Baicalein based nano-delivery system restores mitochondrial homeostasis through PPAR signaling pathway to promote wound healing in diabetes

Abstract

Wound healing in diabetes is a substantial clinical challenge due to the hyperglycemic microenvironment, high pH, bacterial infection, persistent inflammation, and impaired cellular functions, attributed to mitochondrial dysfunction. Here, we have developed an injectable photo-crosslinking nanocomposite hydrogel (BA/GOx@ZIF-8@GelMA, BGZ@GelMA) with baicalein (BA) and glucose oxidase (GOx) loaded Zinc metal-organic framework (ZIF-8) based on methacrylated gelatin (GelMA) to accelerate diabetic infected wound healing by regulating subcellular and cellular functions. The combination of ZIF-8 and BA gives the hydrogel excellent antibacterial properties. A high blood sugar environment triggers the release of GOx in BGZ@GelMA, reducing local glucose and pH, producing hydrogen peroxide (H₂O₂), and releasing BA and Zinc ions (Zn²⁺). This process provides a suitable microenvironment for wound healing. Zn²⁺ can significantly inhibit the proliferation of Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli). The released BA can clear ROS in cells and mitochondria, restore mitochondrial function and stability, and make the hydrogel fundamentally improve the cell function damage induced by hyperglycemia, and ultimately promote cell proliferation, migration and angiogenesis. In general, our multifunctional nanocomposite hydrogel provides a new strategy for diabetes wound healing at the subcellular and cellular functional levels.

Keywords: Baicalein; Diabetic wound healing; Metal-organic framework; Mitochondrion.

Scheme 1

Schematic diagram for the preparation of nanocomposite hydrogel and its function mechanism in diabetic wound treatment

Fig. 1

Characterization of the nanocomposite hydrogel. (A) Schematic diagram of synthesis of nanoparticles and hydrogels. (B) SEM images of ZIF-8, BA@ZIF-8, and BA/GOx@ZIF-8 nanoparticles. (C) Nanoparticle size analysis of the ZIF-8, BA@ZIF-8, BA/GOx@ZIF-8. (D) FTIR spectra of the ZIF-8, BA@ZIF-8, and BA/GOx@ZIF-8 nanoparticles. (E) XRD spectra of ZIF-8, BA@ZIF-8, and BA/GOx@ZIF-8 nanoparticles. (F) ¹H NMR spectra of the GelMA and gelatin. (G) SEM images of the nanocomposite hydrogel. (H) Zeta potential of the ZIF-8, BA@ZIF-8, and BA/GOx@ZIF-8 nanoparticles. (I) The images of injectability of hydrogel

Fig. 2

Biocompatibility evaluation of BGZ@GelMA hydrogel. (A) Proliferation ability of HUVECs determined by the CCK-8 assay after different treatments. (B) Proliferation ability of L929 determined by the CCK-8 assay after different treatments. (C) Representative live/dead staining of HUVECs after incubation for 3 days (scale bar: 100 μm). (D) The representative images of the EDU assay (scale bar: 200 μm). (E) Quantification of the EDU assay

Fig. 3

The antimicrobial effect of BGZ@GelMA hydrogels in vitro. (A) Colonies of different groups of S. aureus and E. coli on agar plates. (B) Antimicrobial ratio of hydrogels against S. aureus. (C) Antimicrobial ratio of hydrogels against E. coli. (D) Representative SEM images of S. aureus and E. coli in different groups (scale bar: 1 μm). (E) Representative images of live/dead fluorescence staining of the S. aureus and E. coli (scale bar: 100 μm)

Fig. 4

Effect of BGZ@GelMA on intracellular ROS and mitochondrial function. (A) Representative Fluorescence images of intracellular ROS (scale bar: 200 μm). (B) Representative Fluorescence images of Mitochondrial ROS (scale bar: 100 μm). (C) Fluorescence images of mitochondrial membrane potential JC-1 staining (scale bar: 50 μm). Quantitative analysis of the (D) intracellular ROS, (E) mitochondrial ROS, and (F) JC-aggregates/monomer fluorescence ratio

Fig. 5

BGZ@GelMA promotes cell migration and angiogenesis in vitro. (A) The representative images of the transwell assay (scale bar: 50 μm). (B) The representative images of scratch migration assay (scale bar: 200 μm). (C) Representative images of tube formation experiments (scale bar: 200 μm). (D) Quantitative analysis of HUVECs transwell migration cells counts. (E) Quantitative analysis of the scratch healing rate of HUVECs. (F) Quantitative analysis of the number of junctions

Fig. 6

BGZ@GelMA hydrogel promotes VEGF expression. (A) Immunofluorescence images of VEGF in different treatment groups, F-actin (red), VEGF(green), DAPI(blue) (scale bar: 50 μm). (B) The protein expression of VEGF in the different groups. (C) Quantitative analysis of VEGF protein. (D) Quantitative analysis relative fluorescence intensity of VEGF

Fig. 7

Regulatory mechanism of BGZ@GelMA in promoting diabetic wound healing. (A) PCA analysis of the global sample. (B) Volcano plot displaying up-regulated and down-regulated genes (fold change ≥ 2 and p < 0.05) in HUVECs co-cultured with BGZ@GelMA. (C) Differentially expressed terms analyzed by the GO enrichment method. (D) Analysis of KEGG-enriched signaling pathways of DEGs and the corresponding genes

Fig. 8

BGZ@GelMA hydrogel promotes diabetic wound healing in vivo. (A) Schematic diagram of the wound treatment process in diabetic rats. (B) Percentage of trauma area in diabetic rats at different time points. (C) Representative digital photos showing the healing progression of diabetic wounds in rats subjected to various treatments (scar bar: 10 mm). (D) Images of diabetic wound traces on days 0, 5, 10, 14, and 21. (E) H&E staining of the collected wound skin tissue in different groups at 7d and 14d. (F) Quantitative analysis of the wound edge length in different groups. (G) Quantitative analysis of collagen deposition in different groups

Fig. 9

The effect of the BGZ@GelMA hydrogel on the three wound healing stages. (A) Masson staining of the collected wound skin tissue in different groups at 7d and 14d. (B) Representative immunohistochemistry staining images of IL-6 and TNF-α in the different groups (scale bar: 50 μm). (C-D) Statistical analysis of the positive area (IL-6 and TNF-α). (E) Representative immunohistochemistry staining images of VEGF and CD31 in the different groups (scale bar: 50 μm). (F-G) Statistical analysis of the positive area (VEGF and CD31)