MnOx nanoflower-based ultrasound-responsive drug delivery for sonodynamic therapy of wound infections

Abstract

The rapid spread of drug-resistant bacterial infections has become a major global health challenge, particularly in the treatment of deep organ abscesses, which often lead to severe and life-threatening infections. Traditional light-responsive and microenvironment-responsive nanoparticle drug delivery systems (DDSs) have limitations in treating deep abscesses. In contrast, ultrasound (US)-driven sonodynamic therapy (SDT), with its non-invasive, targeted radiation and excellent tissue penetration capabilities, offers great potential for eradicating deep bacterial infections. This study proposes an ultrasound-driven manganese-based nanoparticle drug delivery system (AMP) for the effective treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. The core of the system is manganese oxide (MnOx) nanoflowers, which serve as the nanoparticle carrier, loaded with the antimicrobial non-antibiotic drug auranofin, and surface-modified with polyethylene glycol to enhance its biocompatibility and drug delivery performance. AMP demonstrates significant antibacterial activity in vitro and effectively promotes wound healing under US-driven stimulation. Furthermore, the potential antibacterial and wound-healing mechanisms of AMP are revealed for the first time. This study cleverly combines non-antibiotic drugs with inorganic nanomaterials to successfully achieve efficient SDT, providing a new and effective strategy for treating deep bacterial infections.

1. Introduction

Multidrug-resistant (MDR) bacteria have become a major global challenge to public health and human health [[1], [2], [3]]. With the escalating issue of antibiotic overuse, many common pathogens have gradually evolved into so-called “superbugs,” further exacerbating the global public health crisis [4,5]. However, the development of new antibiotics has significantly lagged the evolution of antibiotic resistance, leading to a worsening problem of antimicrobial resistance [[6], [7], [8]]. Therefore, the search for alternative antibacterial strategies has become urgent. In recent years, researchers have explored various alternative therapeutic approaches, including heat, light, electricity, and magnetism, to combat multidrug-resistant bacteria [[9], [10], [11]]. However, these methods still have certain limitations, such as unavoidable damage to normal tissues, insufficient reactive oxygen species (ROS) production, limited tissue penetration depth, and potential systemic adverse reactions [12,13]. Consequently, there is an urgent need to develop safer and more universal antibacterial strategies. Sonodynamic therapy (SDT), owing to its excellent tissue penetration ability and minimal damage to normal tissues, has emerged as a promising approach for both antibacterial and cancer therapies [14,15]. Specifically, SDT activates sonosensitizers using ultrasound (US) to generate highly efficient ROS, such as singlet oxygen (¹O₂) and hydroxyl radicals (·OH), thereby effectively killing bacteria without inducing resistance [[16], [17], [18]]. However, traditional sonosensitizers, such as porphyrins and titanium dioxide, suffer from drawbacks like poor stability and limited biocompatibility, which significantly affect the clinical efficacy of SDT [16,19].

Metal oxide nanoparticles and their hybrids have become important materials in the fields of photocatalysis and biomedicine due to their excellent optical, electronic, photocatalytic, and biological properties [20]. Their non-toxicity, large surface area, appropriate optical bandgap, and high biological activity endow them with broad application potential in these areas [21,22]. In recent years, multivalent manganese oxide (MnOx) nanomaterials have attracted widespread attention. MnOx possesses a narrow bandgap, which makes it prone to the separation of electrons and holes under US stimulation [23]. Additionally, in the absence of external stimuli, MnOx can catalyze the generation of ¹O₂ in acidic environments, a property that may enhance the therapeutic effect of SDT in bacterial infection microenvironments [24]. Therefore, MnOx is considered a promising SDT sonosensitizer [25].

Auranofin (AUR) is an organogold compound used for the treatment of rheumatoid arthritis [26]. Studies have shown that it exhibits relevant biological activities in other clinical applications, including neurodegenerative diseases, tumors, and bacterial infections [27,28]. In Gram-positive bacteria, thioredoxin reductase (TrxR) is an important enzyme that maintains thiol redox homeostasis and reduces ROS-induced cellular damage [29,30]. The antimicrobial mechanism of AUR involves inhibiting bacterial TrxR, thereby reducing the bacterial reduction capacity, increasing bacterial sensitivity to oxidative stress, and enhancing damage caused by ROS [31].

Herein, this study designs an ultrasound-responsive SDT nanodrug delivery system, AUR@MnOx@PEG (AMP), for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. As shown in Scheme. 1, MnOx nanoflowers are first synthesized by reducing potassium permanganate (KMnO₄) with oleic acid (OA), followed by loading the antimicrobial agent AUR, and finally surface modification with methoxy poly(ethylene glycol) amine (mPEG-NH₂) to form the AMP nanocomposite. In this drug delivery system, MnOx generates ¹O₂ and releases Mn²⁺ under ultrasound stimulation. The released Mn²⁺ undergoes a Fenton reaction with hydrogen peroxide in the bacterial infection microenvironment, producing ·OH radicals. Meanwhile, the loaded AUR inhibits the bacterial TrxR enzyme activity, thereby amplifying the lethal effect of ROS on bacteria and significantly enhancing its antimicrobial activity. In vitro antibacterial tests show that AMP, as a sonosensitizer, achieves a 95.8 % bactericidal rate against MRSA at a concentration of 25 μg/mL under ultrasound irradiation. Additionally, in vivo experiments demonstrate that AMP exhibits significant wound healing effects on MRSA-infected mouse wounds and activates wound repair-related mechanisms. This work provides a new approach for the development of non-antibiotic antimicrobial materials and opens new therapeutic avenues for effectively addressing bacterial infections.

Scheme 1.

The synthesis route of AUR@MnOx@PEG and its antibacterial mechanism against MRSA bacteria.

