Ferric Iron/Shikonin Nanoparticle-Embedded Hydrogels with Robust Adhesion and Healing Functions for Treating Oral Ulcers in Diabetes

Abstract

Oral ulcers can be addressed using various biomaterials designed to deliver medications or cytokines. Nevertheless, the effectiveness of these substances is frequently limited in many patients due to poor adherence, short retention time in the mouth, and less-than-optimal drug efficacy. In this study, a new hydrogel patch (FSH3) made of a silk fibroin/hyaluronic acid matrix with light-sensitive adhesive qualities infused with ferric iron/shikonin nanoparticles to enhance healing effects is presented. Initially, this hydrogel forms an adhesive barrier over mucosal lesions through a straightforward local injection, solidifying when exposed to UV light. Subsequently, FSH3 demonstrates superior reactive oxygen species elimination and near-infrared photothermal bactericidal activity. These characteristics support bacterial elimination and regulate oxidative levels, promoting a wound's progression from inflammation to tissue regeneration. In a diabetic rat model mimicking oral ulcers, FSH3 significantly speeds up healing by adjusting the inflammatory environment of the injured tissue, maintaining balance in oral microbiota, and promoting faster re-epithelialization. Overall, the light-sensitive FSH3 hydrogel shows potential for rapid wound recovery and may transform therapeutic methods for managing oral ulcers in diabetes.

Keywords: diabetes; hydrogels; oral ulcers; shikonin, tissue adhesives.

Scheme 1

Depiction of the creation and utilization of FSH3 hydrogel for expedited healing of bacterial infections in wounds of diabetic rats.

Figure 1

Synthesis of matrix hydrogels and evaluation of their adhesive qualities. A) Formation of SFMA, HA‐NB, and the corresponding hydrogels. B) Depiction of ¹H NMR spectra for HA, HA‐NB, SF, and SFMA. C) Illustrative representation of the photo‐induced imine‐crosslinked hydrogel synthesis. D–F) Display of lap‐shear (D), tensile (E), and t‐peel (F) adhesion testing curves for hydrogels varying in HA‐NB levels. G) Overview of testing procedures: lap‐shear, tensile adhesion, and t‐peel tests. H) Results from the t‐peel test using hydrogels with diverse concentrations of HA‐NB. Error bars represent mean ± SD (n = 3).

Figure 2

Analysis of FeSK nanoparticles' physiochemical properties and FSH hydrogels. A,B) SEM imaging (A) and size distribution (B) of FeSK nanoparticles. C,D) Thermal imagery (C) and graphs showing temperature changes (D) in FSH hydrogels. E) Profile of temperature variation in FSH3 hydrogel under different near‐infrared (NIR) irradiation scenarios. F,G) Assessment of swelling (F) and moisture retention capacity (G) in FSH hydrogels. H) Graph illustrating cooling duration in relation to the inverse natural logarithm of the temperature drive during FeSK's cooling stage following 808 nm NIR irradiation (photothermal efficiency = 14.42%). I) SEM images showcasing FSH1 and FSH3 at varied magnifications. J,K) DPPH (J) and ABTS (K) scavenged by FSH1 and FSH3 hydrogels. Error bars represent mean ± SD (n = 3).

Figure 3

Analyzing the mechanics and antioxidant capabilities of FSH hydrogels. A–C) CCK‐8 method assessment of cytotoxic effects exerted by FSH hydrogels on RS1 (A), RAW 264.7 (B), and HGF (C) cells. D) Outcomes of calcein‐AM/PI for tested cells after 72 h of exposure to various treatments. E) Representative images of in vitro tube formation. F–I) Analysis of the rheological characteristics of FSH, including frequency sweeps (F), strain amplitude sweeps (G), dynamic strain steps (H), and shear‐thinning behavior measurements (I). J) Images depicting the application of FSH3 hydrogel on an artificial disc. K) Photographs highlighting the self‐healing properties of the FSH3 hydrogel. Error bars represent mean ± SD (n = 3).

Figure 4

Cellular studies in vitro using FSH hydrogels. A) Migration analysis of RS1 cells following a scratch at 0, 12, and 24 h (scale bar representing 200 µm). B) Remaining area from the scratch test. C–E) Cell viability assessment for RS1 (C), RAW 264.7 (D), and HGF (E) cultures in H₂O₂‐enriched medium with different hydrogel formulations. F–H) Fluorescent imaging of RS1 cells (F), RAW 264.7 macrophages (G), and HGF cells (H) after exposure to H₂O₂ in different sets. Error bars represent mean ± SD (n = 3). *** signifies p < 0.001.

Figure 5

Evaluating the in vitro photothermal antibacterial performance of FSH samples. A) Analysis of antibacterial activity against MRSA and MRPA employing techniques including enumeration, SEM imaging, and fluorescence staining. B,C) Survival rates of MRSA (B) and MRPA (C) in various test samples. D,E) Comparative SYTO9 fluorescence intensity under different experimental setups for MRSA (D) and MRPA (E). F,G) Measurement of biofilm mass for MRSA (F) and MRPA (G). H) Breakdown of established MRSA and MRPA biofilms by various samples, analyzed using crystal violet staining (I), 2D staining (II), and 3D staining (III). Error bars represent mean ± SD (n = 3). *** signifies p < 0.001.

Figure 6

Assessment of the therapeutic efficacy of FSH3 hydrogel on MRSA‐infected dorsal skin lesions in diabetic rats. A) Infrared thermal images of rat wound sites after various treatments. B) Schematic representation of the infected lesion creation and its healing path. C) Representative healing process images of diabetic rat wounds under various treatments, including the extent of wound closure achieved with different therapies on days 1, 3, 7, and 14. D) Day 14 histopathological H&E stain of the lesion area along with detailed images of H&E‐stained sections. E) Masson's trichrome stain at the lesion site on day 14, including magnified views of Masson‐stained sections. F) Immunohistochemical staining for TNF‐α, CD31, CD86, CD206, MPO, and VEGF in the lesion area on day 7.

Figure 7

Assessment of the FSH3 hydrogel for treating MRSA‐induced oral mucosa in diabetic rats. A) Procedure for creating the oral ulcer model. B) Digitized images display the process of ulcer healing among the PBS, Lidocaine, FSH1, FSH1 + NIR, FSH3, and FSH3 + NIR groups over a period from day 1 to day 7. C) Tissue sections undergoing H&E and Masson staining. D) Tissue sections showing staining for TNF‐α, CD31, CD86, CD206, MPO, and VEGF at the oral mucosa ulcer location.

Figure 8

RNA sequencing analysis for NIR‐enhanced FSH3 treatment: A) Quantitative evaluation of DEGs from the data. B) PCA analysis displaying DEGs detected by RNA‐seq between the control and FSH3 + NIR sets. C) Volcano plots displaying DEGs (gray indicates non‐significant genes, red signifies upregulated genes, blue denotes downregulated genes). D) GO enrichment profiling of upregulated genes. E) KEGG pathway profiling for genes that are upregulated. F) Heatmap visualization of the DEGs.