Pollen-based multi-activity mimic enzyme material with immune activation capability as well as preparation method and application of pollen-based multi-activity mimic enzyme material

Abstract

The invention belongs to the technical field of antibacterial materials, and particularly relates to a pollen-based multi-activity mimic enzyme material with immune activation capacity as well as a preparation method and application of the pollen-based multi-activity mimic enzyme material. The mimic enzyme material comprises an amorphous nitrogen-doped carbon substrate derived from natural pollen and vanadium oxide particles which are loaded on the surface of the amorphous nitrogen-doped carbon substrate and are doped with a large number of oxygen vacancy defects, and the mimic enzyme material is named as VAE-Pollen. The amorphous nitrogen-doped carbon substrate derived from the natural pollen provides electrons for the vanadium oxide framework, so that the formation energy of oxygen vacancies is reduced, and the displacement of V atoms in a local structure is about 0.40, so that the geometric structure is optimized, the accessibility of V active sites is improved, and the catalytic efficiency and the multi-functional mimic enzyme activity are remarkably enhanced. Meanwhile, the prepared VAE-Pollen can remove bacteria and activate immune cells through various mechanisms, shows excellent disinfection capacity on wounds infected by staphylococcus aureus, forms immune memory and immune monitoring capacity in organisms, and prevents recurrence of infected wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial materials, and in particular relates to a pollen-based multi-active enzyme-mimicking material with immune activation ability, a preparation method thereof, and an application thereof. Background Art

[0002] Overuse of antibiotics has significantly accelerated the evolution of drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), posing a serious threat to clinical treatment. Recent data show that the average mortality rate associated with MRSA infection is approximately 64% higher than that associated with non-resistant S. aureus infection, highlighting the urgent need for effective medical interventions without inducing further drug resistance. To address this challenge, various artificial enzyme-mimicking materials capable of generating reactive oxygen species (ROS), such as nanocarbon materials, photosensitizers, metal-organic frameworks (MOFs), and metal oxides, have been considered as potential alternatives to antibiotics for combating severe MRSA infections. Among them, vanadium pentoxide (V2O5), a typical vanadium-based haloperoxidase (V-HPO) biomimetic material, has been extensively studied for its ability to catalyze the production of hydroxyl radicals (·OH) and hypochlorous acid (HClO) to kill bacteria.

[0003] However, most current studies focus on their short-term antibacterial effects, often overlooking the inherent potential of materials in regulating inflammation and establishing long-term immune surveillance, which are crucial for preventing recurrence of infection. Therefore, it is particularly urgent to develop a smart antibacterial material that combines rapid bactericidal and immune activation capabilities.

[0004] In humans, natural pollen, due to its highly spiky surface, easily retains and continuously releases allergens in the respiratory tract, triggering a strong allergic reaction. Upon exposure, dendritic cells (DCs) and macrophages (Mφs) phagocytose and process pollen allergens, presenting them to T cells in the spleen. This in turn triggers the release of proinflammatory cytokines, inducing allergic inflammation and asthma. Simultaneously, B cells produce specific antibodies that bind to mast cells and basophils, establishing an "allergic memory" and triggering a more robust immune response upon re-exposure. Inspired by the mechanism of pollen allergy, we hypothesized that the spiky surface morphology of pollen could be exploited to efficiently capture bacteria and adhere to biofilms, thereby inactivating pathogens in situ. Mφs then recognize and process the released bacterial antigens, polarizing toward a proinflammatory phenotype and activating T cells, creating an inflammatory microenvironment conducive to clearing reinvading bacteria.

[0005] Vanadium metal oxides have inherent disadvantages such as low catalytic activity and biological toxicity caused by large dosage. In order to improve their ROS catalytic performance and expand their enzyme-like functions, the crystal surface is regulated, especially the oxygen vacancies (O v) is considered an effective strategy. Therefore, it is crucial to develop new strategies to modulate the coordination structure and bonding microenvironment of catalytic atoms in vanadium metal oxides to achieve efficient ROS catalysis and mimic the immune activation mechanism of pollen allergy. Currently, research in this area has remained largely unexplored. Summary of the Invention

