An acid-sensitive bacteriostatic material, a preparation method and application thereof

Abstract

The application discloses an acid-sensitive antibacterial material and a preparation method and application thereof, and belongs to the field of medical materials. The material is a core-shell structure material in which hollow mesoporous MnO2 nanospheres are wrapped by ZIF-8 and modified by an antibacterial active ingredient AP. In the preparation, the AP is first grafted on the surface of the hollow mesoporous MnO2 nanospheres, and then wrapped by the acid-sensitive ZIF-8. The acid-sensitive antibacterial material organically combines the cell membrane penetration of the AP antibacterial peptide and the chemical dynamic therapy of the CDT to synergistically inhibit bacteria, and combines the high oxidative stress state of cells caused by exogenous manganese oxide nanoparticles with the AP to effectively cope with multi-drug resistant bacterial infections. Meanwhile, the acid-sensitive effect of the ZIF-8 enables the drug to be effectively enriched at the site of bacterial infection, thereby reducing the drug dosage and lowering the drug toxicity in the body. Results show that the material has a significant inhibitory effect on Escherichia coli and Staphylococcus aureus, has good stability and biocompatibility, and can effectively promote wound healing.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical materials technology, specifically relating to an acid-sensitive antibacterial material, its preparation method, and its application. Background Technology

[0002] Slow wound healing due to bacterial infections remains a significant and pressing problem, particularly the issue of bacterial resistance following antibiotic treatment. Currently, antibiotics still play an indispensable role in the field of antibacterial therapy. However, due to the overuse of antibiotics, bacteria develop biofilms that prevent antibiotics from entering cells, leading to antibiotic resistance and seriously threatening health and safety. Statistics indicate that, based on the current rate of antibiotic development and the emergence of resistant bacteria, no antibiotics will be available in 50 years. Therefore, developing novel antibacterial agents to reverse antibiotic resistance is urgently needed. Antimicrobial peptides (APs) possess broad-spectrum antimicrobial activity, and their small size, easy metabolism, and excellent biocompatibility make them attractive potential alternatives to antibiotics.

[0003] Antimicrobial peptides (APs), also known as host defense peptides, have a wide range of sources and multiple functions, such as protecting the host from bacterial infection. First discovered in the early 1980s, antimicrobial peptides are small polypeptides produced by organisms that can effectively inhibit or kill bacteria, fungi, parasites, viruses, and tumor cells. They possess good thermal stability, high-efficiency broad-spectrum antibacterial activity, strong basicity, and anti-infective biological activities. Furthermore, antimicrobial peptides are important immunomodulatory molecules that protect the host from pathogen invasion. Compared to traditional antibiotics that disrupt cell membranes, interrupt DNA replication, or protein synthesis, some APs have been shown to have multiple modes of action with non-specific targets and are less prone to drug resistance due to their diverse mechanisms of action, ranging from membrane damage to interactions with intracellular targets. Therefore, antimicrobial peptides are considered a promising therapeutic option against increasingly drug-resistant pathogens. Different antimicrobial peptides have significantly different amino acid sequences and structures, but they all possess amphiphilic structures, allowing them to bind to other compounds. Therefore, they readily interact with negatively charged components on bacterial cell membranes and integrate into the lipid bilayer. However, their poor stability is one of the main obstacles to the clinical development of many common antimicrobial peptides in antimicrobial therapy. Due to their short half-life and relative toxicity to healthy cells, targeted delivery is necessary to improve their antimicrobial activity and reduce unnecessary consumption. Effectively utilizing nanoscale antimicrobial peptides in therapeutic and diagnostic methods is a strategy for inducing and maintaining their functional conformation. Summary of the Invention

[0004] 1. The problem to be solved

[0005] This application addresses the problems of poor biocompatibility, poor stability, easy inactivation, and relative toxicity to healthy cells of antimicrobial peptides, as well as their insignificant antibacterial effects in applications. It provides an acid-sensitive antimicrobial material, its preparation method, and its application. This antimicrobial material utilizes hollow mesoporous manganese dioxide (MnO2) in a chemokinetic antimicrobial material to nano-modify antimicrobial peptides, achieving a combined antimicrobial therapeutic effect. Finally, a metal-organic framework (ZIF-8) is formed on its surface, enabling it to target the bacterial microenvironment. This material is applied to research on reversing drug resistance in resistant bacteria and promoting wound healing. It is a highly efficient antimicrobial, hemostatic, and wound-healing material with good biocompatibility and high stability.

[0006] 2. Technical Solution

[0007] To solve the above problems, the technical solution adopted in this application is as follows:

[0008] This application provides an acid-sensitive antibacterial material, AP-MnO2@ZIF-8, comprising a zeolite-like imidazole framework (ZIF-8), hollow mesoporous manganese dioxide nanospheres (MnO2), and an antimicrobial peptide (AP). The manganese dioxide nanospheres have a hollow mesoporous core-shell structure, and the antimicrobial peptide is nanoscaled through coordination bonding and electrostatic adsorption. The ZIF-8 encapsulates the antimicrobial peptide modified by the manganese dioxide nanospheres. In this acid-sensitive antibacterial material AP-MnO2@ZIF-8 and its application, the advantages of each material and their combined antibacterial effects can be leveraged.

