Synergistic healing of diabetic wounds through photothermal and peroxidase-like activity of heterogeneous Bi₂S₃/Au nanoparticles

Abstract

Bacterial resistance and biofilm formation around diabetic wounds are major challenges that make the wounds difficult to heal. It is crucial for diabetic wound healing to improve the microenvironment around the wounds. In this study, a novel strategy for diabetic wound healing is developed by combining the peroxidase (POD)-like enzyme activity and photothermal therapy (PTT) to protect against bacterial infections around the wounds. Heterogeneous bismuth sulfide/gold nanoparticles (Bi₂S₃/Au NPs) are synthesized through a two-step wet chemical route. Results show that Bi₂S₃/Au nanozymes display high POD-like enzyme activity and can effectively convert H₂O₂ into ˙OH. The antibacterial rate against S. aureus and E. coli bacteria is 99.8 ± 0.03% and 99.9 ± 0.01%, respectively, in the presence of H₂O₂ under near-infrared light (NIR) irradiation. Animal experiments on infected diabetic wounds demonstrate that the synergistic actions of the Bi₂S₃/Au NPs significantly inhibit the formation of biofilms caused by bacteria, and promote the deposition of collagen and the formation of epithelial and dermal tissue. This study provides a promising solution for innovative therapy of refractory diabetic wounds, which is of great significance for reducing the abuse of antibiotics and the production of drug-resistant bacteria.

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1. Introduction

Diabetes is a chronic metabolic disorder that affects millions of people worldwide. Currently, approximately 25% of diabetic patients suffer from a high risk of chronic nonhealing wounds.¹ Diabetic ulcer characterized by extremely slow or even stagnant wound-healing cascades (hemostasis, inflammation, proliferation and remodeling) that usually form on the feet and legs is an intractable complication of diabetes.² The primary reason for the difficulty in healing chronic diabetic wounds is the presence of biofilms, which are caused by bacteria gathering around the wound area, predominantly Staphylococcus aureus (S. aureus). Bacteria in biofilms are multidrug-resistant, and biofilms act as physical barriers, preventing the penetration of antibiotics or other therapeutic drugs.³ However, hyperglycemia reduces the ability of immune cells to eliminate bacteria while providing enough nutrients for bacteria to grow and reproduce.⁴ Therefore, finding an effective therapeutic strategy to address bacterial biofilm infections and reshaping the surrounding environment has been critical for the treatment of diabetic ulcer.

Photothermal therapy (PTT) has broad applications in the biomedical field, such as cancer therapy and antibacterial treatment, through converting light into heat to destroy microorganisms thermally.^5–7 PTT triggered by near-infrared (NIR) light typically requires a temperature of 50 °C or higher to denature proteins and kill microorganisms, as cell damages at lower temperatures (e.g. 45 °C) can be repaired. Rapid development of nanotechnology provides promising alternative solutions to fight against bacteria through PTT.^8,9 Gold nanoparticles (Au NPs) have attracted considerable attention in the biomedical field due to their good biocompatibility and excellent physicochemical properties, especially their high NIR absorption capability and photothermal conversion ability.¹⁰ It was also reported that Au NPs have strong antibacterial potential due to their capacity to damage bacterial DNA by direct contact and preventing its unwinding during transcription.¹¹ Bismuth sulfide (Bi₂S₃) is an n-type semiconductor and recognized as a superior photothermal agent and photocatalyst with good biocompatibility for wide applications in biosensors and biomedicine.^12–18 Bi₂S₃ can kill bacteria through local photothermal effects.¹⁹ Therefore, it is feasible to combine Au and Bi₂S₃ to construct heterostructures and obtain better photothermal antibacterial performance.¹⁹ However, high temperature may cause inflammation and thermal damage to nearby tissues.^20,21

Nanozymes, as an emerging class of nanoengineered artificial enzymes, can be widely applied in antibacterial treatments because their composition and nanostructure endow them not only with efficient catalytic activities, but also with greater stability and easier scalability than natural enzymes.^22–24 For example, oxidase-like (OXD-like) nanozymes can generate O₂˙⁻ and ˙OH in the presence of oxygen and kill bacteria,^25,26 while POD-like nanozymes can utilize H₂O₂ as a substrate to produce ˙OH realizing sterilization function.^27,28 More importantly, nanozymes designed with different strategies can exhibit diverse enzyme activities.^29–32 It is worth noting that there are still some drawbacks in the use of nanozyme catalyzed production of reactive oxygen species (ROS) for antibacterial therapy, such as the blocking effect of biofilms on ROS permeation, insufficient enzyme activity, and increased bacterial tolerance to single mechanism ROS.