2. Materials and methods

2.1. Material

Potassium permanganate (KMnO₄, 98 %), oleic acid (OA, 96 %), sodium chloride (NaCl, 99 %), dimethyl sulfoxide (DMSO, 98 %), anhydrous ethanol (EtOH, 99 %), and methylene blue (MB, >70 %) were purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd. 1,3-Diphenylisobenzofuran (DPBF, 97 %) and 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO, 97 %) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 2,2,6,6-Tetramethylpiperidine was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Hydrogen peroxide (H₂O₂, 30 %) was purchased from Tianjin Tianli Chemical Reagent Co., Ltd. Agar, tryptone, and yeast extract were purchased from Thermo Fisher Scientific. Bacterial live/dead staining kit, MTT kit, MDA detection kit, and BCA protein concentration detection kit were purchased from Shanghai Biotian Biotechnology Co., Ltd. DMEM medium, F12-K medium, trypsin cell digestion solution, antibiotics, and fetal bovine serum were purchased from Wuhan Biosharp Biotechnology Co., Ltd.

2.2. Synthesis of MnOx nanoflowers

KMnO₄ (200.0 mg) dissolved in distilled water (100 mL) was rapidly stirred for 30 min. OA (2 mL) was then added to the above solution, which was stirred for another 5 h. The brown-black product was collected by centrifugation (8000 r/min) and washed several times with deionized water and alcohol to remove residual reactants. The purified MnOx was then dispersed into deionized water and stored at 4 °C for later use [32].

2.3. Synthesis and characterization of AMP nanoparticles

MnOx (15.0 mg) and AUR (2.0 mg) were dispersed in 5 mL of dimethyl sulfoxide (DMSO) and stirred for 12 h. The product AUR@MnOx (AM) was collected by centrifugation (8000 r/min). Subsequently, AM (10.0 mg) and PEG (10.0 mg) were dispersed in 10 mL of ultrapure water and stirred for another 4 h, and the product AMP was collected by centrifugation (8000 r/min).

The zeta potential and hydrodynamic dimensions of MnOx and AMP were measured by using Nano-ZSZEN3600 (Malvern Instruments). The morphology of MnOx and AMP was observed by TEM. XPS and FTIR were used to characterize AMP. The distribution of elements in AMP nanoparticles was analyzed by elemental Mapping.

2.4. AUR loading efficiency

The loading efficiency of AUR was calculated from UV–vis spectral analysis at the wavelength of 217 nm. The supernatant after stirring and centrifugation of MnOx (15 mg) and AUR (2 mg) in 5 mL of DMSO was collected and preserved, and its UV absorption at 217 nm was measured, and the concentration of AUR in the supernatant was calculated according to the AUR standard curve to evaluate the loading efficiency of AUR.

2.5. Sonodynamic performance of MnOx and AMP in vitro

The DPBF was employed to quantitatively evaluate the generation of ¹O₂ by the samples. DPBF (0.1 mM) was dissolved in DMSO (10 mL) to prepare a 10 mM solution. MnOx (1.0 mg) and AMP (1.0 mg) were added to DMSO (9950 μL) with DPBF (10 mM, 50 μL), respectively. The sample was subjected to US exposure (1.0 MHz, 1.5 W/cm²) for 10 min. Subsequently, the UV–vis absorption spectrum of the DPBF solution was recorded from 350 to 500 nm. The ·OH production was detected using MB color development. The solid powder of MnOx and AMP was dissolved into ultrapure water (nanomaterial concentration: 100 μg/mL), 25 μ M MB solution and 10 mM H₂O₂ solution were added, and US treatment (1.0 MHz, 1.5 W/cm²) was applied for 10 min, and the range of UV sbsorption spectra should be 550–700 nm were tested with the UV-absorption spectra of H₂O₂ were used as a reference to evaluate the effect of generating ·OH.

¹O₂ and ·OH production were detected using an electron spin resonator (ESR). The solid powders of MnOx and AMP were dissolved in ultrapure water (nanomaterial concentration: 1 mg/mL), and then the ROS trapping agents TEMP and DMPO (100 μL, 100 mM) were added, respectively, and the effect of ROS produced by the samples was determined.

2.6. In vitro interaction of AMP and bacterial

In order to study the antimicrobial effect of the materials, MRSA bacterial suspension (100 μL, 1 × 10⁸ CFU/mL) was mixed with different concentrations of AMP nanomaterials (100 μL) and diluted to 1 mL with sterile PBS to achieve final material concentrations of 6.25, 12.5, 25, 50, and 100 μg/mL in the mixture, and the US was processed (1.0 MHz, 1.5 W/cm²) for 10 min, at 37 °C 2 h under the condition of incubation. After co-incubation for 2 h under the condition of 10⁴-fold dilution, 20 μL of the diluted bacterial solution was spread on agar plates, and the plates were placed in a 37 °C constant temperature incubator for overnight incubation. Finally, the number of colonies on the agar plate was counted.

2.7. Bacterial live/dead staining

According to the above procedure, the bacterial solution after co-incubation was centrifuged and washed twice with PBS, and stained with PI (10 μg/mL) and DMAO (10 μg/mL) for 15 min, respectively, and then imaged with a fluorescence microscope to observe the bacterial staining effect and trend.

2.8. TEM and SEM of bacterial

The types of antimicrobial mechanisms were explored by observing the changes in surface morphology of bacteria after different treatments. The material-treated bacterial cells were first fixed with 2.5 % glutaraldehyde, followed by dehydration with a series of ethanol solutions with concentration gradients (15 %, 30 %, 50 %, 75 %, 90 %, 100 %). Finally, the dehydrated bacterial suspension was dropped on copper mesh and silicon wafers, and the bacterial morphology was observed by TEM and sputtering gold spray SEM.