[0009] Zeolite-like imidazole framework (ZIF-8), as a typical size-tunable metal-organic framework material, is economical and readily available; it has extremely high drug loading capacity; and it is degradable in acidic environments. Encapsulating antimicrobial peptide-modified manganese dioxide nanospheres with ZIF-8 allows for targeted drug release in the slightly acidic environment of biofilms. This not only improves drug stability and reduces drug dosage, but also overcomes the hydrophobicity and poor stability of APs and MnO2, reducing the relative toxicity of antimicrobial peptides and manganese dioxide nanospheres.

[0010] Hollow mesoporous manganese dioxide nanospheres (MnO2) possess unique physical and chemical properties, such as large surface area and multiple oxidation states. Manganese dioxide can also serve as an effective material for heterogeneous Fenton catalysts. Exogenous manganese dioxide nanoparticles can, on the one hand, consume intracellular glutathione (GSH), weakening the antioxidant capacity of the cellular antioxidant defense system and inducing oxidative stress. Cells under high oxidative stress combined with antioxidants (APs) can effectively improve treatment efficiency. On the other hand, the reaction of MnO2 with glutathione (GSH) to generate Mn 2+ It can undergo a Fenton-like reaction, through interaction with endogenous cellular metabolites (such as H2O2 and H2O2 / H2O2).+ The reaction generates a large amount of ·OH in the cell, enhancing ROS-mediated apoptosis and thus achieving self-enhanced chemodynamic therapy (CDT). Furthermore, the binding of manganese dioxide to antimicrobial peptides can solve the problem of poor biocompatibility of manganese dioxide. Hollow mesoporous manganese dioxide has more binding sites than traditional spherical manganese dioxide. In this application, nitrogen-manganese coordination bonds are used to link MnO2 to APs to solve the problems of poor stability of antimicrobial peptides and poor biocompatibility of manganese dioxide. Moreover, the reduction product Mn... 2+ It is easily excreted by the liver and kidneys, solving the problems of organ accumulation and biosafety.

[0011] Antimicrobial peptides (APs) can penetrate biofilms and cell membranes, reversing the drug resistance of resistant bacteria. Positively charged APs bind to negatively charged cell membranes and insert their long carbon chains into the cell membrane, leading to cell lysis. Cell damage causes more MnO2 to enter the cell, generating reactive oxygen species (ROS), which enhance the effects of APs and CDTs.

[0012] Furthermore, in the aforementioned antibacterial material AP-MnO2@ZIF-8, the weight ratio of manganese dioxide nanospheres to antimicrobial peptides is (40-60):(60-40).

[0013] Furthermore, in the aforementioned antibacterial material AP-MnO2@ZIF-8, the amino acid sequence of the antimicrobial peptide AP is RRRWWWWV. This antimicrobial peptide is composed of four hydrophobic arginines and one hydrophobic valine. The polypeptide composed of hydrophobic amino acids can aggregate in large quantities in organisms to form a linear structure, which has good penetration into cell membranes, thereby achieving the purpose of antibacterial activity. This antimicrobial peptide can effectively kill MDR pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and MDR Gram-negative bacteria carrying MDR, mcr, and tet(X) genes; it can effectively prevent biofilm formation and eradicate established biofilms and persistent cells; and it maintains its activity under different physiological conditions in vitro.

[0014] This application also provides a method for preparing the above-mentioned acid-sensitive antibacterial material AP-MnO2@ZIF-8, which includes the following steps:

[0015] S1: Synthesis of AP-MnO2: The product AP-MnO2 was prepared by mixing and stirring hollow mesoporous manganese dioxide nanosphere powder with antimicrobial peptide AP.

[0016] S2: Synthesis of AP-MnO2@ZIF-8. In S1, AP-MnO2 microspheres were prepared and mixed with zinc nitrate hexahydrate stock solution. After stirring, 2-methylimidazole solution was added and stirred. The precipitate was washed and dried under vacuum to obtain ZIF-8-encapsulated manganese dioxide composite nanoparticles loaded with antimicrobial peptides.

[0017] Furthermore, the synthesis of the above AP-MnO2@ZIF-8 includes: mixing 50 mg AP-MnO2 with zinc nitrate hexahydrate stock solution and stirring for 1 h (Zn 2+ (Fixed); after adding 2-methylimidazole solution and stirring, it was washed and dried under vacuum.

[0018] Furthermore, in the synthesis of AP-MnO2@ZIF-8, the concentration of zinc nitrate hexahydrate stock solution was 15 mg / ml, and the concentration of 2-methylimidazole stock solution was 28 mg / ml.

[0019] Furthermore, the synthesis of AP-MnO2 includes: dispersing hollow mesoporous manganese dioxide nanosphere powder in water, then adding AP to the suspension, stirring at 37°C for 12 h; collecting the precipitate, washing with water, and drying under vacuum at 60°C.

[0020] Furthermore, the mass ratio of the hollow mesoporous manganese dioxide nanosphere powder to the antimicrobial peptide is 2:1. Even further, relative to 100 mg of hollow mesoporous manganese dioxide nanosphere powder, the volume of its aqueous dispersion solution is approximately 20 mL, and the amount of AP is controlled at approximately 50 mg.

