Multi-response nano-enzyme for bacterial pneumonia as well as preparation method and application of multi-response nano-enzyme

Abstract

The invention discloses an integrated nano-enzyme with multiple response mechanism synergism, and a preparation method and application thereof. A Z-type heterojunction based on boron nanosheets (B NSs) and rare earth doped iron phosphide (Nd < 0.02 > Fe < 1.98 > P) is constructed for the first time, and CTAB is used for surface modification to obtain a ternary composite nano-enzyme (BFC); according to the present invention, the material is endowed with the multi-response ability (POD / GSHOx enzyme activity synergistic activation) on the bacterial infection microenvironment (low pH and high H2O2), and the nano-enzyme has excellent acoustic-magnetic responsiveness, can break through the depth limitation, and can achieve the precise and controllable generation of the reactive oxygen species (ROS). Under the acidic microenvironment of pulmonary infection, the activity of peroxidase (POD) and glutathione oxidase (GSHOx) is activated, so that efficient and controllable synergistic killing is performed on bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical nanomaterials, and in particular to a multi-responsive nanozyme for bacterial pneumonia, its preparation method and application Background Art

[0002] Bacterial pneumonia is one of the main causes of global human death and poses a serious threat to public health. The traditional antibiotic treatment mode used in the past is facing an increasingly severe problem of drug resistance. Due to the adjustable morphology and size, excellent physical and chemical properties, and multi-functional characteristics of nanomaterials, they show great potential in the development of new antibacterial strategies. At present, more and more studies use nanomaterials to combat drug-resistant bacterial infections through photothermal therapy (PTT), photodynamic therapy (PDT), chemodynamic therapy (CDT), sonodynamic therapy (SDT), and magnetic hyperthermia (MHT), etc.

[0003] Although these emerging antibiotic alternative strategies have shown certain advantages, the single treatment mode still has unavoidable defects. For example, the laser penetration depth used in PTT and PDT is limited, SDT can act on deep tissues but the efficiency of reactive oxygen species (ROS) generation is low, and MHT is not affected by tissue depth but the heating effect is poor. These defects greatly reduce their antibacterial efficiency.

[0004] Nanozymes are a series of nanomaterials with mimetic enzyme activities and can perform catalytic reactions of natural enzymes. At present, many nanomaterials have been demonstrated to have mimetic activities such as oxidase (OXD), catalase (CAT), peroxidase (POD), glucose oxidase (GOD), superoxide dismutase (SOD), and glutathione oxidase (GSHOx), etc., and have high biomedical application values.

[0005] Iron-based nanomaterials not only exhibit excellent magnetic hyperthermia effects under AMF, but also have superior POD characteristics as nanozymes and can catalyze H2O2 to generate ·OH radicals in the bacterial infection area with low pH value and high H2O2 level. Ferrous and ferric ions often coexist in iron-based materials. This variable-valence metal has the potential of GSHOx enzyme activity. In the biofilm state at the bacterial infection site, the complex matrix can serve as a physical and metabolic barrier, restricting the supply of nutrients and oxygen and undergoing anaerobic glycolysis, causing the accumulation of bacterial metabolites, thus generating a biofilm microenvironment with hypoxia, weak acidity (pH 4.5 - 6.5), high levels of H2O2, and overexpressed enzymes (esterase, lipase, gelatinase, etc.). Such an infection microenvironment is important for the POD and GSHOx enzyme activities of iron-based nanozymes to play their roles.

[0006] In the prior art, the development of nanozymes mainly utilizes a single treatment mode. Patent CN119700644A discloses an antibacterial material based on nanozyme-enhanced sonodynamic therapy, which includes BiPt nanozymes. The outer side of the BiPt nanozymes is coated with bovine serum albumin (BSA), and protoporphyrin (PpIX) is connected to the BSA to form nanoparticles. The outer side of the nanoparticles is coated with ascorbyl palmitate (AP) hydrogel to obtain the antibacterial material. Currently, there is still a lack of a nanozyme that combines multiple treatment modes to achieve efficient and controllable antibacterial effects. Summary of the Invention

[0007] To solve the above technical problems, the present invention proposes an integrated nanozyme with a synergistic multi-response mechanism, a preparation method, and an application. This nanozyme has excellent acoustic and magnetic responsiveness, can break through the depth limitation, and achieve precise and controllable generation of reactive oxygen species (ROS). In the acidic microenvironment of lung infection, its peroxidase (POD) and glutathione oxidase (GSHOx) activities are activated, thereby achieving efficient and controllable synergistic killing of bacteria.

