Facile synthesis of nanoparticles-stacked Co3O4 nanoflakes with catalase-like activity for accelerating wound healing

Abstract

Delayed wound healing caused by excessive reactive oxygen species (ROS) remains a considerable challenge. In recent years, metal oxide nanozymes have gained significant attention in biomedical research. However, a comprehensive investigation of Co₃O₄-based nanozymes for enhancing wound healing and tissue regeneration is lacking. This study focuses on developing a facile synthesis method to produce high-stability and cost-effective Co₃O₄ nanoflakes (NFs) with promising catalase (CAT)-like activity to regulate the oxidative microenvironment and accelerate wound healing. The closely arranged Co₃O₄ nanoparticles (NPs) within the NFs structure result in a significantly larger surface area, thereby amplifying the enzymatic activity compared to commercially available Co₃O₄ NPs. Under physiological conditions, it was observed that Co₃O₄ NFs efficiently break down hydrogen peroxide (H₂O₂) without generating harmful radicals (·OH). Moreover, they exhibit excellent compatibility with various cells involved in wound healing, promoting fibroblast growth and protecting cells from oxidative stress. In a rat model, Co₃O₄ NFs facilitate both the hemostatic and proliferative phases of wound healing, consequently accelerating the process. Overall, the promising results of Co₃O₄ NFs highlight their potential in promoting wound healing and tissue regeneration.

Keywords: Co3O4; nanoflake; nanozyme; reactive oxygen species; wound healing.

Graphical abstract

Figure 1.

Schematic illustration of the application of Co₃O₄ NFs with CAT-like activity and excellent cytocompatibility for promoting wound healing.

Figure 2.

Characterization of Co₃O₄ NFs. SEM (A-i) and HRTEM (A-ii to A-iv) images of Co₃O₄ NFs; (B) size statistics; (C) schematic of the stacked Co₃O₄ NPs to Co₃O₄ NFs; XPS analysis (D) and XRD spectrum (E) of Co₃O₄ NFs.

Figure 3.

CAT-like ROS-scavenging activities and stability of Co₃O₄ NFs. (A) Clearance of 10 mM H₂O₂ by Co₃O₄ NFs; (B) the generated dissolved oxygen after Co₃O₄ NFs reacted with 100 mM H₂O₂; (C) the observed O₂ bubble generation; (D) determination of the H₂O₂ decomposition products ·OH at pH 7.4; (E) schematic diagram of the catalytic mechanism; H₂O₂ scavenging activity of Co₃O₄ NFs and CAT after autoclaving (F) and 14 days of incubation (G); (H) Co ion release during 14-d incubation. **p < 0.01, ****p < 0.0001, n.s: no significance.

Figure 4.

Co₃O₄ NFs Promote L929 cell proliferation and reduce cellular oxidative stress. (A) Florescent microscopy of L929 cells co-cultured with different concentrations (0, 0.5, 5, 50, 500 and 5000 µg/ml) of Co₃O₄ NFs for 1 and 3 days; (B, C) cell viability of the co-cultured L929 cells on Day 1 or Day 3, respectively; (D) expression of intracellular ROS (DCFH-DA) and live-dead state (AO/EB) of L929 cells co-cultured with 50 µg/ml Co₃O₄ NFs and 800 µM H₂O₂ for 6 h (live cells were represented by green, dead cells by red, nuclei in blue and intracellular ROS in blue-green); (E) statistical analysis of the number of live cells after incubation; (F) schematic illustration of the ROS-scavenging mechanism of Co₃O₄ NFs. ***p < 0.001, ****p < 0.0001.

Figure 5.

Cytocompatibility assessment of Co₃O₄ NFs by ECs and SMCs. Florescent microscopy of ECs (A) and SMCs (D) co-cultured with different concentrations (0, 0.5, 5, 50, 500 and 5000 µg/ml) of Co₃O₄ NFs for 1 and 3 days; cell viability of the co-cultured ECs (B, C) and SMCs (E, F) on Day 1 or Day 3, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6.

In vitro evaluation of Co₃O₄ NFs for wound healing. (A) Schematic representation of animal experiments. (B) Representative images of wound repair on the Day 7 and 14 for the PBS group, 5 µg/ml Co₃O₄ NFs group and 50 µg/ml Co₃O₄ NFs group. (C) Statistical analysis depicting the wound closure. (D) Histological staining (H&E, Masson) of the wound on Day 14: green arrow: fibroblasts, blue arrow: epithelial tissue, yellow arrows: capillaries, black arrows: hair follicles.