In this study, heterogeneous Bi₂S₃/Au NPs with photothermal response and POD-like activity were synthesized for synergistic therapy of diabetic wounds. As shown in Scheme 1, Bi₂S₃/Au NPs are synthesized through a two-step wet chemical route. After adding Bi³⁺ ions to the BSA aqueous solution, the protein molecules can sequester and trap Bi³⁺ to form BSA–Bi³⁺complexes through the free –SH group of cysteine containing BSA molecules. By adjusting the pH to 12.0, BSA molecules denature and lead to the formation of Bi₂S₃ NPs. Subsequently, HAuCl₄ and NaBH₄ are added to Bi₂S₃@BSA solution, respectively, to form heterogeneous Bi₂S₃/Au NPs through a reduction reaction. Excessive BSA molecules are covered on the surface of Bi₂S₃/Au NPs, making them highly stable, biocompatible, and suitable for further modifications. The presence of Au amplifies the Schottky defects of Bi₂S₃ itself, which allows some free electrons to quickly move on the surfaces of Au and Bi₂S₃, endowing Bi₂S₃/Au with POD-like activity, capable of generating abundant ROS such as ˙OH, effectively combating bacteria through synergistic enzyme activity. On the other hand, the plasma surface resonance effect of Au generates some free electrons, and near-infrared light irradiation causes these free electrons to absorb energy and enter the excited state, and then release the energy in the form of heat. Therefore, Bi₂S₃/Au NPs have excellent photothermal conversion efficiency and high POD-like activity, which can synergistically destroy the formation of biofilms, kill the bacteria, and promote the healing of diabetic wounds.

Scheme 1 (A) Synthesis of Bi₂S₃/Au NPs. (B) The POD-like enzyme activity and photothermal response of Bi₂S₃/Au NPs. (C) Healing process of Bi₂S₃/Au NPs for diabetic wounds (CDT: chemodynamic therapy).

2. Materials and methods

2.1 Materials

Tetrachloroauric acid tetrahydrate (HAuCl₄·4H₂O, AR), bovine serum albumin (BSA, BIOFOX), bismuth nitrate pentahydrate (Bi(NO₃)₃·5H₂O, 99.0 wt%), trisodium citrate (C₆H₅Na₃O₇·2H₂O, AR), sodium borohydride (NaBH₄, 98.0 wt%), and sodium hydroxide (NaOH, AR) were used. All materials were used without further purification.

2.2 Synthesis of BSA coated bismuth sulfide (Bi₂S₃@BSA) NPs

250 mg BSA was dissolved in 8.0 mL deionized water, and then 50 mM Bi(NO₃)₃ in 1.0 mL of HNO₃ solution (2.0 M) was slowly added into BSA solution under vigorous stirring. After 30 min, NaOH (2.0 M) was used to adjust the solution pH to 12, and the mixture was allowed to react at 25 °C for 12 h under vigorous stirring. The color of the solution changed from colorless to dark black, and BSA stabilized Bi₂S₃ NPs were thus formed. The resulting Bi₂S₃ NPs were purified by the dialysis of the solution against deionized water for 24 h to remove excess precursors and then stored at 4 °C.

2.3 Synthesis of Bi₂S₃/Au NPs

8 mL of Bi₂S₃@BSA in 42 mL deionized water was mixed with 1.5 mL of HAuCl₄·4H₂O (25 mM) and 9 mL of sodium citrate (1.0 wt%), followed by the dropwise addition of fresh NaBH₄ (0.6 mg mL⁻¹) at room temperature. After 12 h, dialysis was applied to purify the Bi₂S₃/Au heterojunction nanoparticles and then stored at 4 °C.

2.4 Characterization

Transmission electron microscopy (TEM JEM-1400 plus) and field emission scanning electron microscopy (FESEM, JSM-IT800, JEM-F200) were used to identify the size and morphology of the NPs. For energy dispersive spectroscopy (EDS) mapping, an ESCALAB 250Xi was used. A power X-ray diffractometer system (D8 ADWANCE) was used for determining X-ray diffraction (XRD) patterns. UV-vis absorption spectra were acquired using a UV-vis spectrophotometer (T80 double beam spectrophotometer, PG Instruments Limited). An inductively coupled plasma spectrometer (ICP Prodigy 7) was used to measure the residual amount of elements in the body.

2.5 Photothermal response of Bi₂S₃/Au NPs

An 808 nm light with different power densities was used to evaluate the photothermal conversion efficiency of the as-prepared Bi₂S₃/Au NPs. 1 mL of Bi₂S₃/Au NPs at different concentrations was respectively irradiated for 600 seconds compared with water, Au NPs and Bi₂S₃@BSA NPs and Bi₂S₃ NPs + Au NPs. To check the photothermal stability, five cycles were plotted.