[0008] To achieve this purpose, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a preparation method of a multi-response nanozyme for bacterial pneumonia.

[0010] Step (1), preparation of boron nanosheets (B NSs): Mix high-purity boron powder with a solvent to form a mixed solution, and prepare boron nanosheets by ultrasonic exfoliation method. After centrifugal separation and freeze-drying, it is reserved for later use.

[0011] Step (2), preparation of Nd 0.02 Fe 1.98 P nanoparticles: Synthesize rare-earth-doped FeP nanoparticles by a solvothermal method with staged heating under inert gas protection, and obtain the target product through centrifugation, washing, and drying.

[0012] Step (3), preparation of B NSs@Nd 0.02 Fe 1.98 P@CTAB (BFC): In situ load neodymium-doped iron phosphide (Nd 0.02 Fe 1.98 P) on the surface of boron nanosheets (B NSs) by a solvothermal method under inert gas protection, and obtain the BFC nanozyme through CTAB surface modification, centrifugal purification, and freeze-drying.

[0013] Preferably, the solvent described in step (1) is N-methylpyrrolidone.

[0014] Preferably, the specific steps of the ultrasonic exfoliation method in step (1) are as follows: Using a cell disruptor to perform ultrasonic exfoliation at room temperature for 4 - 8 h, with a power of 1000 - 1500 W, an ultrasonic duration of 1 - 3 s, and an interval time of 1 - 3 s. Subsequently, transfer it to an ultrasonic cleaner and perform ultrasonic treatment at 3 - 5 °C for 18 - 32 h.

[0015] Preferably, the conditions for centrifugal separation in step (1) are 10000 - 12000 r / min and a centrifugation time of 10 - 40 minutes.

[0016] Preferably, the rare earth element in step (2) is at least one of Nd, Ce, and Lu; the doping ratio is 0.5 - 4%.

[0017] Preferably, the inert gas in step (2) is argon; for the staged heating, the temperature is 240 - 280 °C and maintained for 0.5 - 2 hours, then 300 - 350 °C and maintained for 1 - 3 h.

[0018] The second object of the present invention is to provide a multi-responsive nanozyme for bacterial pneumonia, which is prepared by the above preparation method.

[0019] The third object of the present invention is to provide an application of the above multi-responsive nanozyme in the preparation of drugs for treating bacterial pneumonia.

[0020] Beneficial effects

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a sound-magnetic integrated nanozyme responsive to the bacterial infection microenvironment. Through the nebulization administration method, while avoiding the systemic toxicity of the drug, it enhances its accumulation at the lesion site to achieve effective treatment of bacterial pneumonia. This sound-magnetic integrated nanozyme can generate heat under the action of an alternating magnetic field, affect bacterial growth, promote SDT to produce ROS, and at the same time enhance the dual enzyme activities of POD and GSHOx in the bacterial infection microenvironment with low pH value and high H2O2 level, promote oxidative stress, and enhance the antibacterial effect. Through the triple synergistic effect of MHT, SDT, and nanozyme activity, the production amount of ROS is maximized to achieve a better treatment effect. In addition, the nanozyme can also reduce the inflammatory response by inducing mitochondrial autophagy. The present invention provides a feasible solution for clinically curing bacterial pneumonia and has certain enlightenment significance and application value.

[0023] Multiresponsive nanozyme design: For the first time, a ternary composite nanozyme (BFC) based on a boron nanosheet (B NSs) substrate, a rare earth-doped iron phosphide (Nd-Fe-P) functional layer, and CTAB surface modification was constructed, endowing the material with multiresponsive ability (synergistic activation of POD / GSHOx enzyme activities) to the bacterial infection microenvironment (low pH, high H2O2).

