Thermosensitive Injectable Hydrogels for Intra-Articular Delivery of Etanercept for the Treatment of Osteoarthritis

Abstract

The intra-articular administration of drugs has attracted great interest in recent decades for the treatment of osteoarthritis. The use of modified drugs has also attracted interest in recent years because their intra-articular administration has demonstrated encouraging results. The objective of this work was to prepare injectable-thermosensitive hydrogels for the intra-articular administration of Etanercept (ETA), an inhibitor of tumor necrosis factor-α. Hydrogels were prepared from the physical mixture of chitosan and Pluronic F127 with β-glycerolphosphate (BGP). Adding β-glycerolphosphate to the system reduced the gelation time and also modified the morphology of the resulting material. In vitro studies were carried out to determine the cytocompatibility of the prepared hydrogels for the human chondrocyte line C28/I2. The in vitro release study showed that the incorporation of BGP into the system markedly modified the release of ETA. In the in vivo studies, it was verified that the hydrogels remained inside the implantation site in the joint until the end of the study. Furthermore, ETA was highly concentrated in the blood of the study mice 48 h after the loaded material was injected. Histological investigation of osteoarthritic knees showed that the material promotes cartilage recovery in osteoarthritic mice. The results demonstrate the potential of ETA-loaded injectable hydrogels for the localized treatment of joints.

Keywords: Pluronic; chitosan; etanercept; intra-articular delivery; osteoarthritis; thermosensitive hydrogels.

Figure 1

Characterization of CS/PF and CS/PF/BGP hydrogels. (A) Photographs of the CS/PF and CS/PF/BGP hydrogel transition from the liquid state at 4 °C (left) to the gel state at 37 °C (right). (B) Gelation times of CS/PF and CS/PF/BGP hydrogels at 37 °C. Values represent mean ± SD (n = 4, standard one-way ANOVA, ** p < 0.01 and **** p < 0.0001). (C) SEM of CS/PF (1) and CS/PF/BGP (2) hydrogels illustrating the microstructure. (D) FTIR spectra of PF (a), CS (b), BGP (c), CS/PF hydrogel (d), and CS/PF/BGP hydrogel (e).

Figure 2

In vitro ETA release profile. (A) Release behavior of the ETA from CS/PF and CS/PF/BGP hydrogels. The error bars represent mean ± SD (n = 3). (B) Comparison of the amount of ETA released at each time point. Values represent mean ± SD (n = 3; two-way ANOVA, Sidak’s multiple comparisons test, ** p < 0.01, *** p < 0.001 and **** p < 0.0001).

Figure 3

Viability assay and morphology of cells grown on hydrogels. (A) Cell viability of C28/I2 cells grown on CS/PF and CS/PF/BGP hydrogels. Values represent mean ± SD (n = 3; two-way ANOVA, Tukey’s multiple comparisons test, * p < 0.05 and ** p < 0.01). (B) LIVE/DEAD^® staining of chondrocytes seeded on CS/PF and CS/PF/BGP hydrogels after 7 and 10 days in culture. Scale bar = 200 µm.

Figure 4

In vivo ETA release profiles. The red and blue curves represent the ETA solution and the hydrogel + ETA, respectively, injected in the knee joint of mice with OA. The error bars represent mean ± SD (n = 6). Statistical significance was determined by two-way ANOVA with multiple comparisons; *** p < 0.001.

Figure 5

Mice knee images after hydrogels injection (A) NIR fluorescence imaging of mice knee joints taken at different time points after intra-articular injection of empty and ETA-loaded hydrogels containing NIR-780. (B) Graph depicting the decrease of fluorescence intensity after injection over the duration of the experiment. Data are presented as mean ± SD (n = 6).

Figure 6

Representative mouse knee images in µ-CT and histological analysis (A) In vivo µ-CT scans of mouse knee joints showing a 3D view of the cartilage surface. From left to right healthy knee (negative control), OA knee with PBS injection (sham treatment/positive control), OA knee treated with empty hydrogel, and OA knee treated with ETA-loaded hydrogel. (B) Histological staining with Safranin O/Fast Green of sections from healthy, sham-treated, and hydrogel-treated mouse knees.