2.6 Enzyme-like activity of Bi₂S₃/Au NPs

The POD-like enzyme activity of Bi₂S₃/Au was evaluated using 1,2-diaminobenzene (OPD) and 3,3′,5,5′-tetramethylbenzidine (TMB) as the chromogenic substrates. In brief, 1 mL of TMB solution (0.48 mg mL⁻¹ in DMSO) was added to 5 mL of acetate buffer solution, and then 100 μL H₂O₂ (0.1 mM in deionized water) was added after adding 1 mL of concentration gradient Bi₂S₃/Au at 40 °C. After reacting for 20 min, UV-vis spectrophotometer was used to measure absorption spectra. Similarly, 0.51 g citric acid and 1.84 g disodium hydrogen phosphate were added to 100 mL deionized water and mixed evenly to form a buffer solution. Subsequently, 40 mg of OPD was added to the aforementioned buffer, and 0.5 mL H₂O₂ (30%) was also added before the experiment. 100 μL of Bi₂S₃/Au NPs were added to 10 mL of the above liquid at room temperature. After reacting for 30 min, 50 μL sulfuric acid solution (2 M) was added to stop the reaction solution, and an UV-vis spectrophotometer was used to measure absorption spectra.

2.7 Hemolysis assay

Rabbit blood and normal saline were mixed in a volume ratio of 1 : 5, centrifuged at a speed of 1500 rpm for 5 min. After removal of the supernatant, the lower layer of red blood cells (RBCs) was washed five times with normal saline until the supernatant was colorless. 50 μL of red blood cell suspension was added to different concentrations of Bi₂S₃/Au (1 mL) solution as the experimental group. Normal saline and deionized water were used as negative and positive control groups, and allowed to stand undisturbed for 2 hours at 37 °C. Immediately before the UV-vis absorbance measurement at 540 nm, each solution was centrifuged for 5 min (1500 rpm). Eqn (1) was used to calculate the hemolysis ratio induced by Bi₂S₃/Au NPs:

2.8 Cell culture

Mouse fibroblast cell line L929 cells were cultured in the DMEM with 10% fetal bovine serum, 100 units per mL of penicillin, and 50 units per mL of streptomycin at 37 °C in an incubator at 95% relative humidity and 5% CO₂.

2.9 Toxicity on healthy cells

The cytotoxicity of Bi₂S₃/Au NPs was determined through CCK-8 assay on L929 cells. Briefly, 5000 cells in the complete culture medium (100 μL) were transferred to a 96-well plate and incubated at 37 °C for 24 hours. The previous culture medium was refreshed with the culture medium in each related well. In the following step, after 24 hours of incubation (37 °C and moisture containing 5% CO₂), CCK-8 (10 μL) was added to each well and the plate was further incubated for 2 hours. Finally, to measure the cell viability, the absorbance was recorded at a wavelength of 450 nm with a microplate reader.

2.10 Calcein AM/PI Staining

Live and dead cells, after different treatments, were imaged following staining with Calcein AM and PI. In a 96-well plate, 5 × 10³ L929 cells per well were seeded, and after incubation for 24 hours, the cells were treated with NPs for 1, 3, and 5 days. There were five experimental groups: PBS, Au NPs, Bi₂S₃@BSA NPs, Bi₂S₃ NPs + Au NPs, and Bi₂S₃/Au NPs. The cells were co-stained with Calcein AM (100 μL, 3 μM) and PI (100 μL, 3 μM) for 30 min, respectively. Finally, the treated plates were imaged by fluorescence microscopy (Leica DM IL LED Fluo).

2.11 Antibacterial performance in vitro

The antibacterial activity of Bi₂S₃/Au NPs against E. coli and S. aureus was evaluated using a co-culture method. After sterilization, Bi₂S₃/Au NPs were dispersed in PBS solution (10 mg mL⁻¹) and mixed with the same volume of E. coli or S. aureus bacterial solution (10⁶ CFU mL⁻¹) before being added in 48-well plates. After incubating for 4 hours, NIR light was applied to the plates, and then incubate for another 2 hours. The appropriate bacterial solution was removed from each well and diluted. 100 μL of the diluted bacterial solution was applied to the solid agar medium, and after incubation for 24 hours, the colonies on the culture medium were counted.

2.12 Diabetic wound healing in vivo

Animal experiments were performed in accordance with the rules outlined in the National Research Council's Guide for the Care and Use of Laboratory Animals, and all animal experiments have been approved by the Experimental Animal Ethics Committee of Wuhan University of Technology (2024-004). All surgical procedures were performed in a sterile room. The experimental animals, male C57BL/6 mice, were obtained from the Hubei Provincial Center for Disease Control and Prevention (China) and randomly divided into different groups.

To evaluate the efficacy of Bi₂S₃/Au NPs in the healing of infected diabetic wounds, a type I diabetic C57BL/6 mice model was established. Briefly, C57BL/6 mice were injected intraperitoneally with STZ (60 mg kg⁻¹) dissolved in citrate buffer (pH = 4.5) multiple times. The successful establishment of the type I diabetic model was confirmed when blood glucose concentrations were ≥16.8 mmol L⁻¹, accompanied by excessive eating and drinking symptoms, weight loss, and increased urination.

