An Injectable Ibuprofen Sustained-Release Composite Hydrogel System Effectively Accelerates Diabetic Wound Healing via Anti-Inflammatory Effects and Angiogenesis

Abstract

Purpose: Excessive inflammation in diabetic wounds, driven by hyperglycemia, prolongs healing, increases the risk of non-healing ulcers, and can lead to severe complications such as amputation or life-threatening infections. Recurrent wound infections and prolonged treatment impose significant economic and psychological burdens, drastically reducing patients' quality of life. Modulating the inflammatory response is a promising strategy to accelerate diabetic wound healing. Ibuprofen (IBU), a widely used anti-inflammatory and analgesic agent, has the potential to promote healing by mitigating excessive inflammation and alleviating wound-associated pain. However, its clinical application is hindered by poor water solubility and a short half-life. Therefore, a controlled and sustained-release system for IBU could enhance its therapeutic efficacy in diabetic wound management.

Materials and methods: Here, we present an in situ multi-crosslinked composite hydrogel system that integrates oxidized alginate (OSA), methacryloylated gelatin (GelMA), and an ibuprofen/amino-modified β-cyclodextrin inclusion complex (IBU/CD-NH₂) via ion crosslinking, photocrosslinking, and Schiff-base reactions.

Results: The optimized hydrogel formulation was synthesized at 35°C, with a P/A molar ratio of 2 and an methacrylamide(MA) volume fraction of 20%. Physicochemical and biocompatibility analyses demonstrated that the IBU-loaded composite hydrogel exhibits enhanced mechanical strength, favorable biocompatibility, tunable degradation, and injectability. This system effectively addresses IBU's solubility and absorption challenges while conforming to wounds of varying shapes and sizes, enabling controlled and sustained drug release. Cellular and animal studies confirmed that the hydrogel continuously and uniformly releases IBU, exerting anti-inflammatory effects while promoting angiogenesis and fibroblast migration. This leads to enhanced granulation tissue formation, collagen deposition, and epidermal regeneration, significantly accelerating wound closure within 14 days.

Conclusion: By simultaneously suppressing inflammation and stimulating tissue regeneration through controlled IBU release, this hydrogel system offers a highly effective strategy for diabetic wound healing and holds strong potential for clinical application.

Keywords: diabetic wound healing; ibuprofen; inflammation; injectable composite hydrogel system; sustained-release.

Figure 1

Schematic of the synthesis and application of the IBU-loaded composite hydrogel system.

Figure 2

Synthesis, modification, and characterization of each hydrogel. (A) Modification method and synthesis diagram of each hydrogel. (B) The highest OD of up to 84.9% of OSA was achieved at a reaction temperature of 35°C and a P/A molar ratio of 2. (C) FTIR results confirmed the successful preparation of OSA. (D) TEM image of the fabricated IBU/CD-NH₂ inclusion complex. Scale bar = 0.02 μm. (E and F). FTIR (E) and ¹H NMR (F) results confirmed the successful modification of GelMA. (G) Results of GelMA with different degrees of methacrylation.

Figure 3

Synthesis and characterization of the composite hydrogel system. (A) Synthesis diagram of the composite hydrogel system. (B) SEM images revealed the microstructure of the composite hydrogel system. (C) Stress‒strain curve revealed that the 20% MA mixture in the composite hydrogel system presented the best mechanical properties. (D) The 20% MA composite hydrogel system presented the lowest swelling ratio. (E) Quantified degradation results of the composite hydrogel system. (F) Injection test of the composite hydrogel system. (G) Release curve of IBU from the composite hydrogel system. (H and I) CCK-8 and 96-hour differential analysis of cell proliferation status (H), live/dead cell staining (I) results revealed the good biocompatibility of the composite hydrogel system. (All the data are shown as the mean ± SD and were analyzed using one-way ANOVA. n=3, *p< 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001).

Figure 4

Gel-IBU promotes the M2 phenotype of macrophages. (A) Schematic diagram of phenotypic changes in macrophages. Lipopolysaccharide (LPS) (B) Morphology of the macrophages changed to the M2 phenotype after Gel-IBU treatment. Scale bar = 50 μm. (C) qPCR analysis of M1-related gene (iNOS, IL-1β) and M2-related gene (Arg1, IL-10) expression in macrophages (D) Immunofluorescence staining results of the control group and different treatment groups. Scale bar = 100 μm. (E) Comparative analysis of the number of cells with positive expression of the corresponding molecular markers in different groups. (All the data are shown as the mean ± SD and were analyzed using one-way ANOVA. n=3, *p< 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001).

Figure 5

Gel-IBU promotes tube formation and fibroblast migration. (A) CCK-8 results for HUVECs treated with different CM. (B) Results of the tube formation assay of MUVECs treated with different Gel-IBU-derived M2-CM. Compared to the control, MUVECs in the CM-M2 group exhibited significantly greater angiogenic capacity, with CM-M2 (25 μg/mL) demonstrating the highest efficiency, forming 29 tubes and 38 nodes. (C) CCK-8 results for L929 cells treated with different CM. (D) Scratch assay results for L929 cells treated with different Gel-IBU-derived M2-CM. (E) Statistical results of the number of tube-forming nodes and tubes formed by HUVECs after treatment with different CM. (F) Statistical results of the L929 migration ratio after treatment with different CM. (All the data are shown as the mean ± SD and were analyzed using one-way ANOVA. n=3-5, *p< 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001).

Figure 6

Gel-IBU accelerates diabetic wound healing in vivo. (A) Schematic diagram of the establishment of a diabetic wound model and the Gel-IBU treatment process. (B) Statistical results of wound-healing assay for the different treatment groups. (C) Analysis of the wound-healing ratio in different treatment groups. (D) H&E staining results for the different treatment groups. Scale bar = 100 μm. (E) Masson’s trichrome staining results for the different treatment groups. Scale bar = 100 μm. (F) Analysis of Masson’s trichrome-stained areas in different treatment groups. (All the data are shown as the mean ± SD and were analyzed using one-way ANOVA. n=3, *p< 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001).

Figure 7

Gel-IBU can promote M2 polarization of macrophages and promote angiogenesis in vivo. (A) Immunofluorescence staining results of CD86/iNOS and CD206/Arg-1 expression in different treatment groups on days 7 and 14. Scale bar = 100 μm. (B) Statistical analysis of the numbers of CD86/iNOS- and CD206/Arg-1-positive cells in different treatment groups on days 7 and 14. (C) Immunofluorescence staining results of CD31 and α-SMA expression in different treatment groups on days 7 and 14. (Scale bar = 100 μm. All the data are shown as the mean ± SD and were analyzed using one-way ANOVA. n=3, *p< 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001).