Pollen-based multi-activity enzyme-like material with immune activation ability and preparation method and application thereof

Abstract

The application belongs to the technical field of antibacterial materials, and particularly relates to pollen-based multi-activity enzyme-like material with immune activation ability and a preparation method and application thereof. The enzyme-like material comprises a natural pollen-derived amorphous nitrogen-doped carbon base and vanadium oxide particles with a large number of oxygen vacancy defects doped on the surface of the base, and is named as VAE-Pollen. Since the natural pollen-derived amorphous nitrogen-doped carbon base provides electrons to the vanadium oxide framework, the formation energy of oxygen vacancies is reduced, the V atom displacement in the local structure is about 0.40 Å, the geometric structure is optimized, the accessibility of V active sites is improved, the catalytic efficiency and multi-function enzyme-like activity are significantly enhanced. Meanwhile, the prepared VAE-Pollen can remove bacteria through various mechanisms, activate immune cells, and exhibits excellent disinfection capacity for staphylococcus aureus infected wounds, and forms immune memory and immune surveillance capacity in the body to prevent the recurrence of infected wounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibacterial materials, and particularly relates to a pollen-based multi-activity enzyme-like material with immune activation capability and a preparation method and application thereof. BACKGROUND

[0002] The overuse of antibiotics has significantly accelerated the evolution of drug-resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), which poses a serious threat to clinical treatment. The latest data shows that the average mortality rate caused by MRSA infection is about 64% higher than that caused by non-drug-resistant S. aureus infection, highlighting the urgent need for effective medical intervention means without inducing further drug resistance. To meet this challenge, various artificial enzyme-like materials capable of generating reactive oxygen species (ROS) are considered as potential antibiotic alternatives to combat severe MRSA infection, such as nanocarbon materials, photosensitizers, metal-organic frameworks (MOFs), and metal oxides. Among them, vanadium pentoxide (V2O5) as a typical vanadium-based haloperoxidase (V-HPO) biomimetic material can catalyze the generation of hydroxyl radicals (·OH) and hypochlorous acid (HClO) to kill bacteria and has been widely studied.

[0003] However, most current studies mainly focus on its short-term antibacterial effect, often ignoring the inherent potential of the material in regulating inflammation and establishing long-term immune surveillance, which is crucial for preventing recurrence of infection. Therefore, it is particularly urgent to develop an intelligent antibacterial material that combines rapid sterilization with immune activation capability.

[0004] In the human body, natural pollen is easy to stay in the respiratory tract and continuously release allergens due to its highly spiky surface, thereby inducing a strong allergic reaction. After contact, dendritic cells (DCs) and macrophages (Mφs) will phagocytose and process pollen allergens, present them to T cells through the spleen, induce the release of pro-inflammatory cytokines, and trigger allergic inflammation and asthma response. At the same time, B cells will produce specific antibodies that bind to mast cells and basophils, establishing "allergic memory" and triggering a more severe immune response upon re-exposure. Inspired by the pollen sensitization mechanism, we hypothesize that the spiky surface morphology of pollen can be used to efficiently capture bacteria and adhere to biofilms, thereby achieving in situ inactivation of pathogens. Subsequently, Mφs can recognize and process leaked bacterial antigens, polarize to the pro-inflammatory type, and activate T cells, forming an inflammatory microenvironment conducive to the clearance of re-invading bacteria.