[0006] To address the above issues, the present invention provides a pollen-based multi-activity enzyme-like material with immune activation capabilities, as well as its preparation method and application. The pollen-based multi-activity enzyme-like material is a natural pollen-derived amorphous nitrogen-doped carbon substrate with vanadium oxide particles on its surface. The vanadium oxide particles have more oxygen vacancy defects than vanadium oxide alone. The pollen-based multi-activity enzyme-like material is named VAE-Pollen in this specification. The natural pollen-derived amorphous nitrogen-doped carbon substrate is loaded with vanadium oxide (VO x ) framework, which helps to reduce the formation energy of oxygen vacancies, accompanied by a displacement of about 0.40 Å of the V atoms in the local structure, thereby optimizing the geometric structure and improving the accessibility of the V active site, significantly enhancing its catalytic efficiency and multifunctional enzyme-like activity. Specifically, VAE-Pollen exhibits enzyme-like performance such as peroxidase (POD), haloperoxidase (HPO) and reduced nicotinamide adenine dinucleotide oxidase (NOX) that is superior to VO2 and current advanced antibacterial materials. In addition, VAE-Pollen exhibits excellent POD enzyme-like reaction kinetics: the maximum reaction rate (V max ) is 2.67 µMs -1 , the conversion number (TON) is 24.75*10 -3 s -1 , which indicates that the prepared pollen-based multifunctional vanadium enzyme-mimicking material has ultrafast and excellent catalytic activity. In addition, systematic biological experimental studies have shown that VAE-Pollen can not only quickly capture bacteria, but also synergistically kill bacteria through multiple mechanisms such as respiratory chain destruction, lipid peroxidation, protein carbonylation and leakage, realizing an "capture-kill" antibacterial mode. In addition, when the antigens of the destroyed bacteria leak out, they can be recognized and processed by macrophages, further promoting the formation of antigen-specific immune memory. When the same bacteria invade again, this memory mechanism can quickly respond and initiate dendritic cell maturation, macrophage polarization, T cell infiltration and B cell differentiation, effectively eliminating pathogens and achieving long-term immune surveillance without additional intervention.

[0007] Specifically, the first technical problem to be solved by the present invention is to provide a pollen-based multi-active enzyme-mimicking material with immune activation ability, wherein the enzyme-mimicking material includes an amorphous nitrogen-doped carbon substrate derived from natural pollen and vanadium oxide particles loaded on its surface, wherein the vanadium oxide particles have more oxygen vacancy defects than vanadium oxide alone.

[0008] Furthermore, the natural pollen-derived amorphous nitrogen-doped carbon substrate donates electrons to the vanadium oxide framework, thereby forming VN bonds.

[0009] Furthermore, due to the introduction of oxygen vacancy defects, the vanadium oxide particles have a V atomic displacement of 0.40 Å in the local structure.

[0010] Furthermore, the enzyme-mimicking material has POD, HPO and NOX enzyme-mimicking activities.

[0011] Furthermore, the POD enzyme reaction kinetics of the enzyme-mimicking material is as follows: the maximum reaction rate is 2.67 μM s -1 , the number of conversions is 24.75*10 -3 s -1 .

[0012] The second technical problem to be solved by the present invention is to provide a method for preparing a pollen-based multi-active enzyme-like material with immune activation ability as described in this article, the preparation method comprising using vanadyl oxalate, hydrogen peroxide and natural pollen as raw materials, and preparing the pollen-based multi-active enzyme-like material with immune activation ability through a hydrothermal reaction.

[0013] Furthermore, the preparation method includes mixing vanadyl oxalate solution, hydrogen peroxide and natural pollen in an alcohol solvent, reacting in a reactor at 150-200°C for 1-6 hours, collecting the reaction product, and obtaining the pollen-based multi-active enzyme-mimicking material with immune activation ability after washing and drying.

[0014] Furthermore, the volume ratio of the vanadyl oxalate solution to hydrogen peroxide is 5:1.

[0015] Furthermore, the mass ratio of the mixed solution of the vanadyl oxalate solution and hydrogen peroxide to the natural pollen is 1:5-20.

[0016] Furthermore, the vanadyl oxalate solution is an aqueous solution of vanadyl oxalate, and its concentration is 0.33 mol / L.

[0017] Furthermore, the hydrogen peroxide is an aqueous solution of hydrogen peroxide with a mass fraction of 30 wt%.

[0018] Furthermore, the natural pollen is defatted pollen. The defatting method is well known to those skilled in the art, for example, the defatting process can be performed as described in the detailed description of the present invention.

[0019] Furthermore, the natural pollen can be, but is not limited to, natural sunflower pollen. Those skilled in the art will appreciate that the present invention is also applicable to other types of natural pollen.

[0020] Furthermore, the alcohol solvent is ethanol.

[0021] The third technical problem to be solved by the present invention is to provide the use of the pollen-based multi-active enzyme-mimicking material with immune activation ability as described herein in the preparation of materials for generating reactive oxygen species, antibacterial and / or inducing macrophage polarization.

[0022] Furthermore, bacteria may include but are not limited to drug-resistant Staphylococcus aureus, and macrophages may include but are not limited to mouse mononuclear macrophage leukemia cells.

[0023] The fourth technical problem to be solved by the present invention is to provide the use of the pollen-based multi-active enzyme-mimicking material with immune activation ability as described herein in the preparation of products for treating bacterial wound infections and preventing recurrence of infections.

[0024] Furthermore, the bacterial infection may include but is not limited to Staphylococcus aureus infection.

[0025] As used herein, recurrence of infection includes recurrence of infection caused by secondary bacterial infection.

