A porous carbon biocatalytic material loaded with rhodium-iron bimetallic active sites, and a preparation method and application thereof

Abstract

This invention belongs to the field of catalytic materials technology, specifically relating to a porous carbon biocatalytic material loaded with rhodium-iron bimetallic active sites, its preparation method, and its applications. The porous carbon biocatalytic material comprises a spiky porous carbon substrate, and rhodium clusters and iron single atoms uniformly loaded on its surface, forming Rh-Fe bimetallic active sites with charge transfer effects. The porous carbon biocatalytic material of this invention exhibits peroxidase-like and NADH oxidase-like activities under acidic conditions, and glucose oxidase-like, catalase-like, and superoxide dismutase-like activities under neutral conditions. It can efficiently kill bacteria and adsorb bacterial antigens, preventing macrophages from excessively polarizing to a pro-inflammatory state, ultimately promoting tissue regeneration and achieving ultra-rapid healing of inflammatory diabetic ulcers, with good biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of catalytic materials technology, specifically relating to a porous carbon biocatalytic material supported on rhodium-iron bimetallic active sites, its preparation method, and its application. Background Technology

[0002] Infectious diabetic wound healing remains a significant clinical challenge, primarily due to persistent bacterial infection, escalating antibiotic resistance, chronically high blood sugar levels, and impaired angiogenesis. These factors collectively lead to severe hypoxia and oxidative stress at the wound site. These pathological conditions ultimately result in chronic inflammation and tissue necrosis, significantly increasing the risk of amputation and death in diabetic patients, while also imposing a heavy economic and social burden on global public health.

[0003] To address this issue, artificial enzyme-mimicking materials capable of catalyzing the generation of reactive oxygen species (ROS) from hydrogen peroxide have emerged as a promising alternative to antibiotics for treating severe bacterial infections. For example, recently reported nanomaterials, photosensitizers, metal-organic frameworks, and inorganic materials have all demonstrated significant antibacterial effects in in vitro and in vivo experiments. However, these therapeutic strategies still face two key challenges. First, the ROS-based bactericidal process inevitably disrupts bacterial cell membranes, leading to the leakage of large amounts of bacterial antigens. These released antigens can be phagocytosed by macrophages, further activating the immune response, potentially exacerbating inflammation and hindering wound healing. Second, after bacterial clearance and antigen-induced inflammation, the pH of the wound microenvironment gradually shifts from acidic to near neutral. Under these conditions, most currently reported enzyme-mimicking antibacterial materials exhibit weak anti-inflammatory regulatory capabilities due to limited biological functions. Therefore, there is an urgent need to develop effective therapeutic strategies with multiple biological functions to address the complex wound microenvironment.

[0004] In biological systems, glucose oxidase (GOD), natural antioxidant enzymes (such as catalase (CAT) and superoxide dismutase (SOD)), and natural oxidases (such as peroxidase (POD) and NADH oxidase (NOX)) work together to maintain redox homeostasis and metabolic balance. However, the widespread application of these natural enzymes in therapy is limited by their short circulating half-life, high production cost, poor structural stability, and potential immunogenicity risks. Therefore, designing artificial enzyme-mimicking biocatalytic materials that can mimic the activities of various natural enzymes under different pH conditions holds promise as an effective strategy to achieve synergistic regulation of efficient antibacterial activity and subsequent tissue repair in infectious diabetic wounds. However, the development of artificial enzyme-mimicking materials still faces significant challenges: single-metal active sites typically exhibit limited catalytic diversity due to low electron transfer efficiency and suboptimal catalytic configurations in complex multi-electron oxygen reactions. Consequently, such catalytic systems often result in insufficient antibacterial efficacy, bacterial residue, or inadequate tissue regeneration, accompanied by persistent chronic inflammation.

[0005] In contrast, bimetallic catalysts typically exhibit superior and more diverse catalytic performance in biocatalytic reactions compared to monometallic systems, primarily due to the synergistic effect between the two metal components. By combining metal single atoms (SAs) with nanoclusters (NCs), the electronic structure can be effectively modulated, forming bimetallic active sites dynamically regulated by direct interactions. This structural feature helps optimize the adsorption strength of reaction intermediates, lowers the energy barrier of catalytic reactions, and thus enhances multifunctional catalytic performance. Recent studies have shown that rhodium (Rh) possesses glucose oxidase (GOD)-like activity, while iron (Fe) is widely used as the active center for peroxidase (POD)-like catalysis. However, how to construct an Rh-Fe bimetallic active configuration with strong electronic coupling effects to achieve a synergistic effect of "highly efficient sterilization" and "immunomodulatory healing promotion" has not yet been reported in existing technologies. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of the prior art and provide a multifunctional biocatalytic material that can adapt to changes in the pH of the wound microenvironment and has both efficient bactericidal, anti-inflammatory and healing-promoting functions. It also provides a method for preparing the material and its application in the preparation of antibacterial materials and drugs for treating infected wounds.

[0007] In a first aspect, the present invention provides a porous carbon biocatalytic material loaded with rhodium-iron bimetallic active sites, the porous carbon biocatalytic material comprising a spiky porous carbon substrate, and rhodium clusters and iron single atoms uniformly loaded on the surface of the spiky porous carbon substrate, wherein the rhodium clusters exist in the form of nanoclusters, the iron single atoms exist in the form of single atoms, and Rh-Fe bimetallic active sites with charge transfer effect are formed between the rhodium clusters and the iron single atoms.

[0008] Furthermore, the spiky porous carbon substrate is obtained as follows: a zinc-based metal-organic framework is subjected to a hydrothermal reaction in the presence of urea, followed by carbonization and acid etching to obtain the spiky porous carbon substrate.