Thirty diabetic mice were randomly divided into three groups, i.e., the control group, the Bi₂S₃/Au NPs group, and Bi₂S₃/Au NPs + NIR irradiation group. The experimental mice were rendered unconscious by administering an intraperitoneal injection of a pentobarbital sodium solution at a dosage of 50 mg kg⁻¹. The back hair was shaved, and two full circular skin wounds (10 mm in the diameter) were made on the back of each mouse. Each wound was inoculated with 10 μL of S. aureus at a 10⁹ CFU mL⁻¹ concentration. 0.5 hours later, 50 μL of PBS with Bi₂S₃/Au NPs was injected into the wound. The Bi₂S₃/Au NPs + NIR group was illuminated with 808 nm NIR for 5 min.

2.13 Statistical analysis

The data are represented by the mean ± standard deviation, and the statistical significance differences between groups were analyzed using one-way analysis of variance (ANOVA) using SPSS 22.0 software. A significance level of p < 0.05 indicates a statistically significant difference, while a significance level of p < 0.01 indicates a highly statistically significant difference.

3. Results and discussion

3.1 Synthesis and characterization of Bi₂S₃/Au NPs

Fig. 1(A) and (B) show TEM images of Bi₂S₃@BSA NPs and Bi₂S₃/Au NPs, respectively. Both of them were spherical. Compared with the slight aggregation of Bi₂S₃@BSA NPs, Bi₂S₃/Au NPs displayed better uniform and dispersion. The HR-TEM image of Bi₂S₃/Au NPs (Fig. 1(C)) reveals the presence of Bi₂S₃ and Au components in the heterodimer. Due to the high electron density of Au, the contrast of the Au rich portion in the heterodimer is deeper than that of the Bi₂S₃ portion. There were two types of lattice stripes in Bi₂S₃/Au NPs, one of them can be indexed to the (111) plane of Au (0.23 nm) and the other one can be indexed to the (221) plane of Bi₂S₃, which is consistent with that in Bi₂S₃@BSA NPs (Fig. S1(C), ESI†).

Fig. 1 Structural characterization of Bi₂S₃/Au NPs. (A) TEM image of Bi₂S₃@BSA NPs. (B) TEM image of Bi₂S₃@BSA/Au NPs. (C) HR-TEM image of Bi₂S₃@BSA/Au NPs. (D) XRD patterns of BSA, Bi₂S₃@BSA NPs, and Bi₂S₃/Au NPs. (E) Size distribution of Bi₂S₃/Au NPs. (F) Surface charge of Au NPs, Bi₂S₃@BSA NPs, and Bi₂S₃/Au NPs. (G) XPS survey wide scans of Bi₂S₃@BSA NPs and Bi₂S₃/Au NPs. (H) XPS spectra of Bi 4f in Bi₂S₃@BSA NPs and Bi₂S₃/Au NPs. (I) XPS spectrum of Au 4f in Bi₂S₃@BSA/Au NPs. (J) EDS elemental mapping of Bi₂S₃/Au NPs.

Fig. 1(D) shows XRD patterns of BSA, Bi₂S₃@BSA NPs, and Bi₂S₃/Au NPs. The broad diffraction peak near 21° is derived from the BSA layer. The diffraction peaks at 24.9°, 28.6°, and 35.6° can be assigned to the (130), (211), and (240) plane of Bi₂S₃ (JCPDS, 17-0320), respectively. While the diffraction peaks at 38.2°, 44.4°, and 64.6°, can be indexed to the (111), (200), and (220) plane of Au, respectively, which matched well with the standard XRD card of Au (JCPDS. 04-0784), respectively. Fig. 1(F) shows that the average size was 4.56 ± 0.02 nm for Bi₂S₃@BSA NPs and 4.73 ± 0.06 nm for Au NPs (Fig. 1(E)). Then Bi₂S₃ NPs and Au NPs were combined together to form a dimer, and several dimers gather together to form Bi₂S₃/Au NPs. The surface of both Bi₂S₃@BSA NPs and Bi₂S₃/Au NPs was negatively charged (Fig. 1(F)), and the presence of BSA on the surface of NPs was also confirmed through UV-vis spectra due to characteristic absorption at 270 nm (Fig. S2, ESI†). These results suggest that Bi₂S₃/Au NPs have high stability in water.

