Insulin/PHMB-grafted sodium alginate hydrogels improve infected wound healing by antibacterial-prompted macrophage inflammatory regulation

Abstract

Background: Non-healing chronic wounds with high susceptibility to infection represent a critical challenge in modern healthcare. While growth factors play a pivotal role in regulating chronic wound repair, their therapeutic efficacy is compromised in infected microenvironments. Current wound dressings inadequately address the dual demands of sustained bioactive molecule delivery and robust antimicrobial activity.

Results: In this study, we developed a sodium alginate hydrogel (termed P-SA/Ins), which incorporated polyhexamethylene biguanide (PHMB) grafting and long-acting glargine insulin loading. P-SA/Ins exhibited the favorable physicochemical performance, biocompatibility and antibacterial efficacy against both Gram-negative and Gram-positive pathogens through inhibition of bacterial proliferation and biofilm formation. Glargine insulin was applied to prolonged insulin delivery. P-SA/Ins treatment attenuated S. aureus induced pro-inflammatory cytokine cascades in macrophages. The evaluation in vivo using a rat model with S. aureus infected wound demonstrated that P-SA/Ins significantly enhanced wound healing and optimized skin barrier through antimicrobial-mediated modulation of macrophage polarization and subsequent inflammatory cytokine profiling.

Conclusions: Our findings demonstrate that P-SA/Ins promotes wound healing and restores epidermal barrier integrity, indicating its potential as a therapeutic dressing for chronic wound healing, particularly in cases with infection risk.

Keywords: Infected wound healing; Inflammation regulation; Insulin; PHMB; Skin barrier.

Scheme 1

Schematic illustration of P-SA/Ins promoted wound healing and optimized the skin barrier by antibacterial-prompted macrophage inflammatory regulation

Fig. 1

Characterization of P-SA/Ins. A: Schematic and general view of P-SA/Ins. B: SEM images of each group. The scales bars: 500 μm and 100 μm. C: FI-TR spectra of gels of each group. D: The swelling ratio of each group in 37℃ PBS within 48 h. E: The cumulative released amount of insulin from SA/Ins and P-SA/Ins incubated in 37℃ PBS

Fig. 2

Biocompatibility of P-SA/Ins. A-C: Representative images and statistical data of Cell Toxicity assay of HDF and HUVEC cell for 24 h. (ns: p > 0.05, scales bars: 0.5 mm). D-E: The statistical data of cell growth of HDF (D) and HUVEC (E). (** p ≤ 0.01, * p ≤ 0.05) F-G: Wound healing assay and statistical data of HDF(F-G) and HUVEC (H-I). (Scale bar: 1 mm. *** p ≤ 0.001, * p ≤ 0.05)

Fig. 3

Antibacterial and inflammation regulation of P-SA/Ins in vitro. A: Schematic of antibacterial assay of P-SA/Ins. B: The statistical data of OD value counting assay of S. aureus. (***p ≤ 0.001, n = 12). C: The statistical data of crystal violet assay for S. aureus biofilm formation (*** p ≤ 0.001, n = 9). D: The statistical data of the diameters of inhibition zones for S. aureus and E. coli. E: Representative SEM images showed S. aureus. The scales bars: 1 μm. Red arrow: the good condition and aggregation of S. aureus; Yellow arrow: scattered and crimpled S. aureus. F: Schematic of antibacterial and inflammation regulation of P-SA/Ins (scar bar: 1 μm). G: Heatmap of inflammatory cytokines of leaching culture medium cultured with macrophage and S. aureus. H-L: The statistical data of IL-1β, TNF-α, IL-1α, IFN-γ, IL-13 in leaching culture medium of cultured with macrophage and S. aureus. (ns: p > 0.05, ** p ≤ 0.01, * p ≤ 0.05.)

Fig. 4

Effect of wound healing of P-SA/Ins. A: The establishment of rat model with infected full thickness excision dorsal wounds. B: Photographs of healing wounds of rats. C: Quantitative analysis of percentage of wound area. (SA vs. SA/Ins., ^#P < 0.05; SA vs. P-SA/Ins, ***P < 0.01, **P < 0.01, *P < 0.05, n = 6). D-E: H&E (D) and Masson (E) staining of rat wounds on 14 days after treatment (Scale bar: 1 mm). F: The statistical data of the length of epidermal tongue (ns: p > 0.05, ** p ≤ 0.01, * p ≤ 0.05, n = 6). G: The statistical data of the collagen content (ns: p > 0.05, **** p ≤ 0.0001, *** p ≤ 0.001, n = 6)

Fig. 5

Antibacterial effect of P-SA/Ins in wound healing. A: The representative images of Gram stain to detect S. aureus (Yellow arrow) in wound tissue on 14 days after treatment (Scale bar: 1 mm). B: The amount of S. aureus in wound tissue after treatment for 0, 5, 9, and 14 days. Ctrl vs. SA/Ins, # P < 0.05. Ctrl vs. P-SA/Ins, ***P < 0.01, **P < 0.01; n = 6). C: The insulin concentration in wound. (***P < 0.01, **P < 0.01, *P < 0.05, n = 6)

Fig. 6

Regulation of Infected Wound Inflammation by P-SA/Ins. A: Multi-color IHC staining show CD68, iNOS, Arg-1 in rat wounds. Scale bar: 50 μm and 25 μm. B-E: The statistical data of IL-1β, IL-13, IL-10, TNF-α level in rat wounds. (ns: p > 0.05, ***P < 0.01, **P < 0.01, *P < 0.05, n = 3)

Fig. 7

Optimized healing of infected wounds by P-SA/Ins. A: The skin nails in zoom-in of Masson staining. Scale bar: 250 μm. Black dashed line: the epidermis. Red arrow: basement membrane and skin nails. B: Immunofluorescent staining showed K14, ZO-1in rat wounds on 14 days after treatment. Scale bar: 50 μm. White dashed line: basement membrane. White arrow: Expression and distribution of K14, ZO-1 C: The statistical data of skin nails in rat wounds. (ns: p > 0.05, **P < 0.01, n = 5) D: The statistical data of K14 positive rate cell in rat wounds. (ns: p > 0.05, **P < 0.01, n = 5). E: The statistical data of ZO-1 positive rate cell in rat wounds. (ns: p > 0.05, *P < 0.05, n = 5)