[0021] Furthermore, the preparation method of the above-mentioned hollow mesoporous manganese dioxide nanospheres is as follows: manganese dioxide is generated on the surface of silica nanospheres, and then the silica is etched to obtain hollow mesoporous manganese dioxide nanospheres, specifically including the following steps:

[0022] The silica precipitate was dispersed in water; sodium hydroxide solution and hexadecyltrimethylammonium bromide (CTAB) were added to the mixture and gently stirred; potassium permanganate was then slowly added to the mixture to form a dispersion, followed by the addition of ethanol; the dispersion was stirred at 35°C for 12 h, and then the temperature was increased to 50°C and stirred for another 6 h; the mixture was centrifuged, and the supernatant was collected, yielding a brown SiO2 / MnO2 aqueous dispersion; the precipitate was redispersed in sodium carbonate solution, and air was removed from the flask; the mixture was stirred at 60°C for 20 h; centrifuged and washed with water to obtain hollow mesoporous manganese dioxide nanospheres.

[0023] Furthermore, in the above-mentioned method for preparing hollow mesoporous manganese dioxide nanospheres, the amount of aqueous solution is controlled at 80ml-100ml relative to 100mg of silica nanospheres, the amount of sodium hydroxide is controlled at 1ml-2ml, the amount of CTAB is controlled at 1g-1.5g, the amount of potassium permanganate is 40mg-50mg, the amount of ethanol is 1ml-2ml, the dropping time is controlled at 40min-80min, and the amount of sodium carbonate solution is controlled at about 50ml.

[0024] Furthermore, in the above method for preparing hollow mesoporous manganese dioxide nanospheres, the mass ratio of CTAB to Na2CO3 in the dispersion system is 1:(10.5-11). Even further, the concentration of Na2CO3 in the dispersion system is 2 mol / L.

[0025] Furthermore, the synthesis of the above-mentioned antimicrobial peptides includes: selecting Wang resin as the carrier for amino acid binding, using a mixture of N,N-diisopropylethylamine (DIEA), 1-hydroxybenzotriazole (HOBt), and N,N'-diisopropylcarbodiimide (DIC) as the condensation reagent, and using the Fomc solid-phase synthesis method to synthesize from the carboxyl terminus to the amino terminus according to the amino acid sequence of RRRWWWWV.

[0026] Furthermore, the synthesis of the above-mentioned antimicrobial peptide includes: using Fomc-protected L-amino acids as raw materials, Wang resin as a carrier, and DIC as a catalyst, the target peptide is synthesized from the C-terminus to the N-terminus through a standard solid-phase peptide synthesis method. Then, the synthesized peptide is cleaved from the resin using a cleavage solution and centrifuged in ether to obtain a crude peptide, which is then purified to obtain the target peptide.

[0027] Furthermore, the synthesis of the above-mentioned antimicrobial peptides includes the following steps:

[0028] First, the reaction tube was ultrasonically cleaned, then rinsed with pure water, and finally washed with DCM (dichloromethane). It was then placed on the experimental platform, and excess water was removed from the reaction tube. The Wang resin was then soaked in DCM for a period of time (20 minutes) to allow it to fully swell, and then dried. The designed SD of the Wang resin was 0.5. The amount of amino acids added was calculated using the formula: Amount of amino acids = 1g (resin amount) × 0.5 (SD) × M. W Calculate the amount of the first amino acid, glycine, by multiplying the relative molecular mass of the amino acid by 1.33 and dividing by 1000. Then, calculate the amount of 1-hydroxybenzotriazole (HOBt) using the formula: 1g (resin amount) × 0.5 (SD) × 135 × 3 ÷ 1000. Finally, calculate the mass of dimethylaminopyridine (DMAP) using the formula: 1g (resin amount) × 0.5 (SD) × 122 × 1.5 ÷ 1000.

[0029] Add 1 ml of N,N'-diisopropylcarbodiimide (DIC) and react for 4 h; after drying, seal the head, add dimethylformamide (DMF), 2 ml of acetic anhydride and diisopropylethylamine (DIEA), react for 30 min to 60 min, then wash three times with DMF; add piperidine solution for deprotection, react for 10 min, then dry the solution, add piperidine solution again and react for 5 min, then dry the solution again; take a small amount of resin and place it in a detection tube, then add two drops of ninhydrin and one drop of phenol solution, heat the reaction for 1 min, and observe the solution color; if the solution is blue or brown, the deprotection is successful.

[0030] According to the calculation formula, add 0.4313g of the first amino acid, arginine. The formula is: Amount of amino acid = 1g (resin amount) × 0.5 (SD) × M W (Amino acid relative molecular mass) × 3 ÷ 1000 to obtain the amount of the remaining amino acids and HOBt to be added. Add DIC and react for 1 hour. After the condensation reaction is complete, wash three times alternately with DMF (dimethylformamide), methanol, and DMF (dimethylformamide). Then, take a small amount of resin and place it in a detection tube. Add two drops of ninhydrin and one drop of phenol solution, heat for 1 minute, and observe the color of the solution. If there is no color, it means that the amino acid has been successfully condensed onto the resin and the next step can be carried out. If the solution is colored, it is blue or brown (proline may be pale yellow), it means that the condensation is unsuccessful and the feeding step needs to be returned to and the condensation restarted. If the condensation is complete, repeat the above steps until the last amino acid is successfully condensed. After the last amino acid is detected and colored, it means that all amino acid sequences have been linked to the resin.