The chemical state of Bi₂S₃/Au NPs was characterized through XPS spectra, as shown in Fig. 1(G)–(I). The presence of C 1s, N 1s, and O 1s characteristic peaks is derived from BSA molecules, while the Bi 4f and Au 4f characteristic peaks prove the existence of Bi and Au elements. The Bi 4f core-level XPS spectrum of Bi₂S₃@BSA NPs shows spin orbit double baryons at 158.91 eV and 164.11 eV, belonging to Bi 4f_7/2 and Bi 4f_5/2, respectively. In the XPS spectrum of Bi₂S₃/Au NPs, Bi 4f_7/2 and Bi 4f_5/2 became wider and significantly decomposed into two components. The S atoms of Bi₂S₃ at the interface of the heterojunction preferred to bond to the Au atom rather than Bi as Pauling's electronegativity of Au is larger. As the incorporation of Au into Bi₂S₃ deviates S atoms from the lattice, Bi atoms occupy the S atom sites. This results in the generation of BiS anti-site (Bi replacing S) defects. The anti-site Bi atoms located in the defects are more electropositive compared to the lattice Bi atoms. Thus, they appeared at the higher binding energy of the Bi 4f core-level XPS spectrum. In the Au 4f XPS spectrum, two peaks were located at 83.99 eV and 87.69 eV, corresponding to Au 4f_7/2 and Au 4f_5/2, respectively.

Compared with the standard peak, there was a certain broadening and splitting trend, which means that some Au atoms may bind with S atoms at the interface of Bi₂S₃.³³Fig. 1(J) shows TEM and EDS mapping images of Bi₂S₃/Au NPs. It is obvious that Bi, Au, and S elements were uniformly dispersed in Bi₂S₃/Au NPs. The red regions were derived from the Au element, the green regions were derived from the Bi element, and the yellow regions were derived from the S element. The merged image shows that the Au element is distributed around the Bi and S elements.

3.2 POD-like enzyme activity of Bi₂S₃/Au NPs

Bi₂S₃/Au NPs is a heterojunction composed of n-type semiconductor Bi₂S₃ NPs and noble metal Au, which also has a Schottky barrier. The Schottky barrier can serve as an efficient electron trap preventing electron–hole recombination in photocatalysis, which often results in an enhanced photocatalytic performance at the interface of the two materials. The transfer of electrons from high Fermi levels to low Fermi levels catalyzes the production of ˙OH from H₂O₂ in the surrounding environment (Fig. S4, ESI†), resulting in Bi₂S₃/Au NPs exhibiting POD-like activity. In detail, the POD-like enzyme activity of Bi₂S₃/Au NPs was evaluated using OPD and TMB as the substrates. Fig. 2(A) shows the catalytic mechanism of the POD enzyme. In the presence of the POD enzyme, oxygen atoms are transferred from H₂O₂ molecules to the amino groups in OPD and TMB molecules, resulting in the oxidation of OPD and TMB to form ox-OPD and ox-TMB, causing colorless OPD and TMB to turn yellow (ox-OPD) and blue (ox-TMB), respectively. Fig. 2(B) and (C) show the absorbance changes of OPD and TMB solutions in the presence of H₂O₂ and Bi₂S₃/Au NPs. The results show that, compared with Au NPs, Bi₂S₃@BSA NPs, and Bi₂S₃ NPs + Au NP mixture groups, Bi₂S₃/Au NPs displayed significantly higher POD-like enzyme activity to oxidize OPD and TMB in the presence of H₂O₂. On increasing the concentrations of Bi₂S₃/Au NPs, the catalytic oxidation of OPD and TMB increased.

Fig. 2 POD-like enzyme activity of Bi₂S₃/Au NPs. (A) Schematic diagram of POD-like enzyme activities. (B) UV-vis spectra of Bi₂S₃@BSA NPs, Au NPs, Bi₂S₃ NPs + Au NPs (mixture), and Bi₂S₃/Au NPs showing the POD-like enzyme activity using TMB as the substrate. (C) UV-vis spectra of Bi₂S₃@BSA NPs, Au NPs, Bi₂S₃ NPs + Au NPs (mixture), and Bi₂S₃/Au NPs showing POD-like enzyme activity using OPD as the substrate. (D) Temperature variation curves of Bi₂S₃@BSA NPs, Au NPs, Bi₂S₃ NPs + Au NPs (mixture), and Bi₂S₃/Au NPs under 808 nm irradiation. (E) The photothermal profiles of Bi₂S₃/Au NPs over five successive cycles under 808 nm (1 W cm⁻²) irradiation and linear cooling time of Bi₂S₃/Au NPs and –Ln(θ).

These results demonstrate that the formation of the heterogeneous structure endows Bi₂S₃/Au NPs with high POD-like enzyme activity. Because there is a certain amount of H₂O₂ in the diabetic wound,³⁴ Bi₂S₃/Au NPs can catalyze H₂O₂ to generate ˙OH through POD-like activity, thus playing a crucial role in antibacterial activity.

