Bacteria responsive polyoxometalates nanocluster strategy to regulate biofilm microenvironments for enhanced synergetic antibiofilm activity and wound healing

Abstract

Backgroud: Nowadays, biofilms that are generated as a result of antibiotic abuse cause serious threats to global public health. Such films are the primary factor that contributes to the failure of antimicrobial treatment. This is due to the fact that the films prevent antibiotic infiltration, escape from innate immune attacks by phagocytes and consequently generate bacterial resistance. Therefore, exploiting novel antibacterial agents or strategies is extremely urgent. Methods: Herein, we report a rational construction of a novel biofilm microenvironment (BME)-responsive antibacterial platform that is based on tungsten (W)-polyoxometalate clusters (POMs) to achieve efficient bactericidal effects. Results: On one hand, the acidity and reducibility of a BME could lead to the self-assembly of POMs to produce large aggregates, which favor biofilm accumulation and enhance photothermal conversion under near-infrared (NIR) light irradiation. On the other hand, reduced POM aggregates with BME-induced photothermal-enhanced efficiency also exhibit surprisingly high peroxidase-like activity in the catalysis of bacterial endogenous hydrogen peroxide (H₂O₂) to produce abundant reactive oxygen species (ROS). This enhances biofilm elimination and favors antibacterial effects. Most importantly, reduced POMs exhibit the optimal peroxidase-like activity in an acidic BME. Conclusion: Therefore, in addition to providing a prospective antibacterial agent, intelligent acid/reductive dual-responsive POMs will establish a new representative paradigm for the areas of healthcare with minimal side effects.

Keywords: Biofilm; acid/reductive-responsive; peroxidase-like activity; photothermal antibacterial effect; polyoxometalate.

Scheme 1

Schematic illustration of pH/GSH responsiveness of GdW₁₀O₃₆ NCs as a combined paradigm for peroxidase catalyst-photothermal effect to regulate BMEs for enhancing antibiofilm activity and wound healing.

Figure 1

Synthesis and characterization of oxidized and reduced GdW₁₀O₃₆ NCs. (A) Schematic diagram of synthesis. (B-D) TEM images of nanoclusters at pH = 7.4, 6.4 and 4.0. (E) DLS measurements of GdW₁₀O₃₆ NCs with continuous acidification from pH = 7.4 to 4.0. UV-vis spectra and corresponding photographs of GdW₁₀O₃₆ clusters before and after reduction in the presence of various concentrations of GSH (0, 10, 15, and 20 mM) on the 2nd (F) and 5th day (G). (H) XPS spectra of GdW₁₀O₃₆ NCs. (I-J) XPS spectra of W 4f in GdW₁₀O₃₆ NCs before and after reduction.

Figure 2

Photothermal effects of reduced GdW₁₀O₃₆ NCs in vitro. (A) Temperature evaluation of reduced GdW₁₀O₃₆ NCs (5.3 ppm Gd) as a function of irradiation time (0-10 min) using an 808 nm laser at different power densities (0.2, 0.4, 0.6, 0.8, and 1.0 W/cm²). (B) Temperature evaluation of reduced GdW₁₀O₃₆ NCs that were incubated with different concentrations of GSH (50, 100, and 150 mM) as a functional of irradiation time (0-10 min) using an 808 nm laser at a power density of 1.0 W/cm². Acidity-dependent photothermal effects (C) and the corresponding IR images (D) of reduced GdW₁₀O₃₆ NCs (5.3 ppm Gd) under the irradiation of an 808 nm NIR laser at a power density of 1.0 W cm^-2 for 10 min.

Figure 3

Peroxidase-like activity of reduced GdW₁₀O₃₆ NCs is dependent on pH, temperature, H₂O₂, and concentrations. (A) pH-dependent and (B) temperature-dependent activities with TA (0.5 mM), H₂O₂ (33 mM), and reduced GdW₁₀O₃₆ NCs (33 µg/mL). Reduced GdW₁₀O₃₆ NCs and HRP show an optimal pH of 4.0-5.0 and optimal temperature around 40-50 °C. (C) H₂O₂ concentration-dependent peroxidase-like activity with reduced GdW₁₀O₃₆ NCs (33 µg/mL) and TA (0.5 mM). Reduced GdW₁₀O₃₆ NCs require a higher H₂O₂ concentration than HRP to reach maximal peroxidase activity. (D) Reduced GdW₁₀O₃₆ NCs-dependent peroxidase-like activity with TA (0.5 mM) and H₂O₂ (33 mM). The maximum point in each curve (A-D) was set as 100 %. The insets show the fluorescence spectra of the corresponding reduced GdW₁₀O₃₆ NCs reaction system.