[0024] Acoustic-magnetic integrated synergistic therapy: Innovatively integrating magnetic hyperthermia therapy (MHT), sonodynamic therapy (SDT), and nanozyme-catalyzed therapy, heat is generated by an alternating magnetic field (MHT) to inhibit bacterial growth, ultrasound activates a photosensitizer (SDT) to produce ROS, and nanozyme catalysis enhances oxidative stress, achieving synergistic enhancement of triple antibacterial mechanisms.

[0025] Microenvironment regulation and inflammation alleviation: The nanozyme regulates the host immune response by inducing mitophagy, reducing excessive inflammatory damage, and breaking through the limitation of "bactericidal-inflammation imbalance" in traditional antibacterial therapy.

[0026] Targeted delivery and safety optimization: Using the aerosol inhalation administration method, taking advantage of the enhanced permeability and retention effect (EPR) in the lungs to enhance lesion accumulation and avoid systemic toxicity; CTAB modification improves the dispersion stability and targeting of the material in biological media. Description of the Drawings

[0027] Figure 1 Schematic diagram of the preparation process and reaction mechanism of the present invention

[0028] Figure 2 Structural characterization of BFC prepared in Example 1: Figure 2 Figure A is the SEM image of BFC; Figure 2 Figure B is the XRD pattern of BFC; Figure 2 Figure C is the FTIR spectrum of BFC; Figure 2 Figure D is the Zetapotential diagram of BFC; Figure 2 Figure E is the schematic diagram of the energy band structure of BFC.

[0029] Figure 3 Magnetic hyperthermia heating curve and real-time infrared thermogram of BFC prepared in Example 1;

[0030] Figure 4 Sonodynamic performance characterization of BFC prepared in Example 1: Figure 4 Figure A is the DPBF degradation comparison diagram of BFC; Figure 4 Figure B is the ESR spectrum of BFC generating 1 O2 under US;

[0031] Figure 5 Enzyme activity performance characterization of BFC prepared in Example 1:Figure 5 Figure A of [ID] is the TMB colorimetric spectrum; Figure 5 Figure B of [ID] is the DTNB spectrum of BFC consuming GSH under different pH conditions; Figure 5 Figure C of [ID] is the Michaelis-Menten kinetic analysis of the POD enzyme activity of BFC; Figure 5 Figure D of [ID] is the Lineweaver-Burk fitting diagram of the POD enzyme activity of BFC.

[0032] Figure 6 Evaluation of the in vitro antibacterial performance of BFC prepared in Example 1: Figure 6 Figure A of [ID] is the colony photo and antibacterial rate quantification diagram of BFC by plate coating; Figure 6 Figure B of [ID] is the 3D biofilm microscopy imaging and statistical quantification diagram of BFC.

[0033] Figure 7 CLSM images of mitochondrial autophagy of RAW267.4 cells of BFC prepared in Example 1.

[0034] Figure 8 Therapeutic effect of BFC prepared in Example 1 on pneumonia in vivo: Figure 8 Figure A of [ID] is the biocompatibility diagram of BFC; Figure 8 Figure A of [ID] is the hemolysis rate diagram of BFC; Figure 8 Figure C of [ID] is the in vivo fluorescence imaging situation monitored over time after pulmonary physical and chemical administration of BFC-ICG; Figure 8 Figure D of [ID] is the photo of the pulmonary homogenate plate coating and the H&E stained section of the lung on the third day of BFC treatment; Figure 8 Figure E of [ID] is the immunofluorescence staining diagram of pulmonary macrophages on the third day of BFC treatment. Detailed implementation method

[0035] Next, the technical solutions in the present invention will be clearly and completely described in combination with the embodiments in the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0036] Example 1

[0037] A preparation method of a multi-responsive nanozyme for bacterial pneumonia,

[0038] The specific preparation steps are as follows:

[0039] 1. Preparation of boron nanosheets (B NSs):