2.9. Bacterial ROS staining and confocal imaging

Detection of ROS in bacteria: Take MRSA bacterial solution (100 μL, 1 × 10⁸ CFU/mL), incubate the material-treated bacteria for 2 h, centrifuge the supernatant, and wash with PBS twice. According to the procedure of Reactive Oxygen DCFH-DA Detection Probe Kit, 10 µM DCFH-DA solution was added and incubated with MRSA bacteria for 0.5 h. After staining, the bacteria were washed twice with PBS, and CLSM was used to observe the distribution of ROS inside the bacteria and to compare the ability of the different components of the nanomaterials to produce ROS.

2.10. In vitro cytotoxicity assay

To test the cytotoxicity of AMP using the MTT assay, LO2 human normal hepatocytes were inoculated in 96-well cell culture plates at a density of 1 × 10⁴ per well, and then the well plates were cultured in a biochemical incubator (5 % CO₂, 37 °C). After the cells at the bottom of the well plate grew to about 60 % of the bottom, the culture medium was removed from the well plate. Then, 100 μL of AMP (6.25, 12.5, 25, 50, and 100 μg/mL) of different concentrations were added to each well, and a blank with only DMEM (10 % FBS, 1 % double antibody) was set up as a control group, and incubated in the incubator overnight. Subsequently, 20 μL of MTT (5 mg/mL) was added to each well and incubation was continued for 4 h. At the end of the incubation, the supernatant was carefully removed, and then 150 μL of DMSO was added to each well to dissolve the mezanine at the bottom of the small wells, and the well plate was subsequently placed on a shaker and shaken to dissolve it fully. The absorption value at 490 nm was measured by using an enzyme marker and the cell viability was calculated according to the formula. The cytotoxicity assessment of A549 cells follows the same experimental procedure as described above.

$$\mathit{Cellviability}(\%)=\frac{{\mathit{OD}}_{490}(experiment)}{{\mathit{OD}}_{490}(control)}\times 100\%$$

2.11. In vivo acute toxicity assessment

All animal experiments were conducted in accordance with the animal ethical procedures and guidelines of the People's Republic of China and approved by the Animal Ethics Committee of Huazhong Agricultural University (HZAUMO-2025-0035). AMP dispersion (400 μg/mL), was injected subcutaneously into BALB/c mice and kept for 24 h. Controls were injected with PBS. Blood was obtained from mice by eyeball removal and euthanasia, and the obtained blood was preserved in EDTA or sodium citrate anticoagulation tubes for routine blood analysis.

2.12. Hemolysis assay

Whole blood was obtained from healthy BALB/c mice using the eyeball removal method, collected in anticoagulated tubes containing EDTA or sodium citrate, shaken well and centrifuged (3000 r/min, 10 min) to obtain erythrocytes, and washed by centrifugation with PBS buffer solution until the supernatant was free of visible red color. Then 2 % erythrocytes were mixed with different concentrations of AMP dispersions (6.25, 12.5, 25, 50, and 100 μg/mL), and the supernatant was incubated at room temperature for 4 h. After the incubation, the supernatant was centrifuged (3000 r/min for 10 min), and then the supernatant was analyzed by using an enzyme marker at 540 nm. The AMP hemolysis rate was calculated by using the absorbance value at 540 nm with an enzyme marker and according to the formula. The PBS group was used as a negative control and the Water group as a positive control.

$$\mathit{Hemolysisrate}(\%)=\frac{{\mathit{OD}}_{540}(experiment)}{{\mathit{OD}}_{540}(control)}\times 100\%$$

2.13. In vivo sonodynamic antibacterial therapy

All animal experiments were conducted in accordance with the animal ethical procedures and guidelines of the People's Republic of China and approved by the Animal Ethics Committee of Huazhong Agricultural University . To evaluate the antimicrobial capacity of AMP in vivo and its ability to promote wound healing, a mouse model of MRSA wound infection was established. BALB/c mice (6–8 weeks, weighing 16–18 g) were selected, and a circular skin wound with a diameter of 10 mm was constructed on the back of the mice by surgical means and infected with a drop of 100 μL of MRSA bacterial solution (1 × 10⁸ CFU/mL) for 24 h. After successful establishment of the infection model, the mice were divided into six groups, namely, PBS, PBS + US, MnOx, MnOx + US. AMP and AMP + US, 6 mice in each group, and the US group was treated (1.0 MHz, 1.5 W/cm²) for 10 min [33]. Then, the infected wound area of each mouse was treated with different nanomaterials every 1 d, and the repair of the wounds was recorded with a digital camera at different time points (day 0, 2, 4, 6, and 8), and the residual bacteria in the epidermis were collected on day 8. At the same time, the mice were monitored for the Body weight changes. After the experiments were completed, the mice were euthanized, and the blood of both treated and healthy mice was analyzed for blood biochemical indexes. The collected skin tissue samples were fixed with 4 % paraformaldehyde and then subjected to H&E staining, Masson staining analysis and immunohistochemical analysis. The collected samples of mouse viscera treated with different materials were fixed with 4 % paraformaldehyde and then continued with H&E staining to assess whether the mouse viscera showed lesions.

To evaluate the in vivo antimicrobial capacity of AMP and its role in promoting wound healing, a mouse model of MRSA-induced skin abscess infection was established. BALB/c mice (6–8 weeks old, weighing 16–18 g) were selected, and 100 μL of MRSA bacterial suspension (1 × 10⁸ CFU/mL) was subcutaneously injected into the experimental mice using a medical syringe to induce abscess formation. After 48 h, the mice were randomly divided into 6 groups, namely the PBS, PBS + US, MnOx, MnOx + US, AMP, and AMP + US, with 6 mice in each group. The US groups were treated under the conditions of 1.0 MHz and 1.5 W/cm² for 10 min. Subsequently, the infected wound areas of each mouse were treated with different nanomaterials every other day, and the wound repair was recorded with a digital camera at different time points (day 0, 2, 4, 6, 10, and 14). Residual bacteria in the abscess area were collected on day 14. Meanwhile, the body weight changes of the mice were monitored.