[0031] At this point, add methanol solution and agitate the resin to ensure thorough mixing. After thorough mixing, remove most of the methanol, leaving a small amount, but do not dry the resin completely. Then add more methanol solution and agitate the resin again to ensure thorough mixing. Finally, dry the methanol solution until the resin is granular. Transfer the dried resin to a 50ml centrifuge tube and add 10ml of cleavage reagent (TFA (trifluoroacetic acid): water: EDT (ethylene dithiol): TMSBr (methylsilane) = 95:2:2:1) and react for 2 hours. After the cleavage reaction is complete, transfer the resin-peptide solution to a new reaction tube and filter it into another clean 50ml centrifuge tube using a syringe rubber bulb. Add ice-cold ether to the centrifuge tube containing the peptide solution for sedimentation, centrifuge at 3000 rpm for 5 minutes, and repeat three times. The precipitate at the bottom of the centrifuge tube is the target crude peptide. Dry and store the obtained crude peptide.

[0032] The crude peptide obtained from the cleavage step was dissolved in ultrapure water (peptide sequences containing many hydrophobic amino acids are difficult to dissolve in water; a small amount of acetonitrile can be added to aid dissolution), aiming to make the peptide solution as saturated as possible. The resulting peptide solution was filtered through a 0.45 μm pore size aqueous filter membrane (if acetonitrile was added previously, an organic phase filter membrane should be used). A small amount of the filtered solution was analyzed by mass spectrometry. The resulting ion mass-to-charge ratio (MMR) values ​​were compared with the molecular weight of the synthesized peptide sequence. If the obtained MMR corresponded to the amino acid molecular weight, the corresponding sequence was successfully synthesized. A small amount of the filtered peptide solution was analyzed using high-performance liquid chromatography (HPLC). The peak concentration was determined by analyzing the resulting HPLC chromatogram (e.g., if the organic phase concentration range in HPLC is 5% to 95% over 45 minutes, and the elution time is assumed to be 20–30 minutes, the organic phase concentration at elution can be calculated to be 45%–65%). The obtained peak concentration was used to set the organic phase range for preparative HPLC, and all remaining dissolved peptide samples were purified. A small amount of the purified solution was analyzed by mass spectrometry (MS). The obtained ion mass-to-charge ratio (MBC) value was compared with the peptide's molecular weight to confirm the synthesis of the target peptide sequence. The purified solution was then lyophilized to obtain pure peptide powder. A small portion of the lyophilized pure peptide powder was dissolved, filtered, and analyzed using analytical HPLC to determine the purity of the synthesized peptide. The obtained pure peptide was stored at 4°C for long-term preservation.

[0033] Furthermore, in the synthesis of the above-mentioned antimicrobial peptides, relative to 1g of Wang resin, the amount of the first arginine is controlled at 0.43g to 0.44g, the amount of the remaining amino acids is controlled at 1.29g to 1.3g, the amount of HOBt is controlled at about 0.2025g, the amount of DMAP is controlled at about 0.0915g, and the amount of DIC is controlled at 1ml to 1.5ml.

[0034] Furthermore, in the synthesis of the above-mentioned antimicrobial peptides, the concentration of phenol solution was controlled at 4 g / ml, the concentration of ninhydrin solution was controlled at 0.05 g / ml, and the solvent was anhydrous ethanol.

[0035] Furthermore, in the synthesis of the above-mentioned antimicrobial peptides, the piperidine solution is prepared by mixing piperidine and DMF in a 1:4 ratio, measuring 100 ml of piperidine into a wash bottle, and then measuring 400 ml of DMF into it and mixing for later use.

[0036] This application also provides the application of the aforementioned acid-sensitive antibacterial material in the treatment of drug-resistant bacterial infections.

[0037] This application also provides the application of the aforementioned acid-sensitive antibacterial material in promoting wound healing.

[0038] This application also provides the application of the aforementioned acid-sensitive antibacterial material in treating drug-resistant bacterial infections and promoting wound healing.

[0039] This application also provides the application of the above-mentioned acid-sensitive antibacterial material in the preparation of drugs for treating drug-resistant bacterial infections.

[0040] This application also provides the application of the above-mentioned acid-sensitive antibacterial material in the preparation of drugs that promote wound healing.

[0041] This application also provides the application of the above-mentioned acid-sensitive antibacterial material in the preparation of drugs for treating drug-resistant bacterial infections and promoting wound healing.

