Biodegradable Janus sonozyme with continuous reactive oxygen species regulation for treating infected critical-sized bone defects

Chemicals

N, N-dimethylformamide (DMF), ethanol, tetrakis (4-carboxyphenyl) porphyrin (TCPP), Manganese tetrakis (4-carboxyphenyl) porphyrin (TCPP(Mn)), Manganese chloride tetrahydrate (MnCl₂^.4H₂O), Copper nitrate trihydrate (Cu(NO₃)₂^.3H₂O), and trifluoroacetic acid (TFA) were purchased from Aladdin. Polyvinylpyrrolidone (PVP) and que were purchased from Sigma-Aldrich.

Preparation of MON(Cu), MON(CuMn) and MON(CuMn)-Q

TCPP (16 mg, 0.02 mmol) was added to 48 ml DMF solution and heated with stirring until the solution was free of particles. Cu(NO₃)₂^.3H₂O (14.4 mg, 0.06 mmol) and TFA (0.16 mmol) were then added to 16 ml of ethanol and stirred well. Finally, the mixture of DMF and ethanol solution was placed in a muffle furnace, heated to 80 °C at 2 °C/min and held for 4 h. The products were washed with ethanol, centrifuged three times (7500 × g for 10 min each time) and finally dried. In order to obtain MON(CuMn), the TCPP in the above step could be replaced with TCPP(Mn) (17 mg, 0.02 mmol). To prepare MON(CuMn)-Q loaded with different concentrations of que, 25 µg/mL, 50 µg/mL, and 100 µg/mL of que were mixed with 200 µg/mL of MON(CuMn), respectively, and stirred for a period exceeding 12 hours at room temperature. Finally, the probe sonication (JY92-IIN, Ningbo Scientz Biotechnology Co. LTD) was employed for 30 min at power density of 130 W to generate the smaller nanosheets.

Characterization

A filed-emission scanning electron microscope (FE-SEM, S-4800, Hitachi) and TEM (JEOL-2100F, Japan Electronics Co., Ltd) were used to obtain morphology characterization. To measure the thickness of each nanosheet, the AFM (Agilent 5500, Bruker Dimension Icon) was applied. The elemental composition and chemical state of the nanosheets were determined by XPS (Escalab 250Xi, Thermo Fisher Scientific).

Que-metal chelate measurement

Firstly, que solutions of 20, 50, 100, 200 and 500 µg/mL were prepared by uniformly dispersing que in PBS. Subsequently, the preparation of que and Cu²⁺ chelates was conducted by the addition of Cu(NO₃)₂^.3H₂O to a solution of que at a concentration of 100 µg/mL. The molar ratios of que to Cu²⁺ were varied, with the ratios set at 2:1, 5:1, 10:1, 20:1, 50:1, and 100:1. The que and Mn²⁺ chelates were prepared in a manner analogous to that described above, with MnCl₂^.4H₂O added to a solution of que at a concentration of 100 µg/mL. The molar ratios of que and Mn²⁺ were varied, with the aim of investigating the effects of these ratios on the properties of the chelates. Molar ratios of 2:1, 5:1, 10:1, 20:1, 50:1, and 100:1 were employed. Finally, to prepare the chelates of que, Cu²⁺ and Mn²⁺, a solution of 100 µg/mL que was used and MnCl₂^.4H₂O and Cu(NO₃)₂^.3H₂O were added to achieve molar ratios of 10:1:1, 20:1:1, and 50:1:1 for que, Cu²⁺, and Mn²⁺, respectively. The absorbance of the solutions was quantified using a fluorescence spectrophotometer (Shanghai Mepda Instrument Co., Ltd.). The instrument was operated with a PMT voltage of 700 V, a scanning speed of 1,500 nm/min, and a response time of 0.4 s. Excitation light at 425 nm and emission light between 450 nm and 800 nm were employed.