[0040] Disperse 200 mg of high-purity boron powder in 200 mL of N-methylpyrrolidone. Ultrasonically exfoliate the above mixture at room temperature for 6 h using a cell disruptor, with the parameters set as follows: power 1200 W, ultrasonic duration 2 s, and interval time 2 s. Subsequently, transfer it to an ultrasonic cleaner and ultrasonicate at 4 °C for 24 h. After the ultrasonic treatment, centrifuge the liquid at 3000 r / min for 5 min and take the supernatant; centrifuge the supernatant at 11000 r / min for 30 min, discard the supernatant, and retain the precipitate; add ultrapure water to the obtained product, centrifuge at 11000 r / min for 30 min, and repeat twice; put the obtained product into a -20 °C refrigerator for pre-freezing for 3 h, and then put it into a freeze dryer for freeze-drying for 12 h to obtain the B NSs product.

[0041] 2. Nd 0.02 Fe 1.98 Preparation of PNPs:

[0042] Add 349.6 mg of iron(III) acetylacetonate Fe(acac)3 and 4.4 mg of neodymium(III) acetylacetonate Nd(acac)3 to 50 mL of oleylamine OM. Gradually heat to 260 °C under an argon atmosphere and maintain for 1 h; quickly add 3.3 mL of trioctylphosphine TOP and heat to 320 °C for 2 h. After the reaction, wait until it cools to room temperature, centrifuge at 12000 rpm for 30 min, and discard the supernatant; wash the product three times with ethanol and cyclohexane, and finally dry it in a vacuum oven at 60 °C to obtain Nd 0.02 Fe 1.98 P product.

[0043] 3. B NSs@Nd 0.02 Fe 1.98 Preparation of P@CTAB (BFC):

[0044] Put the ultrasonically exfoliated B NSs into 50 mL of OM and stir at room temperature for 2 h. Then add 349.6 mg of iron(III) acetylacetonate Fe(acac)3 and 4.4 mg of neodymium(III) acetylacetonate Nd(acac)3 to 50 mL of oleylamine OM. Gradually heat to 260 °C under an argon atmosphere and maintain for 1 h; quickly add 3.3 mL of trioctylphosphine TOP and heat to 320 °C for 2 h. After the reaction, wait until it cools to room temperature, centrifuge at 12000 rpm for 30 min, and discard the supernatant; disperse the product in cyclohexane, and then slowly add it dropwise to the ethanol solution of CTAB under ultrasonic action, ultrasonicate for 10 min, place it in a shaker overnight, centrifuge at 12000 rpm and discard the supernatant, wash it three times with water, and obtain the final product B NSs@Nd 0.02 Fe 1.98 P@CTAB (BFC) after freeze-drying.

[0045] The structure and performance of Example 1 will be analyzed and described below for the present invention.

[0046] 1. Structure characterization of BFC:

[0047] The SEM image of the BFC prepared in Example 1 is as shown in Figure 2 Figure A. Morphological observation shows that the BFC is a thin sheet covered with spherical particles on the surface. Figure 2 The XRD crystal structure shown in Figure B indicates that the characteristic peaks match the crystal structure of the previously synthesized single-component nanoparticles, confirming the successful synthesis of BFC. Figure 2 Figure C shows that, compared with individual components, the composite material has the characteristic peaks of all single components, indicating the successful synthesis of BFC. Figure 2 The Zeta potential in Figure D shows that the BF potential is negative before CTAB modification, and positively charged BFC is obtained after modification. The schematic diagram of the energy band structure of BFC is as shown in Figure 2 Figure E, and the two may form a Z-scheme heterojunction, suggesting that O2 can be converted into 1 O2 under ultrasonic action.

[0048] 2. Magnetothermal performance of BFC:

[0049] BFC was dispersed in ultrapure water to prepare a series of BFC dispersions with different concentrations, and by applying an external alternating magnetic field (AMF), an infrared thermal imager was used to record its temperature rise. The results are as shown in Figure 3 Figure. Under an AMF of 1.6 mT and 498 kHz, the aqueous solution without BFC has a temperature rise of no more than 5 °C within 10 min, while the 4 mg / mL BFC dispersion rises from room temperature to 54 °C.