2.14. Statistics

All date in this work were displayed as means ± SD (Standard Deviation). Statistical significance was determined with a p-value: P < 0.05(*), P < 0.01(**), P < 0.001(***).

3. Results and discussion

3.1. Preparation and characterization of AMP

MnOx nanoflowers with homogeneous morphology were synthesized by referring to the MnOx synthesis method of Zhang et al [32]and improving it. As shown in Fig. 1A, its morphology was observed by TEM, and the MnOx showed a nanoflower-like structure with a size of 100 ± 10 nm and good dispersion (PDI of 0.025). AUR was loaded on MnOx nanoflowers by electrostatic adsorption and AMP was obtained by surface modification with mPEG-NH₂. As shown in Fig. 1B, its morphology was observed by TEM. the AMP still showed nanoflower-like structure with a size of 100 ± 30 nm and good dispersion (PDI of 0.183). Further, to investigate whether the AUR was successfully loaded on MnOx nanoflowers, the material was characterized by high-angle annular dark-field scanning transmission and surface scanning of the corresponding elements. As shown in Fig. 1C, there is a clear co-localization of Au elements on top of Mn elements, indicating that AUR was loaded on the MnOx nanoflowers. Dynamic light scattering (DLS) studies showed (Fig. 1D) that the particle sizes of MnOx are around 180 nm, while that of AUR@MnOx is around 200 nm. The material's Zeta potential indicated (Fig. 1E) that the negative surface charge of the material changed from -27.3 mV to -25.7 mV after loading AUR; and the negative surface charge of the material changed to -22.3 mV after modifying mPEG-NH₂, which initially indicated the successful loading of AUR and the successful modification of mPEG-NH₂. Furthermore, the successful synthesis of the materials was verified by ultraviolet-visible (UV-vis) absorption spectroscopy. As shown in Fig. 1F, both AUR and mPEG-NH₂ exhibited distinct absorption peaks in the 200-220 nm range. Compared with MnOx, AMP demonstrated significantly enhanced absorbance in this wavelength region, further confirming the successful fabrication of the composite material. Then, the organic functional groups of mPEG-NH₂, AUR, MnOx and AMP were analyzed by Fourier Transform Infrared (FT-IR) spectrometer. As shown in Fig. 1G, the carbonyl and ether bond stretching vibration peaks appeared at the vicinity of 1750 cm^-1 and 1010 cm^-1 for AUR and AMP, respectively, whereas no obvious peaks were found for MnOx at these two locations, which also indicated the successful loading of AUR. Next, the loading rate of AUR was determined by using UV-vis absorption and calculated from the standard curve to be 11.4 % (SI. 1). Finally, the elemental valence states of AMP were analyzed by X-ray photoelectron spectroscopy (XPS). The distribution of elemental species tested by XPS was consistent with the surface-scanning results, and the analysis of the elemental valence states of Mn indicated that the prepared nanoparticles were double elongated crystalline MnO₂ with MnOx on the surfaces (Fig. 1H, 1I, SI. 2, SI. 3). In summary, we successfully synthesized AMP [24].

Fig. 1.

AMP materials synthesis and characterization. (A) TEM image of MnOx (scale bar: 100 nm). (B) TEM image of AMP (scale bar: 100 nm). (C) Mapping images of AMP (scale bar: 50 nm). (D) Dynamic light scattering particle size. (E) Zeta potential. (F) UV–vis absorption spectrum. (G) FT-IR absorption spectrum. (H) XPS spectra of AMP. (I) XPS spectrum of Mn element in AMP. The data are presented as the mean ± SD, n = 3 (Technical repetitive experiment).

3.2. Sonodynamic performance of AMP in vitro

Under US stimulation, MnOx can convert O₂ into toxic ¹O₂, which causes damage to bacteria. 1,3-Diphenylisobenzofuran (DPBF) is a commonly used reagent for degrading ¹O₂. Therefore, the sonodynamic production of ¹O₂ by the material was evaluated by using DPBF (Fig. 2A). After 10 min of US stimulation, a significant decrease in the absorbance of DPBF can be observed for the MnOx and AMP groups. In contrast, the DPBF without added materials did not show a significant decreasing trend in absorbance after 10 min of US treatment (Fig. 2A, 2B, SI. 4, SI. 5). This indicated that MnOx had excellent sonodynamic properties and the AMP-loaded AUR and mPEG-NH₂ do not significantly affect the sonodynamic properties of the material. And the ¹O₂ produced by the material has a significant time-dependence, which increases with the increase of US treatment time, which suggests that the material has a long-lasting sonodynamic performance.

Fig. 2.

Performance testing and characterization of AMP materials. (A) ¹O₂ generation activity of different materials. (B) ¹O₂ generation activity of AMP at different time intervals. (C) ESR characterization of ¹O₂ generation under US conditions. (D) ·OH generation activity of different materials. (E) ·OH generation activity of AMP at different concentrations. (F) ·OH generation activity under US conditions. (G) ·OH generation activity of AMP at different H₂O₂ concentrations. (H) ESR characterization of ·OH generation under US conditions. (I) The consumption of hydrogen peroxide by AMP at different concentrations. The data are presented as the mean ± SD, n = 3 (Technical repetitive experiment).