[0026] Beneficial effects of the present invention

[0027] The present invention has developed a pollen-based multi-activity enzyme-mimicking material loaded with vanadium oxide doped with a large number of oxygen vacancy defects, named VAE-Pollen. The catalytic material obtained by the present invention has excellent POD-like enzyme activity and good reaction kinetics: the maximum reaction rate V max 2.67 µM s -1 , the conversion number TON is 24.75*10 -3 s -1 The VAE-Pollen obtained by the present invention can quickly capture and remove bacteria, and the leaked substances can cause macrophages to respond quickly and polarize toward a pro-inflammatory type, forming a pro-inflammatory microenvironment that is beneficial to killing bacteria.

[0028] In vivo experiments have shown that VAE-Pollen exhibits excellent bacterial disinfection capabilities against bacterially infected wounds and can activate the body's immune response, generating immune memory. This rapidly triggers an immune response upon re-infection, enabling long-term immune surveillance. This eliminates bacteria from wounds and promotes wound healing without the need for additional materials. This invention provides an effective nanomedicine for catalyzing reactive oxygen species, accelerating wound healing, and preventing recurrence, offering a new avenue for the development of non-antibiotic antimicrobial strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the synthesis of VAE-Pollen and its structural diagram of the present invention.

[0030] Figure 2 SEM images of (a) Flat pollen, (b) Pollen, (c) VAE-Pollen (1:5), (d) VAE-Pollen (1:10), and (e) VAE-Pollen (1:20).

[0031] Figure 3 This is the XRD pattern of VAE-Pollen obtained in Example 1 of the present invention.

[0032] Figure 4 Figure 3: (a) HAADF-STEM image of the atomic array of the VAE-Pollen crystallized region; (b) high-resolution HAADF-STEM image of the VAE-Pollen defect; and (c) the corresponding atomic distribution map obtained from the area highlighted by the line without arrows in (b).

[0033] Figure 5 Figure 2: STEM spectrum imaging of VAE-Pollen (a) and EDX elemental mapping showing the distribution of V, N, O and C elements (b).

[0034] Figure 6 Figure 1 shows the electronic structure analysis results of VO2 for VAE-Pollen and Comparative Example 1: (a) Electron paramagnetic resonance spectrum of VAE-Pollen. (b) and (c) High-resolution XPS spectra of VAE-Pollen in the (a) V 2p and (b) N 1s regions, respectively.

[0035] Figure 7 Performance statistics of VO2 and VAE-Pollen in catalyzing the production of reactive oxygen species: (a) is a test graph of POD-like enzyme activity of VO2 and VAE-Pollen; (b) is a reaction rate curve with changes in H2O2 concentration and (c) the maximum reaction rate V of VO2 and VAE-Pollen max and Michaelis constant K m(d) Histogram of the HPO-like enzyme activities of VO2 and VAE-Pollen; (e) Graph of the NADH oxidase-like activities of VO2 and VAE-Pollen; (f) Radar chart of the multi-enzyme activities and free radical generation capacity of VO2 and VAE-Pollen. The control group refers to a blank control group without any catalytic material (VO2 and VAE-Pollen); the hydrogen peroxide group refers to a control group with only hydrogen peroxide added without any catalytic material. In this disclosure, all references to the control group refer to the blank control group without the addition of enzyme-mimicking materials, and all references to the hydrogen peroxide group refer to the control group with only hydrogen peroxide added without any catalytic material. All other testing and incubation conditions were the same; n = 3 independent experiments, data are expressed as mean ± SD.

[0036] Figure 8 Statistical results of POD enzyme-mimicking activity of enzyme-mimicking materials obtained in Examples 1 to 3 and Comparative Examples 1 to 3.

[0037] Figure 9 Plate coating image of Staphylococcus aureus and Escherichia coli after co-incubation with VO2 and VAE-Pollen; scale bar: 9 cm.

[0038] Figure 10 Live / dead fluorescence staining of Staphylococcus aureus and Escherichia coli after co-incubation with VO2 and VAE-Pollen. SYTO-9 staining signals represent live bacteria, and PI staining signals represent dead bacteria. Scale bar: 15 µm.

[0039] Figure 11 Live / dead fluorescence staining of Staphylococcus aureus and Escherichia coli biofilms after co-incubation with VO2 and VAE-Pollen. Fluor-647 staining signals represent biofilms, SYTO-9 staining signals represent live bacteria, and PI staining signals represent dead bacteria; scale bar: 40 µm.

[0040] Figure 12 The supernatant extracted after co-incubation of Staphylococcus aureus with VO2 and VAE-Pollen was added to macrophages to test the polarization state of macrophages. The CD206 staining signal represents the secretion factor of M2, and the iNOS staining signal represents the secretion factor of M1; scale bar: 100 µm.

[0041] Figure 13 Cytotoxicity tests of VO2 and VAE-Pollen; scale bar: 100 µm.

[0042] Figure 14Figure 3. The effect of VAE-Pollen on wound healing in the treatment of Staphylococcus aureus infection: (a) Representative images of the wound area under different treatment methods on days 0, 1, 3, 7, and 11; (b) Time evolution of wound size after different treatments; (c) On the first day after infection, fluid was collected from the wound for plate smearing; (d) H&E and Masson staining of the epidermis of the different treatment groups. Figure 14 In the table, I represents the control group, II represents the hydrogen peroxide group, III represents the vancomycin group, and IV represents the VAE-Pollen group.