[0005] Vanadium metal oxides have inherent drawbacks such as low catalytic activity and biological toxicity caused by large dosage. To enhance their ROS catalytic performance and expand their enzyme-like functions, crystal face regulation, especially oxygen vacancies (O vThe introduction of vanadium oxides is considered an effective strategy. Therefore, developing a new strategy to modulate the coordination structure and bonding microenvironment of catalytic atoms in vanadium metal oxides to obtain highly efficient ROS catalytic activity and mimic the immune activation response induced by pollen allergy is crucial. Currently, research in this area has not yet been explored. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a pollen-based multi-active enzyme-mimicking material with immune activation capabilities, its preparation method, and its applications. The pollen-based multi-active enzyme-mimicking material is a natural pollen-derived amorphous nitrogen-doped carbon substrate with vanadium oxide particles loaded on its surface. The vanadium oxide particles have more oxygen vacancy defects compared to vanadium oxide alone. This pollen-based multi-active enzyme-mimicking material is named VAE-Pollen in this specification. The natural pollen-derived amorphous nitrogen-doped carbon substrate is converted to vanadium oxide (VO₂O₃)... x The framework provides electrons, which helps lower the formation energy of oxygen vacancies, accompanied by a V atom shift of approximately 0.40 Å in the local structure. This optimizes the geometry and improves the accessibility of the V active site, significantly enhancing its catalytic efficiency and multifunctional enzyme-mimicking activity. Specifically, VAE-Pollen exhibits superior enzyme-mimicking performance compared to VO2 and current advanced antimicrobial materials such as peroxidase (POD), haloperoxidase (HPO), and reduced nicotinamide adenine dinucleotide oxidase (NOX). Furthermore, VAE-Pollen demonstrates excellent reaction kinetics of POD-like enzymes: the maximum reaction rate (V... max The value is 2.67 µMs. -1 The number of transformations (TON) is 24.75 * 10. -3 s -1 This indicates that the prepared pollen-based multifunctional vanadium-based enzyme material possesses ultrafast and excellent catalytic activity. Furthermore, systematic biological experiments have shown that VAE-Pollen not only rapidly captures bacteria but also synergistically kills them through multiple mechanisms, including respiratory chain disruption, lipid peroxidation, protein carbonylation, and leakage, achieving a "capture-kill" antibacterial mode. Moreover, when the antigens of the disrupted bacteria are leaked, they can be recognized and processed by macrophages, further promoting the formation of antigen-specific immune memory. When the same bacteria re-invade, this memory mechanism can rapidly respond and initiate dendritic cell maturation, macrophage polarization, T cell infiltration, and B cell differentiation, effectively clearing pathogens and achieving persistent 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 capability, the enzyme-mimicking material comprising 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 compared with vanadium oxide alone.

[0008] Further, the natural pollen derived amorphous nitrogen-doped carbon substrate provides electrons to the vanadium oxide framework, resulting in the formation of V-N bonds.

[0009] Further, 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] Further, the enzyme-mimicking material has POD, HPO and NOX enzyme-mimicking activities.

[0011] Further, the enzyme-mimicking material has a POD-like enzyme reaction kinetics with a maximum reaction rate of 2.67 µM s -1 , a turnover number of 24.75*10 -3 s -1 .

[0012] The second technical problem to be solved by the present application is to provide a preparation method of the pollen-based multi-activity enzyme-mimicking material with immune activation ability as described herein, which comprises using vanadyl oxalate, hydrogen peroxide and natural pollen as raw materials to prepare the pollen-based multi-activity enzyme-mimicking material with immune activation ability by hydrothermal reaction.

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

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

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

[0016] Further, the vanadyl oxalate solution is an aqueous solution of vanadyl oxalate with a concentration of 0.33 mol / L.

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

[0018] Further, the natural pollen is pollen treated by defatting. The method of defatting treatment is well known to those skilled in the art, for example, defatting treatment can be carried out as described in the specific embodiment part of the present application.

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

[0020] Further, the alcohol solvent is ethanol.

[0021] A third technical problem to be solved by the present application is to provide the use of the pollen-based multi-activity enzyme-like material with immune activation ability as described herein in the preparation of a material for producing active oxygen, antibacterial and / or inducing macrophage polarization.

[0022] Further, the bacteria can include but are not limited to drug-resistant Staphylococcus aureus, and the macrophages can include but are not limited to mouse monocyte macrophage leukemia cells.

[0023] A fourth technical problem to be solved by the present application is to provide the use of the pollen-based multi-activity enzyme-like material with immune activation ability as described herein in the preparation of a product for treating bacterial infection of a wound and preventing recurrence of infection.

[0024] Further, the bacterial infection can 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] Advantages of the present application

[0027] The present application develops a pollen-based multi-activity enzyme-like material loaded with vanadium oxide doped with a large number of oxygen vacancy defects, named VAE-Pollen. The catalytic material obtained by the present application has excellent POD-like enzyme activity, better reaction kinetics performance: maximum reaction rate V max is 2.67 µM s -1 , and the conversion number TON is 24.75*10 -3 s -1 The VAE-Pollen obtained by the present application can quickly capture and eliminate bacteria, and the leaked substances can make macrophages respond quickly, polarize to the pro-inflammatory type, form a pro-inflammatory microenvironment, and facilitate bacterial killing.