[0009] Furthermore, the organic ligand of the zinc-based metal-organic framework is 2,5-dihydroxyterephthalic acid.

[0010] Furthermore, in the porous carbon biocatalytic material, the molar ratio of rhodium to iron is 1:1 to 1:2.

[0011] Furthermore, the porous carbon biocatalytic material has a positively charged surface.

[0012] Furthermore, the porous carbon biocatalytic material exhibits peroxidase-like (POD) and NADH oxidase-like activities under acidic conditions (e.g., pH 4.5-6.5), catalyzing the generation of large amounts of reactive oxygen species (ROS) and oxidizing NADH to NAD⁺; under neutral conditions (e.g., pH 7.4), it exhibits glucose oxidase-like (GOD), catalase (CAT), and superoxide dismutase (SOD) activities, effectively scavenging hydrogen peroxide and superoxide anion free radicals.

[0013] Furthermore, the maximum reaction rate (Vmax) of the porous carbon biocatalytic material under peroxidase-like catalytic reaction at pH 4.5 is 4.7059 µM s⁻¹, and the transformation number (TON) is 113.559 s⁻¹.

[0014] In a second aspect, the present invention provides a method for preparing porous carbon biocatalytic materials as described herein, comprising the following steps:

[0015] (1) In solvent I, Zn salt is reacted with organic ligand 2,5-dihydroxyterephthalic acid to form a zinc-based metal-organic framework;

[0016] (2) The zinc-based metal-organic framework is subjected to hydrothermal treatment in urea solution, followed by first carbonization and acid etching to obtain a spiky porous carbon substrate;

[0017] (3) Mix 2-methylimidazolium, Zn salt and spiky porous carbon substrate in solvent II, then add Rh salt and Fe salt, stir the reaction, centrifuge, wash and dry to obtain the precursor;

[0018] (4) The precursor is subjected to a second carbonization and acid etching treatment to obtain the porous carbon biocatalytic material.

[0019] Further, the Zn salt mentioned in step (1) is at least one of zinc nitrate, zinc acetate, zinc chloride, or their hydrates.

[0020] Furthermore, solvent I is an alcohol solvent, including methanol or ethanol.

[0021] Furthermore, the reaction between the Zn salt and the organic ligand is carried out under ultrasound for 0.5-3 h.

[0022] Furthermore, after the reaction of Zn salt with organic ligand is completed, the precipitated reaction product is collected by centrifugation and washed with water.

[0023] Furthermore, the mass concentration of the urea solution in step (2) is 0.1-0.3%.

[0024] Furthermore, the hydrothermal treatment temperature is 170-180 ℃, and the reaction time is 12-36 h;

[0025] Furthermore, in the first carbonization and acid etching process, the carbonization temperature is 1000-1200 ℃, the reaction time is 1-3 h, and the acid etching uses 1M hydrochloric acid at 70-90 ℃ for 1-3 h.

[0026] Furthermore, step (2) also includes washing (including, for example, water washing and alcohol washing) and drying the product after hydrothermal treatment.

[0027] Furthermore, step (2) also includes washing and drying the product after the first carbonization and acid etching treatment.

[0028] Further, in step (3), the molar ratio of Rh salt to Fe salt is 1:1, and the molar ratio of the total amount of Rh salt and Fe salt to Zn salt is 1:(30-50).

[0029] Furthermore, the Zn salt is at least one of zinc nitrate, zinc acetate, zinc chloride, or their hydrates.

[0030] Furthermore, the Rh salt is at least one of rhodium chloride, rhodium nitrate, rhodium acetylacetonate, or their hydrates.

[0031] Furthermore, the Fe salt is at least one of ferric nitrate, ferric chloride, ferric acetylacetone, or their hydrates.

[0032] Furthermore, solvent II is an alcohol solvent, including methanol or ethanol.

[0033] Furthermore, in step (4), the second carbonization and acid etching process is carried out at a carbonization temperature of 900-1100 ℃, preferably 1000 ℃, and a reaction time of 1-3 h, preferably 2 h. The acid etching process uses 1M hydrochloric acid and the etching time is 1-3 h, preferably 1 h.

[0034] Furthermore, step (4) also includes washing and drying the product after the second carbonization and acid etching treatment.

[0035] In the preferred technical solution, the first carbonization temperature of step (2) is 1100℃, the holding time is 1 hour, and the acid etching is 1M hydrochloric acid at 80℃ for 3 hours; the second carbonization temperature of step (4) is 1000℃, the holding time is 2 hours, and the acid etching is 1M hydrochloric acid for 1 hour.

[0036] Furthermore, the first and second carbonization processes are carried out under an inert atmosphere.

[0037] In a third aspect, the present invention provides the application of porous carbon biocatalytic materials as described herein in the preparation of antibacterial materials.

[0038] Furthermore, the antibacterial activity includes anti-Staphylococcus aureus (such as methicillin-resistant Staphylococcus aureus) and / or anti-Escherichia coli (extended-spectrum β-lactamase (ESBL)-producing Escherichia coli).

[0039] In a fourth aspect, the present invention provides the use of porous carbon biocatalytic materials as described herein in the preparation of medicaments for treating bacterial wound infections and inflammation caused by bacterial wound infections, as well as promoting the healing of bacterial wounds.

[0040] Furthermore, the wound in question is a wound belonging to a diabetic patient.

[0041] Furthermore, the bacteria include Staphylococcus aureus (such as methicillin-resistant Staphylococcus aureus) and / or Escherichia coli (Escherichia coli that produces extended-spectrum β-lactamase (ESBL)).