[0043] Figure 15 Figure 11: Inflammatory factor levels in epidermal histological sections of different treatment groups on day 11: Fluorescence staining of inflammatory factors IL-1β and TNF-α; scale bar: 80 µm.

[0044] Figure 16 H&E-stained sections of rat visceral tissues after different treatments 11 days later. Scale bar: 100 µm.

[0045] Figure 17 Figure 3: The therapeutic effect of VAE-Pollen on the recurrence of bacterially infected wounds after the first successful treatment of wound infection by establishing immune memory. (a) Representative images of wound areas treated with different treatments on days 20 (day 0 of the second modeling), 24, and 32; (b) Plate smear of fluid taken from wounds treated with different treatments and colony counts; (c) Dendritic cell activation response levels in epidermal histological sections of different treatment groups on day 3 after infection; (d) T cell activation response levels in epidermal histological sections of different treatment groups on day 3 after infection; (e) H&E and Masson staining of the epidermis of different treatment groups.

[0046] Figure 18 These are H&E staining images of visceral tissue sections of diabetic rabbits after different treatments 11 days later. DETAILED DESCRIPTION

[0047] The present invention has developed a pollen-based multi-activity enzyme-like material loaded with vanadium oxide doped with a large number of oxygen vacancy defects, named VAE-Pollen, which can be used to improve ROS catalytic performance and has multiple enzyme-like activities. x ) framework provides electrons, which helps to reduce the formation energy of oxygen vacancies, accompanied by a displacement of about 0.40 Å of the V atoms in the local structure, thereby optimizing the geometric structure and improving the accessibility of the V active site, significantly enhancing its catalytic efficiency and multifunctional enzyme-like activity, with a maximum reaction rate of V max 2.67 µM s-1 And the conversion number TON is 24.75*10 -3 s -1 At the same time, the prepared VAE-Pollen can eliminate bacteria and activate immune cells through multiple mechanisms, showing excellent disinfection ability for wounds infected with Staphylococcus aureus, and forming immune memory and immune surveillance capabilities in the body to prevent the recurrence of infected wounds.

[0048] The present invention is further described below with reference to specific examples, which, however, are not intended to 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 the art.

[0049] In the Examples, Comparative Examples, and Test Examples of the present invention, all reagents were obtained from the following sources: Vanadyl oxalate was prepared in-house in the laboratory. Specifically, V2O5 (6.598 mmol / 1.2 g) and H2C2O4 (19.794 mmol / 1.782 g) were dissolved in 40 mL of pure water and stirred in an oil bath at 80°C for three hours. The solution turned clear blue, yielding vanadyl oxalate (0.33 mol / L). Hydrogen peroxide (H2O2) was obtained from Aladdin. Pure water (18.2 MΩ·cm) used in the experiments was obtained from a Milli-Q academic system (Millipore Corp., Billerica, MA, USA). All chemicals were used directly without further purification.

[0050] Example 1:

[0051] VAE-Pollen is synthesized by hydrothermal method. The schematic diagram of its formation process and its structural model are shown in Fig. Figure 1 Briefly, natural sunflower pollen was first defatted. Subsequently, a 0.33 mol / L VOC₂O₄ solution and 30% hydrogen peroxide (H₂O₂, mass fraction) were mixed in a 5:1 volume ratio. The resulting mixture, along with 20 mg of defatted pollen, was added to 15 mL of ethanol at a 1:5 mass ratio and stirred for 15 minutes. The mixture was then transferred to a polytetrafluoroethylene-lined autoclave and reacted at 170°C for 2 hours. After the reaction, a black product was obtained by centrifugation, washed thoroughly with deionized water and ethanol, and dried overnight under vacuum at 60°C. The resulting product was named VAE-Pollen (1:5), where 1:5 refers to the mass ratio of the VOC₂O₄ solution to the hydrogen peroxide solution to the pollen.

[0052] Example 2:

[0053] Except that the mass ratio of the mixed solution of VOC2O4 solution and hydrogen peroxide to pollen was 1:10, the remaining steps were the same as those in Example 1, and the obtained product was named VAE-Pollen (1:10).

[0054] Example 3:

[0055] Except that the mass ratio of the mixed solution of VOC2O4 solution and hydrogen peroxide to pollen was 1:20, the remaining steps were the same as those in Example 1, and the obtained product was named VAE-Pollen (1:20).

[0056] Comparative Examples 1-2

[0057] Watermelon pollen and sunflower pollen were purchased directly from Taobao. After purchase, 10 g of each was stirred in 100 mL of ether for 4 hours. The pollen was then suspended in 100 mL of phosphoric acid and heated at 70 °C for 10 hours. Finally, the final products were washed sequentially with deionized water, acetone, deionized water, ethanol, and deionized water to complete the defatting process. The resulting products were named flat pollen (for watermelon pollen) and pollen (for sunflower pollen), respectively.