2,2,6,6-Tetramethylpiperidine (TEMP) is a commonly used ¹O₂ trapping agent. Subsequently, TEMP was used to monitor the ROS species produced by the material under US treatment. Fig. 2C (SI. 6, SI. 7) shows the results of the EPR analysis of ¹O₂, and both the AMP + US group and the MnOx + US group show distinct ¹O₂ characteristic peaks (1:1:1). In addition, the ·OH production of the materials was also evaluated by Methylene blue (MB). The production of ·OH could cause the discoloration of MB solution. Fig. 2D, 2F show that even without the addition of H₂O₂ and US, MnO_x and AMP alone can generate ·OH to discolor MB, and the addition of 1 mM H₂O₂ could produce more ·OH. After US treatment, the production of ·OH could be further increased. Moreover, the ·OH produced by AMP exhibits a material concentration-dependent and H₂O₂ concentration-dependent variation (Fig. 2E, 2G). Subsequently, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to monitor the ·OH produced by the materials. Fig. 2H (SI. 8, SI. 9) shows the EPR analysis results of ·OH, and it could be seen that both the MnO_x group and the AMP group display distinct characteristic peaks of ·OH (with a ratio of 1:2:2:1). Finally, the titanium sulfate colorimetric assay verified the consumption of H₂O₂ by AMP·H₂O₂ could react with titanium sulfate to form a yellow peroxide-titanium complex, and the absorbance at 410 nm is directly proportional to the H₂O₂ concentration. The experimental results indicate that the consumption of H₂O₂ by AMP exhibits a concentration-dependent variation (Fig. 2I, SI. 10).

3.3. Antibacterial of AMP in vitro

Using the dilution plate coating method, we evaluated the effectiveness of AMP in inhibiting free MRSA bacteria in vitro [34]. As shown in Fig. 3A, 3B, the inhibitory effect of AMP on MRSA bacteria after SDT at different concentrations was investigated. The results showed that after a single ultrasound treatment (1.5 W/cm², 10 min), the inhibition rate of MRSA bacteria reached 95.8 % when the AMP concentration was 25 μg/mL, showing a significant inhibition trend compared to the untreated MRSA. Therefore, a concentration of 25 μg/mL AMP was chosen for subsequent experiments. Next, we investigated the inhibitory effects of different control groups on MRSA bacteria after ultrasound treatment. As shown in Fig. 3C, 3D, the effects of SDT with the same concentration (25 μg/mL) of MnOx and AMP on MRSA are shown. The results showed that a single US treatment (1.0 MHz, 1.5 W/cm²) for 10 min had no significant inhibitory effect on the bacteria (bacterial survival rate of 97.3 %). In contrast, after US treatment (1.0 MHz, 1.5 W/cm²) for 10 min, the AMP group showed good antibacterial effect, with almost complete disappearance of surface colonies and an antibacterial rate of 97.9 %. It is hypothesized that the antibacterial effect comes from two aspects: on the one hand, the ROS generated by the ultrasonic response of AMP can damage the bacteria, and on the other hand, the AUR released by AMP amplifies the damage caused by the ROS to the bacteria by decreasing the activity of the TrxR enzyme of the bacteria and attenuating the reducing ability of the bacteria so as to achieve the effect of killing the bacteria completely. The AMP group without US treatment and the MnOx group with US treatment also showed some antibacterial effects (antibacterial rates of 80.0 % and 90.1 %, respectively), and it was hypothesized that the former was the antibacterial effect exerted by AUR, while the latter was the antibacterial effect exerted by the ROS generated by the sonodynamic of MnOx. It is noteworthy that the MnOx group without US treatment also showed a weak antimicrobial effect (antimicrobial rate of 44.1 %), which is hypothesized to be the weak antimicrobial effect exerted by the Fenton-like reaction of Mn²⁺ released by the material with the small amount of H₂O₂ produced by bacteria to produce ·OH.

Fig. 3.

In vitro antibacterial activity of AMP against MRSA bacteria. (A) Plate images of MRSA bacteria treated with different concentrations of AMP. (B) Quantification of antibacterial efficacy of AMP against MRSA at different concentrations. (C) Plate images of MRSA bacteria after treatment with different materials. (D) Quantification of bacterial count after MRSA treatment with different materials. (E) CLMS images of bacterial live/dead staining (Green: DMAO, Red: PI, scale bar: 50 μm). (F) TEM images of MRSA after treatment with different materials (scale bar: 250 nm). (G) SEM images of MRSA after treatment with different materials (scale bar: 250 nm). (H) CLMS images of bacterial ROS levels staining (Green: DCFH-DA, scale bar: 50 μm). (I) Quantification of bacterial ROS levels. (J) MDA levels of MRSA bacteria. (K) Protein levels in the supernatant after treating MRSA with different materials. The data are presented as the mean ± SD, n = 3 (Technical repetitive experiment); p-values are assessed by one-way analysis of variance (ANOVA) analysis; *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4. Bacterial live/dead staining

To further confirm the antimicrobial activity of AMP, this study evaluated its inhibitory effect on MRSA using live/dead fluorescent staining. Propidium Iodide (PI) and N, N-dimethylaniline N-oxide (DMAO) are two commonly used fluorescent dyes for detecting the activity and death status of bacteria. They work as follows: PI can penetrate damaged bacterial cell membranes, bind to DNA and emit red fluorescence, thus marking dead bacteria; whereas DMAO can penetrate intact bacterial cell membranes and is used to stain live bacteria, which shows blue fluorescence under UV light to help differentiate live bacteria [35]. As evidenced by the previous experiments, ROS and AUR released in response to AMP ultrasound demonstrated a good synergistic effect, which could effectively play an antibacterial activity. As shown in Fig. 3E, from the fluorescence imaging, It can be seen that the AMP + US group can produce a large amount of red fluorescence, which has good antimicrobial activity compared with PBS.