[0050] 3. Sonodynamic performance of BFC:

[0051] The ROS generation ability of BFC was detected using a ROS probe. Nd 1 Fe 0.02 P, B NSs, and BFC solutions containing DPBF (a molecular probe commonly used to detect the presence of 1.98 O2) were respectively exposed to ultrasonic irradiation (1.0 MHz, 1.0 W cm -2 and 50% duty cycle). The results are as shown in Figure 4 Figure A, showing that the absorption value of DPBF at 414 nm gradually decreases with the increase of ultrasonic time, indicating that Nd 0.02 Fe 1.98 P, B NSs, and BFC can all generate 1O2. Among them, the SDT performance of B NSs may be attributed to the excellent ultrasonic response ability of generally two-dimensional structured sonosensitizers. The heterostructure of BFC inhibits the recombination after electron-hole separation under ultrasound, 1 resulting in a higher generation efficiency of O2, thus enhancing the SDT performance. In addition, due to the magnetothermal effect of BFC, when an AMF is applied to raise the temperature to 42 °C, since thermal energy increases the entropy of chemical reactions and accelerates the electron transfer rate, the effective separation of electron-hole pairs is promoted, and the SDT performance is further enhanced.

[0052] In addition, using TEMP as 1 the scavenger of O2, the ability of each component to generate 1 O2 under US was further verified by electron spin resonance (ESR). The results are shown in Figure 4 Figure B, showing that the BFC + US 42 °C group has a more distinct 1:1:1 1 O2 characteristic peak, that is, the ability to generate 1 O2 is stronger, which is consistent with the test results of the DPBF probe.

[0053] 4. Enzymatic activity performance of BFC:

[0054] The POD activity was determined by the TMB color reaction. 100 μL of the BFC aqueous solution with a concentration of 200 μg / mL was added to 840 μL of acetic acid-sodium acetate buffer solutions with different pH values (5.4, 6.5, 7.4) respectively. Then, 10 μL of the DMSO solution containing TMB (20 mg / mL) was added, and finally, 50 μL of the hydrogen peroxide aqueous solution with a concentration of 5 mM was added. After mixing evenly, the absorbance value of the mixed solution at λ = 652 nm at different time intervals was recorded by a full-wavelength microplate luminescence detector.

[0055] According to Figure 5 the test results shown in Figures A and B, BFC has a more excellent ·OH generation ability under acidic conditions and can oxidize TMB, indicating better POD enzymatic activity. By setting a series of hydrogen peroxide aqueous solutions with different concentrations, the POD enzyme kinetics of BFC and BFC under magnetothermal conditions were evaluated and fitted, and the corresponding Michaelis constant Km and maximum reaction rate Vmax were calculated. The Km of BFC is 3.38 mM, and the Vmax is 159 nMs -1 , while under the condition of applying AMF, the Km of BFC (42 °C) decreases to 1.28 mM, and the Vmax increases to 274 nMs -1 , indicating that magnetothermal treatment improves the affinity of BFC for the substrate H2O2, accelerates the chemical reaction rate, increases the maximum reaction rate, and the POD enzyme activity can be enhanced under magnetothermal conditions.

[0056] To prove that the BFC prepared above also has GSH-Ox activity, the content of GSH was detected using DTNB to reflect the consumption of GSH by BFC. According to Figure 5 As shown in Figure C of

[0057] 5. Antibacterial performance of BFC in vitro:

[0058] A Gram-positive bacterium: methicillin-resistant Staphylococcus aureus (MRSA) and a Gram-negative bacterium: multidrug-resistant Klebsiella pneumoniae (MDR-Kp) were respectively selected, and the plate coating method was used to visually show the inactivating ability of BFC against these two bacteria. The results are as shown in Figure 6 Figure A of