Antioxidant performance

The nanosheets were homogeneously dispersed into PBS at a concentration of 200 µg/ ml. Subsequently, the ·O₂^– scavenging ratio and CAT-like activity were measured according to the manufacturer’s protocols (BC0205, Solarbio; S0109, Beyotime). In addition, to further investigate the ·O₂⁻ and ·OH scavenging abilities of nanosheet and que-metal chelates, XPS was performed using DMPO as the spin trapping reagent. Briefly, 10 mM xanthine, 0.5 U XO, 10 mM DMPO and each nanosheet were added into the methyl alcohol solutions for ·O₂^– detection. Then an aliquot of the solution (100 µL) was transferred into the quartz tube for ESR (JES-FA200, Japan Electronics Co., Ltd) measurements. To detect ·OH, 5 mM H₂O₂, 2 mM FeSO₄, 50 mM DMPO, and each nanosheet were added into the PBS solutions and 100 µL of the solution was used for ESR measurement. For que-metal chelates, a 2 mg/mL solution of que was first prepared with water or methanol. After that, 1 mL of que solution was taken and 0.13 ml of 0.2 mg/mL MnCl₂^.4H₂O solution and 0.16 ml of 0.2 mg/mL Cu(NO₃)₂^.3H₂O solution were added. After mixing, 10 µL was taken for ESR determination. To further investigate the ability of nanosheets or chelates to scavenge ROS at different PH values, we added 1 M HCl or NaOH and used a pH meter to adjust the final solution pH to 5.8, 7.8, and 9.8 before measuring with the kits or ESR.

Ion release measurement

The MON(Cu), MON(CuMn) and MON(CuMn)-Q solutions (200 µg/mL) were incubated at 37 °C for short (0, 2, 12 and 24 h) and long (3, 5, 7, 14, 21 and 28 d) periods. Afterwards, the supernatants were collected and the release of ions was measured using inductively coupled plasma atomic emission spectrometry (ICP-AES, Optimal 8000, Perkin-Elmer), inductively coupled plasma optical emission spectrometry (ICP-OES, Vista-MPX, Varian) and inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7800 Series). In order to detect the content of Cu²⁺ and Mn²⁺ in MON(Cu), MON(CuMn) and MON(CuMn)-Q, concentrated nitric acid was added to a solution of nanosheets at a concentration of 200 µg/mL and finally stored at 60 °C for 12 hours. Following this, the solution was measured using the instrument.

In order to ascertain whether US has an impact on ion release, we proceeded to take the nanosheet solutions both before and after US, and subsequently incubate them at 37 °C for a period of 2, 12 and 24 hours. The US parameter was: 1.5 W/cm², 50% duty cycle, 15 min. After centrifugating at 25,000 × g for 15 min, the resulting supernatants were then employed for ICP measurements.

Quercetin release measurement

Firstly, the spectra of standard que solutions were measured using a fluorescence spectrophotometer (Shanghai Mepda Instrument Co., Ltd.). Fluorescence intensity was recorded at an excitation light of 425 nm and an emission light of 450 nm to 800 nm. The MON(Cu), MON(CuMn) and MON(CuMn)-Q solutions (200 µg/mL) were incubated at 37 °C for varying periods of time, namely 0, 2, 12 and 24 hours for the short incubation period, and 3, 5, 7, 14, 21 and 24 hours for the long incubation period. The precipitate was removed by centrifugation at 25,000 × g for 15 minutes, after which the supernatant was taken for spectroscopic determination. The que content was calculated by measuring the maximum fluorescence intensity of the supernatant.

To ascertain the quantity of que loaded on the MON(CuMn)-Q, MON(CuMn)-Q solution was incubated for a period of 6 hours and subsequently subjected to centrifugation at 25,000 × g for 15 minutes. The supernatant was then taken for spectroscopic determination. The que loaded on the MON(CuMn)-Q was estimated by subtracting the amount of que present in the supernatant from the total amount of feed.

In order to ascertain the impact of US on the release of que from the nanosheets, 200 µg/mL of nanosheets were employed in the experiments. The US parameters employed were as follows: 1.0 MHZ, 1.5 W/cm², 50% duty cycle, 15 min. The MON(CuMn)-Q solution were then incubated at 37 °C for a period of 2, 12 and 24 hours. Following this, the supernatants were collected for fluorescence intensity measurement.