3.5. Antimicrobial mechanism of AMP is revealed

Bacterial cell membranes serve to maintain bacterial shape, osmoregulation, protection against mechanical external forces, and resistance to infection, so damage to bacterial cell membranes may result in their dysfunction and leakage of bacterial contents, ultimately leading to bacterial death. Previously, it was confirmed by plate coating and bacterial live/dead staining that ROS produced by AMP stimulated by US could lead to bacterial death. Further, the bacterial morphology was observed using electron microscopy to elucidate the potential antibacterial mechanism. As shown in Fig. 3F, 3G, it can be clearly seen that the nanomaterials aggregated around the bacteria and had direct contact with them. In TEM, it can be found that the shape of the MRSA changed after AMP + US treatment compared with the PBS and US groups, and the surrounding of the bacteria became fuzzy, showing obvious membrane damage oxidative stress and content leakage. The phenomenon of bacterial cell membrane collapse was also clearly observed in SEM. This can indicate that the change in the shape of the bacteria led to the death of the bacteria.

ROS play an important role in cell signaling and homeostasis in vivo. However, the rapid accumulation of ROS can cause bacterial oxidative stress, resulting in severe damage or even death to bacteria31. Therefore, DCFH-DA fluorescent probe was utilized to label ROS in bacteria, and the distribution of ROS within bacteria was observed by CLSM (Fig. 3H). At the same time, quantify the green fluorescence of ROS inside the MRSA bacteria after treatment with the material, as shown in the Fig. 3I. The results showed that the AMP + US group exhibited the strongest green fluorescence, i.e., AMP has SDT-responsive properties, enabling it to generate a large amount of ROS and effectively kill bacteria.

In the preliminary stage, we investigated the antimicrobial mechanism of AMP by analyzing bacterial morphology and using the DCFH-DA probe to label the ROS levels inside MRSA bacteria [36]. ROS act on the bacterial cell membrane, oxidizing the lipids within the membrane, leading to membrane rupture. Malondialdehyde (MDA), a product of lipid oxidation, is the most direct parameter for detecting cellular lipid oxidation. Therefore, we used an MDA assay kit to measure MDA levels in bacterial cell membranes (SI. 12), reflecting the degree of lipid oxidation in the bacterial cell membrane. As shown in Fig. 3J, the MDA level was highest in the AMP + US group, indicating the most significant lipid oxidation, which directly reflects the ROS levels and the extent of bacterial cell membrane rupture, consistent with the results of previous experiments. In conclusion, AMP exhibits SDT-responsive characteristics, capable of inducing lipid oxidation in bacterial cell membranes, leading to bacterial rupture and subsequent bacterial death.

Proteins play a crucial role in maintaining the normal physiological functions of bacteria. The rupture of the bacterial cell membrane leads to the leakage of intracellular proteins, thereby disrupting the physiological activities of the bacteria. To assess this process, we employed the BCA protein quantification assay kit to measure the protein content in the supernatant of MRSA bacteria following treatment. As shown in Fig. 3K (SI. 11), the protein content in the supernatant of MRSA in the AMP + US group was the highest. This result is consistent with previous experiments and suggests that the large amounts of ROS generated by AMP + US directly target the bacterial cell membrane, causing lipid oxidation within the membrane, which in turn induces membrane rupture, resulting in the leakage of intracellular proteins and the disruption of MRSA bacterial physiological functions, ultimately leading to bacterial death.

3.6. In vitro and in vivo safety tests

AMP showed outstanding antimicrobial efficacy and its effect on cells was further evaluated by methyl thiazolyl tetrazolium (MTT) assay. As shown in Fig. 4A, 4B, the in vitro cytotoxicity of AMP was evaluated by co-incubating AMP with LO2 cells and A549 cells. The results showed that as the concentration of AMP increased, the survival rate of LO2 cells and A549 cells remained above 80 % even at a concentration of 100 μg/mL, indicating that AMP had no significant in vitro cytotoxicity. Next, the in vitro hemolysis rate of AMP was evaluated (Fig. 4C, SI. 13). Different concentrations of AMP solutions and erythrocyte suspensions were contacted, co-incubated and then centrifuged to test the hemoglobin content in the supernatant, using the PBS group as a negative control and the Water group as a positive control. The supernatants of the different AMP concentration groups were as clear as those of the PBS group and did not show hemolysis. However, the supernatant of erythrocytes treated in the Water group showed obvious hemoglobin leakage and showed obvious hemolysis. This indicates that AMP has good hemocompatibility.

Fig. 4.

In vitro biological safety evaluation of AMP. (A) Cytotoxicity evaluation of AMP on LO2 cells. (B) Cytotoxicity evaluation of AMP on A549 cells. (C) Compatibility of AMP with mouse erythrocytes. Complete blood count analysis. (D) WBC. (E) RBC. (F) GR#. (G) Lym#. (H) Mid#. Liver function. (I) ALT. (J) AST. Kidney function. (K) BUN. (L) CR. (M) H&E staining sections of mouse five major organs (Heart, Liver, Spleen, Lung, Kidney. scale bars: 200 μm). The data are presented as the mean ± SD, n = 3 (Biological experiment repetition); p-values are assessed by one-way analysis of variance (ANOVA) analysis; *P < 0.05, **P < 0.01, ***P < 0.001.

To further evaluate the potential acute biological toxicity of AMP, subcutaneous injection of AMP was used to assess acute toxic effects in mice. After 24 h, the mice were euthanized, and blood was collected from the eyeball for routine blood analysis (SI. 14) and liver and kidney function tests [37]. As shown in Fig. 4D-4L, blood biochemical parameters (WBC, RBC, GR#, Lym#, Mid#, ALT, AST, BUN, CR, etc.) were analyzed and compared with those of normal mice. The experimental results indicated that the blood parameters of the mice injected with AMP remained within the normal range.