3.3 Photothermal response of Bi₂S₃/Au NPs

Due to the structure of Bi₂S₃/Au NPs, the separation and transfer speed of free electrons on the surface of Bi₂S₃ NPs is enhanced by doping Au NPs, which suppresses the recombination of electron–hole pairs and improves the photothermal response performance of Bi₂S₃ NPs. Au has a strong ability to absorb infrared light, and the increase in surface electrons enhances its LSPR effect, which allows it to generate a large amount of heat in near-infrared light. Fig. 2(D) shows photothermal performance of Bi₂S₃/Au NPs. Bi₂S₃/Au NPs at a concentration of 5 mg mL⁻¹ (the content of the Bi element is 2.09 μg mg⁻¹, and the content of the Au element is 53.46 μg mg⁻¹) were irradiated with 808 nm NIR light at 0.5 W cm⁻², 1 W cm⁻², 1.5 W cm⁻², and 2 W cm⁻² for 10 min, the temperature increased by 17.3 °C, 27.9 °C, 39.9 °C, and 51.7 °C, respectively. Under NIR irradiation at a power of 1 W cm⁻², the temperature of the solution increased with the increase of the Bi₂S₃/Au NP concentration, which can reach 51.6 °C and 59.8 °C and the temperature of the solution at a Bi₂S₃/Au NP concentrations of 5 mg mL⁻¹ and 10 mg mL⁻¹ after irradiation for 10 min, respectively. Although Au NPs, Bi₂S₃@BSA NPs, and their mixture (Bi₂S₃ NPs + Au NPs) also displayed a certain photothermal response, their temperature after NIR irradiation for 10 min was significantly lower than that of Bi₂S₃/Au NPs at the same concentration. Moreover, Bi₂S₃/Au NPs still maintained their original photothermal response after 5 cycles, indicating that it has good photothermal stability (Fig. 2(E)). These results indicate that the formation of heterogeneous structure endows Bi₂S₃/Au NPs with excellent photothermal performance.

3.4 In vitro biocompatibility and photothermal antibacterial ability of Bi₂S₃/Au NPs

Considering the small particle size of Bi₂S₃/Au NPs, which can easily enter the bloodstream through capillaries, both blood compatibility and cytocompatibility of Bi₂S₃/Au NPs were evaluated in vitro. Red blood cells are damaged when the hemolysis value is higher than 5%, as required by the ASTM E2524-08 standard. The hemolytic activity of Bi₂S₃/Au NPs at different concentrations (1.25, 2.5, 5, and 10 mg mL⁻¹) was examined, and the lysis profiles of red blood cells were expressed as a percentage of hemoglobin release compared with positive and negative controls.

Fig. 3(A) shows that even at the highest dose of 10 mg mL⁻¹, no significant hemolysis was observed, suggesting good blood compatibility of Bi₂S₃/Au NPs. The cytocompatibility of Bi₂S₃/Au NPs was assessed on L929 cells using the CCK-8 kit and fluorescent staining, as shown in Fig. 3(B)–(D). After co-incubation for 1 day, no significant cytotoxicity was observed in the Bi₂S₃/Au NP group at a concentration of 5 mg mL⁻¹. But when the concentration increased to 10 mg mL⁻¹, L929 cells were damaged and apoptosis began. Therefore, 5 mg mL⁻¹ was used as the concentration for follow-up experiments. After co-incubation for 5 days, the cell viability was 79.9 ± 6.6% for Bi₂S₃/Au NPs, 34.9 ± 3.4% for Au NPs, 47.1 ± 6.3% for Bi₂S₃@BSA NPs, and 48.6 ± 7.2% for Bi₂S₃ NPs + Au NPs (mixture), respectively. After staining with the Calcein-AM dye, the live cells show green fluorescence. It is obvious that the amount of live cells in the Bi₂S₃/Au NP group cells was more than that in the Au NPs, Bi₂S₃@BSA NPs, and Bi₂S₃ NPs + Au NP (mixture) groups. These results demonstrate that Bi₂S₃/Au NPs have good blood compatibility and cytocompatibility.

Fig. 3 Blood compatibility and cytocompatibility of Bi₂S₃/Au NPs. (A) Hemolysis of Bi₂S₃/Au NPs. (B) Cell viability of Bi₂S₃/Au NPs on L929 cells. (C) The effect of Bi₂S₃/Au NPs on the proliferation of L929 cells. (D) Live/dead staining of L929 cells co-incubated with Bi₂S₃/Au NPs at different time intervals (n = 5, mean ± SD, **p < 0.01, ***p < 0.001).

Diabetic wounds are prone to infection in the hyperglycemic environment, S aureus is one of the most common strains in the wound.³⁵ In this study, Gram-positive S. aureus and Gram-negative E. coli bacteria were used to evaluate the antibacterial ability of Bi₂S₃/Au NPs. As shown in Fig. 4, under the NIR irradiation for 5 min (1 W cm⁻²), Bi₂S₃/Au NPs showed an excellent antibacterial ability, compared to other experimental groups. The antibacterial rate of Bi₂S₃/Au NPs against S. aureus and E. coli was 97.4 ± 4.1% and 99.9 ± 0.03%, respectively.