Theoretical calculations

Spin-polarised first-principle calculations were performed using the density functional theory (DFT) with the Vienna Ab-initio Simulation Package (VASP)⁵⁸. The electronic exchange and correlation effects were described using the generalized gradient approximation (GGA) with the Perdew-Burke-Ernzerhof (PBE) functional^59,60,61. A uniform G-centered k-points mesh with a resolution of 2π × 0.05 Å⁻¹ and Methfessel-Paxton electronic smearing were employed for the integration in the Brillouin zone during the geometric optimisation process. throughout the computations, a cutoff energy of 500 eV was utilised. These settings ensure convergence of the total energies to within 1 meV per atom. The structure was relaxed until all forces on the atoms were less than 10 meV Å⁻¹, and the total stress tensor was within 0.03 GPa of the target value. A vacuum distance of 12 Å was set to ensure sufficient vacuum and avoid interactions between two periods. The van der Waals corrections to the total Kohn-Sham energy were incorporated into the calculations. The van der Waals corrections were applied using the DFT-D3 method⁶². The Mulliken charge and the potential energy distribution (PED) were calculated by vibrational energy distribution analysis (VEDA 4)⁶³.

Piezocatalytic property test

The eletrochemical property were measured using an electrochemical workstation (CHI 660E, Shanghai CH Instruments Co., Ltd.) under 1.5 W/cm² US irradiation. In addition, a three-electrode system was constructed in a quartz glass cell in 0.5 M Na₂SO₄ aqueous solution, using a Pt plate as the counter electrode, an Ag/AgCl electrode as the reference electrode and an experimental sample as the working electrode. Singlet oxygen (¹O₂) was detected by employing ESR (JES-FA200, Japan Electronics Co., Ltd) with 2,2,6,6-tetramethylpiperidine (TEMP, Aladdin) as the trapping agent.

In vitro antibacterial test

MRSA was purchased from American Type Culture Collection (ATCC, Manassas, VA). To explore the antimicrobial capacity of the material itself, 10⁶ CFU/mL MRSA was used for evaluation. 10 µL of MRSA suspension was taken and diluted 200 times with each nanosheet and vancomycin solution (200 µg/mL). Then, the mixture was shaken at 37°C for 24 h, and 20 µL suspension is taken at 0, 4, 8, 16 and 24 h for spread plate experiments. The groups were: Control, Van, MON(Cu), MON(CuMn) and MON(CuMn)-Q.

In order to investigate the sonodynamic antibacterial ability of the material, 10⁶ CFU/mL MRSA solution was used for spread plate experiments. Specifically, 10 µL of MRSA suspension was taken and diluted 200 times with each nanosheet solution (200 µg/mL). Additionally, 200 µg/mL vancomycin solution was used for comparison with the nanosheet solutions. The mixture in the US group and each nanosheet group was then treated for 15 minutes under US irradiation (1.0 MHZ, 1.5 W/cm², 50% duty cycle). Next, 20 µL of suspension was collected for spread plate experiments. Finally, the plates were placed in a incubator at 37 °C for 24 hours and then counted. The experimental groups were as follows: Control, US, Van, MON(Cu) + US, MON(CuMn) + US and MON(CuMn)-Q + US. To observe the morphology of the bacteria, the FE-SEM (S-4800, Hitachi) was applied.

Hemolysis test

1 mL of rat 5% erythrocytes (Shanghai Yuanye Biotechnology Co., LTD) was transferred to a 1.5 mL EP tube and subjected to centrifugation at 200 × g for 15 minutes, after which the supernatant was removed. The erythrocytes were resuspended following three washes of the precipitate with saline. The MON(CuMn)-Q was added to the erythrocyte suspension in order to achieve final concentrations of 25 µg/mL, 50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, and 250 µg/mL. 20 µL of RIPA lysate (BL504A, Biosharp) was added to another tube of erythrocyte suspension, which served as the positive control. Another tube of erythrocyte suspension was prepared without any added material and served as the negative control. Given that the nanosheets exhibit a colour that could potentially influence the determination of the final absorbance, we have additionally configured the MON(CuMn)-Q solution with the corresponding concentration as the background. The EP tubes of each group were incubated at 37°C for 1 h and then centrifuged at 200 × g for 15 minutes. Subsequently, 100 µL of the supernatant was taken and the absorbance at 540 nm was measured. The percentage of haemolysis was calculated using the following formula:

Cell culture

hBMSCs were obtained from bone marrow blood of healthy volunteers. These volunteers were recruited from the department of orthopaedic surgery of Wuhan Union Medical College in compliance with the ethical committee of Tongji Medical College of Huazhong University of Science and Technology (HUST) (No. S347). Briefly, the steps are to mix bone marrow blood, lymphocyte separation solution and sterile PBS and then centrifuge to obtain hBMSCs. Immortalised HUVECs and THP1 were bought from the companies (Hunan Fenghui Biotechnology Co., Ltd, Shanghai Biyuntian Biological Co., Ltd.). The short tandem repeat (STR) technology was employed for cell identification. hBMSCs and HUVECs were cultured in DMEM/F12 medium (Boster Biological Technology Co., Ltd). THP1 was cultured in 1640 medium (Boster Biological Technology Co., Ltd). The medium was also supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution (Boster Biological Technology Co., Ltd) and all cells were cultured at 37 °C.

CCK8 assay

Firstly, A total of 1 × 10⁴ cells per well were seeded in 24-well plates and co-cultured with different concentrations of nanosheets. Furthermore, after the set incubation period, the culture medium with or without nanosheets was removed with 10% CCK8 solution (AC11L054, Life-iLab) and incubated at 37 ^◦C for 1 h. Finally, liquid (100 µL) from each well was transferred to 96-well plates, and the absorbance at 450 nm was recorded.

To investigate the impact of US on cell viability, 5 × 10⁵ hBMSCs were seeded in six-well plates and co-cultured with 150 µg/mL MON(CuMn)-Q before the US. The parameter of US is: 1.0 MHZ, 1.5 W/cm², 50% duty cycle, 15 min. Cells before US and on day 1, day 3, day 7 and day 14 after US were subjected to CCK8 assay.

Quantitative real-time fluorescence polymerase chain reaction

HUVECs were seeded in 6-well plates and then incubated for three days under the different conditions. The groups were Control, MON(Cu), MON(CuMn) and MON(CuMn)-Q. After incubation, RNA was extracted using the FastPure® Cell/Tissue Total RNA Isolation Kit V2 (Vazyme Biotech Co., Ltd), and the procedure was carried out in accordance with the instructions. The extracted RNA was then quantified using an ultra-micro spectrophotometer (Applied Biosystems). To obtain complementary DNA (cDNA), HiScript III RT SuperMix (R323-01, Vazyme) was added to the RNA solution and the mixture was reverse transcribed into cDNA using a reverse transcription instrument (Applied Biosystems). Finally, the prepared cDNA, primer, SYBR fluorescent dye (Q712-02, Vazyme), and DEPC water (BL510A, Biosharp) were mixed and a real-time fluorescence quantitative PCR system (BioRad Laboratories) was applied to detect each target gene. The primer sequences were listed in Supplementary Table 5. The Cq values were normalised using GAPDH, and the expression was calculated according to the 2-^ΔΔCT method. The reaction procedure involved a denaturation step at 95 °C for 30 seconds, followed by an annealing step at 95 °C for 5 seconds and an extension step at 55 °C for 10 seconds, repeated for 40 cycles.

Western blot

Total cellular proteins were collected by radio-immunoprecipitation assay (RIPA, BL504A, Biosharp) using a mixture of protease and phosphoproteinase inhibitors (BL615A and BL612A Biosharp). Protein samples were separated by sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred to the membrane. The membrane was then blocked with 0.1% Tris Buffered Saline Tween (TBST) blocking solution containing 5% bovine serum albumin (BSA, BS114, Biosharp) for 1 h. Primary antibodies were incubated overnight at 4°C and the membrane was washed three times with PBS. The membrane was washed three times with 0.1% TBST and then incubated with the appropriate secondary antibodies at room temperature for 1 h. The membrane was then visualised, photographed and analyzed semiquantitatively on a chemiluminescence imaging system (Tanon 5200, Tanon). The information of antibody was presented in Supplementary Table 6.