[0042] 3. Beneficial effects

[0043] Compared with the prior art, the advantages of this application are as follows:

[0044] (1) This application provides an acid-sensitive antibacterial material, AP-MnO2@ZIF-8, which is a core-shell structure of manganese dioxide nanospheres modified with antibacterial active ingredients and encapsulated by ZIF-8. The antibacterial active ingredients include antimicrobial peptides. ZIF-8 has an extremely high drug loading rate, is degradable in acidic environments, and is economically readily available. Encapsulating manganese dioxide composite nanoparticles with ZIF-8 allows for targeted drug release in the slightly acidic environment of biological membranes, which not only improves drug stability and reduces drug dosage but also acts on the bacterial microenvironment, directly increasing the antibacterial effect. Manganese dioxide (MnO2) has unique physical and chemical properties, such as a large surface area and multiple oxidation states. Manganese dioxide can also be used as an effective material for multiphase Fenton catalysts. Exogenous manganese dioxide nanoparticles can consume intracellular GSH, weaken the antioxidant capacity of the cell's antioxidant defense system, and cause oxidative stress, reducing the product MnO2. 2+ It is easily excreted by the liver and kidneys, solving the problems of organ accumulation and biosafety. Furthermore, the MnO2 reacts with glutathione (GSH) to produce Mn... 2+Highly toxic hydroxyl radicals (·OH) can be generated from H2O2 via a Fenton-like reaction, thereby achieving self-enhanced chemodynamic therapy (CDT). Combining MnO2 with antimicrobial peptides can effectively improve treatment efficiency in cells under high oxidative stress, providing a new strategy for combination therapy. In addition, the combination of manganese dioxide and antimicrobial peptides can solve the problem of poor biocompatibility of manganese dioxide. Hollow mesoporous manganese dioxide has more binding sites than traditional spherical manganese dioxide. In this application, MnO2 and AP are linked by coordination bonds to solve the problems of poor stability of antimicrobial peptides and poor biocompatibility of manganese dioxide. Antimicrobial peptides (AP) can penetrate biological membranes and cell membranes, reverse the drug resistance of drug-resistant bacteria. Positively charged AP binds to negatively charged cell membranes and inserts the long carbon chain of AP into the cell membrane, thereby causing cell lysis. Cell damage leads to more MnO2 entering the cell to generate ROS, enhancing the effects of AP and CDT.

[0045] (2) The acid-sensitive antibacterial material AP-MnO2@ZIF-8 provided in this application has a particle size of 100-150 nm for manganese dioxide nanospheres and 120-180 nm for AP-MnO2@ZIF-8, which are relatively uniform in size and well distributed.

[0046] (3) The preparation method and application of the acid-sensitive antibacterial material AP-MnO2@ZIF-8 provided in this application are economical and readily available, and the preparation is simple. It has high stability, no side effects, and can effectively promote the ablation of the coating and treat wounds infected by drug-resistant bacteria. It also has good biocompatibility, high safety, and good clinical application prospects. Attached Figure Description

[0047] Figure 1 These are the material characterization results during the preparation of AP-MnO2@ZIF-8 in this application, where: A is the SEM image of MnO2 nanospheres; B is the SEM image of AP-MnO2; C is the SEM image of AP-MnO2@ZIF-8; D is the UV-Vis spectrum of MnO2, AP-MnO2, and AP-MnO2@ZIF-8; E is the FT-IR spectrum of AP, MnO2, AP-MnO2, and AP-MnO2@ZIF-8; F is the Zeta potential of MnO2, AP-MnO2, and AP-MnO2@ZIF-8; and G is the energy spectrum of AP-MnO2@ZIF-8.

[0048] Figure 2This application presents a study on the AP loading capacity and stability of AP-MnO2@ZIF-8. Wherein: A represents the size and polydispersity index of manganese dioxide; B represents the size and polydispersity index of AP-MnO2; C represents the size and polydispersity index of AP-MnO2@ZIF-8; D represents the size and polydispersity index of AP-MnO2@ZIF-8 at pH 7.4; E represents the size and polydispersity index of AP-MnO2@ZIF-8 at pH 5.5; F represents the particle size change of AP-MnO2@ZIF-8 at specific time intervals (0, 3, 6, 9, 12, 15 days); G represents the size of MnO2, AP-MnO2, and AP-MnO2@ZIF-8; H represents the size of AP-MnO2@ZIF-8 at different pH values; and I represents the AP standard curve.

[0049] Figure 3 This paper presents the in vitro antibacterial effect of AP-MnO2@ZIF-8 as described in this application. A shows representative plate photographs, with the horizontal control being the drug concentration AP-MnO2@ZIF-8 and the vertical controls being MDR E. coli and MDR S. aureus; B shows representative plate photographs, with the horizontal control being different materials (drugs) and the vertical controls being MDR E. coli and MDR S. aureus; C shows the relative survival rate of bacteria corresponding to the number of colonies counted on the plates; D shows the relative survival rate of bacteria corresponding to the number of colonies counted on the counting plate. (Data are expressed as mean ± SD, n = 3, *p < 0.05, **p < 0.005).

[0050] Figure 4 This is an assessment of intracellular reactive oxygen species detection in AP-MnO2@ZIF-8 in this application.

[0051] Figure 5 This is a CLSM image of the bacterial cells in this application.

[0052] Figure 6 These are electron micrographs of MDR E. coli and MDR S. aureus cells treated with AP-MnO2@ZIF-8 according to this application.