[0042] The porous carbon biocatalytic material functions through the following mechanisms: in the acidic microenvironment of bacterial infection, it catalyzes the generation of ROS through POD-like and NADH oxidase-like activities and disrupts the bacterial respiratory chain, thus efficiently killing bacteria; in the neutral microenvironment after the bacteria are cleared, it removes excess ROS through CAT-like and SOD-like activities, and efficiently adsorbs antigenic substances (such as LTA and LPS) leaked after bacterial death through its positive surface charge and high specific surface area, blocking the uptake of antigens by macrophages, thereby inhibiting M1 macrophage polarization, avoiding chronic inflammation, and promoting wound healing.

[0043] Beneficial effects of the present invention

[0044] This invention develops a porous carbon biocatalyst with Rh clusters and Fe single atoms supported on its surface, named Rh-Fe / JAEP. The catalytic material obtained in this invention exhibits excellent POD-like and NADH oxidase-like activities and favorable reaction kinetic parameters under acidic conditions: maximum reaction rate (Vt).max The value was 4.7059 µM s. -1 The number of transformations (TON) was 113.559 s. -1 It exhibits excellent GOD- and CAT-like enzyme activities under neutral conditions. The Rh-Fe / JAEP obtained in this invention can inhibit bacterial growth by disrupting the respiratory chain metabolism and can also block the uptake of antigens leaked after bacterial death by macrophages, thus avoiding the formation of an oxidative stress environment. In vivo experiments show that Rh-Fe / JAEP demonstrates excellent bacterial clearance and subsequent ROS elimination capabilities against diabetic bacterial infections. In other words, this invention provides an effective nanomedicine for catalyzing the clearance of bacteria by reactive oxygen species, preventing the formation of inflammatory wounds, and promoting ultra-rapid healing of skin wounds. It also provides a new approach for designing biocatalytic metal compounds to replace traditional antibiotics in the treatment of skin wound infections. Attached Figure Description

[0045] Figure 1 The figure shows the statistical results of POD-like enzyme activity of samples from Examples 2-4(a) and Examples 5-7(b), n = 3 independent experiments, and the data are expressed as mean ± SD.

[0046] Figure 2 This is a schematic diagram of the structure of Rh-Fe / JAEP of the present invention.

[0047] Figure 3 SEM images of JAEP (a) and Rh-Fe / JAEP (b).

[0048] Figure 4 The XRD patterns are of the samples obtained in Examples 2-4(a), Examples 5-7(b), and Comparative Examples 1-3(c).

[0049] Figure 5 The image shows the Zeta potential data of Rh-Fe / JAEP, Rh / JAEP, Fe / JAEP and JAEP obtained in Example 5 and Comparative Examples 1-3 of this invention.

[0050] Figure 6 The image shows the HR-TEM image and elemental distribution of Rh-Fe / JAEP obtained in Example 5 of this invention.

[0051] Figure 7 This is the HR-TEM image and elemental distribution diagram of Rh / JAEP obtained in Comparative Example 1 of this invention.

[0052] Figure 8 The image shows the HR-TEM image and elemental distribution of Fe / JAEP obtained in Comparative Example 2 of this invention.

[0053] Figure 9High-resolution HAADF-STEM image of the Rh-Fe / JAEP crystal region obtained in Example 5 and its Fourier transform (a); High-resolution HAADF-STEM image showing the distribution and distance of Rh clusters and Fe single atoms (b); STEM spectral imaging of Rh-Fe / JAEP and its elemental distribution (c).

[0054] Figure 10 High-resolution XPS spectra of the electronic structure of Rh-Fe / JAEP. (a) Fe 2p XPS spectra of Rh-Fe / JAEP and Fe / JAEP; (b) Rh 3d XPS spectra of Rh-Fe / JAEP and Rh / JAEP; (c) N 1s XPS spectra of Rh-Fe / JAEP, Fe / JAEP and Rh / JAEP.

[0055] Figure 11 XAS results for the Rh-Fe / JAEP coordination environment.

[0056] Figure 12 The figures show statistical results of POD-like enzyme activities for different samples, n = 3 independent experiments, and data are expressed as mean ± SD (a); steady-state kinetic test graphs for samples from Example 5 (b), Comparative Example 1 (c), and Comparative Example 2 (d), n = 3 independent experiments, and data are expressed as mean ± SD; and the maximum reaction rate V for samples from Example 5, Comparative Example 1, and Comparative Example 2. max Michaelis constant K m A comparison chart of the value and the number of transformations (TON) (e).

[0057] Figure 13 This is a comparison of NADH-like oxidase activities in different samples, n = 3 independent experiments. Data are expressed as mean ± SD.

[0058] Figure 14 Oxygen production concentration was measured using a dissolved oxygen meter for different samples and hydrogen peroxide (a); CAT-like enzyme activity test graph and relative H2O2 clearance rate at 30 minutes were obtained, n = 3 independent experiments, and data are expressed as mean ± SD (b); SOD-like enzyme activity test graph (c) and DPPH clearance rate (d) were obtained, n = 3 independent experiments, and data are expressed as mean ± SD.

[0059] Figure 15 Photographs of surviving colonies of Staphylococcus aureus and Escherichia coli after treatment with hydrogen peroxide, Rh / JAEP+hydrogen peroxide, Fe / JAEP+hydrogen peroxide, and Rh-Fe / JAEP+hydrogen peroxide, respectively, followed by plate coating.

[0060] Figure 16SEM images of Staphylococcus aureus and Escherichia coli after treatment with hydrogen peroxide, Rh / JAEP+hydrogen peroxide, Fe / JAEP+hydrogen peroxide, and Rh-Fe / JAEP+hydrogen peroxide, respectively; scale bar: 2 μm, 2.5 μm.

[0061] Figure 17 Live / dead fluorescence staining images of Staphylococcus aureus and Escherichia coli after co-incubation with hydrogen peroxide, Rh / JAEP+hydrogen peroxide, Fe / JAEP+hydrogen peroxide, and Rh-Fe / JAEP+hydrogen peroxide, respectively; Scale bar: 15 μm.