[0058] Comparative Example 3

[0059] Commercial vanadium dioxide (VO2) was used, obtained from Aladdin.

[0060] Experimental Example 1: Structural Characterization of VAE-Pollen:

[0061] Scanning electron microscopy (SEM) images were obtained using a Thermo Fisher Scientific (FEI) Apreo S HiVoc, and the gold coating was approximately 1 nm. Figure 2 As shown, SEM images of Flat pollen (a) and Pollen (b) prepared in Comparative Examples 1 and 2, and VAE-Pollen (1:5) (c), VAE-Pollen (1:10) (d) and VAE-Pollen (1:20) (e) prepared in Examples 1-3 are respectively shown. It can be seen that Examples 1-3 successfully prepared pollen-based amorphous nitrogen-doped carbon substrates with surface-loaded vanadium oxide.

[0062] Next, the VAE-Pollen (1:10) obtained in Example 2 of the present invention was further characterized. Unless otherwise specified, VAE-Pollen hereinafter refers to VAE-Pollen (1:10).

[0063] The crystal structure of VAE-Pollen was analyzed using an X-ray diffractometer (XRD, DX-2700BH, Haoyuan Instrument Co., Ltd., China) under Cu Kα radiation in the 2θ range of 10° to 80°. Figure 3 As shown: VAE-Pollen has no obvious crystallization peak and basically retains the peak shape of Pollen.

[0064] Scanning transmission electron microscopy (STEM) images and energy-dispersive X-ray spectroscopy (EDX) elemental mapping were obtained on a cs-corrected STEM (FEI Titan Cubed Themis G2 300). Electron paramagnetic resonance (EPR) measurements were performed using a Bruker EPR EMX Plus instrument (Bruker Beijing Technology Co., Ltd., USA) at 9.8 GHz (microwave power: 1 mW). VAE-Pollen angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images are shown in Figure 2. Figure 4 As shown. First, the atomic resolution structure of VAE-Pollen was described by HAADF-STEM images. Figure 4 As shown in a, the atomic resolution STEM image of the nanocrystal region of VAE-Pollen shows a regular and bright array of VO2 atoms. Secondly, another STEM image ( Figure 4 b) A large number of vanadium and oxygen vacancy defects can be observed. Figure 4 The histogram of c proves the existence of defects. STEM spectral imaging ( Figure 5 a) and atomic-level selected-area energy-dispersive X-ray spectroscopy (EDX) elemental mapping ( Figure 5 b) Showing the uniform distribution of V atoms on the pollen surface.

[0065] The valence state and electronic structure of VAE-Pollen were investigated by electron paramagnetic resonance (EPR) and X-ray photoelectron spectroscopy (XPS) on a K-Alpha™+ X-ray photoelectron spectrometer system (ThermoScientific) using a hemispherical 180° double-focusing analyzer with a 128-channel detector. Figure 6 As shown in a, VAE-Pollen has more vacancy defects than VO2. Figure 6 The V 2p and N 1s spectra of VAE-Pollen in b and c show that compared with VO2, the V 2p peak of VAE-Pollen shifts toward low binding energy, and correspondingly, the pyridine N peak in N 1s shifts toward high binding energy, which is mainly attributed to the introduction of amorphous nitrogen-doped carbon substrate derived from natural pollen, which provides electrons to V and forms VN bonds.

[0066] Experimental Example 2: Enzyme Activity Evaluation of VAE-Pollen

[0067] (1) POD enzyme imitation test:

[0068] 1.1 ROS production test:

[0069] The material solution (10 mg / mL, 10 μL) was added to NaOAc-HOAc buffer (100 mM, pH = 4.5), and then 25 μL of TMB solution (10 mg / mL) was added. The final volume of the mixed solution was 2 mL. Subsequently, the solution was used for UV-visible spectroscopy at a wavelength of 652 nm ( Figure 7 a).

[0070] 1.2 Kinetic parameter test:

[0071] Michaelis constant (K m ) is calculated based on the Michaelis saturation curve. The initial reaction rate (V0) is calculated from the absorbance change according to the Beer-Lambert law formula (1), where [S] represents the concentration of each H2O2. The Michaelis constant (K m ) and the maximum reaction rate (V max ) is obtained by plotting the Lineweaver-Burk plot by performing a double reciprocal treatment on formula (2). In addition, the turnover number (TON) is calculated according to formula (3), where [E0] is the molar concentration of the doped metal in the nanozyme. Figure 7 b and c are the reaction rate curves with the change of H2O2 concentration and the maximum reaction rate V of VO2 and VAE-Pollen, respectively. max and Michaelis constant K m A histogram of values.

[0072] (1)

[0073] (2)

[0074] (3)

[0075] (2) HPO enzyme-mimicking test:

[0076] 2.1 Hypochlorous acid production test:

[0077] The HPO enzyme activity was tested using lapis lazuli blue. 1930 μL of lapis lazuli blue solution (200 μM), 60 μL of enzyme material solution (10 mg / mL), and 10 μL of H2O2 solution (0.1 M) were mixed. After 30 minutes of reaction, the catalytic activity was determined by measuring the absorbance change of lapis lazuli blue in the wavelength range of 645 nm to 520 nm. Figure 7 d).