Finally, at the end of the in vivo antimicrobial experiments in mice, the major internal organs of the mice were sectioned and the long-term biotoxicity of the materials was assessed by H&E staining. As shown in Fig. 4M, none of the major organs of the mice treated with the material showed obvious lesions. In summary, AMP has good biosafety and biocompatibility in the treatment of mouse models.

3.7. SDT ability of AMP in vivo

AMP exhibits excellent in vitro antimicrobial activity and has a favorable biosafety profile. To investigate its in vivo antimicrobial activity and potential future medical applications, we established a mouse epidermal wound infection model and evaluated the in vivo antimicrobial activity according to the experimental procedure shown in Fig. 5A. We photographed the wounds of the mice at different time points and quantified the wound area by using Image J image processing technology.

Fig. 5.

In vivo antimicrobial activity evaluation of AMP. (A) AMP animal experiment flowchart. (B) Digital camera images of mouse wound healing after treatment with different materials and bacterial residue plate images on day 8 of the wound (scale bars: 2 m m). (C) Quantification chart of wound healing trends. (D) Quantification chart of bacterial residue on the wound on day 8. (E) Mouse body weight monitoring during material treatment. The data are presented as the mean ± SD, n = 3 (Biological experiment repetition); p-values are assessed by one-way analysis of variance (ANOVA) analysis; *P < 0.05, **P < 0.01, ***P < 0.001.

The antimicrobial and wound healing ability of nanomaterials was assessed by the healing of epidermal wounds in mice [38], as shown in Fig. 5B, 5C. It can be found that with the passage of time, the wound of each group showed a significant healing trend, and the degree of the healing trend of each group was different, and the AMP + US group had the best degree of wound healing during the treatment, whereas the healing effect of the PBS group, the US group, and the MnOx group was the poorest. On day 8 of treatment, the wound healing rate of the AMP + US group reached 72.2 %, while the wound healing rate of the PBS control group was only 32.6 %, which, in combination with the above experimental results, indicates that the AMP + US group has a good ability to promote wound healing.

Bacterial residue at the site of wound infected tissue is also a way to assess wound repair, and bacteria at the site of wound infection were collected from each group of mice on day 8. The amount of wound bacterial residue was illustrated by bacterial culture and agar plate coating. The results are shown in Fig. 5B, 5D. The agar plates demonstrated significant differences in bacterial residues. Compared to the PBS group, the AMP + US group showed the most obvious trend of bacterial reduction, with a statistically significant antibacterial rate of 98.2 %. The AMP and MnO_x + US groups also showed more obvious bacterial reduction trends, with antimicrobial rates of 75.6 % and 78.1 %, respectively, which was like the in vitro antimicrobial effect. And as shown in Fig. 5E, the body weight of mice did not show obvious fluctuation during the whole treatment process, and the overall trend was slightly increased. In summary, AMP nanomaterials have excellent in vivo antimicrobial properties after US irradiation.

Previously, AMP demonstrated excellent antibacterial performance in an in vitro epidermal infection model. To further evaluate the antibacterial capability of AMP, we established a subcutaneous abscess infection model. As illustrated in Fig. 6A, prior to the initiation of the experiment, all infected groups were subcutaneously pre-injected with strains one day in advance, and formal treatment was commenced after 48 h of initial pustule formation.

Fig. 6.

In vivo evaluation of AMP against purulent subcutaneous infection. (A) Schematic diagram of establishment of subcutaneous infection model and AMP treatment process. (B) Representative images of the skins treated by PBS, PBS + US, MnOx, MnOx + US, AMP, and AMP + US respectively at different time points and bacterial residue plate image on day 14 of the wound (scale bars: 1 cm). (C) Mouse body weight monitoring during material treatment. (D) Quantification chart of abscess area. (E) Quantification chart of bacterial residue on the wound on day 14. The data are presented as the mean ± SD, n = 3 (Biological experiment repetition); p-values are assessed by one-way analysis of variance (ANOVA) analysis; *P < 0.05, **P < 0.01, ***P < 0.001.

As shown in Fig. 6B, 24 h after subcutaneous injection of MRSA, all groups of mice exhibited obvious rejection reactions: distinct milky white areas were observed at the lesion sites, indicating disruption of the local microenvironment. After three rounds of treatment, the abscess areas in the AMP group, MnOx + US group, and AMP + US group gradually decreased. Notably, the wounds in the AMP + US group almost healed by day 14, with the abscess area reduced to only 0.19 cm², suggesting that the infection had been effectively controlled (Fig. 6D). The abscess areas in the AMP group and MnOx + US group were also relatively smaller compared to the PBS group; however, larger wounds remained on day 14, with abscess areas of 0.92 cm² and 0.58 cm², respectively, indicating inferior therapeutic efficacy compared to the AMP + US group. In contrast, the abscess area in the PBS group continued to expand until day 6, while those in the US group and MnOx group expanded gradually until day 4. By day 14, the abscess areas were 1.42 cm², 1.05 cm², and 0.95 cm², respectively (Fig. 6D).

On day 14, bacterial samples were collected from the abscess areas, and bacterial quantification was performed using the spread plate method. As shown in Fig. 6B, 6E, compared with the PBS group, nearly all bacteria in the AMP + US group were eradicated, with a bacterial survival rate of only 0.99 %. A small number of bacteria remained in the MnOx + US group, with a survival rate of 31.47 %. In contrast, many bacteria persisted in the US group, MnOx group, and AMP group, with survival rates of 97.04 %, 71.65 %, and 55.52 %, respectively. These results demonstrate that the AMP + US group can generate ROS through ultrasound and simultaneously release AUR to inhibit bacterial TrxR activity, thereby amplifying the bacterial damage caused by ROS and achieving effective bacterial eradication, which in turn promotes the healing of abscess areas. Neither the MnOx + US group (generating ROS alone) nor the AMP group (releasing AUR alone) can achieve the desired antibacterial effect. And as shown in Fig. 6C, the body weight of mice did not show obvious fluctuation during the whole treatment process.