[0062] Figure 18 After adding the supernatants of Staphylococcus aureus and Escherichia coli co-incubated with hydrogen peroxide, Rh / JAEP+hydrogen peroxide, Fe / JAEP+hydrogen peroxide and Rh-Fe / JAEP+hydrogen peroxide respectively to macrophages, the polarization state of macrophages was characterized. (a) The content of two antigens was tested, n = 3 independent experiments, and the data are expressed as mean ± SD; (b) The level of inflammatory factors secreted by macrophages was detected, n = 3 independent experiments, and the data are expressed as mean ± SD; (c) The phenotype of macrophages was determined by flow cytometry.

[0063] Figure 19 The therapeutic effects of Rh-Fe / JAEP on diabetic bacterial wounds are shown in the following figures: (a) Representative images of wound areas under different treatment methods on days 0, 3, 7 and 11; (b) Heat maps of wound recovery rates after different treatments, n = 3 independent experiments, data are expressed as mean ± SD; (c) Photographs of bodily fluids collected from the wound on day 2 and plate smears; (d) Statistical graph of bacterial counts on plate smears, n = 3 independent experiments, data are expressed as mean ± SD.

[0064] Figure 20 TNF-α / IL / 1β fluorescence images of epidermal histological sections from different treatment groups on day 11; scale bar: 1 mm.

[0065] Figure 21 M1 polarization level of macrophages in epidermal histological sections of different treatment groups on day 11; scale bar: 1 mm.

[0066] Figure 22 M2 polarization levels of macrophages in epidermal histological sections of different treatment groups on day 11; scale bar: 1 mm; and M1 / M2 ratio statistic, n = 3 independent experiments, data are expressed as mean ± SD.

[0067] Figure 23The graph shows the cell viability after co-culturing with different treatment groups and mammalian cells. n = 3 independent experiments. Data are expressed as mean ± SD.

[0068] Figure 24 Organ histological sections from different treatment groups on day 11, scale bar: 100 μm. Detailed Implementation

[0069] This invention develops a biocatalyst, named Rh-Fe / JAEP, with a uniformly doped spiky porous carbon surface containing single-atom Fe and clustered Rh. Under acidic conditions, it catalyzes the generation of large amounts of ROS and exhibits oxidation of NADH; under neutral conditions, it can be used for ultrafast and broad-spectrum catalytic ROS removal. Due to the electron transfer interaction between the Fe single atom and the Rh cluster, unique Rh-Fe active sites are formed, thereby improving the reversible redox properties of the active sites. This results in the prepared catalytic material possessing excellent enzyme-like activity, with the maximum reaction rate V of the POD-like enzyme reaching a certain level. max (4.7059 µM s) -1 ) and the transformation number TON (113.559 s -1 The prepared Rh-Fe / JAEP exhibits excellent in vitro bacterial clearance capabilities. Simultaneously, its high specific surface area and positively charged surface allow for rapid and efficient adsorption of negatively charged antigens released from dead bacteria, blocking macrophage uptake and preventing the formation of an inflammatory microenvironment. In in vivo experiments, Rh-Fe / JAEP demonstrated excellent bacterial clearance capabilities against diabetic bacterial infections, subsequently rapidly eliminating excess reactive oxygen species in the wound, preventing long-term chronic inflammation, and promoting wound healing.

[0070] In the embodiments, comparative examples, and experimental examples of this invention, the reagents used were all obtained from the following sources: zinc nitrate (II) hexahydrate (Zn(NO3)2•6H2O), ferric nitrate (III) nonahydrate (Fe(NO3)3•9H2O), and 2-methylimidazole were obtained from Aladdin. Rhodium(III) chloride hydrate (RhCl3•xH2O) was produced by Anage Chemicals. The pure water (18.2 MΩ·cm) used in the experiments was obtained from Milli-Q Academic Systems (Millipore Corp., Billerica, MA, USA). All chemicals were used directly without further purification.

[0071] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0072] Example 1: Preparation of spiky porous carbon substrate JAEP

[0073] (1) Add 0.22 g of zinc acetate and 60 mg of 2,5-dihydroxyterephthalic acid to 70 mL of methanol and sonicate for 30 minutes. Wash the resulting precipitate three times with pure water, place it in a hydrothermal reactor, add 75 mL of pure water and 150 mg of urea, and react at 175 °C for 24 hours. After washing with pure water and ethanol and drying, place the product in a tube furnace and heat it to 1100 °C at a rate of 5 °C / min under a nitrogen atmosphere and hold for 1 hour for carbonization treatment.

[0074] (2) The carbonized product was etched with 1 M hydrochloric acid at 80°C for 3 hours. After cleaning and drying, the spiky porous carbon substrate was obtained and named JAEP.

[0075] Examples 2-4: Preparation of Rh-Fe / JAEP at different carbonization temperatures

[0076] (1) 0.564 g of 2-methylimidazole, 0.25 g of Zn(NO3)2·6H2O and 70 mg of JAEP prepared in Example 1 were stirred and mixed in 60 mL of methanol. Then 2.2 mg of RhCl3·xH2O and 4.25 mg of Fe(NO3)3·9H2O were added, and the mixture was stirred at room temperature for 24 hours. The black product was collected by centrifugation, washed and dried to obtain the precursor.

[0077] (2) The precursor was placed in a tube furnace and heated to 900°C (Example 2), 1000°C (Example 3) and 1100°C (Example 4) at a rate of 5°C / min under a nitrogen atmosphere, and then fired at high temperature for 2 hours.