[0078] (3) NADH enzyme test:

[0079] 3.1 NADH oxidation test:

[0080] 10 μL of enzyme-like material solution (final concentration 50 μg / mL) and 400 μL of NADH solution (2 mM) were added to 1590 μL of HEPES buffer (10 mM, pH = 6.5). After 30 minutes of reaction, the absorbance change was measured by UV-visible spectrometer in the wavelength range of 250–500 nm ( Figure 7 e).

[0081] The above test results are as follows Figure 7 As shown in af. As can be seen from the figure: within 10 minutes, VAE-Pollen has a higher ROS production activity than VO2 ( Figure 7 a); Under the same hydrogen peroxide substrate concentration, VAE-Pollen has a faster reaction rate ( Figure 7 b). The Michaelis constant (K m ), maximum reaction speed (V max ) and turnover number (TON, the maximum number of substrates converted per unit of active catalytic atom), such as Figure 7 As shown in c, VAE-Pollen shows a larger V compared to VO2 max (2.67 µM s -1 ) and higher TON (24.75*10 -3 s -1 ), indicating that VAE-Pollen exhibits more effective H2O2 catalytic kinetics. Subsequently, the present invention systematically combined VAE-Pollen with the latest reported active oxygen catalytic materials, including V-Fe2O3, V2O5, Ru NPs, etc. max The results were compared with the TON values ​​(Table 1), and the results showed that VAE-Pollen exhibited the best POD enzyme-mimicking activity among these established enzyme-mimicking materials.

[0082] In addition, VAE-Pollen has better HPO enzyme-like performance than VO2 ( Figure 7 d) and NADH oxidase mimetic activity ( Figure 7 e): HPO mimetic enzyme can catalyze hydrogen peroxide and chloride ions to produce hypochlorous acid with disinfection ability, while NADH oxidase can oxidize NADH to oxidized state NAD + , destroying the normal respiratory chain metabolism of bacteria and eventually leading to bacterial death. Figure 7f is a radar chart of the multi-type enzyme activities and free radical generation capabilities of VO2 and VAE-Pollen, which more intuitively demonstrates the excellent performance of VAE-Pollen prepared by the present invention relative to VO2.

[0083] Table 1: Comparison of the enzyme-mimicking material obtained in Example 1 of the present invention with other enzyme-mimicking materials reported in the prior art

[0084] Artificial POD enzyme <![CDATA[V max (µM s -1 )]]> <![CDATA[TON (10 -3 s -1 )]]> Ref VAE-Pollen 2.67 24.75 The present invention <![CDATA[V-Fe2O3]]> 1.07 22.38 Angew. Chem. Int. Ed. 263, e202310811 (2024) <![CDATA[V2O5]]> 0.49 2.57 Angew. Chem. Int. Ed. 263, e202310811 (2024) CoO 1.14 8.55 ACS Sustainable Chem. Eng. 7, 13989-13998 (2019). <![CDATA[CeO2]]> 0.18 3.1 Chem. Soc. Rev. 48, 1004-1076 (2019). CuO 0.28 2.23 Biosens. Bioelectron. 61, 374-378 (2014). <![CDATA[Mn2O3]]> 1.01 7.98 J. Mater. Chem. B 8, 1191-1201 (2020). Fe-NC 0.62 3.99 ACS Catal. 10, 6422-6429 (2020). Cu-NC 0.06 3.3 Langmuir 38, 6860-6870 (2022). Co-NC 0.17 9.58 ACS Catal. 10, 6422-6429 (2020). Ru NPs 0.18 5.5 J. Colloid Interface Sci. 631, 86-95 (2023). Pt cubes 0.25 0.01 ACS Appl. Mater. Interfaces 9, 10027-10033 (2017).

[0085] The enzyme mimicking properties of the enzyme-mimicking materials obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3 were further tested, and the results were as follows: Figure 8 As shown, it shows that VAE-Pollen shows the best performance.

[0086] Test Example 3: Antibacterial Experimental Test of VAE-Pollen

[0087] Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 25922, Gram-positive bacteria) and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli ATCC 53104, Gram-negative bacteria) were used as representative pathogens to evaluate the bacterial capture and killing capabilities of the VAE-Pollen material. VAE-Pollen and other control samples containing H2O2 were mixed with 1 mL (10 6 CFU / mL) of bacterial suspension was co-cultured at 37°C for 12 hours. The final concentrations of the material and H2O2 in the experiment were 60 μg / mL and 0.2 mM, respectively. The cultured bacterial suspension was diluted 10 5 The ability to inhibit colony formation was assessed by plating on agar plates and performing count culture (incubation overnight at 37°C). Figure 9 As shown in the figure, the VAE-Pollen treatment group showed excellent antibacterial activity against both types of bacteria compared to the other control groups. Subsequently, the bacteria were stained using the Live / Dead BacLight bacterial activity staining kit (SYTO-9 for live bacteria and propidium iodide PI for dead bacteria). Dextran Fluor-647 can stain the biofilm formed by planktonic bacteria, and the results were analyzed and observed using a fluorescence microscope. Figure 10 and 11 As shown ( Figure 10 For planktonic bacteria, Figure 11Compared to VO2, VAE-Pollen not only caused a significant number of bacterial deaths, but also observed fluorescent aggregation, indicating bacterial aggregation around the material, demonstrating the material's excellent bacterial capture capabilities. These results demonstrate that VAE-Pollen can catalyze the production of large amounts of ROS, causing bacterial oxidative stress and death, and has a universal antibacterial effect against both Gram-positive and Gram-negative bacteria.