Immunofluorescence

Cells were seeded in six-well plates, incubated for a period of time and then the medium was removed. The cells were then fixed with 4% paraformaldehyde for 30 minutes, followed by incubation with 0.5% Triton X-100 for 20 minutes at room temperature, rinsed three times with PBS, and then blocked with immunofluorescence blocking solution (P0102, Beyotime) for 30 minutes at room temperature, followed by the addition of a specific amount of primary antibody prepared with TBST and incubated overnight at 4 °C in a humidity box. The next day, the cell well plates were washed three times on a horizontal shaker. The cells were covered with primary antibody of the appropriate genus and incubated at room temperature for 60 min, washed with 0.1% Phosphate Buffer Saline Tween (PBST), then DAPI stain (C1002, Beyotime) was added to the slides and incubated at room temperature for 10 min, protected from light. Finally, the images were observed and captured under a fluorescence microscope. In addition, confocal (LSM900, Zeiss) was used to observe the polarization of macrophages.

Cytoskeleton tracking was conducted using the Tubulin-Tracker kit (C2215S, Beyotime). The procedure was as follows: the Tubulin-Tracker Deep Red staining working solution was initially configured, the cell culture was removed, the cells growing on the crawler slides were washed with PBS, and after addition of the working solution, the cells were incubated at 37 °C for 1 h. Finally, the cells were photographed using confocal microscopy.

Alizarin Red S staining

hBMSCs were cultured in for 21 days in culture medium containing 50 µg/mL MON(Cu), 100 µg/mL MON(CuMn) and 150 µg/mL MON(CuMn)-Q, respectively. hBMSCs cultured in medium without nanosheet for 21 days were set as the control group. Then cells were washed with PBS and fixed in 4% paraformaldehyde solution for 30 min before staining. Appropriate amount of 0.2% Alizarin Red S solution (G1450, Solarbio) was added, evenly cover the cells, and stain at room temperature for 30 min. Then the cells were washed with PBS thoroughly, and can then be viewed and photographed under a microscope.

Alkaline phosphatase staining

hBMSCs were cultured in for 14 days in culture medium containing 50 µg/mL MON(Cu), 100 µg/mL MON(CuMn) and 150 µg/mL MON(CuMn)-Q, respectively. hBMSCs cultured in medium without nanosheet for 14 days were set as the control group. Then cells were washed with PBS and fixed in 4% paraformaldehyde solution for 30 min before staining. Appropriate amount of ALP staining solution (C3206, Beyotime) was added, evenly cover the cells, and stain at room temperature for 30 min. Then the cells were washed with PBS thoroughly, and can then be viewed and photographed under a microscope.

RNA sequencing

The hBMSCs were divided into two groups: the Control and the MON(CuMn)-Q groups. The cells in the MON(CuMn)-Q group were cultured with 150 µg/mL of the nanosheet for 21 days. Cells in the control group were cultured for 21 days in a medium containing no material. Moreover, total RNA was extracted using TRIzol Reagent (15596026, Invitrogen). RNA samples were quantified after being tested the quality and integrity. Then an RNA sequencing library was built. Raw sequencing data were analyzed with the assistance of QL Bio (Beijing, China) using Novaseq 6000 PE150 (Illumina, USA) with default parameters. GO analysis and KEGG enrichment analysis of differentially expressed genes were performed with a P-value cutoff of 0.05.

Cell flow cytometry

9 × 10⁶ THP1 were seeded in T25 culture flasks and 50 ng/mL PMA was added to induce THP differentiation into macrophages for 3 days. To detect the immunomodulatory ability of the nanosheets, 1 ug/mL LPS and adherent THP1 were co-cultured for 2 days, followed by the addition of 1 mmol/L DMTU, 50 µg/mL MON(Cu), 100 µg/mL MON(CuMn) and 200 µg/mL MON(CuMn)-Q to the medium for 2 days. Macrophages were scraped from the culture flasks to detect macrophage polarisation, and a fixation/permeabilization kit (BD) was used prior to permeabilization to facilitate intracellular bicolor staining. The staining of CD86 and CD206 were achieved using fluorescein-labelled anti-mouse antibodies (BD) (Supplementary Table 6). Mitochondrial membrane potential and ROS staining were performed using the JC-1 staining kit and the ROS staining kit, respectively (S0033S, C2005, Beyotime).