Figure 4

Enzyme-like properties of reduced GdW₁₀O₃₆ NCs. (A, B) Absorbance spectra and visual color changes of TMB (1 mM) and OPD (1.85 mM) in different reaction systems: (1) H₂O+TMB, (2) 808 nm+TMB, (3) Reduced GdW₁₀O₃₆ NCs+TMB, (4) Reduced GdW₁₀O₃₆ NCs+808 nm+TMB, (5) H₂O₂+TMB, (6) Reduced GdW₁₀O₃₆ NCs+H₂O₂+TMB, and (7) Reduced GdW₁₀O₃₆ NCs+808 nm+H₂O₂+TMB. (C) Fluorescence spectra of terephthalic acid for hydroxyl radical detection at the maximum emission wavelength of 435 nm. (D) Different color reactions and the mechanism scheme of reduced GdW₁₀O₃₆ NCs catalysis in the presence of H₂O₂ and various peroxidase substrates represented by AH. (E) Scheme of the catalytic mechanism of reduced GdW₁₀O₃₆ NCs combined with irradiation from an 808 nm laser in the presence of H₂O₂.

Figure 5

Photographs and antibacterial activity of reduced GdW₁₀O₃₆ NCs after different treatments. (A, C) Photographs and (B, D) survival rates of treated E. coli and S.aureus after exposed to (I) Control, (II) Reduced GdW₁₀O₃₆, (III) H₂O₂, (IV) Reduced GdW₁₀O₃₆+ H₂O₂, (V) Control+NIR, (VI) Reduced GdW₁₀O₃₆+NIR, (VII) H₂O₂ +NIR, and (VIII) GdW₁₀O₃₆ + H₂O₂ +NIR. Conditions: 200 µg/mL reduced GdW₁₀O₃₆, 200 µM H₂O₂, 1 W/cm² 808 nm laser and 15 min. P values were based on the Student's test: *P < 0.05, and **P < 0.01.

Figure 6

Bacterial biofilm elimination effect and images of reduced GdW₁₀O₃₆ NCs after different treatments. (A, C) Bacterial biofilm eradication effect of E. coli and S.aureus bacteria treated with reduced GdW₁₀O₃₆ NCs after after treated with (I) PBS (control), (II) Reduced GdW10, (III) H₂O₂, (IV) Reduced GdW₁₀ + H₂O₂ in the presence or absence of NIR. (B, D) OD₅₉₀ value for evaluating changes in biofilm biomass of E. coli and S.aureus bacteria after different treatments. (E) 3D confocal laser scanning microscopy (CLSM) of biofilms formed from E. coli and S.aureus bacteria treated with reduced GdW₁₀O₃₆ NCs after different treatments. Image sizes: 212.1×212.1 µm. The biofilms were stained with CA. P values were based on the Student's test: *P < 0.05, and **P < 0.01.

Figure 7

Morphology and live/dead staining imaging of E. coli and S. aureus bacteria after different treatments. SEM images (A, C) and fluorescent staining photographs (B, D) of E. coli and S. aureus display bacterial membrane changes after different treatments. (I) Control, (II) Reduced GdW₁₀O₃₆, (III) H₂O₂, (IV) Reduced GdW₁₀O₃₆+H₂O₂, (V) Control+NIR, (VI) Reduced GdW₁₀O₃₆+NIR, (VII) H₂O₂ +NIR, and (VIII) GdW₁₀O₃₆ + H₂O₂ +NIR. (A, C: Scale bar represents 2 µm; B, D: Scale bar represents 20 µm).

Figure 8

Wound-healing studies of reduced GdW₁₀O₃₆ NCs after different treatments in vivo. (A) Relative wound area and (B) weight of mice infected with E.coil after different treatments. (C) Photographs and histologic analyses (D) of E. coli- infected wound treated with (I) PBS (control), (II) H₂O₂ , (III) Reduced GdW₁₀, (IV) Reduced GdW₁₀ +NIR, (V) Reduced GdW₁₀ +H₂O₂, and (VI) Reduced GdW₁₀ +NIR+H₂O₂ at days 4 and 10 and their corresponding histologic analyses. P values were based on the Student's test: *P < 0.05, and **P < 0.01.