[0088] Experimental Example 4: VAE-Pollen Promotes Macrophage Polarization Potential

[0089] The present invention also validates the potential of VAE-Pollen to promote antibacterial and pro-inflammatory macrophage polarization. If VAE-Pollen can induce macrophage polarization toward a pro-inflammatory state by catalyzing hydrogen peroxide to produce ROS, this could trigger the body's immune response and favor the formation of an inflammatory microenvironment that facilitates the clearance of invading bacteria. To test this hypothesis, the present invention first conducted in vitro experiments to induce macrophage polarization.

[0090] RAW 264.7 macrophages (2 × 10 5 5×10 cells / well) were inoculated into 24-well plates and cultured overnight. Subsequently, 200 μL of Staphylococcus aureus (MRSA, 5×10 9 The supernatant of cells was incubated with VAE-Pollen and other control groups for 3 hours and then co-cultured for 24 hours. Immunofluorescence staining was then performed. During the polarization of macrophages, iNOS and CD206 are considered to be specific markers of macrophages (iNOS is a pro-inflammatory marker, and CD206 is an anti-inflammatory marker). Figure 12 As shown, the iNOS intensity of other treatment groups and the control group was very limited, and there was no significant difference in CD206 fluorescence intensity among the groups. However, the macrophages in the VAE-Pollen-treated group showed enhanced iNOS fluorescence signals, indicating that a large amount of antigenic substances existed in the supernatant of the bacterial solution treated with VAE-Pollen, inducing macrophage polarization toward a pro-inflammatory type.

[0091] Before conducting in vivo experiments, the biocompatibility of the enzyme-mimicking materials (VAE-Pollen and VO2) prepared by the present invention on human umbilical vein endothelial cells (HUVECs) was tested. Figure 13 As shown by live / dead staining, none of the treatment groups caused obvious toxicity to the cells within three days, demonstrating the good biocompatibility of the material.

[0092] Experimental Example 5: Evaluation of in vivo treatment of wounds infected with Staphylococcus aureus

[0093] The present invention establishes a Staphylococcus aureus infected wound model to evaluate the bacterial clearance, wound healing and immune surveillance establishment of the material in skin tissue. Figure 14 a, b summarize the photos of the wound healing process and the wound healing area records. Among them, the wound healing rate of the VAE-Pollen group and the vancomycin group was significantly higher than that of the other groups, and was almost completely healed on the 11th day, while the wound surface of the control group and the hydrogen peroxide group was still exposed and covered with scab. On the first day after modeling, the wound surface liquid was taken and smeared on a flat plate ( Figure 14 c) The VAE-Pollen and vancomycin groups almost eliminated all bacteria, while a large number of residual bacteria remained on the skin surface of the rats in the control and hydrogen peroxide groups, which led to slow wound recovery in both groups on the 11th day. Subsequently, hematoxylin-eosin (H&E) and Masson staining were used to observe the histological condition of the wound on the 11th day after treatment. Figure 14 As shown in Figure d, collagen was significantly reduced in the damaged skin tissue, leading to poor wound healing, impaired tissue remodeling, and increased scar size and epidermal thickness index. On day 11, only a small number of collagen fibers were present in the control and hydrogen peroxide groups, and the collagen fibers were loose and disordered. In contrast, the VAE-Pollen group demonstrated a lower epidermal thickness index and greater collagen deposition. The collagen fibers in the skin tissue were denser, thicker, and better aligned, similar to those in normal skin and the dermis.