While stimulating hBMSCs with 100 µmol/L H₂O₂, cells were co-cultured with 1 mmol/L DMTU, 50 µg/mL MON(Cu), 100 µg/mL MON(CuMn) and 150 µg/mL MON(CuMn)-Q for 3 days, respectively. The cells were then digested with pancreatic enzyme (C0201, Beyotime) and stained with JC-1 and ROS kits. After three times of PBS washing, the cells were detected by flow cytometry (FACSCalibur, BD Biosciences).

Tube formation experiment

5 × 10⁵ HUVECs were seeded on six-well plates and cultured for three days by adding medium with or without nanosheets. The matrix gel (354230, Corning) should be placed in a refrigerator at 4°C overnight, one day prior to the experiment, in order for the gel to melt at a gradual pace. The cell culture medium should be removed and 300 µL of matrix gel was added to each well in a dropwise manner, with care taken to avoid the formation of air bubbles. The cells were then incubated for a period of 6 hours, after which they were photographed for observation.

Scratch assay

Firstly, a marker pen was used on the back of the six-well plate to draw even horizontal lines about every 0.5~1 cm across the hole. Cells in logarithmic growth phase were trypsinised into single cell suspension and seeded into six-well culture plates. The cell plate was 60,000 cells/well, and the inoculation principle was that the fusion rate reached 100% after overnight, and the final amount of medium per well was 2 ml. The cells were cultured at 37 °C in a 5% CO₂ incubator for 24 hours. Then, the plate was scratched parallel or perpendicular to the horizontal line behind the gun head with a 200-microlitre gun head. Then the cells were washed with PBS and the serum-free medium was added. Finally, the cross-line scratches with a marker behind the 6-well plate was erased and photos were taken at 0 and 6 time points.

Preparation and characterization of the hydrogel

In order to configure Je-1 hydrogel, Je-1 stock solution was first prepared at a concentration of 100 mg/mL of DMSO. Afterwards, the Je-1 stock solution was diluted to different concentrations using 1× PBS and incubated at 37°C for more than 15 min to form gels. To prepare MON(CuMn)-Q-J ointment, the Je-1 stock solution was diluted with a solution containing MON(CuMn)-Q to give a final concentration of MON(CuMn)-Q of 200 µg/mL. The vial tilt experiments were carried out to assess the formation of hydrogels. In addition, FE-SEM (S-4800, Hitachi) was used to observe the surface morphology of the gels.

Animal experiment

Spraque-Dawley (SD) Rats purchased from the laboratory animal centre of HUST were selected as the experimental animal model. All rats were male and weighed between 180 and 220 g. All experiments were approved by the animal research committee of HUST (No. S3946) and complied with international animal welfare standards. The SD rats were raised in accordance with standardised conditions of a specific pathogen-free environment, with a constant temperature of 21–24 °C and a 1:1 dark:light cycle. The animals were randomly and blindly assigned to groups for in vivo experiments. Rats were first anaesthetized with 3% pentobarbital (0.3 mg/kg) by intraperitoneal injection, and then the skin was prepared locally on the rat skull for surgery. The skin, subcutaneous tissue, muscle and periosteum were dissected with a scalpel from the bridge of the nose to the base of the skull to fully expose the parietal bone, and an 6 mm round full-thickness skull defect was created in the parietal bone using a 6 mm diameter trephine, which was flushed and cooled with physiological saline while maintaining the integrity of the dura mater. In the MRSA, the Van and the MON(CuMn) + US groups, 100 µL 10⁸ CFU/mL of MRSA suspension was injected into the defects of rats. In the Control group, 100 µL of sterile saline was injected into the defects instead of the bacterial solution. After three days of surgery, the wounds in the MRSA-infected rats were reopened. 100 µL PBS or MON(CuMn)-Q-J was injected to the defects of the rats in the MRSA group and the MON(CuMn) + US group, respectively. 40 mg/kg vancomycin was injected into the tail vein of rats in the Van group. Then rats in the MON(CuMn) + US were treated by US. The US parameters were 1.0 MHZ, 1.5 W/cm² and 50% duty cycle, 1 MHz for 15 min. On the 14th day after treatment, SD rats were sacrificed for H&E staining and IF. Images around the wound were taken on the 14th day after treatment. Masson staining, H&E staining, IHC and IF were performed on the skulls of the rats after 28 days of treatment. The heart, liver, spleen and kidney were also collected for H&E staining. Renal function assessment was performed on on day 28 following the treatment.

