Transdermal microneedle integrating a biomimetic self-enhancing Fenton reaction nano-reactor for alleviating rheumatoid arthritis by inflammatory microenvironment remodeling

Abstract

Rationale: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, and persistent inflammation in multiple joints is an important sign for the progression of RA. To this end, we developed the transdermal microneedle integrating biomimetic self-enhancing Fenton reaction nano-reactor, for the purposes of eliminating reactive oxygen species, reducing hypoxia and inflammation, and regulating macrophage phenotype. Methods: A novel biomimetic self-enhanced Fenton reaction nano-reactor was synthesized using an M1 macrophage cell membrane-coated tannic acid-modified iron oxide nanoparticle (IO-NH₂-TA TNPs@M1). The regulatory mechanisms of the IO-NH₂-TA TNPs@M1 were investigated by evaluating ROS scavenging, degree of hypoxia, adsorption of pro-inflammatory factors, and M2 macrophage polarization. Then, the nano-reactor was incorporated into a dissolving microneedle, utilizing enzyme-cut oligomeric sodium hyaluronate, and subsequently assessed for pharmacodynamics and safety. Results: In vitro mechanisms of IO-NH₂-TA TNPs@M1 included eliminating ROS, inhibiting the expression of HIF-1α, decreasing the content of pro-inflammatory factors (IL-6 and TNF-α), and inducing macrophage M2 polarization. Pharmacodynamic and in vitro mechanistic studies showed that IO-NH₂-TA TNPs@M1DM maximally alleviated joint swelling and fever, protected joint cartilage, improved the local hypoxia environment and promoted macrophage M2 polarization. Cytotoxicity assays and HE staining showed that IO-NH₂-TA TNPs@M1DM displayed good biocompatibility. Conclusions: This study designed and synthesized an innovative biomimetic self-enhancing Fenton reaction nano-reactor, and utilized microneedles for the transdermal delivery, providing a scientific and effective new strategy for the precise treatment of RA.

Keywords: M2 macrophage polarization; ROS scavenging; inflammation; reducing hypoxia.

Figure 1

Schematic illustration of preparation and mechanisms of IO-NH₂-TA TNPs@M1DMs.

Figure 2

UV spectra of IO-NH₂ NPs and IO-NH₂-TA NPs (A). TEM images of IO-NH₂ NPs and IO-NH₂-TA NPs (B). Drug loading and encapsulation efficiency values of NPs formed at diverse ratios (w/w) of IO-NH₂-TA TNPs (C). SDS-PAGE protein profile of M1, M1M, and IO-NH₂-TA TNPs@M1 (D). Size distribution, zeta potential and stability of IO-NH₂ NP, IO-NH₂-TA NPs, IO-NH₂-TA TNPs and IO-NH₂-TA TNPs@M1 (E-G). Drug release of IO-NH₂-TA TNPs and IO-NH₂-TA TNPs@M1 at different concentrations of H₂O₂ (H).

Figure 3

Flow cytometry analysis (A-B) and confocal laser scanning microscopy images (C) of ROS in MH7A cells under various treatments. Scale bars, 20 µm. Immunofluorescence staining of HIF-1α (D) in MH7A cells under various treatments. Scale bars, 20 µm.

Figure 4

Expression levels of IL-6 (A) and TNF-α (B) in MH7H cells under various conditions. Flow cytometry analysis of CD86 and CD206 expression in macrophages (C) and quantitative analysis (D).

Figure 5

(A) Digital microscope images (scale bar, 1 mm), optical microscope images, and SEM images of IO-NH₂-TA TNPs@M1DM (scale bar, 50 µm). (B) EDS mapping of IO-NH₂-TA TNPs@M1DM. Scale bar, 50 µm. (C) Mechanical properties of microneedles.

Figure 6

(A) Penetration of microneedles loaded with various microneedles (scale bar, 1 mm). (B) Mechanical properties of microneedles. Digital microscope images of IO-NH₂-TA TNPs@M1DM at 0, 2, 5, 10 and 30 min after piercing into the rat skin in vivo (scale bar, 50 µm), and the percentage of undissolved volume of IO-NH₂-TA TNPs@M1DM (n = 3). (C) Amounts of Fe in penetrated skin (n = 3). (D) Skin penetration and retention amounts of Tof (n = 3).

Figure 7

Body weight change (A), paw thickness (B), paw temperature (C) and arthritis joint scores of rats (D) under different treatments (n = 6). Rat joints at the end of different treatments (E).

Figure 8

(A) HE staining, Safranin O solid green staining of rat joints following different treatments. Scale bar, 100 µm. (B) HIF-1α immunofluorescence staining of rat joints following different treatments. Scale bar, 200 µm. (C) Flow cytometry analysis of CD86 and CD206 expression in synovial tissue of rat joints.

Figure 9

(A) Cytotoxicity of the different NPs on MH7A and RAW 264.7 cells. (B) Histopathological staining of major organs. Scale bar, 25 µm.