Construction of heparin-based hydrogel incorporated with Cu5.4O ultrasmall nanozymes for wound healing and inflammation inhibition

Abstract

Excessive production of inflammatory chemokines and reactive oxygen species (ROS) can cause a feedback cycle of inflammation response that has a negative effect on cutaneous wound healing. The use of wound-dressing materials that simultaneously absorb chemokines and scavenge ROS constitutes a novel 'weeding and uprooting' treatment strategy for inflammatory conditions. In the present study, a composite hydrogel comprising an amine-functionalized star-shaped polyethylene glycol (starPEG) and heparin for chemokine sequestration as well as Cu_5.4O ultrasmall nanozymes for ROS scavenging (Cu_5.4O@Hep-PEG) was developed. The material effectively adsorbs the inflammatory chemokines monocyte chemoattractant protein-1 and interleukin-8, decreasing the migratory activity of macrophages and neutrophils. Furthermore, it scavenges the ROS in wound fluids to mitigate oxidative stress, and the sustained release of Cu_5.4O promotes angiogenesis. In acute wounds and impaired-healing wounds (diabetic wounds), Cu_5.4O@Hep-PEG hydrogels outperform the standard-of-care product Promogram® in terms of inflammation reduction, increased epidermis regeneration, vascularization, and wound closure.

Keywords: Hydrogels; Inflammatory chemokines; Nanozymes; Reactive oxygen species; Wound healing.

Graphical abstract

Fig. 1

Schematic of Cu_5.4O@Hep-PEG hydrogels in the treatment of diabetic wounds. (A) Preparation of Cu_5.4O@Hep-PEG hydrogels. I The Hep-PEG gel network is prepared by covalent cross-linking between the carboxyl groups in heparin sodium and the amino groups in 4-arm PEG-NH₂. II The Cu_5.4O@Hep-PEG hydrogel is prepared by soaking Hep-PEG hydrogel in a Cu_5.4O USNP solution for 2 h. (B) In diabetic wounds, high levels of inflammation generate intracellular ROS, which modulates the production of chemokines associated with inflammation. Furthermore, the pro-inflammatory factors generated induce the migration and infiltration of more inflammatory cells. The infiltrated inflammatory cells produce excessive ROS to further aggravate inflammation, leading to a feedback cycle. To break this cycle completely, it is essential to disrupt their mutual interaction by simultaneously scavenging ROS and capturing pro-inflammatory factors. Cu_5.4O@Hep-PEG can release Cu_5.4O USNPs to scavenge ROS, capture pro-inflammatory factors, improve the environment of wound sites, and accelerate diabetic wound healing.

Fig. 2

(A) SEM and elemental mapping images of Cu_5.4O@Hep-PEG hydrogel. (B) In vitro cumulative release of Cu_5.4O USNPs from Cu_5.4O@Hep-PEG over time. (C) Young's moduli and mesh sizes of Hep-PEG and Cu_5.4O@Hep-PEG hydrogels. (D) Binding kinetics of MCP-1 and (E) IL-8 for hydrogels with different Cu_5.4O USNP loading amounts over 24 h of co-incubation. (F) MCP-1 and (G) IL-8 binding amounts for Cu_5.4O@Hep-PEG hydrogels after co-incubation for 24 h at different chemokine concentrations. (H) Chemokine saturation of Cu_5.4O@Hep-PEG and the binding sites required for chemokines in chronic wound sites. (I) In vitro DPPH, H₂O₂, ·OH, and O₂·^- scavenging capabilities of Cu_5.4O@Hep-PEG hydrogels over time.

Fig. 3

Cellular pro-inflammatory-factor capture with Cu_5.4O@Hep-PEG. (A) Schematic of the transmigration assay procedure used to evaluate pro-inflammatory-factor capture by Cu_5.4O@Hep-PEG. A transwell is inserted into the well of cell culture plate. The small brown circles represent immune cells, the small yellow dots represent pro-inflammatory factors, and the grey semi-ellipse at the bottom represents the hydrogel. (B) The number of macrophages and (C) neutrophils in the lower chamber of the transwell incubated with human IL-8 (5 ng mL⁻¹) and mouse MCP-1 (10 ng mL⁻¹) under different treatment conditions. (D) Concentrations of MCP-1 and (E) CXCL-1 incubated with 100 μM H₂O₂ under different treatment conditions. (F) The number of macrophages and (G) neutrophils in the lower chamber of a transwell incubated with conditioned medium collected after incubation with 100 μM H₂O₂ under different treatment conditions. The data in (B)–(G) are means ± s.d. from six independent replicates. (*p < 0.05; **p < 0.01; n.s.: no significance, One-way ANOVA).

Fig. 4

Efficiency of Cu_5.4O@Hep-PEG for diabetic wound healing. (A) Representative images of diabetic wounds at different time points (blue 6-mm-diameter disc provided for scale reference). (B) Area percentages of closed wounds. (C) Representative histological images and (D) lengths of regenerated epidermis 14 days after surgery (blue arrows indicate regenerated epidermis) (Scale bar: 500 μm). (E) Masson's trichrome staining results for diabetic wounds under different treatment regimens (Scale bar: 100 μm). (F) Collagen index measurements performed with Image J software. (G) CD31 (red) and DAPI (blue) staining results for diabetic wound tissues from each group (Scale bar: 100 μm). (H) CD31 IHC staining of diabetic wound tissues from each group. The red dots indicate blood vessels (Scale bar: 100 μm). (I) Statistical analysis of the number of blood vessels per wound field. The data in (B), (C), and (E) are mean ± s.d. from six independent replicates. (*p < 0.05; **p < 0.01; n.s.: no significance, One-way ANOVA).

Fig. 5

Anti-inflammatory and anti-oxidative-stress effects of Cu_5.4O@Hep-PEG in diabetic wounds. (A) Representative confocal images and (B) statistical analysis of the fluorescence intensity of DHE (red) in diabetic wounds from each group. (Scale bar: 100 μm) (C) Representative images and (D) statistical analysis of the CD11b staining (red) and DAPI staining (blue) of diabetic wounds from each group at different time points. (Scale bar: 100 μm) (E) Concentrations of MCP-1 and (F) CXCL-1 in wounds at different times. (G) Volcano plots showing the proteins upregulated and downregulated by Cu_5.4O@Hep-PEG. (H) PCA results for differentially expressed proteins in the wound tissues in the wound-dressing and Cu_5.4O@Hep-PEG groups. Each data point corresponds to PCA analysis result for a single sample. (I) Heat maps showing the proteins involved in inflammation and oxidative stress that are significantly upregulated and downregulated upon Cu_5.4O@Hep-PEG treatment, and (J) relative expression of these proteins. The data in (B) and (D–F) are means ± s.d. from six independent replicates (One-way ANOVA). The data in (J) are means ± s.d. from three independent replicates (t-test). (*p < 0.05; **p < 0.01).