To eliminate the potential confounding influence of Je-1’s antimicrobial activity on the observed outcomes, we conducted an independent assessment of the therapeutic efficacy of Je-1 and MON(CuMn)-Q-J. Three days following the cranial injection of MRSA into the rats, the wounds were reopened and 100 µl of PBS, Je-1 and MON(CuMn)-Q-J were administered. A total of five rats that had been administered PBS were selected to receive additional US treatment, defined as the US group. Another five rats that had been administered PBS were selected to be the MRSA group. The rats that were administered Je-1 and MON(CuMn)-Q-J were subjected to US treatment, resulting in the formation of two distinct groups: the Je-1 + US group and the MON(CuMn)-Q-J + US group. On days 1, 3, and 7 following the administration of the treatment, tissues from the defects were collected for the purpose of spread plate experiment. Furthermore, tissues around defects were obtained on the initial day for Gram staining. A CT examination was conducted on the skull on day 28 post-treatment.

Acute toxicity test

Following the creation of a defect at the apex of the rat skull and the subsequent injection of MRSA, hydrogels comprising varying concentrations of MON(CuMn)-Q were employed to fill the aforementioned defect. Following a seven-day treatment period, the rats were sacrificed, and their serum was collected for subsequent analysis to assess liver and kidney function.

In order to ascertain the degree of skin irritation caused by the material, an intradermal irritation test was employed to determine the toxicity of the material^64,65. The animal experiment has been approved by institutional animal care and use committee (IACUC) (No. 2024100902). nine female New Zealand rabbits, with a body weight exceeding 2 kg, were selected for the study. The rabbits were raised under a standard and specific pathogen-free environment with a constant temperature of 15–25 °C and a 1:1 dark:light cycle. The animals were randomly and blindly assigned to groups for in vivo experiments. Following a one-week period, during which the rabbits were fed, the hair on both sides of the spine on the back of each rabbit was removed 16 hours prior to the commencement of the test, to serve as test and observation sites. Ten points were selected on the left and right sides of the rabbit’s back, with each point spaced at moderate intervals. The top five holes on the left were allocated to the sample group, the top five holes on the right were assigned to the 0.9% saline solution (polar negative control), the bottom five holes on the left were designated for the 5% SDS solution group (positive control), and the bottom five holes on the right were allocated to the sesame oil (non-polar negative control) group. The local and surrounding skin tissue reactions at the injection site, including erythema, oedema, and necrosis, were observed and photographed at 24, 48 and 72 hours following intradermal injection.

In vivo antibacterial test

To further evaluate the antibacterial capacity of MON(CuMn)-Q, 2 mm × 1 mm granulation tissues were collected from the defects for bacterial examination after 1, 3, and 7 days of the treatments. Subsequently, the samples were put into 10 ml of sterile PBS and homogenized for 5 min. Then 20 µL of the solution was used for spread plate experiments, and the plates were incubated for 24 h at 37 °C. Then viable bacterial counts were taken. After 1, 7, and 14 days of treatment, rats were sacrificed, and the skull was collected for Gram staining.

Routine blood test

The blood of rats was collected from the orbital vein on 14th day after treatment, and analyzed by an animal hematology analyzer (BC-2800vet, Mindray).

Micro-CT evaluation

The infected skull tissue was scanned by a micro-CT imaging system (skyscan1176, Bruker). Then CT-Vox software and SkyScan CT software were performed for 3D reconstruction and analysis,

Histological analysis

All samples except for the heart, liver, spleen, and kidney were decalcified for 21 days before slicing. The slices were then stained with H&E, Masson, and Gram staining. The slices were observed by an optical microscope.

Statistical analysis

The mean ± standard deviation was used to express our data. All experiments were performed at least three times. Two-tailed ANOVA and two-tailed unpaired Student’s t test were used to assess the significant difference between group means. It was considered that *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 were statistically significant, while p > 0.05 was considered non-significant (ns). The investigators were blinded to group allocation during data collection and analysis. No data or samples were excluded from the study.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-54894-8