[0028] In vivo experiments show that VAE-Pollen exhibits excellent bacterial disinfection capacity for bacterial infected wounds, and can activate the immune response in vivo, produce immune memory, quickly start the immune response in vivo when the wound is infected again, achieve long-term immune surveillance, and achieve bacterial elimination and promote wound healing of the wound without the need for additional materials. The present application provides an effective nanodrug for catalytic active oxygen, rapid healing of infected wounds and prevention of recurrence, and provides a new way for the development of non-antibiotic antibacterial strategies. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a synthesis diagram of the VAE-Pollen of the present application and a structural diagram thereof.

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

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

[0032] Figure 4 HAADF-STEM images of: (a) Atomic array of crystalline region of VAE-Pollen; (b) High resolution HAADF-STEM image of defect of VAE-Pollen; (c) Corresponding atomic distribution map obtained from the area highlighted with unarrowed line in the area of (b).

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

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

[0035] Figure 7 Performance statistics of VO2 and VAE-Pollen in catalyzing production of reactive oxygen: (a) is the test chart of POD-like enzyme activity of VO2 and VAE-Pollen; (b) is the reaction rate curve chart with the change of H2O2 concentration and (c) is the maximum reaction rate Vmax of VO2 and VAE-Pollen max and Michaelis constant K mBar graphs of values; (d) Bar graphs of HPO-like enzyme activity of VO2 and VAE-Pollen; (e) Line graphs of NADH oxidase-like enzyme activity of VO2 and VAE-Pollen; (f) Radar plots of various enzyme-like activities and radical generation capacity of VO2 and VAE-Pollen; wherein control group refers to a blank control group without adding any catalytic material (VO2 and VAE-Pollen); hydrogen peroxide group refers to a control group without adding any catalytic material but adding only hydrogen peroxide. In the present application, all the mentioned control groups refer to blank control groups without adding enzyme-like material, and all the mentioned hydrogen peroxide groups refer to control groups without adding any catalytic material but adding only hydrogen peroxide. Other test, cultivation, and the like conditions are the same; n = 3 independent experiments, data are expressed as mean ± SD.

[0036] Figure 8 POD enzyme-like activity statistical result graph of the enzyme-like material obtained in Examples 1-3 and Comparative Examples 1-3.

[0037] Figure 9 Plate coating graph 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 graph of Staphylococcus aureus and Escherichia coli after co-incubation with VO2 and VAE-Pollen, SYTO-9 staining signal represents live bacteria, and PI staining signal represents dead bacteria; scale bar: 15 µm.

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

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

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

[0042] Figure 14Figure for the effect of VAE-Pollen on wound healing in the treatment of S. aureus infection: (a) Representative images of wound area under different treatment means at day 0, day 1, day 3, day 7 and day 11; (b) Time evolution of wound size after different treatments; (c) At day 1 after infection, liquid taken from the wound was plated; (d) Epidermis of different treatment groups was taken for H&E and Masson staining. Figure 14 In the figure, I represents the control group, II represents the hydrogen peroxide group; III represents the vancomycin group; IV represents the VAE-Pollen group.

[0043] Figure 15 Figure for the level of inflammatory factors of epidermis histological sections of different treatment groups at day 11: fluorescence staining images of inflammatory factors IL-1β and TNF-α; scale bar: 80 µm.

[0044] Figure 16 Figure for H&E staining images of visceral tissue sections of rats after different treatments at day 11. Scale bar: 100 µm.

[0045] Figure 17 Figure for the treatment effect of the immune memory established by VAE-Pollen on the recurrence of bacterial infection wounds after the first successful treatment of wound infection: (a) Representative images of wound area under different treatment means at day 20 (day 0 of the second modeling), day 24, day 32; (b) Plate counting chart of liquid taken from the wound under different treatment means and bacterial colonies; (c) Level of dendritic cell activation response of epidermis histological sections of different treatment groups at day 3 after infection; (d) Level of T cell activation response of epidermis histological sections of different treatment groups at day 3 after infection; (e) H&E and Masson staining of epidermis of different treatment groups.