[0059] The biofilm models were constructed using MRSA and MDR-Kp to evaluate the in vitro antibiofilm activity of BFC NPs. After applying different stimuli, the constructed biofilms were stained with SYTO9 / PI and three-dimensional photos were taken using a confocal microscope to observe the survival of bacteria in each biofilm. According to Figure 6 the 3D biofilm staining in Figure B of

[0060] 6. Anti-inflammatory effect of BFC at the cellular level:

[0061] To study the anti-inflammatory situation of BFC, the macrophage RAW267.4 was stained with the mitochondrial marker Mito-Tracker and the lysosomal marker Lyso-Tracker to reflect whether mitochondria and lysosomes were co-localized, so as to reflect whether mitophagy occurred. The occurrence of mitophagy can inhibit the release of inflammatory factors, thereby playing an anti-inflammatory role. The results are as shown in Figure 7 As shown, at a comparable level of mitochondrial expression, the expression of lysosomes in the group with the addition of BFC was significantly increased, indicating an enhanced occurrence of mitophagy, suggesting an enhanced anti-inflammatory effect.

[0062] 7. Therapeutic effect of BFC on pneumonia in vivo:

[0063] Mouse embryonic fibroblasts (L929) were first used to verify the biocompatibility of BFC. The cell compatibility of different concentrations of BFC was quantitatively tested by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, i.e., thiazolyl blue (MTT). As Figure 8 shown in Figure A of

[0064] , as the concentration of BFC gradually increased, the cell viability values of L929 cells were close to those of the control group. When the concentration reached 400 μg / mL, the cell survival rate was still over 80%, proving its good cell safety. Figure 8 In addition to considering the cell compatibility of BFC, its hemolytic ability on red blood cells was also detected. The results are shown in Figure B of

[0065] . On the premise of using PBS and deionized water as negative control and positive control respectively, the hemolysis rate of BFC gradually increased with the increase of concentration. When the material concentration reached as high as 4 mg / mL, the hemolysis rate was still not more than 1%, far lower than the safety value of 5%. Therefore, it was proved that the BFC nanozyme had excellent blood safety. Figure 8 After coupling ICG dye with BFC, it could be used to monitor the in vivo metabolism of BFC. The fluorescence intensity at different times after pulmonary physical and chemical administration was analyzed by a small animal in vivo imaging instrument. The results are shown in Figure C of

[0066] . At 0 h after administration, it mainly accumulated in the lungs and trachea. It had good pulmonary retention within 48 h, and the fluorescence signal completely disappeared from the lungs at 56 h. The above results showed that the synthesized BFC nanozyme showed good pulmonary retention ability within 48 h after administration and could exert its therapeutic effect. Figure 8 The typical hospital and community infection pathogen MDR-Kp was used to establish a BALB / C pneumonia mouse model to evaluate the in vivo anti-infection ability of the BFC nanozyme. The establishment and treatment monitoring process of the pneumonia mouse model was carried out according to this process: on day 0, the suspension of MDR-Kp was evenly sprayed on the lungs of mice by aerosol inhalation. One day later, the mice showed coughing and increased respiratory mucus, indicating the successful establishment of the infection model (day 1). Then, different groups were stimulated for treatment, and the treatment process lasted for 3 days. After different treatments, lung tissue homogenates were collected on day 3 after treatment and plated. According to Figure 8 the results in Figure D of Figure 8 , the bacterial load in the lungs of the BFC+AMF+US group was significantly reduced, and the antibacterial rate reached 100%. H&E staining was used to analyze the pathological changes of lung tissue sections of infected mice. A large number of neutrophil infiltrations and obvious pulmonary fibrosis were observed in the Control group; with the application of treatment conditions, the number of neutrophils gradually decreased, and the degree of pulmonary fibrosis in the BFC+AMF+US group was significantly reduced and was comparable to that of the healthy group, indicating its excellent therapeutic effect.

