Reversing inflammatory microenvironment by a single intra-articular injection of multi-stimulus responsive lipogel to relieve rheumatoid arthritis and promote joint repair

Abstract

Rheumatoid arthritis (RA) is a common chronic disease dominated by inflammatory synovitis, which is characterized with hyperplastic synovium, up-regulated matrix metalloproteinase (MMP) expression, hypoxic joint cavity and excessive reactive oxygen species (ROS) accumulation. Such local adverse microenvironment in RA joints further exacerbates the infiltration of synovial inflammatory cells, especially M1-type macrophages. Regulating intra-articular pathological conditions, eliminating excess M1 macrophages or converting them to an anti-inflammatory M2 phenotype may break the vicious progression circle. Herein, we develop a multi-stimulus responsive lipogel as effective platform to relieve RA symptoms and promote articular cartilage recovery via reversing its inflammatory microenvironment. The injectable lipogel is fabricated by loading polydopamine nanoparticles and methotrexate into a thermosensitive gel, and intra-articularly injected to form the therapeutic depot (PDA/MTX@TSG) in situ. The gel degrades slowly under esterase hydrolysis, and maintains sustained drug release in physiological conditions. Meanwhile, it can 1) induce a reversible gel-sol phase transition upon mild photothermal treatment (external NIR light control), and 2) specifically respond to MMP-rich RA microenvironment (internal enzymatic hydrolysis effect). Such stimulus-responsive system can deliver therapeutic components in a controllable manner, and significantly reverse adverse inflammatory microenvironment of RA joints through ROS eliminating, hypoxia alleviating, and M1-M2 macrophage polarization effects. Animal experiments indicate that observable RA relief and joint repair are realized after a single lipogel injection combined with NIR irradiation. Our study highlights the importance of altering local RA microenvironment via anti-inflammatory macrophage polarization, and therefore presents a potent therapeutic strategy for RA treatment in clinical intervention.

Keywords: Drug delivery; Lipogel; Macrophage polarization; Methotrexate; Reactive oxygen species; Rheumatoid arthritis.

Graphical abstract

Scheme 1

Schematic illustration for the fabrication of PDA/MTX@TSG lipogel, its enzyme-erosion/NIR photothermal-assisted drug releasing behavior, and in vivo therapeutic mechanism on RA relief.

Fig. 1

Characterization of PDA/MTX@TSG. a) Photograph of gelatinous PDA/MTX@TSG when exposed to PBS (left), and schematic diagram for its internal structure (right). b) Hydrodynamic size distribution and TEM image (inset) of the synthesized PDA nanoparticles. Scale bar: 200 ​nm. c) SEM image of the blank TSG sample. d) SEM image of the PDA/MTX@TSG sample. e) Infrared thermal images of PBS, TSG and PDA@TSG upon 10-min NIR laser irradiation (808 ​nm, 1.0 ​W/cm⁻²). f) Temperature changes of different samples under NIR irradiation. g) Temperature-induced G′ and G″ modulus variations of the PDA/MTX@TSG prepared with lecithin/diolein ratio at 35:65. h) Rheological measurements of the sample viscosity and shear stress changes versus the shear rate. i) Photographs displaying the reversible phase transition behavior (left) and the injectable PDA/MTX@TSG precursor through a syringe #5 needle (right). j) Oxygen production in different groups as indicated when incubated with H₂O₂ solution. k) Representative photographs showing the gel morphology changes after incubated with PBS, MMP-3, or esterase. l) The corresponding weight changes of PDA/MTX@TSG during the 28-day degradation process.

Fig. 2

In vitro drug release, biocompatibility, ROS scavenging and hypoxia-alleviating properties. a) Cumulative MTX release from PDA/MTX@TSG upon 1 time and 5 times of enzyme stimulation or b) NIR irradiation. c) The viability of HSF, BMSC or RAW264.7 ​cells with/without LPS activation after different treatments for 7 days. d) Fluorescence images of LPS-activated RAW264.7 ​cells after various treatments as indicated for 4 ​h. Scale ​= ​100 ​μm. Local magnification scale ​= ​30 ​μm. e) Fluorescent staining of RAW264.7 ​cells showing intracellular ROS (scale bar: 100 ​μm) and HIF-1α (scale bar: 50 ​μm) expression in different groups. Quantitative analysis on the corresponding positive area of intracellular f) ROS and g) HIF-1α staining.

Fig. 3

In vitro M1 to M2 polarization of macrophages induced by PDA/MTX@TSG. a) Immunofluorescence staining of CD68 (macrophage marker), CD86 (M1 marker), CD206 (M2 marker) and cell nucleus (DAPI) in LPS-activated RAW264.7 ​cells after different treatments. Scale bar ​= ​100 ​μm. Expression of b) M1-type and c) M2-type macrophage markers measured by qRT-PCR assay.

Fig. 4

Inhibition of inflammation and hypoxia microenvironment on AIA rats after intra-articular injection of PDA/MTX@TSG. a) Immunofluorescence staining of M1 (CD86, green) and M2 (CD206, red) macrophages in the synovium of AIA rats' joints after different treatments. Scale bar: 100 ​μm. Levels of b) M1 markers (TNF-α, IL-6), and c) M2 markers (Arg-1, IL-10) in synovial tissues quantitatively analyzed by qRT-PCR assay. d) HIF-1α (red) expression in the femur of AIA rats' knee joints after different treatments for six weeks. Scale bar: 100 ​μm. e) Quantitative analysis of the HIF-1α levels in synovial tissues by qRT-PCR assay.

Fig. 5

Evaluation of in vivo therapeutic effect. a) Schematic diagram for the treatment process on AIA rats. b) Photographs of the paw appearance after 6 weeks, and the 3D micro-CT images of rat ankle joints after 1, 3, and 6 weeks, respectively. The changes of c) ankle joint diameter and d) AI score on AIA rats in different groups during 6 weeks. Analysis on bone parameters including e) BV/TV, f) Tb. Sp, and g) Tb·Th of the rat knee joints after 6-week treatments. h) H&E staining, safranin-O staining and TNF-α (red, blue: DAPI) immunofluorescence staining of rat knee cartilages. Abbreviations: hyaline cartilaginous lining (HC), bone (B), pannus (P). Scale bars: 100 ​μm. i) Cartilage thickness and j) cartilage retention rate of the rat knee joints in different groups after 6 weeks. k) TNF-α expression in the rat synovium after 6-week treatments measured via ELISA method.