[0078] The obtained samples were designated as Rh-Fe / JAEP (900℃), Rh-Fe / JAEP (1000℃), and Rh-Fe / JAEP (1100℃), respectively. These samples were not etched with hydrochloric acid and were used to investigate the effect of carbonization temperature on the formation of Rh-Fe active sites and catalytic performance.

[0079] Examples 5-7: Preparation of Rh-Fe / JAEP after acid etching

[0080] The samples (Rh-Fe / JAEP (1000℃)) calcined at 1000℃ in Example 3 were further subjected to acid etching. Specifically, the samples were placed in 1 M hydrochloric acid and etched at room temperature for 1 hour (Example 5), 2 hours (Example 6), and 3 hours (Example 7), respectively. After etching, the samples were cleaned and dried, and the resulting samples were designated as Rh-Fe / JAEP (1 h), Rh-Fe / JAEP (2 h), and Rh-Fe / JAEP (3 h), respectively.

[0081] As shown in Experiment 1 below and Figure 1 As shown, screening using POD-like enzyme activity revealed that in Examples 2-4, the material performance was optimal when the firing temperature was 1000℃; and in Examples 5-7, the performance improvement was highest when the etching time was 1 hour. Therefore, the optimal preparation process was determined to be: carbonization temperature 1000℃ and acid etching time 1 hour. Unless otherwise specified, "Rh-Fe / JAEP" used in subsequent performance characterization and in vitro / in vivo experiments refers to the sample obtained in Example 5 (i.e., carbonized at 1000℃ and etched for 1 hour).

[0082] Comparative Examples 1-3: Preparation of Single Metal or Blank Control Samples

[0083] The preparation method is the same as in Example 5, except that the metal salt is added in step (1):

[0084] Comparative Example 1: Rh / JAEP was prepared by adding only RhCl3·xH2O and not Fe(NO3)3·9H2O.

[0085] Comparative Example 2: Fe / JAEP was prepared by adding only Fe(NO3)3·9H2O and not adding RhCl3·xH2O.

[0086] Comparative Example 3: Pure JAEP (i.e., the product of Example 1) was obtained without adding any metal salts.

[0087] Experimental Example 1

[0088] This experimental example is used to detect the peroxidase-like (POD) activity of samples obtained under different preparation conditions in order to verify the effectiveness of different preparation processes.

[0089] Test method: The material solution (10 mg / mL, 10 μL) was added to NaOAc-HOAc buffer (100 mM, pH = 4.5), followed by the addition of 25 μL of TMB solution (10 mg / mL), resulting in a final mixed solution volume of 2 mL. Subsequently, UV-Vis spectroscopy was performed at 652 nm to monitor the formation of oxidized TMB.

[0090] Result: As Figure 1 As shown, in Examples 2-4 where carbonization temperature was examined, the material exhibited the highest POD-like enzyme activity when the firing temperature was 1000°C (Example 3). Figure 1 a). Therefore, 1000℃ was selected as the optimal carbonization temperature for subsequent acid etching. In Examples 5-7, which examined etching time, the performance improvement was most significant when the etching time was set to 1 hour (Example 5). Figure 1 b).

[0091] Based on the above activity test results, the optimal preparation process was determined to be: carbonization temperature of 1000℃ and acid etching time of 1 hour. Unless otherwise specified, "Rh-Fe / JAEP" in the following text and figures refers to the sample obtained in Example 5 (i.e., carbonized at 1000℃ and etched for 1 hour).

[0092] Experimental Example 2: Structural and Morphological Characterization

[0093] The crystal structure of the catalyst was analyzed using X-ray diffraction (XRD, DX-2700BH, Haoyuan Instruments Co., Ltd., China) under 2θ conditions with Cu Kα radiation ranging from 10 to 80°. Scanning electron microscopy (SEM) images of the approximately 1 nm gold coating were obtained using a Thermo Scientific (FEI) Apreo S HiVoc. Elemental mapping using scanning transmission electron microscopy (STEM) and energy-dispersive X-ray spectroscopy (EDX) was performed on a cs-corrected STEM (FEI Titan Cubed Themis G2 300). The surface charge of the microspheres was tested using a Malvern Nano-ZS instrument. The valence state and electronic structure of Rh-Fe / JAEP were determined using X-ray photoelectron spectroscopy (XPS) on a K-Alpha™+ X-ray photoelectron spectroscopy system (Thermo Scientific) with a hemispherical 180° double-focusing analyzer equipped with a 128-channel detector. X-ray absorption spectra were acquired on the BL07A1 beamline at NSRRC, and the radiation was monochromated using a Si (111) dual-crystal monochromator. XANES data preprocessing and analysis were performed using Athena software.

[0094] Figure 2 The conceptual diagram illustrates the electronic structure of Rh-Fe / JAEP, whose unique Rh-Fe catalytic sites ultimately enhance biocatalytic activity. Electron microscopy (SEM) images show that after loading Rh and Fe metal sites, Example 5 ( Figure 3 b) Compared to Comparative Example 3 ( Figure 3 a) No obvious deformation occurred; the particles exhibited a micron-sized, spherical, porous, spiky shape. Powder X-ray diffraction (PXRD) patterns showed that Examples 2-7 and Comparative Examples 1-3 exhibited similar crystal forms, indicating that Rh and Fe doping did not produce obvious metal cluster peaks and did not significantly alter the structure of the porous carbon surface. Figure 4 ac). Zeta potential test ( Figure 5 The results show that after introducing the two metal salts Rh and Fe, the potential of the substrate JAEP and the two comparative samples increased the most, and the positive charge was increased, which is beneficial for adsorbing the negatively charged antigen.