[0053] Figure 7 This is an experiment on the promotion of mouse wound healing by AP-MnO2@ZIF-8 in this application, where: A is the change in mouse body weight during treatment; B is the quantification of the wound area in mice; C is a photograph of the mouse wound, with the groups being Blank, AP, MnO2, AP-MnO2, and AP-MnO2@ZIF-8, respectively; and D is the H&E staining results of the wound tissue of mice in different treatment groups. Detailed Implementation

[0054] The present application will be further described below with reference to specific embodiments.

[0055] It should be noted that terms such as "upper", "lower", "left", "right", and "middle" used in this specification are only for clarity of description and are not intended to limit the scope of implementation. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered as within the scope of this application.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0057] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0058] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0059] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0060] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as “less than about 4.5,” which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0061] Example 1

[0062] This embodiment provides an acid-sensitive antibacterial material AP-MnO2@ZIF-8 and its preparation method. The AP-MnO2@ZIF-8 comprises a zeolite-like imidazole framework (ZIF-8), manganese dioxide nanospheres (MnO2), and an antimicrobial peptide (AP). The manganese dioxide nanospheres have a hollow mesoporous core-shell structure. The antimicrobial peptide modifies the manganese dioxide nanospheres by connecting them through coordination bonds and / or electrostatic adsorption. The zeolite-like imidazole framework encapsulates the antimicrobial peptide-modified manganese dioxide nanospheres. The weight ratio of manganese dioxide nanospheres to antimicrobial peptides is (40-60):(60-40). The preparation method specifically includes the following steps:

[0063] S1: Synthesis of hollow mesoporous manganese dioxide nanospheres (MnO2)

[0064] The silica (SiO2) precipitate was dispersed in 100 mL of water;

[0065] Add 1.3 mL of sodium hydroxide solution (100 mmol) -1 ) and 1.0 g of cetyltrimethylammonium bromide (CTAB) were added to the above mixture and stirred gently;

[0066] 40 mg of potassium permanganate (KMnO4) was slowly added to the above mixture to form a dispersion;

[0067] After 60 minutes, 1.5 mL of ethanol (CH3CH2OH) was added dropwise to the above dispersion.

[0068] The dispersion was stirred at 35°C for 12 hours, and then the temperature was increased to 50°C and stirred for 6 hours.

[0069] Centrifuge the mixture and then remove the upper liquid. The SiO2 / MnO2 aqueous dispersion is brown in color.

[0070] Collect the SiO2 / MnO2 aqueous dispersion by centrifugation;

[0071] The precipitate was redispersed in 50 mL of sodium carbonate solution (2.0 mol / L), and the air in the flask was removed.

[0072] The above mixture was stirred at 60°C for 20 hours;

[0073] Centrifugation and washing with water yielded hollow mesoporous manganese dioxide nanospheres (MnO2).

[0074] S2: Synthesis of antimicrobial peptides (AP)

[0075] The antimicrobial peptide provided in this embodiment has the amino acid sequence RRRWWWWV. Its preparation method includes: selecting Wang resin as the carrier for amino acid binding, using a mixture of N,N-diisopropylethylamine, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide as the condensation reagent, and using the Fomc solid-phase synthesis method to synthesize it from the carboxyl terminus to the amino terminus according to the amino acid sequence of RRRWWWWV.

[0076] S3: Synthesis of AP-MnO2

[0077] 100 mg of manganese dioxide nanospheres prepared in S1 were dispersed in 20 ml of water, and then 50 mg of AP prepared in S2 was added to the suspension, i.e., the mass ratio of manganese dioxide nanospheres to antimicrobial peptides was 2:1; the mixture was stirred at 37 °C for 12 h, the precipitate was collected, washed with water, and dried under vacuum at 60 °C to obtain manganese dioxide-loaded antimicrobial peptides (AP-MnO2).

[0078] S4: Synthesis of AP-MnO2@ZIF-8

[0079] 50 mg of AP-MnO2 microspheres prepared in S3 were mixed with zinc nitrate hexahydrate stock solution (0.12 g zinc nitrate hexahydrate dissolved in 8.0 g water) and stirred for 1 h (Zn 2+ (Fixed); Add 2-methylimidazole solution (2.23 g 2-methylimidazole dissolved in 80 g water) and stir for 24 h. Then wash and dry the precipitate under vacuum to obtain AP-MnO2@ZIF-8.

[0080] Results analysis:

[0081] (1) Synthesis results and characterization analysis of AP-MnO2@ZIF-8

[0082] Scanning electron microscopy (SEM) revealed that the hollow mesoporous manganese dioxide nanospheres exhibited a uniform spherical shape with a size ranging from 100 nm to 150 nm. Figure 1 A) and no aggregation occurred, demonstrating good dispersibility; Figure 1 B is a scanning electron microscope image of AP-MnO2, which shows obvious changes on the smooth surface of the manganese dioxide nanospheres, with linear structural material loaded on the "core" of the manganese dioxide nanospheres. Figure 1 B) becomes irregular because hydrophilic and hydrophobic antimicrobial peptides do not form particularly regular shells, which also indicates that the antimicrobial peptides were successfully adsorbed onto the surface of manganese dioxide nanospheres; after coating ZIF-8 onto the surface of manganese dioxide nanoparticles loaded with AP, the material size was controlled between 120nm and 180nm. Figure 1 C) It meets the requirements of the EPR effect in the human body, enabling nanoparticles to successfully reach the site of action.