3.8. Skin tissue section staining analysis

In the mouse epidermal wound infection model, AMP demonstrated good antibacterial activity and tissue repair ability. To further evaluate the repair effect on the infected tissue, we isolated mouse skin tissue on the 8th day of treatment and performed histological staining using H&E, Masson’s trichrome, immunohistochemistry (IHC), and immunofluorescence (IF). The repair effect and inflammatory factors were analyzed.

As shown in Fig. 7A, H&E staining results showed that after PBS and US treatment, there was obvious tissue loss at the infection site of the mice, with a large number of collagen fibers and inflammatory cell infiltration in the dermis, and the MnOx + US group and the AMP group had a certain ability to repair the skin, but there was still part of the phenomenon of tissue loss and inflammatory cell infiltration, and at the same time there were part of the follicles and sebaceous glands, while in the AMP + US Masson staining can indirectly illustrate the skin repair, collagen fibers became blue after Masson staining, as shown in Fig. 7B. The blue color was most prominent in the AMP + US group, with many collagen fibers deposited, while tissue defects and gaps were observed in both the PBS and US groups. Combined with the above experimental results, it showed that AMP had the ability to promote the repair of infected tissues.

Fig. 7.

Skin tissue section analysis. (A) H&E staining analysis (scale bar: 200 µm). (B) Masson staining analysis (scale bar: 200 µm). (C) IL-6 IHC staining analysis (scale bar: 100 µm). (D) TNF-α IHC staining analysis (scale bar: 100 µm). (E) CD86 immunofluorescence staining analysis (Blue: DAPI, Red: CD86, scale bar: 50 µm). (F) CD206 immunofluorescence staining analysis (Blue: DAPI, Green: CD206, scale bar: 50 µm). (G) IL-6 quantitative analysis. (H) TNF-α quantitative analysis. (I) CD86 fluorescence quantitative analysis. (J) CD206 quantitative analysis. The data are presented as the mean ± SD, n = 3 (Technical repetitive experiment); p-values are assessed by one-way analysis of variance (ANOVA) analysis; *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The pleiotropic cytokine interleukin-6 (IL-6), a key mediator of the chemokine network, serves critical functions in immune response potentiation and acute-phase protein induction, with its serum concentration dramatically elevated during inflammatory processes or tissue damage [39]. Tumor necrosis factor-α (TNF-α), a prototypical proinflammatory cytokine secreted primarily by activated macrophages, orchestrates inflammatory cascades through T lymphocyte activation and subsequent cytokine production. Immunohistochemical (IHC) analysis of dermal specimens across treatment groups (Fig. 7C, 7D) demonstrated the remarkable anti-inflammatory efficacy of AMP nanoparticle, as evidenced by substantial mitigation of wound site inflammation. Quantitative assessment (Fig. 7G, 7H) revealed statistically significant suppression of inflammatory mediators relative to PBS controls, with TNF-α and IL-6 expression attenuated by 86.24 % and 71.64 %.

The dynamic regulation of macrophage polarization constitutes a fundamental mechanism governing tissue repair progression. Pro-inflammatory M1 macrophages (CD86 + ) dominate the initial inflammatory phase in infected tissues, while alternatively activated M2 macrophages (CD206 + ) mediate inflammation resolution and tissue regeneration. Our investigation of AMP-mediated SDT's effects on macrophage polarization (Fig. 7E, 7I, 7J) yielded clinically relevant findings: Both PBS and US-only control groups exhibited maximal CD86 expression with concomitant CD206 suppression, reflecting persistent M1-dominant inflammation. Strikingly, the AMP + US therapeutic intervention induced significant M1-to-M2 phenotype switching, this phenotypic transition marks the critical shift from pro-inflammatory to reparative microenvironment, initiating the proliferative phase of wound healing.

3.9. Long-term toxicity assessment

Preliminary experiments indicated that AMP exhibited no cytotoxicity and did not show signs of acute toxicity in mice. In subsequent experiments, we evaluated the long-term toxicity of AMP in mice. First, during the treatment period, no significant fluctuations in body weight were observed (Fig. 5E). Subsequently, H&E staining of the major organs from each group of mice revealed no apparent pathological lesions (SI. 15). Taken together, these results demonstrate that AMP possesses good biosafety and holds potential for clinical applications.

4. Conclusion

In summary, we have proposed an ultrasound-driven manganese-based nanoparticle drug delivery system (AMP) for sonodynamic antibacterial therapy and remodeling of the infected microenvironment to promote wound healing. MnOx, containing manganese in multiple oxidation states, catalyzes the generation of ROS from H₂O₂ within the infection microenvironment. Additionally, MnOx exhibits a sonocavitation effect, which, under US, produces more ROS, thereby enabling a synergistic therapeutic effect of both chemical kinetics and sonodynamics. At the same time, US can trigger the release of AUR from the AMP platform, which, in conjunction with ROS, enhances oxidative stress damage to bacteria, thus demonstrating excellent antibacterial performance. In vitro experimental results show that AMP + US at a concentration of 50 µg/mL can achieve a 100 % inhibition rate against MRSA bacteria. Furthermore, this system can modulate the immune response of cells at the infection site and promote wound healing. This study utilizes the unique properties of MnOx to develop an ultrasound-driven nanoparticle platform that can effectively address wound infections and other related diseases, providing new insights for the development of novel sonosensitive agents.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.