[0094] Subsequently, the present invention used interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) staining to detect the relief of VAE-Pollen on oxidative stress and inflammation in the wound surface after 11 days. Figure 15 As shown, the unhealed wound area showed a large amount of interleukin-1β and tumor necrosis factor-α signals; the VAE-Pollen group had almost no inflammatory factors, indicating that VAE-Pollen can effectively promote wound healing after clearing bacteria. In addition, the present invention also evaluated the biosafety of the prepared enzyme-mimicking material (VAE-Pollen) through H&E staining of major organs (heart, liver, spleen, lungs, and kidneys). Figure 16 As shown, no major organs and tissues showed obvious damage or abnormalities, indicating that the enzyme-mimicking material prepared by the present invention has low cytotoxicity. After the wounds of the rats in the four groups were completely healed, the animals were subjected to a second Staphylococcus aureus infection wound model without any treatment to verify whether VAE-Pollen established long-term immune surveillance through immune activation in the body to cope with possible recurrence of infection. Figure 17As shown in a and b, after 11 days, the wound recovery in the VAE-Pollen group was the best, and the bacteria in the skin tissue were cleared by factors and antibodies secreted by the immune cells in the body; on the 3rd day, the spleen was taken for immunofluorescence staining, and the VAE-Pollen group had the most mature dendritic cells and T cells infiltration ( Figure 17 c and d), verifying the immune activation ability of VAE-Pollen. Hematoxylin-eosin (H&E) and Masson staining were used to observe the histological status of the wounds in each group on day 11: the group previously treated with VAE-Pollen had the best recovery effect ( Figure 17 e). Finally, the present invention also proved that the secondary infection did not cause pathological effects on the main organs (heart, liver, spleen, lung, kidney) by H&E staining. Figure 18 ).

[0095] In summary, the above research results confirm that the pollen-based enzyme-mimetic material synthesized in this invention, containing vanadium oxide particles with numerous oxygen vacancy defects, is an ideal antibacterial material for highly efficient and ultrafast ROS catalysis. The introduction of defects results in a local structural displacement of the V atoms by approximately 0.40 Å, thereby optimizing the geometry and increasing the accessibility of the V active site, significantly enhancing its catalytic efficiency and multifunctional enzyme-mimetic activity. The VAE-Pollen enzyme-mimetic material prepared in this invention exhibits excellent POD enzyme-mimetic reaction kinetics and HPO and NADH oxidase-mimetic activities. In vitro and in vivo experiments have demonstrated that, upon bacterial invasion, VAE-Pollen can promote pro-inflammatory polarization of macrophages, forming an inflammatory microenvironment conducive to bactericidal activity and instilling immune memory. This material not only promotes bacterial clearance and rapid healing of wounds after initial bacterial infection, but also establishes long-term immune surveillance. Upon re-invasion of bacteria, immune cells in the body rapidly respond, rapidly clearing bacteria from the wound without additional intervention, promoting wound healing.

[0096] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features can be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the description of the present invention. Furthermore, it is obvious to those skilled in the art that improvements or changes can be made based on the above description, and all such improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A pollen-based multi-active enzyme-mimicking material with immune activation ability, characterized in that: The enzyme-mimicking material includes an amorphous nitrogen-doped carbon substrate derived from natural pollen and vanadium oxide particles supported on the surface of the carbon substrate, wherein the vanadium oxide particles have more oxygen vacancy defects than vanadium oxide alone.

2. The enzyme-mimicking material according to claim 1, characterized in that The natural pollen-derived amorphous nitrogen-doped carbon substrate donates electrons to the vanadium oxide framework, resulting in the formation of VN bonds.

3. The enzyme-mimicking material according to claim 1, characterized in that Due to the introduction of oxygen vacancy defects, the vanadium oxide particles have a V atomic displacement of 0.40 Å in the local structure.

4. The enzyme-mimicking material according to claim 1, characterized in that The enzyme-mimicking material has POD, HPO and NOX enzyme-mimicking activities; The POD enzyme reaction kinetics of the enzyme-mimicking material is as follows: the maximum reaction rate is 2.67 μM s -1 , the number of conversions is 24.75*10 -3 s -1 .

5. A method for preparing a pollen-based multi-activity enzyme-mimicking material with immune activation ability according to any one of claims 1 to 4, characterized in that: The preparation method comprises the steps of using vanadyl oxalate, hydrogen peroxide and natural pollen as raw materials, and preparing the pollen-based multi-active enzyme-mimicking material with immune activation ability through a hydrothermal reaction.

6. The preparation method according to claim 5, characterized in that The preparation method comprises mixing a vanadyl oxalate solution, hydrogen peroxide and natural pollen in an alcohol solvent, reacting the mixture in a reactor at 150-200° C. for 1-6 hours, collecting the reaction product, and washing and drying the reaction product to obtain the pollen-based multi-active enzyme-mimicking material with immune activation ability.

7. The preparation method according to claim 6, characterized in that The volume ratio of vanadyl oxalate solution to hydrogen peroxide is 5:1; The mass ratio of the mixed solution of vanadyl oxalate solution and hydrogen peroxide to natural pollen is 1:5-20; The concentration of the vanadyl oxalate solution is 0.33 mol / L; The mass fraction of the hydrogen peroxide is 30 wt %.

8. The preparation method according to claim 6 or 7, characterized in that The natural pollen is defatted pollen; The alcohol solvent is ethanol.

9. Use of the pollen-based multi-activity enzyme-mimicking material with immune activation ability according to any one of claims 1 to 4 in the preparation of materials for generating reactive oxygen species, antibacterial and / or inducing macrophage polarization.

10. Use of the pollen-based multi-active enzyme-mimicking material with immune activation ability according to any one of claims 1 to 4 in the preparation of a product for treating bacterial infection of wounds and preventing recurrence of infection.

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