[0083] The ultraviolet-visible (UV-vis) spectrum of the above materials is as follows: Figure 1 As shown in Figure D, manganese dioxide nanoparticles, as metal particles, do not exhibit obvious absorption peaks between 200 nm and 800 nm. AP-MnO2 shows an absorption peak at 190 nm, indicating that AP was successfully modified on the surface of manganese dioxide nanospheres. The characteristic absorption peak of ZIF-8 appears around 210 nm. AP-MnO2@ZIF-8 shows characteristic peaks at both 190 nm and 210 nm, indicating that the acid-sensitive antibacterial material AP-MnO2@ZIF-8 was successfully prepared.

[0084] Fourier transform infrared (FT-IR) spectroscopy Figure 1 E) indicates that AP-MnO2 at 455 cm⁻¹ -1 The presence of characteristic peaks for nitrogen-manganese coordination bonds at 1204 cm⁻¹ indicates that manganese dioxide successfully modified the peptide. AP and manganese dioxide showed peaks at 1204 cm⁻¹. -1 and 579cm -1 The characteristic peak at 2925 cm⁻¹ is related to the vibrations of CN and Mn-O, indicating that AP and manganese dioxide have been successfully synthesized. -1 The characteristic peak at the point shows an aliphatic bond, indicating that AP-MnO2 is encapsulated by ZIF-8, forming a complete composite nanoparticle AP-MnO2@ZIF-8.

[0085] Figure 1 F is the Zeta potential diagram of AP-MnO2@ZIF-8. The manganese dioxide nanospheres are negatively charged with a potential of -24.5±1.0 mV. The potential of AP after electrostatic adsorption with a positive charge is 12.8±1.6 mV. After encapsulation with positively charged ZIF-8, the potential increases to 17.2±1.4 mV, indicating that the synthesis of the composite material AP-MnO2@ZIF-8 is very successful.

[0086] In addition, elements such as C, O, Mn, and Zn present in AP-MnO2@ZIF-8 were also detected by energy dispersive spectroscopy (EDS). Figure 1 G), the detection of these elements can more intuitively show the elemental composition of the composite material and the successful synthesis of the composite material.

[0087] Example 2

[0088] This embodiment provides a stability analysis of AP-MnO2@ZIF-8.

[0089] AP-MnO2@ZIF-8 particles dispersed in neutral PBS and bacterial culture medium for over 360 hours showed minimal particle size variation and good stability in biological culture media, with size distributions as shown below. Figure 2As shown, the average particle sizes of the obtained MnO2, AP-MnO2, and AP-MnO2@ZIF-8 are 159 nm, 244 nm, and 301 nm, respectively. Figure 2 A, 2B, 2C, 2G), as time goes on, such as Figure 2 As shown in F, the material may exhibit agglomeration, which would slightly increase the size, but the increase is not significant, thus demonstrating the good stability of this material.

[0090] The effect of different pH values ​​on the stability of AP-MnO2@ZIF-8 was measured. The size of AP-MnO2@ZIF-8 cells was detected in vitro to assess their stability in different environments. Specific dimensions and polydispersity index (PDI) were determined as follows: Figure 2 As shown in D, 2E, and 2H, the material of this application has significantly smaller dimensions under weakly acidic conditions than under neutral conditions. Figure 2 As shown in F, the material size decreases significantly over time in a weakly acidic environment, indicating that the material will decompose and collapse over time, providing a pathway for material release and demonstrating that the material in this application has acid-sensitive properties.

[0091] The amount of AP released into the solution was determined by measuring the absorbance at 280 nm using UV-Vis spectroscopy at different time points. Figure 2 I). This example demonstrates that AP achieves a nanoparticle release rate of up to 80%.

[0092] In summary, these results indicate that the synthesis of AP-MnO2@ZIF-8 was successful, and AP-MnO2@ZIF-8 exhibits good stability.

[0093] Example 3

[0094] This embodiment provides an in vitro antibacterial test of AP-MnO2@ZIF-8.

[0095] The inhibitory effects of AP-MnO2@ZIF-8 on *Escherichia coli* (MDR E. coli) and *Staphylococcus aureus* (MDRS. aureus) were observed using the CFU method. A blank control and three concentration gradients of AP-MnO2@ZIF-8 (50, 100, and 150 μg / ml) were set up. After bacterial activation to the logarithmic growth phase, the mixture was diluted, and 10 μg / ml of each drug was used. -5 0.5 ml of CFU-concentrated bacterial suspension was mixed with 0.5 ml of acid-sensitive antibacterial material of different concentrations, incubated, and then treated. Three replicates were performed for each group. The results of the in vitro antibacterial test are as follows: Figure 3As shown, A is a representative plate photograph, with the horizontal control being the drug concentration AP-MnO2@ZIF-8 and the vertical controls being MDR E. coli and MDR S. aureus; B is a representative plate photograph, with the horizontal control being different materials (drugs) and the vertical controls being MDR E. coli and MDR S. aureus; C is the relative survival rate of bacteria corresponding to the number of colonies counted on the plate; D is the relative survival rate of bacteria corresponding to the number of colonies counted on the counting plate. (Data are expressed as mean ± SD, n = 3, *p < 0.05, **p < 0.005).