[0095] High-resolution TEM (HRTEM) images of Rh-Fe / JAEP, Rh / JAEP, and Fe / JAEP are shown below. Figure 6 , Figure 7 and Figure 8 As shown. First, TEM images demonstrate that Example 5 and Comparative Examples 1-2 of this invention have similar spiky particle morphology, with Rh existing in the form of nanoclusters and Fe existing in the form of single atoms, both uniformly distributed on the surface of the spiky porous carbon. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images are shown below. Figure 9 As shown. Figure 9 As shown in (a), lattice fringe measurements confirm that the cluster is an Rh cluster; and Figure 9 (b) Measurements indicate that the distance between Fe and Rh is 0.301 nm, suggesting a potential interaction. Atomic-level selected-region energy-dispersive X-ray spectroscopy (EDX) elemental mapping ( Figure 9 c) This further confirms that Rh is uniformly distributed on the substrate surface in the form of clusters and Fe is uniformly distributed in the form of single atoms.

[0096] X-ray photoelectron spectroscopy (XPS) was used to further understand the chemical and electronic structure of the Rh-Fe / JAEP biocatalyst. Figure 10 In (a), the Fe 2p peak of Rh-Fe / JAEP shifts to a higher binding energy by 0.51 eV compared to Fe / JAEP. Meanwhile, Figure 10 (b) The Rh 3d peak of Rh-Fe / JAEP shifts to a lower binding energy by 0.2 eV compared to Rh / JAEP, while the Metal-N peaks of the three do not show a significant shift. Figure 10 c), demonstrating that charge transfer exists between the Rh clusters and Fe single atoms in Rh-Fe / JAEP. Figure 11 XAS data demonstrate that Rh-Fe / JAEP forms unique Rh-Fe sites, which may be beneficial for enhancing biocatalytic performance.

[0097] Experimental Example 3: Evaluation of Enzyme-like Activity and ROS Scavenging Activity

[0098] (1) Free radical generation test:

[0099] 1.1 Catalytic ROS test:

[0100] The material solution (10 mg / mL, 10 μL) was added to NaOAc-HOAc buffer (100 mM, pH = 4.5), followed by the addition of 25 μL of TMB solution (10 mg / mL). The final mixed solution volume was 2 mL. This solution was then used for UV-Vis spectroscopy at 652 nm.

[0101] like Figure 12 As shown in (a), Example 5 of the present invention was compared with Comparative Examples 1-3, and Rh-Fe / JAEP exhibited the best ROS production activity; subsequently, the Michaelis constant (K) was calculated. m ), maximum reaction rate (V) max The values ​​of the transform number (TON, the maximum number of substrates that can be transformed per unit of active catalytic atom) and the transform number (TON). Figure 12 As shown in (bd) and (e), Rh-Fe / JAEP has the highest reaction rate and conversion number, indicating that Rh-Fe / JAEP exhibits more efficient H2O2 catalytic kinetics.

[0102] (2) NADH oxidation test:

[0103] 2.1 Catalytic oxidation of NADH to NAD+ experiment:

[0104] 10 μL of catalyst (final concentration: 50 μg / mL) and 400 μL of NADH (2 mM) were added to 1590 μL of HEPES buffer solution (10 mM, pH = 6.5). After reacting for 30 min, the absorption change was measured in the wavelength range of 250–500 nm using UV-Vis spectrophotometry.

[0105] like Figure 13 As shown, Rh-Fe / JAEP exhibits the highest NADH oxidation rate.

[0106] (3) CAT-like enzyme test:

[0107] 3.1 H2O2 removal:

[0108] A total of 10 mM H2O2 and 50 μg / mL Rh-Fe / JAEP were mixed in PBS (pH=7.4) to a final volume of 2 mL. Then, 50 μL of the above solution was mixed with 100 μL of titanium sulfate solution (13.9 mM), and the absorbance at 405 nm was recorded every 10 minutes until the reaction time was 60 minutes. At 30 minutes of reaction, the absorbance of the solution at 405 nm was measured to evaluate the H2O2 scavenging capacity of the biocatalyst.

[0109] 3.2 O2 generation determination:

[0110] A total of 100 mM H2O2 and 10 μg / mL Rh-Fe / JAEP were mixed in PBS (pH=7.4) to a final volume of 20 mL. O2 concentration was then measured every 5 seconds using a dissolved oxygen meter (INESA, JPSJ-605F) until 300 s. To analyze the biocatalytic kinetics of O2 generation, 10 μg / mL of the biocatalyst and different concentrations of H2O2 (100, 200, 300, 400, 500, and 600 mM, respectively) were mixed in PBS to a final volume of 20 mL. O2 concentration was then measured every 5 seconds until 100 s.

[0111] (4) Free radical scavenging test:

[0112] 4.1 •O2 - Cleanup test:

[0113] Dissolve 1 mg KO2 in 1 mL of dimethyl sulfoxide (DMSO, containing 3 mg / mL 18-crown-6-ether) to generate and stabilize •O2. − Then, Rh-Fe / JAEP was dispersed in the above KO2 / DMSO solution at a final concentration of 50 μg / mL; after reacting for 5 minutes, residual •O2 − The nitroblue tetrazolium (NBT)-DMSO solution (10 μL, 10 mg / mL) was captured; the absorbance of the solution at 680 nm was measured, and then reacted with •O2. − The original concentrations were compared to obtain the •O2 − Its ability to clear debris.

