Intelligent ROS therapy driven by iron-based nanozyme with controllable catalytic activity for infected wound healing

Abstract

Therapeutic generation of reactive oxygen species (ROS) through catalytic therapy demonstrates antibacterial efficacy against wound infections. However, prolonged and unregulated ROS production risks inducing intolerable oxidative stress alongside exacerbated inflammatory responses, creating a microenvironment counterproductive to wound healing. Here, inspired by rechargeable batteries, we have developed a catalytic activity-controllable nanozyme by integrating Fe^(II) and Fe^(III) within metal-organic frameworks (FeNZ). Specifically, the overexpressed glutathione in the infective wound can increase the Fe^(II) fraction in FeNZ and endow FeNZ with peroxidase (POD)-like activity, which can convert hydrogen peroxide (H₂O₂) into hydroxyl radicals (•OH) for effective eradication of both drug-sensitive and drug-resistant bacteria (Staphylococcus aureus, 97.9% of antibacterial rate; methicillin-resistant S. aureus (MRSA), 93.2% of antibacterial rate) by disrupting bacterial membranes. Of note, the catalytic performance of FeNZ declined in parallel with the increase in Fe^(III) content during the •OH generation process, resulting in a low inflammatory microenvironment for infected wound healing and faster wound healing (95.5% of healing rate for FeNZ + H₂O₂ group, 83.5% of healing rate for Control group, day 16). The activity-controllable FeNZ thus holds promise as an effective agent for bacterial elimination and enhanced wound repair, presenting a novel strategy for the management of infected wounds.

Keywords: Adjustable enzyme activity; Bacterial infection; Chemodynamic therapy; Nanozyme; Wound repair.

Scheme 1

The synthetic illustration of FeNZ and its therapeutic effect in infected wound. (a) The synthesis procedure of FeNZ by hydrothermal method. (b) The controllable catalytic activity of FeNZ is regulated by GSH or H₂O₂. (c) In infected wounds, FeNZ leverages GSH to enhance its enzymatic activity and decomposes H₂O₂ to produce •OH for bacteria elimination. Concurrently, the catalytic activity of FeNZ gradually diminishes, which helps to mitigate oxidative stress and inflammatory responses, facilitating wound healing

Fig. 1

Characterization of FeNZ. (a) TEM images of FeNZ. The red arrowheads represent spherical structures, and the blue arrowheads represent lamellar structures. (b) Size distribution of FeNZ. (c) Elemental mapping of FeNZ. (d) XPS spectrum of FeNZ. (e, f) O 1s and Fe 2p XPS spectra of FeNZ. (g, h) Fe 2p XPS spectra of FeNZ treated with H₂O₂ (FeNZ-l) and FeNZ treated with GSH (FeNZ-h). (i) Valence proportion diagram of Fe in FeNZ, FeNZ-h and FeNZ-l. (j, k) TEM images of FeNZ-h and FeNZ-l

Fig. 2

In vitro catalytic activity of FeNZ. (a-d) •OH generation of FeNZ at different concentrations with H₂O₂ (10 mM) at pH 5.0 (TMB and MB spectrophotometer assay). (e) ESR spectra using DMPO as the spin trap: i) DMPO, ii) H₂O₂ (100 µM), iii) FeNZ (100 µg mL^− 1), iv) FeNZ + H₂O₂. (f) Oxidase-like activity of FeNZ at pH 5.0. (g) The production of H₂O₂ by FeNZ (100 µg mL^− 1) at different pH conditions. (h) The production of •OH by FeNZ (100 µg mL^− 1) without H₂O₂ at different pH conditions. (i) Changes of GSH and GSSG contents under different concentrations of FeNZ. (j) GSH/GSSG ratios calculated from i. (k, l) •OH generation effects of FeNZ-h, FeNZ, and FeNZ-l (50 µg mL^− 1) at pH 5.0. (m, n) H₂O₂ generation effects of FeNZ-h, FeNZ, and FeNZ-l (50 µg mL^− 1) at pH 5.0. (o) Schematic diagram of the “charging-discharging process” on the catalytic activity of FeNZ. Statistical significances were denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

In vitro antibacterial activity of FeNZ. (a-d) Representative images of bacterial colonies and antibacterial ratios of (a, b) S. aureus or (c, d) MRSA treated with different concentrations of FeNZ. (e-h) Representative images of bacterial colonies and antibacterial ratios of (e, f) S. aureus or (g, h) MRSA treated with FeNZ with or without H₂O₂ (100 µM). (i-l) Representative images of bacterial colonies and antibacterial ratios of (i, j) S. aureus or (k, l) MRSA treated with FeNZ or FeNZ-h or FeNZ-l with H₂O₂ (100 µM). Statistical significances were denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Fig. 4

Mechanism for the antibacterial effect of FeNZ. (a) SEM images of MRSA after different treatments. The yellow arrowheads represent ruptures and deep hollows appeared on the membrane, and the red arrowheads represent cytoplasmic leakage in the surrounding area. (b, c) Representative confocal images and its statistical analysis of MRSA after different treatments. DAPI and PI were used to stain the cell nucleus and dead cells, respectively. (d) Flow cytometry results of MRSA stained with PI. (e, f) DNA and protein leakage of MRSA in the supernatants after different treatments. Flow cytometry analysis of ROS and O₂·⁻ in MRSA using (g) DCFH-DA and (h) DHE as fluorescence indicators. (i) Intracellular GSH/GSSG ratios. (j) The corresponding antibacterial ratios of FeNZ and (or) H₂O₂ in the presence of mannitol (20 mM [70]). (k) Representative images of different processing corresponding to j. (l, m) The inhibition of biofilm formation and eradication of established after different treatments examined by crystal violet staining. Statistical significances were denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant

Fig. 5

In vivo antibacterial assay. (a) Schematic diagram of antibacterial model in vivo. (b, c) Representative photographs and corresponding quantitative analysis of bacterial colonies obtained from infected wounds. (d) Schematic diagram of the in vivo wound healing experimental design. (e) Representative photographs of infected wounds at different time intervals after treatments. Statistical significances were denoted as ***p < 0.001, ns, not significant

Fig. 6

In vivo wound healing. (a) Relative wound healing rate calculated based on Fig. 5e. (b) Relative body weight of mice. (c) Survival rates with different treatments for 16 days. H&E and Masson-staining images of infected skin tissues on (d) day 16 and (e) day 23, the yellow arrowheads represent hair follicles, and the red arrowheads represent sebaceous glands

Fig. 7

Pro-oxidation effects of FeNZ. (a) CLSM images and (b) corresponding flow cytometry analysis of the intracellular ROS levels of L929 cells treated with PBS, FeNZ-h (100 µg mL^− 1), FeNZ (100 µg mL^− 1), and FeNZ-l (100 µg mL^− 1) at air condition. (c) DHE and (d) immunofluorescence staining images of skin tissues treated for 96 h. TNF-α: Green, IL-6: Red, Hoechst: Blue

Fig. 8

Biosafety of FeNZ. (a) Live (green)/dead (red) assay of HUVECs exposed to different concentrations of FeNZ. (b) Hemolysis activity of FeNZ at different concentrations. (c) Flow cytometry analysis of apoptotic HUVECs under different concentrations of FeNZ. (d) The apoptotic ratios of HUVECs under different concentrations of FeNZ. (e) Typical H&E-staining images of the major organs from mice after different treatments on day 16