Fig. 4 Antibacterial ability of Bi₂S₃/Au NPs. (A) Image of S. aureus and E. coli bacterial colonies with and without NIR irradiation (5 min, 1 W cm⁻²). The antibacterial rate of Bi₂S₃/Au NPs against S. aureus (B) and E. coli (C) in the absence of H₂O₂. (D) Image of S. aureus and E. coli bacterial colonies with and without NIR. (E) The antibacterial rate of Bi₂S₃/Au NPs against S. aureus and E. coli in the presence of H₂O₂. (F) SEM images of S. aureus and E. coli bacteria treated with Bi₂S₃/Au NPs before and after NIR irradiation (n = 3, mean ± SD, ***p < 0.001).

In the initial stage of the diabetic wound, the concentration of H₂O₂ increases while the corresponding enzyme activity does not.³⁴ Therefore, a series of experiments were designed to detect the influence of POD-like enzyme activity of Bi₂S₃/Au NPs on antibacterial ability. After adding H₂O₂ at a concentration of 0.2 mM, compared with the blank group and the control group (only H₂O₂ added), the antibacterial ability of the Bi₂S₃/Au NPs group was significantly enhanced, and the inhibition rates against E. coli and S. aureus were 73.9 ± 7.2% and 87.6 ± 2.1%, respectively. After further NIR irradiation, the inhibition rate of bacteria increased up to 99.9 ± 0.01% and 99.8 ± 0.03%, respectively. After irradiation with NIR at 1 W cm⁻² for 5 min, the temperature can rise to 47.9 °C. At the same time, Bi₂S₃/Au NPs catalyze H₂O₂ and produce numerous ˙OH. The synergistic action of the thermal and oxidative stress can destroy bacterial proteins and nucleic acids, and thus kill bacteria. Fig. 4(F) shows the shape of E. coli and S. aureus bacteria. Both of the bacteria in the control group maintained good morphology, smooth surface, and intact structure. While in the Bi₂S₃/Au NPs + NIR group, both of the bacteria showed significant deformation and bacterial cell content outflow. These results provide strong evidence for the synergistic antibacterial ability of Bi₂S₃/Au NPs through combining photothermal response and the POD-like enzyme activity.

3.5 Healing of infected diabetic wounds by Bi₂S₃/Au NPs

To evaluate the healing effect of Bi₂S₃/Au NPs on infected diabetic wounds, animal experiments were conducted using a STZ-induced C57BL/6 diabetic mouse model, as shown in Fig. 5. Type I diabetic mice (TIDM) were established by injecting low dose STZ solution (60 mg kg⁻¹) intraperitoneally several times and giving sucrose water as the sole water source at a concentration of 10%. After being injected with STZ solution for two weeks, the blood glucose level of STZ-induced mice was 22.3 ± 3.7 mmol L⁻¹, which was higher than the critical value of 16.8 mmol L⁻¹ and implied the successful establishment of the diabetic mouse model. The blood glucose level of normal mice was 7.3 ± 0.5 mmol L⁻¹. It is worth noting that the body weight of STZ-induced diabetic mice was lower than that of healthy mice, which was only 79.4% of that of healthy mice on the 14th day. The wound region in each group was photographed and recorded on the days 0, 3, 7, 10, and 14 after treatment, as shown in Fig. 5(B). The wounds of diabetic mice in each group healed to varying degrees within 14 days, and the wounds in the Bi₂S₃/Au NPs + NIR group displayed the fastest healing rate. On the 3rd day, the wounds in the Bi₂S₃/Au NPs + NIR group were crusted, while those in the control group were still exposed to the environment. The average size of the wounds in the Bi₂S₃/Au NPs + NIR group was 29.4 ± 4.9% of the initial size, much lower than 45.2 ± 8.5% in the Bi₂S₃/Au NPs group and 68.6 ± 17.4% in the control group. On the 7th and 10th day, the healing rate of the wounds in the Bi₂S₃/Au NPs + NIR group decreased, but it was still higher than that in the control group and the Bi₂S₃/Au NP group. These results suggest that the main effect of Bi₂S₃/Au NPs on diabetic wounds is to eliminate bacteria adsorbed on the surface, which can promote the rapid formation of the scabs in the wounds and is conducive to early wound healing. On the 14th day, the mice in the Bi₂S₃/Au NPs + NIR group had more hair growth at the wound site compared to the control group, and most of the wounds were covered by hair.

