxi University of Chinese Medicine (Grant No. 2023XKJS-26).

Disclosure

The authors report no conflicts of interest in this work.