[0046] Figure 18 Figure for H&E staining images of visceral tissue sections of diabetic rabbits after different treatments at day 11. DETAILED DESCRIPTION

[0047] The present application develops 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 the ROS catalytic performance and has multiple enzyme-like activities. Since the amorphous nitrogen-doped carbon matrix derived from natural pollen provides electrons to the vanadium oxide (VO x ) framework, it helps to reduce the formation energy of oxygen vacancies, accompanied by displacement of V atoms in the local structure by about 0.40 Å, thereby optimizing the geometric structure and improving the accessibility of V active sites, significantly enhancing its catalytic efficiency and multiple functional enzyme-like activities, with a maximum reaction rate V max of 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 remove bacteria through multiple mechanisms, activate immune cells, show excellent disinfection ability to Staphylococcus aureus infected wounds, and form immune memory and immune surveillance ability in the body to prevent the recurrence of infected wounds.

[0048] The present application will be further described in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0049] In the examples, comparative examples and test examples of the present application, the reagents used are as follows: vanadyl oxalate is obtained from the laboratory itself, and the specific process is as follows: V2O5 (6.598 mmol / 1.2 g) and H2C2O4 (19.794 mmol / 1.782 g) are dissolved in 40 mL of pure water, and stirred in an oil bath at a temperature of 80°C for three hours. The solution becomes clear blue, and vanadyl oxalate (0.33 mol / L) is obtained. Hydrogen peroxide (H2O2) is obtained from Aladdin. The pure water (18.2MΩ·cm) used in the experiment is from Milli-Q academic system (Millipore Corp., Billerica, MA, USA). All chemicals are used directly without further purification.

[0050] Example 1:

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

[0052] Example 2:

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

[0054] Example 3:

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

[0056] Comparative Examples 1-2

[0057] Watermelon pollen and sunflower pollen were directly purchased from Taobao. After purchase, 10 g of watermelon pollen and sunflower pollen were stirred in 100 mL of diethyl ether for 4 hours. Then, it was suspended in 100 mL of phosphoric acid and heated at 70 °C for 10 hours. Finally, defatting treatment was completed by washing with deionized water, acetone, deionized water, ethanol and deionized water in turn to obtain the final product. The prepared products were named Flat pollen (pollen of watermelon) and pollen (pollen of sunflower), respectively.

[0058] Comparative Example 3

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

[0060] Test Example 1: Structural characterization of VAE-Pollen:

[0061] Scanning electron microscope (SEM) images were obtained using Thermo Fisher Scientific (FEI) Apreo S HiVoc, with a gold coating of about 1 nm. The results, as shown in Figure 2 Fig. 1, respectively show the 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. It can be seen that pollen-based amorphous nitrogen-doped carbon substrates loaded with vanadium oxide on the surface were successfully prepared in Examples 1-3.

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

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

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

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

[0066] Test Example 2: Enzymatic activity evaluation of VAE-Pollen

[0067] (1) POD enzymatic test:

[0068] 1.1 ROS production test:

[0069] The material solution (10 mg / mL, 10 μL) was added into NaOAc-HOAc buffer (100 mM, pH = 4.5), and then 25 μL of TMB solution (10 mg / mL) was added, respectively. The final mixed solution volume was 2 mL. Subsequently, the solution was used for ultraviolet-visible spectrum test at 652 nm wavelength (UV- 2450, Shimadzu, Japan). Figure 7 a).

[0070] 1.2 Kinetic parameter test:

[0071] Michaelis constant (K m ) was calculated according to Michaelis saturation curve. The initial reaction rate (V0) was calculated according to the Beer-Lambert law formula (1) from the absorbance change, where [S] represents the concentration of each H2O2. Michaelis constant (K m ) and maximum reaction rate (V max ) were obtained by double-reciprocal processing of formula (2) to draw Lineweaver-Burk graph. In addition, turnover number (TON) was calculated according to formula (3), where [E0] is the molar concentration of doped metal in nanoszyme. Figure 7 b and c are the reaction rate curve graph and the column chart of maximum reaction rate V max and Michaelis constant K m value of V0 2 and VAE-Pollen accompanying the change of H2O2 concentration, respectively.

