Delivered baicalein immunomodulatory hydrogel with dual properties of pH-responsive and anti-infection orchestrates pro-regenerative response of macrophages for enhanced hypertrophic scars therapy

Abstract

Antibiotic-resistant bacterial infections in skin wounds can cause persistent inflammatory responses, which may lead to severe hypertrophic scarring. In this study, a pH-responsive antibacterial hydrogel composed of phenylboronic acid-grafted chitosan (PBCS) and tannic acid (TA) was developed to achieve controlled and long-lasting release of baicalein (BA) to address the critical challenges of bacterial infection, wound healing, and scarring. The composite hydrogel (BA@PBCS-TA) not only demonstrates excellent injectability, self-healing properties, and robust mechanical performance but also exhibits favorable biological characteristics. Through pH-responsive release of BA, it effectively eliminates Methicillin-resistant Staphylococcus aureus (MRSA). In vivo experiments further confirm its ability to significantly inhibit fibroblast activation and reduce abnormal collagen deposition, effectively preventing excessive scar formation. Additionally, network pharmacology has identified Glycogen Synthase Kinase 3 Beta (GSK3β) as a key target for BA in inhibiting hypertrophic scar formation. Cellular experiments further demonstrate that the BA@PBCS-TA hydrogel can suppress GSK3β expression, activate the Wnt/β-catenin signaling pathway to repolarize macrophages into the M2 phenotype, and exhibit significant immunomodulatory effects. These results highlight the BA@PBCS-TA hydrogel's ability to harness the excellent properties of biomaterials and optimize BA's pharmacological effects, ultimately promoting wound healing and offering a strategic solution for scar reduction.

Keywords: Bacterial infection; Baicalein; GSK3β; Hydrogel; Hypertrophic scars; M2 macrophage.

Graphical abstract

Scheme 1

Schematic illustration of the multifunctional hydrogel dressing system designed to promote wound healing in infected environments and inhibit hypertrophic scar formation through pH-responsive drug release and macrophage repolarization.

Fig. 1

BA inhibited M1 macrophage polarization in vitro. (A) chemical structure of BA. RAW264.7 were treated without or with LPS and different concentrations of BA. (B) the expression of inflammatory (TNF-α, IL-6 and IL-1β) and anti-inflammatory cytokines (IL-10 and TGF-β) were quantified by ELISA. (C) immunofluorescence staining and (D) flow cytometry assay was used to examine the expression of CD86 (M1 macrophage marker) and CD206 (M2 macrophage marker). n = 3, ####p < 0.0001 vs. control group; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.001 vs. model group; BA: baicalein; IL-10, interleukin 10; TGF-β, Transforming growth factor-β; IL-6, interleukin 6; TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β.

Fig. 2

Characterization and property of BA@PBCS-TA hydrogel. (A) 1H NMR spectra of chitosan, PB, and BA@PBCS. (B) FTIR spectra of chitosan, PB, and PBCS. (C) XRD spectra of chitosan, PB, and PBCS. (D) UV–Vis spectra of PBCS, BA-TA and BA@PBCS-TA hydrogel. (E) FTIR spectra of PBCS, TA, BA, BA-TA and BA@PBCS-TA hydrogel. (F) XRD spectra of PBCS, TA, BA, BA-TA and BA@PBCS-TA hydrogel. (G) XPS survey scan of the BA@PBCS-TA hydrogel. (H) High-resolution XPS spectra of C 1s. (I) High-resolution XPS spectra of B 1s. (J) Scanning electron microscopy image of BA@PBCS-TA hydrogel. (K) Rheological behavior of BA@PBCS-TA hydrogel. (L) Oscillation strain sweep of the hydrogel. (M) Shear-thinning behavior of the hydrogel. (N) Hydrogel drug release across different pH levels. (O) BA@PBCS-TA hydrogel's self-Healing capability. (P) Hydrogel adhesion performances on diverse surfaces and its spreadability on human or porcine skin.

Fig. 3

Functional performance and biocompatibility evaluation of hydrogels. (A) Photographs of MRSA and E.coil colonies, and (B, C) corresponding quantitative calculation of antibacterial rates after treatments of different hydrogels. (D) Representative scratch assay images of L929 cells treated with different hydrogels and (E) corresponding quantitative analysis of cell migration rates based on scratch area. ELISA was performed to assess the expression of CD31 (F) and VEGF (G) in HUVEC cells following treatment with various hydrogels. (H) Photographs of hemolysis assays and (I) statistically analyzed hemolysis rates. (J) Cytocompatibility of hydrogels and in vivo hematological parameters, including (K)white blood cell (WBC) count -, (L)red blood cell (RBC) count , and (M)platelet count. n = 3, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Hydrogels suppressed HTS formation in MRSA-infected wounds in rabbit ears. (A) Scheme of the HTS experiment. Representative images of (B) the dynamic healing process and (C) MRSA colonies of wound treated with different hydrogels, and (D) corresponding quantitative calculation. n = 3, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.

Fig. 5

Histological analysis of scar tissues in rabbit ears. (A) H&E staining, Masson staining, α-SMA and CD206/86 immunofluorescence staining for each group. Quantitative analysis of (B) thickness and (C) SEI in H&E staining, (D) CVF in Masson staining, (E) α-SMA and (F) CD206/86 in immunofluorescence staining. Flow cytometry assay was used to examine the expression of (G) TNF-α (inflammatory cytokines) and (H) TGF-β (anti-inflammatory cytokines). n = 3, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.

Fig. 6

Identification of intersection targets shared by BA and HTS. (A) Venn diagram of Targets regulated by BA and HTS, respectively. (B) 50 intersection targets of BA and HTS. (C) The PPI network of HTS and BA intersection targets. (D) The 10 core targets of BA with average BC, CC, and degree values higher than the average. GO functional enrichment analysis (E) and KEGG pathway analysis (F) of BA in the treatment of HTS.

Fig. 7

A highlight pathway map of BA against HTS.

Fig. 8

BA promoted M2 macrophage polarization via GSK3β/β-catenin signaling pathways. (A–D) Visualization and (E) molecular dynamics of molecular docking results of BA complexed with GSK3β. (F) Western blot of the protein levels and (G) corresponding quantitative analysis in RAW264.7 with or without BA treatment in different condition.