A Multifunctional Nanozyme Integrating Antioxidant, Antimicrobial and Pro-Vascularity for Skin Wound Management

Abstract

Background: Skin wounds are a prevalent issue that can have severe health consequences if not treated correctly. Nanozymes offer a promising therapeutic approach for the treatment of skin wounds, owing to their advantages in regulating redox homeostasis to reduce oxidative damage and kill bacteria. These properties make them an effective treatment option for skin wounds. However, most of current nanozymes lack the capability to simultaneously address inflammation, oxidative stress, and bacterial infection during the wound healing process. There is still great potential for nanozymes to increase their therapeutic functional diversity and efficacy.

Methods: Herein, copper-doped hollow mesopores cerium oxide (Cu-HMCe) nanozymes with multifunctional of antioxidant, antimicrobial and pro-vascularity is successfully prepared. Cu-HMCe can be efficiently prepared through a simple and rapid solution method and displays sound physiological stability. The biocompatibility, pro-angiogenic, antimicrobial, and antioxidant properties of Cu-HMCe were assessed. Moreover, a full-thickness skin defect infection model was utilized to investigate the wound healing capacity, as well as anti-inflammatory and pro-angiogenic properties of nanozymes in vivo.

Results: Both in vitro and in vivo experiments have substantiated Cu-HMCe's remarkable biocompatibility. Moreover, Cu-HMCe possesses potent antioxidant enzyme-like catalytic activity, effectively clearing DPPH radicals (with a scavenging rate of 80%), hydroxyl radicals, and reactive oxygen species. Additionally, Cu-HMCe exhibits excellent antimicrobial and pro-angiogenic properties, with over 70% inhibition of both E. coli and S. aureus. These properties collectively promote wound healing, and the wound treated with Cu-HMCe achieved a closure rate of over 90% on the 14th day.

Conclusion: The results indicate that multifunctional Cu-HMCe with antioxidant, antimicrobial, and pro-angiogenic properties was successfully prepared and exhibited remarkable efficacy in promoting wound healing. This nanozymes providing a promising strategy for skin repair.

Keywords: Cu-HMCe nanozyme; antibacterial; antioxidation; vascularization; wound healing.

Scheme 1

Schematic illustration showing the copper-doped hollow mesopores cerium oxide nanozyme preparation and its role in the treatment of skin wounds.

Figure 1

Characterization of Cu-HMCe. (A) The SEM image and (B) TEM image of Cu-HMCe. (C) SEM images of Cu-HMCe and energy dispersive X-ray mapping of Cu, Ce, Cu and N elements. (D) XPS spectra of Cu-HMCe. The high-resolution spectrum of (E) Cu 2p and (F) Ce 3d. (G) The particle size distribution, (H) hydrodynamic size and (I) zeta potential of the Cu-HMCe.

Figure 2

(A) Cell viability of HUVECs co-cultured with different concentrations of Cu-HMCe. (B) Cell viability of HUVECs co-cultured with control, HMCe, and Cu-HMCe for 1, 3, and 5 days. (C) Live/dead assays. (D) CLSM images of HUVECs morphology after 3 days of control, HMCe and Cu-HMCe treatment. (E) Hemolysis ratio and (F) Images of fresh mouse erythrocytes co-cultured with different concentrations of Cu-HMCe. Significance levels of *p< 0.05 and ***p<0.001 were applied.

Figure 3

(A) migration assays and (B) tube formation assays of HUVECs co-cultured with control, HMCe and Cu-HMCe. Quantified analysis of (C) migration assays and (D) tube formation assays. Significance levels of *p< 0.05, **p< 0.01, and ***p<0.001 were applied.

Figure 4

Antibacterial sensitivity of nanozymes. (A) The antibacterial rate of S. aureus and (B) E. coli. (C) pH-dependent release of Cu in Cu-HMCe. (D) Time-dependent scavenging of free radicals of Cu-HMCe. (E) Free radical eliminating activity of control, HMCe or Cu-HMCe. (F) Quantified analysis of DPPH assays. (G) and (H) OH scavenging ability of control, HMCe or Cu-HMCe. (I) ROS staining of control, HMCe or Cu-HMCe. Significance levels of, **p< 0.01 and ***p<0.001 were applied.

Figure 5

(A) The wound closure rates within 14 days after surgery. Corresponding quantitative analysis of the (B) wound re-epithelization and (C) collagen deposition on 14 days. (D) Histological analysis of wound tissues at 14 days. Significance levels of *p< 0.05, **p< 0.01, and ***p<0.001 were applied.

Figure 6

(A) Immunohistochemical staining images for TNF-α, IL-6 or CD31 of the wound sections after 10-day treatment. Quantified analysis of the relative expression of (B) IL-6 and (C) TNF-α. (D) Quantified analysis of the number of vessels. (E) Immunofluorescence staining images for CD31 and α-SMA of the wound sections after 14-day treatment. Significance levels of *p< 0.05, **p< 0.01, and ***p<0.001 were applied.