[0072] (1)

[0073] (2)

[0074] (3)

[0075] (2) HPO enzymatic test:

[0076] 2.1 Hypochlorous acid production test:

[0077] HPO enzymatic activity was tested by azurite blue. 1930 μL of azurite blue solution (200 μM), 60 μL of enzymatic 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 judged by measuring the absorbance change of azurite blue in the wavelength range of 645 nm to 520 nm (Figure 7 d).

[0078] (3) NADH enzyme-mimicking assay:

[0079] 3.1 NADH oxidation test:

[0080] 10 μL of enzyme-mimicking 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 reacting for 30 minutes, the absorbance change was measured using a UV-Vis 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 the figure, within 10 minutes, VAE-Pollen exhibits higher ROS production activity compared to VO2. Figure 7 a); At the same hydrogen peroxide substrate concentration, VAE-Pollen exhibits a faster reaction rate ( Figure 7 b). The Michaelis constant (K) was then calculated. m ), maximum reaction rate (V) max The values ​​of the transformation number (TON, the maximum number of substrates that can be transformed per active catalytic atom) and the transformation number (TON, the maximum number of substrates that can be transformed per active catalytic atom), such as Figure 7 As shown in c, VAE-Pollen exhibits a larger V compared to VO2. max (2.67 µM s) -1 ) and higher TON (24.75*10 -3 s -1 This indicates that VAE-Pollen exhibits more efficient H2O2 catalytic kinetics. Subsequently, this invention systematically integrates VAE-Pollen with recently reported reactive oxygen species catalytic materials, including V-Fe2O3, V2O5, Ru NPs, etc. max The TON values ​​were compared (Table 1), and the results showed that VAE-Pollen exhibited the best POD enzyme-mimicking activity among these established enzyme-mimicking materials.

[0082] Furthermore, VAE-Pollen exhibits superior HPO enzyme-mimicking performance compared to VO2. Figure 7 d) and NADH oxidase mimicry activity ( Figure 7 e): HPO enzyme can catalyze the reaction of hydrogen peroxide and chloride ions to produce hypochlorous acid, which has disinfecting properties, while NADH oxidase can oxidize NADH to oxidized NAD. + This disrupts the normal respiratory chain metabolism of bacteria, ultimately leading to bacterial death. Figure 7f is a radar graph showing the various enzyme activities and free radical generation capabilities of VO2 and VAE-Pollen, which more intuitively demonstrates the superior performance of VAE-Pollen prepared in this invention compared 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 V max (µM s -1 )]]> TON (10 -3 s -1 )]]> Ref VAE-Pollen 2.67 24.75 The invention V-Fe203 1.07 22.38 Angew. Chem. Int. Ed. 263, e202310811 (2024) [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). CeO2 0.18 3.1 Chem. Soc. Rev. 48, 1004-1076 (2019). CuO 0.28 2.23 Biosens. Bioelectron. 61, 374-378 (2014). Mn203 1.01 7.98 J. Mater. Chem. B 8, 1191-1201 (2020). Fe-N-C 0.62 3.99 ACS Catal. 10, 6422-6429 (2020). Cu-N-C 0.06 3.3 Langmuir 38, 6860-6870 (2022). Co-N-C 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 mimicry characteristics of the enzyme-mimicking materials obtained in Examples 1-3 and Comparative Examples 1-3 of the present invention were further tested, and the results are as follows: Figure 8 As shown, this indicates that VAE-Pollen exhibits the best performance.

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

[0087] Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 25922, Gram-positive) and extended-spectrum β-lactamase-producing Escherichia coli (E. coli ATCC 53104, Gram-negative) were used as representative pathogens to evaluate the bacterial capture and killing ability of VAE-Pollen material. VAE-Pollen 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 60 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 9 As shown, compared to other control groups, the VAE-Pollen treatment group exhibited excellent antibacterial activity against both types of 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 Dextran Fluor-647 could stain the bacterial film formed by airborne bacteria. The staining was then analyzed and observed using a fluorescence microscope. Figure 10 and 11 As shown ( Figure 10 It is a planktonic bacterium. Figure 11(For bacterial biofilm), VAE-Pollen not only caused the death of a large number of bacteria compared to VO2, but also showed fluorescence aggregation, indicating that bacteria gathered around the material, demonstrating the material's excellent bacterial capture ability. These results show that VAE-Pollen can catalyze the generation of large amounts of ROS, causing bacterial oxidative stress and death, and has a general antibacterial effect against both Gram-positive and Gram-negative bacteria.