Fig. 5 Healing of infected diabetic wounds by Bi₂S₃/Au NPs. (A) Schematic diagram of the experimental process. (B) Wounds in different groups of mice at different time intervals. (C) Weight changes in diabetic mice and normal mice. (D) Changes of blood glucose in diabetic mice and normal mice. (E) The wound size of mice in each group at different time intervals. (F) Schematic diagram of wound contraction of mice in each group (n = 4, mean ± SD, *p < 0.05, **p < 0.01).

Fig. 5(E) shows the relative changes of the wound areas in each group within 14 days, which were represented as the ratio of the wound area at different time intervals to the initial wound area on day 0. There was a significant difference between the Bi₂S₃/Au NPs + NIR group and other groups at the early stage of wound healing. Despite not being exposed to NIR irradiation, Bi₂S₃/Au NPs still had a certain inhibitory effect on bacterial growth, which can be attributed to the POD-like enzyme activity of the Bi₂S₃/Au NPs.

Fig. 6 shows the H&E staining and Masson staining images of the newly generated epithelial tissue. On the 7th day, there was a significant differentiation of keratinocytes in the basal layer, thickening of the spinous layer, and a more obvious granular layer in the Bi₂S₃/Au NPs + NIR group. The dermis layer gradually formed with a large proliferation of fibroblasts and a large number of collagen fibers and elastic fibers filling the intercellular spaces. At the same time, some capillaries were also formed. In the blank group, the thinner spinous layer, fewer keratinocytes, and slower fibroblast division were observed, and the wounds were not completely filled. On the 14th day, the collagen fiber content in the dermis in the Bi₂S₃/Au NPs + NIR and Bi₂S₃/Au NPs group significantly increased, and more capillaries and some hair follicles formed while the blank group displayed fewer fibroblasts and thinner dermis layer.

Fig. 6 H&E staining and Masson staining of skin tissue in different groups on the 7th and 14th day.

Collagen is related to the formation of new blood vessels, while the formation of a vascular network can accelerate cellular metabolism and promote the synthesis and deposition of collagen fibers.³⁶ Masson staining was used to evaluate collagen deposition in the wounds at each time interval. The results show that, on the 7th and 14th day, collagen deposition at the wound sites in the Bi₂S₃/Au NP group was more pronounced than that in the blank group. The quantitative analysis (Fig. S8, ESI†) shows that there was a significant increase in collagen volume fraction in the Bi₂S₃/Au NPs + NIR group in the early stage of wound healing.

To explore the biological safety of Bi₂S₃/Au NPs, on the 14th day, H&E staining was performed on tissue sections of the heart, liver, spleen, lungs, and kidneys, and the content of Bi and Au elements in different tissues was also measured. As shown in Fig. 7, no significant organ damage or inflammatory lesions were observed, and the content of Bi and Au elements in different organizations was within the safe dose range (Fig. S9, ESI†), indicating the good biological safety of the Bi₂S₃/Au NPs.

Fig. 7 H&E staining images of various visceral tissue sections in different groups on the 14th day.

4. Conclusions

In summary, a facile strategy has been developed to synthesize the heterogeneous Bi₂S₃/Au NPs with high POD-like enzyme activity and photothermal conversion efficiency. The Schottky defects of Bi₂S₃ are amplified due to the formation of the Bi₂S₃/Au NPs, which promotes the fast movement of free electrons on the surfaces of Au and Bi₂S₃, endowing the Bi₂S₃/Au NPs with POD-like enzyme activity, capable of generating abundant ROS such as ˙OH in the presence of H₂O₂ and effectively protecting against bacterial infections. On the other hand, the plasma surface resonance effect of Au generates free electrons, which can absorb energy and enter the excited state under NIR irradiation, and then release the energy in the form of heat. The in vitro experimental results show that, the antibacterial rate of the Bi₂S₃/Au NPs was 99.8 ± 0.03% and 99.9 ± 0.01% against E. coli and S. aureus bacteria, respectively, under NIR irradiation. Animal experiments on the infected diabetic wounds demonstrate that the treatment of Bi₂S₃/Au NPs under NIR irradiation significantly promoted the proliferation of fibroblasts, collagen deposition, and the formation of epithelial and dermal tissue. The synergistic actions of the POD-like enzyme activity and photothermal effect of the Bi₂S₃/Au NPs can effectively inhibit the formation of biofilms caused by the bacteria and promote the rapid formation of scabs in the wounds, which is conducive to early wound healing. This study provides a promising solution for innovative therapy of refractory diabetic wounds, which is of great significance for reducing the abuse of antibiotics and the production of drug-resistant bacteria.

Data availability

All data generated or analyzed during this study are included in the main text and the ESI.†

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

The authors gratefully acknowledge financial support from the National Key Research and Development (R&D) Program of China (No. 2024YFF0508601).

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Footnote

† Electronic supplementary information (ESI) available: Fig. S1–S9. See DOI: https://doi.org/10.1039/d5tb00446b

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