[0114] 4.2 DPPH Scavenging Test:

[0115] The scavenging ability of the biocatalyst obtained in this invention for 1,1-diphenyl-2-picrylhydrazine radicals (DPPH•) was evaluated by measuring the absorption wavelength at 519 nm. A total of 50 μg / mL of DPPH• ethanol solution and a certain concentration of biocatalyst (50 μg / mL) were mixed and the volume was adjusted to 2 mL; the mixture was then allowed to stand for 10 min before the λ was measured. max Absorbance at 519 nm;

[0116] The enzyme activity and free radical scavenging ability of the biocatalysts obtained in Example 5 and Comparative Examples 1-3 of this invention are as follows: Figure 14 As shown in the figure; it can be seen from the figure that Rh-Fe / JAEP has the highest H2O2 removal efficiency (reaction time 30 minutes) Figure 14 b). Simultaneously, oxygen generation tests also verified that the Rh-Fe / JAEP biocatalyst can effectively decompose H2O2 substrates to generate a large amount of O2 (b). Figure 14 a). SOD enzymes also catalyze the removal of •O2.- It plays an important role in anti-ROS systems. This invention uses the nitrotetrazole blue chloride method to study the effect of the prepared catalyst on •O2. - Free radical scavenging ability. Notably, Rh-Fe / JAEP exhibits significant •O2 scavenging ability. - Cleaning efficiency (cleaning rate reaches ~80% within 5 minutes) Figure 14 c) DPPH• is a commonly used reagent for evaluating the ability of biocatalysts to scavenge RNS. For example... Figure 14 As shown in (d), the DPPH• scavenging efficiency of Rh-Fe / JAEP is significantly higher than that of the other two comparative examples. In summary, this invention reveals that the Rh-Fe / JAEP biocatalyst exhibits strong hydrogen peroxide and reactive oxygen species scavenging capabilities.

[0117] Experiment Example 4: Antibacterial Test

[0118] Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 25922, Gram-positive) and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli ATCC 53104, Gram-negative) were used as representative pathogens to evaluate the bacterial clearance ability of Rh-Fe / JAEP materials.

[0119] Rh-Fe / JAEP was compared with other control samples containing H2O2, and 1 mL (10 6 A bacterial suspension (CFU / mL) was co-cultured at 37°C for 12 hours. The final concentrations of the material and H₂O₂ were 240 μg / mL and 0.2 mM, respectively. The cultured bacterial suspension was then diluted 10... 5 The sample was spread on agar plates and incubated for counting (overnight incubation at 37°C) to assess its ability to inhibit colony formation. Figure 15 The results showed that, compared to other control groups, the Rh-Fe / JAEP treatment group exhibited excellent antibacterial ability against both types of bacteria, completely eliminating residual bacteria. The treated bacterial solution was then cured with freshly prepared 2.5 wt% glutaraldehyde for 12 h. The cured bacteria were then dehydrated using a gradient of alcohol concentrations (0, 30%, 50%, 70%, 80%, 90%, 95%, and 100%, each concentration held for 15 min). After dehydration, the bacteria were dropped onto a silicon wafer, allowed to air dry, and then subjected to SEM testing. Figure 16 As shown, the Rh-Fe / JAEP treatment group captured a large number of bacteria, and the bacteria exhibited significant morphological changes, demonstrating the destructive effect of Rh-Fe / JAEP on the bacteria. Subsequently, the bacteria were stained using the Live / Dead BacLight bacterial viability staining kit (SYTO-9 for live bacteria, propidium iodide PI for dead bacteria), and analyzed and observed using a fluorescence microscope. Figure 17(The text appears to be incomplete and contains errors. A more accurate translation would require the full context.) The Rh-Fe / JAEP treatment group caused the greatest bacterial death compared to other treatment groups. These results indicate that the Rh-Fe / JAEP treatment group can induce bacterial oxidative stress and death by catalyzing the production of large amounts of ROS, exhibiting a broad-spectrum antibacterial effect against both Gram-positive and Gram-negative bacteria.

[0120] Experiment Example 5: Test of the ability to prevent macrophages from polarizing towards a pro-inflammatory mode

[0121] This invention also verifies the ability of Rh-Fe / JAEP to prevent macrophages from polarizing to a pro-inflammatory state and causing chronic inflammation after bacterial clearance by adsorbing antigens leaked from the bacteria. The specific antigens of Staphylococcus aureus and Escherichia coli are lipoteichoic acid (LTA) and endotoxin (LPS), respectively. Figure 18 As shown in (a), with increasing Rh-Fe / JAEP concentration, the uptake of both types of antigens increased, and eventually the antigen levels returned to the levels of the negative group. Because antigen adsorption avoided macrophage uptake, the secretion level of inflammatory factors was lowest. Figure 18 (b) During macrophage polarization, CD163 and CD86 are considered to be M2 and M1 type-specific markers, respectively. Figure 18 As shown in (c), the proportion of M1 type gradually decreases with increasing Rh-Fe / JAEP concentration gradient. These results indicate that Rh-Fe / JAEP can prevent pro-inflammatory polarization of macrophages by capturing antigens, thus avoiding the occurrence of chronic inflammation.

[0122] Experimental Example 6: Evaluation of in vivo treatment for bacterial wound healing in diabetic patients