[0088] Experiment 4: VAE-Pollen promotes the polarization potential of macrophages

[0089] This invention also verifies the potential of VAE-Pollen in promoting the antibacterial and pro-inflammatory polarization of macrophages. During the process of VAE-Pollen catalyzing the production of ROS from hydrogen peroxide, if it can induce macrophage polarization towards a pro-inflammatory state, it can initiate the body's immune response, tending to create an inflammatory microenvironment that is conducive to the clearance of invading bacteria. To verify this hypothesis, this invention first conducted in vitro experiments to induce macrophage polarization.

[0090] RAW 264.7 macrophages (2 × 10⁻⁶) 5 Inoculate each well with 5 × 10⁶ cells / well into a 24-well plate and incubate overnight. Then, add 200 µL of Staphylococcus aureus (MRSA, 5 × 10⁶ cells / well) to each well. 9 The supernatant (CFU / mL) was co-incubated with VAE-Pollen and other control groups for 3 hours and then co-cultured for 24 hours. Immunofluorescence staining was then performed. During macrophage polarization, iNOS and CD206 are considered specific markers for 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, macrophages in the VAE-Pollen treatment group showed enhanced iNOS fluorescence signal, indicating that there were a large amount of antigenic substances in the supernatant of the bacterial culture treated with VAE-Pollen, which induced macrophages to become pro-inflammatory.

[0091] Prior to in vivo experiments, the biocompatibility of the enzyme-mimicking materials (VAE-Pollen and VO2) prepared in this invention with human umbilical vein endothelial cells (HUVECs) was tested. Figure 13 The live / dead staining results showed that none of the treatment groups caused significant toxicity to cells within three days, demonstrating the good biocompatibility of the material.

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

[0093] This invention establishes a Staphylococcus aureus infection wound model to evaluate the bacterial clearance, wound healing, and establishment of immune surveillance in skin tissue using materials. Figure 14 a and b summarize the photographs of the wound healing process and the recorded wound healing area. The VAE-Pollen group and the vancomycin group showed significantly higher wound healing rates than other groups, achieving almost complete healing by day 11. In contrast, the wounds in the control group and the hydrogen peroxide group remained exposed and covered by scabs. On the first day after modeling, wound surface fluid was collected and applied to a plate. Figure 14 c) The VAE-Pollen and vancomycin groups almost completely eliminated bacteria, while the control and hydrogen peroxide groups still had a large number of residual bacteria on their skin surfaces, resulting in slower wound healing in both groups on day 11. Subsequently, the histological condition of the wounds on day 11 after treatment was observed using hematoxylin-eosin (H&E) and Masson staining. 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 amount of collagen fibers were observed in the control group and the hydrogen peroxide group, and these fibers were loose and disordered. In contrast, the VAE-Pollen group exhibited a lower epidermal thickness index and more collagen deposition, with denser, thicker, and better-arranged collagen fibers in the skin tissue, similar to normal skin and the dermis.

