Hyaluronic Acid Hydrogels Hybridized With Au-Triptolide Nanoparticles for Intraarticular Targeted Multi-Therapy of Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by synovial inflammation in multiple joints. Triptolide (TP) is a disease-modifying anti-rheumatic drug (DMARD) highly effective in patients with RA and has anti-inflammatory properties. However, its clinical application has been limited owing to practical disadvantages. In the present study, hyaluronic acid (HA) hydrogel-loaded RGD-attached gold nanoparticles (AuNPs) containing TP were synthesized to alleviate the toxicity and increase therapeutic specificity. The hydrogels can be applied for targeted photothermal-chemo treatment and in vivo imaging of RA. Hydrogel systems with tyramine-modified HA (TA-HA) conjugates have been applied to artificial tissue models as surrogates of cartilage to investigate drug transport and release properties. After degradation of HA chains, heat was locally generated at the inflammation region site due to near-infrared resonance (NIR) irradiation of AuNPs, and TP was released from nanoparticles, delivering heat and drug to the inflamed joints simultaneously. RA can be penetrated with NIR light. Intraarticular administration of the hydrogels containing low dosage of TP with NIR irradiation improved the inflamed conditions in mice with collagen-induced arthritis (CIA). Additionally, in vitro experiments were applied to deeply verify the antirheumatic mechanisms of TP-PLGA-Au@RGD/HA hydrogels. TP-PLGA-Au@RGD/HA hydrogel treatment significantly reduced the migratory and invasive capacities of RA fibroblast-like synoviocytes (RA-FLS) in vitro, through the decrease of phosphorylation of mTOR and its substrates, p70S6K1, thus inhibiting the mTOR pathway.

Keywords: NIR imaging; TP-PLGA-Au@RGD/HA hydrogels; mTOR/p70S6K pathway; photothermal-chemo treatment; rheumatoid arthritis.

FIGURE 1

Preparation process of TP-PLGA-Au@RGD/HA hydrogels and schematic illustration of the anti-inflammatory effect of TP-PLGA-Au@RGD/HA hydrogels in CIA mice.

FIGURE 2

(A) Size distribution and (B) zeta potential of TP-PLGA NPs, TP-PLGA-Au NPs, and TP-PLGA-Au@RGD NPs. (C) Zeta potential of TP-PLGA-PEI NPs. (D) FT-IR spectra of the TP-PLGA NPs and TP-PLGA-Au@RGD NPs. (E) UV–vis–NIR spectra of TP-PLGA-Au@RGD NPs. (F) TEM image of TP-PLGA-Au@RGD NPs. (G) TEM image showed that TP-PLGA-Au@RGD NPs were embedded within the matrix of hydrogels.

FIGURE 3

(A) Synthesis of TA-HA. (B) 1H NMR spectra of modified HA. (C-1) The morphology of the freeze-dried TA-HA, was white flocculent. (C-2) SEM image of TA-HA. (C-3) The morphology of freeze-dried TA-HA in PBS solution was transparent, colorless, and fluid. (C-4) Under the catalysis of HRP and H2O2, TA-HA formed hydrogels after 3 minutes, and the morphology of hydrogels had a transparent and non-fluid semi-solid nature.

FIGURE 4

(A) Temperature curves of TP-PLGA-Au@RGD/HA hydrogels (808 nm). (B) Temperature evolution of TP-PLGA-Au@RGD/HA hydrogels over 4 on-off cycles with 808 nm laser irradiation. (C) Thermal images of TP-PLGA-Au@RGD/HA hydrogels under 808 nm laser irradiation at different time points.

FIGURE 5

(A) Profiles of TP release from TP-PLGA-Au@RGD/HA hydrogels and TP-PLGA-Au@RGD NPs under initial NIR irradiation for 10 min. (B) Profiles of TP release from TP-PLGA-Au@RGD/HA hydrogels with or without NIR irradiation for 10 min. Data are expressed as mean values for n = 3, and error bars represent the standard deviation of the means (*p < 0.05).

FIGURE 6

(A) Anti-proliferative effect of TP-PLGA-Au@RGD/HA hydrogels on RA-FLS. The data were obtained from three independent experiments and are expressed as mean ± SD. (B) TP-PLGA-Au@RGD/HA hydrogels reduced levels of phospho-mTOR (p-mTOR), with a p-mTOR/total median reduction of 54%, and levels of phosphorylation of mTOR targets with a phospho-p70S6K (p-p70S6K/total median reduction of 38%, indicating inhibition of the mTOR pathway. Data are expressed as mean ± SEM values. *P < 0.05 compared with the blank group.

FIGURE 7

(A) Fluorescence images of inflamed paws of CIA mice of non-injection, 12 h post-injection, and 24 h post-injection of TP-PLGA-Au@RGD-Cy7/HA hydrogels, and mice were intraarticularly administered. (B) Arthritis index versus time for CIA mice. Arthritis indices were significantly different among groups (*p < 0.05).

FIGURE 8

(A) Histological findings of synovial tissues from healthy mice and CIA mice on day 28 after different treatments. H&E (synovial inflammation, original magnifications × 100, and immunohistochemical staining for IL-1β, IL-6, and TNF-α, original magnifications × 20) (B) Semiquantitative analysis of histopathological evaluation (synovial inflammation and immunohistochemical staining for IL-1β, IL-6, and TNF-α). Asterisks (*) represent significance compared with the untreated mice at *p < 0.05.

FIGURE 9

(A) Micro-computed tomography images of hind paws of CIA mice in different treatment groups. (B) Effects of G4 on bone destruction in CIA mice. Bone volume/total volume (BV/TV; %) in CIA mice. *p < 0.05 versus G1; **p < 0.01 versus G1. (C) Histological sections of major organs extracted on day 28 after administration of health (top), TP-PLGA-Au@RGD/HA hydrogels combined with NIR irradiation (bottom, G4) and G2. Images were acquired under ×20 magnification.