[0123] This invention establishes a rat model of bacterial skin wound infection in diabetic patients to evaluate angiogenesis, anti-inflammation, and wound healing in diabetic infected wounds. Figure 19 (a) and (b) summarize the photographs of the wound healing process and the wound healing rate. Among them, the wound healing rate of the Rh-Fe / JAEP group was higher than that of the other groups, and it was almost completely healed by day 11, while the wounds of the other treatment groups were still exposed and covered by scabs. Figure 19 (c) and (d) show photographs and colony counts of wound tissue samples after plating. Rh-Fe / JAEP completely eliminated bacteria in the wound, laying the foundation for successful wound healing. Subsequently, immunofluorescence staining was used to observe the histological condition of the wound on day 11 after treatment. This invention uses interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), F4 / 80, CD11c, and CD206 staining to detect the mitigation of oxidative stress and inflammation in the wound by Rh-Fe / JAEP. Figure 20As shown, on day 11, the Rh-Fe / JAEP group had the lowest secretion of inflammatory factors, which was most conducive to wound healing. Figure 21 and Figure 22 Immunofluorescence staining of M1 and M2 macrophages in the wound revealed that the Rh-Fe / JAEP group had the lowest M1 / M2 ratio, indicating the lowest degree of inflammation. Furthermore, the biosafety of the prepared biocatalyst (Rh-Fe / JAEP) was evaluated using CCK-8 cell assays and H&E staining of major organs (heart, liver, spleen, lung, and kidney). Figure 23 As shown, the present invention has no significant effect on cell activity; Figure 24 The results indicate that no significant damage or abnormalities were observed in the major organs and tissues, suggesting that the biocatalyst prepared in this invention has low cytotoxicity. These animal experimental results demonstrate that Rh-Fe / JAEP can serve as an effective and safe nanomedicine to combat oxidative stress and achieve ultra-rapid healing of diabetic bacterial wounds.

[0124] In summary, the above research results confirm that the Rh-Fe / JAEP biocatalyst synthesized in this invention, possessing multiple biological functions, is an ideal and highly efficient ultrafast, broad-spectrum catalytic antibacterial and anti-inflammatory nanoplatform for the generation and scavenging of ROS. The introduction of Rh-Fe active sites enhances electron transfer and strengthens its biocatalytic activity. The Rh-Fe / JAEP biocatalyst prepared in this invention exhibits excellent antibacterial and anti-inflammatory properties. In vitro and in vivo experiments have demonstrated that Rh-Fe / JAEP can significantly capture antigens leaked after bacterial killing, preventing macrophages from excessively polarizing to a pro-inflammatory state, ultimately promoting tissue regeneration and achieving ultrafast healing of inflammatory diabetic ulcers.

[0125] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A porous carbon biocatalytic material supported on rhodium-iron bimetallic active sites, characterized in that, The porous carbon biocatalytic material includes a spiky porous carbon substrate, and rhodium clusters and iron single atoms uniformly loaded on the surface of the spiky porous carbon substrate. The rhodium clusters exist in the form of nanoclusters, and the iron single atoms exist in the form of single atoms. Furthermore, the rhodium clusters and iron single atoms form Rh-Fe bimetallic active sites with charge transfer effects. The spiky porous carbon substrate is obtained as follows: a zinc-based metal-organic framework is subjected to a hydrothermal reaction in the presence of urea, followed by carbonization and acid etching to obtain the spiky porous carbon substrate. The organic ligand of the zinc-based metal-organic framework is 2,5-dihydroxyterephthalic acid.

2. The porous carbon biocatalytic material according to claim 1, characterized in that, In the porous carbon biocatalytic material, the molar ratio of rhodium to iron is 1:1 to 1:

2.

3. The porous carbon biocatalytic material according to claim 1 or 2, characterized in that, The porous carbon biocatalytic material has a positively charged surface and exhibits peroxidase-like and NADH oxidase-like activities under acidic conditions, and glucose oxidase-like, catalase-like, and superoxide dismutase-like activities under neutral conditions.

4. A method for preparing a porous carbon biocatalytic material according to any one of claims 1-3, characterized in that, Includes the following steps: (1) In solvent I, Zn salt is reacted with organic ligand 2,5-dihydroxyterephthalic acid to form a zinc-based metal-organic framework; (2) The zinc-based metal-organic framework is subjected to hydrothermal treatment in urea solution, followed by first carbonization and acid etching to obtain a spiky porous carbon substrate; (3) Mix 2-methylimidazolium, Zn salt and spiky porous carbon substrate in solvent II, then add Rh salt and Fe salt, stir the reaction, centrifuge, wash and dry to obtain the precursor; (4) The precursor is subjected to a second carbonization and acid etching treatment to obtain the porous carbon biocatalytic material.

5. The preparation method according to claim 4, characterized in that, The Zn salt mentioned in step (1) is at least one of zinc nitrate, zinc acetate, zinc chloride, or their hydrates; Solvent I is an alcohol solvent, including methanol or ethanol; The mass concentration of the urea solution in step (2) is 0.1-0.3%; The hydrothermal treatment temperature is 170-180 ℃, and the reaction time is 12-36 h; In the first carbonization and acid etching process, the carbonization temperature is 1000-1200 ℃, the reaction time is 1-3 h, and the acid etching uses 1M hydrochloric acid at 70-90℃ for 1-3 h.

6. The preparation method according to claim 4, characterized in that, The molar ratio of Rh salt to Fe salt in step (3) is 1:1, and the molar ratio of the total amount of Rh salt and Fe salt to Zn salt is 1:(30-50). The Zn salt is at least one of zinc nitrate, zinc acetate, zinc chloride, or their hydrates; The Rh salt is at least one of rhodium chloride, rhodium nitrate, rhodium acetylacetonate, or their hydrates; The Fe salt is at least one of ferric nitrate, ferric chloride, ferric acetylacetone, or their hydrates; Solvent II is an alcohol solvent, including methanol or ethanol.

7. The preparation method according to claim 4, characterized in that, In step (4), the second carbonization and acid etching process is carried out at a carbonization temperature of 900-1100 ℃ and a reaction time of 1-3 h. The acid etching process uses 1M hydrochloric acid and the etching time is 1-3 h.

8. The application of the porous carbon biocatalytic material as described in any one of claims 1-3 in the preparation of antibacterial materials.

9. The use of the porous carbon biocatalytic material as described in any one of claims 1-3 in the preparation of a medicament for treating bacterial wound infections and inflammation caused by bacterial wound infections, and for promoting the healing of bacterial wounds.

10. The application according to claim 9, characterized in that, The wound in question is that of a diabetic patient.

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