[0094] Subsequently, this invention used interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) staining to detect the effect of VAE-Pollen on the reduction of oxidative stress and inflammation in the wound after 11 days. Figure 15 As shown, the unhealed wound area exhibited abundant interleukin-1β and tumor necrosis factor-α signals; the VAE-Pollen group showed almost no inflammatory factors, indicating that VAE-Pollen can effectively promote wound healing after bacterial clearance. Furthermore, the biocompatibility of the prepared enzyme-mimicking material (VAE-Pollen) was evaluated by H&E staining of major organs (heart, liver, spleen, lung, and kidney). Figure 16 As shown, no significant damage or abnormalities were observed in the major organs and tissues, indicating that the enzyme-mimicking material prepared in this invention has low cytotoxicity. After the wounds of rats in all four groups had completely healed, a second model of Staphylococcus aureus infection was established in the animals without any treatment to verify whether VAE-Pollen establishes long-term immune surveillance through immune activation in the body to cope with possible infection recurrence. Figure 17a and b, after 11 days, the wound recovery of the VAE-Pollen group was the best, and the bacteria in the skin tissue were cleared by the factors and antibodies secreted by the immune cells in the body; the spleen was taken for immunofluorescence staining at 3 days, and the mature dendritic cells and T cells of the VAE-Pollen group were the most infiltrated Figure 17 c and d, the immune activation ability of VAE-Pollen was verified. Hematoxylin-eosin (H&E) and Masson staining were used to observe the histological conditions of the wounds of each group at 11 days: the group treated with VAE-Pollen had the best recovery effect Figure 17 e). Finally, the present application also proved that the main organs (heart, liver, spleen, lung, kidney) were not affected by the secondary infection by H&E staining of the main organs Figure 18 .

[0095] In summary, the pollen-based enzyme-like material of vanadium oxide particles with a large number of oxygen vacancy defects synthesized by the present application is an ideal efficient superfast antibacterial material for catalyzing ROS. The introduction of defects causes the displacement of V atoms in the local structure by about 0.40 Å, thereby optimizing the geometric structure and improving the accessibility of V active sites, significantly enhancing its catalytic efficiency and multiple functional enzyme-like activity. The VAE-Pollen enzyme-like material prepared by the present application has excellent POD enzyme-like reaction kinetics, HPO and NADH oxidase enzyme-like activity. And through in vitro and in vivo experiments, it is confirmed that when bacteria invade, VAE-Pollen can promote the pro-inflammatory polarization of macrophages, forming an inflammatory microenvironment conducive to killing bacteria, and forming immune memory. Not only can it promote the bacterial clearance of the wound surface of the primary bacterial infection and rapid healing, but also establish long-term immune surveillance, and when bacteria invade again, the immune cells in the body respond quickly, and the bacteria in the wound can be quickly cleared without additional intervention, promoting wound healing.

[0096] It should be noted that the present application is described in the specification and drawings of the present application, but the present application can be implemented in many different forms, and is not limited to the embodiments described in the specification. These embodiments are not additional limitations on the content of the present application, and the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Furthermore, each of the above technical features continues to be combined, forming various embodiments not listed above, which are considered to be within the scope of the present application. Furthermore, for those skilled in the art, the above description can be improved or changed, and all these improvements and changes should be within the scope of the appended claims of the present application.

Claims

1. A pollen-based multi-active enzyme-mimicking material with immune-activating capabilities, characterized in that, The enzyme-mimicking material comprises 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 compared to vanadium oxide alone; and the enzyme-mimicking material is prepared by hydrothermal reaction using vanadium oxalate, hydrogen peroxide and natural pollen as raw materials.

2. The enzyme-mimicking material according to claim 1, characterized in that, The amorphous nitrogen-doped carbon substrate derived from natural pollen donates electrons to the vanadium oxide framework, enabling 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 exhibit a V atom shift of 0.40 Å in the local structure.

4. The enzyme-mimicking material according to claim 1, characterized in that, The enzyme-mimicry material exhibits POD, HPO, and NOX enzyme-mimicry activities; The POD-like enzyme reaction kinetics of the described enzyme-mimicking material are as follows: the maximum reaction rate is 2.67 µM s. -1 The number of transformations is 24.75 × 10. -3 s -1 .

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

6. The preparation method according to claim 5, characterized in that, The preparation method includes mixing vanadium oxalate solution, hydrogen peroxide and natural pollen in an alcohol solvent, reacting in a reaction vessel at 150-200℃ 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.

7. The preparation method according to claim 6, characterized in that, The volume ratio of vanadium oxalate solution to hydrogen peroxide is 5:1; The mass ratio of the mixed solution of vanadium oxalate and hydrogen peroxide to natural pollen is 1:5-20. The concentration of the vanadium 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 pollen that has undergone defatting treatment; The alcohol solvent is ethanol.

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

10. Use of the pollen-based multi-active enzyme mimicry material with immune-activating capabilities as described in any one of claims 1-4 in the preparation of products for treating bacterial wound infections and preventing recurrence of infection.

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