[0067] The lungs of mice after different treatments were stained using immunofluorescence staining to observe the changes in inflammation-related macrophages. Figure 8 Panel E of Figure 8 showed strong green fluorescence (CD86 positive, M1 macrophages) and weak yellow fluorescence (CD206 positive, M2 macrophages) in the lung tissues of the Control group, indicating that there was still severe inflammation in the lungs. As the treatment conditions were continuously applied, the green fluorescence gradually weakened and the yellow fluorescence gradually increased. Strong yellow fluorescence was observed in the lung tissues treated with BFC+AMF+US, comparable to that of the healthy group, indicating that BFC effectively reversed the inflammatory environment in the lungs and exerted excellent anti-inflammatory regulation.

[0068] As can be seen from the above examples and performance analysis, the multifunctional responsive nanozyme prepared by the present invention, through aerosol drug delivery, avoids systemic drug toxicity while enhancing its accumulation at the lesion site to achieve effective treatment of bacterial pneumonia. The acoustic-magnetic integrated nanozyme can generate heat under the action of an alternating magnetic field, affect bacterial growth, promote SDT to produce ROS, and simultaneously enhance the dual enzyme activities of POD and GSHOx in the bacterial infection microenvironment with low pH value and high H2O2 level, promoting oxidative stress and enhancing the antibacterial effect. Through the triple synergistic effects of MHT, SDT, and nanozyme activity, the production of ROS is maximized to achieve better therapeutic effects. In addition, the nanozyme can also reduce the inflammatory response by inducing mitophagy, providing a feasible solution for the clinical cure of bacterial pneumonia and having certain enlightenment significance and application value.

[0069] In the description of this specification, the description with reference to terms such as "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0070] The above is only the best implementation mode of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several modifications or equivalent replacements can be made to the technical solution of the present invention, and the same technical effects can also be achieved, which should also be regarded as belonging to the protection scope of the present invention.

Claims

1. A preparation method of a multi-responsive nanozyme for bacterial pneumonia, characterized in that, It includes the following steps: (1) Preparation of boron nanosheets (B NSs): Mix high-purity boron powder with a solvent to form a mixed solution. Prepare boron nanosheets by ultrasonic exfoliation method, followed by centrifugal separation and freeze-drying for later use. (2) Nd 0.02 Fe 1.98 Preparation of P nanoparticles: Rare earth-doped Fe2P nanoparticles were synthesized by a solvothermal method with staged heating under inert gas protection, and the target product was obtained by centrifugation, washing, and drying. (3)B NSs@Nd 0.02 Fe 1.98 Preparation of P@CTAB (BFC) nanozyme: Under the protection of inert gas by solvothermal method, neodymium-doped iron phosphide (Nd 0.02 Fe 1.98 P) was in-situ loaded on the surface of boron nanosheets (B NSs), and then surface-modified with CTAB, centrifugally purified and freeze-dried to obtain BFC nanozyme.

2. The preparation method according to claim 1, wherein: The solvent described in step (1) is N-methylpyrrolidone.

3. The preparation method according to claim 1, wherein: The specific steps of the ultrasonic exfoliation method described in step (1) are as follows: Use a cell disruptor to ultrasonically exfoliate for 4 - 8 h at room temperature, with a power of 1000 - 1500 W, an ultrasonic duration of 1 - 3 s, and an interval time of 1 - 3 s. Then transfer it to an ultrasonic cleaner and ultrasonically exfoliate at 3 - 5 °C for 18 - 32 h.

4. The preparation method according to claim 1, characterized in that: The conditions for the centrifugal separation in step (1) are 10000 - 12000 r / min and a centrifugation time of 10 - 40 minutes.

5. The preparation method according to claim 1, characterized in that: The rare earth element in step (2) is at least one of Nd, Ce, and Lu; the doping ratio is 0.5 - 4%.

6. The preparation method according to claim 1, characterized in that: The inert gas in step (2) is argon; for the staged heating, the temperature is 240 - 280 °C and maintained for 0.5 - 2 hours, then 300 - 350 °C and maintained for 1 - 3 h.

7. A nanozyme prepared by the method for preparing a multi-responsive nanozyme for bacterial pneumonia according to any one of claims 1 - 6.

8. Use of the multi-responsive nanozyme according to claim 7 in the preparation of a drug for treating bacterial pneumonia.

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