[0096] Results analysis:

[0097] The MIC values ​​of AP-MnO2@ZIF-8 against MDR bacteria were 20 μg / mL and 39 μg / mL, respectively. The bacterial survival rate of AP-MnO2@ZIF-8 at 150 μg / mL was almost zero, indicating that the nanocomposite material has effective bacterial inhibitory effects. Single-factor CFU results ( Figure 3 The results showed that the survival rate of drug-resistant bacteria treated with AP-MnO2@ZIF-8 (100 μg / mL) was less than 5%, indicating that the antibacterial effect of CDT / AP was better than that of CDT and AP alone.

[0098] Example 4

[0099] This embodiment provides an evaluation of the chemokinetic antibacterial effect of AP-MnO2@ZIF-8.

[0100] AP-MnO2@ZIF-8 (100 μg / mL) solution was mixed with GSH (2.0 mM) of the same concentration and incubated at 37 °C for different times (0 min, 20 min, and 40 min). Unreacted glutathione was then calibrated using DTNB (5,5'-dithiobenzenemethyl ester (2-nitrobenzoic acid)). The reaction of manganese dioxide with GSH reduced the characteristic absorption peak of DTNB, resulting in a new absorption peak at 412 nm (the product being 5-thio-2-nitrobenzoic acid). DTNB requires light protection, so all operations were performed under light-protected conditions. The obtained data were then plotted as curves.

[0101] A solution of 1,3-diphenylisobenzene (DPBF) containing AP-MnO2@ZIF-8 (6.0 μg / mL) was mixed with the same concentration of GSH (2.0 mM) and then incubated for different times (0 min, 3 min, 6 min, 9 min, 12 min, 15 min, 18 min, 21 min, and 24 min). The absorbance change of DPBF at 410 nm was observed, confirming the formation of ·OH.

[0102] The strain was inoculated into test tubes and placed on a shaker at 37°C for 12 h to reach the logarithmic growth phase. The activated strain was then incubated with PBS, manganese dioxide (200 μg / mL), and AP-MnO2@ZIF-8 (200 μg / mL) at 37°C for 12 h. The cells were then washed three times with PBS, and 1 mL of 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe solution was added, followed by co-incubation for 30 min. Excess DCFH-DA probe was then aspirated, and the cells were washed three times with PBS before observation using a fluorescence microscope. The amount of reactive oxygen species (ROS) in the cells could be determined by the intensity of the green fluorescence produced by the probe binding to ROS, or by immediately digesting the collected cells with digestion solution and measuring the fluorescence intensity by flow cytometry.

[0103] Results Analysis

[0104] The reaction of manganese dioxide with glutathione (GSH) reduces the characteristic absorption peak of DTNB (the product is 5-thio-2-nitrobenzoic acid), and a new absorption peak appears at 412 nm. For example... Figure 4 As shown in Figure A, the change peak of unreacted GSH at 412 nm in the UV-Vis adsorption curve of DTNB indicates that the manganese dioxide on AP-MnO2@ZIF-8 (100 μg / mL) is consumed by GSH (approximately 2 mM).

[0105] Mn 2+ The mediated Fenton-like reaction has been confirmed in previous reports, and the scavenging effect of GSH on ·OH has been studied. Mn is generated via the reaction of manganese dioxide and GSH. 2+ Ions, then through Mn 2+ Hydrogen peroxide reacts with hydrogen peroxide to generate ·OH. The generated ·OH was detected using DPBF (absorption peak at 410 nm). Therefore, GSH of the same concentration was added to AP-MnO2@ZIF-8 solution containing hydrogen peroxide at different times to explore ROS formation. After 20 min, DPBF was added to the solution as a ·OH indicator at a set time. The effect of ·OH-induced DPBF degradation was observed using changes in the UV-Vis absorption peak at 410 nm. Figure 4 As can be seen from B, the DPBF degradation caused by the Fenton-like reaction indicates that AP-MnO2@ZIF-8 has chemokinetic activity against multidrug resistance.

[0106] The results showed that the composite nanomaterial AP-MnO2@ZIF-8, formed by encapsulating manganese dioxide with MOF, still exhibited good chemokinetic therapeutic effects. H2DCFH-DA reacts with ·OH to generate DCFH-DA fluorescence, leading to the formation of intracellular ROS. Figure 4C) indicates that the drug-treated bacterial cells exhibit strong green fluorescence.

[0107] Example 5

[0108] This experimental example provides a live / dead cell assay for AP-MnO2@ZIF-8.

[0109] Logarithmic-phase cultures of *E. coli* and *Staphylococcus aureus* were incubated with AP-MnO2@ZIF-8 in a shaking incubator for 12 h. Bacteria treated with PBS served as a blank control. At the end of incubation, *Staphylococcus aureus* bacteria were harvested by centrifugation and stained in the dark for 30 min using a live-dead bacteria double staining kit. After staining, the cells were washed three times with PBS to remove excess staining solution. Samples were prepared, and bacterial staining was observed under the red and green channels of a fluorescence microscope.

[0110] In this study, bacteria treated with SYTO9 / PI were stained. Live cells stained with SYTO9 showed green fluorescence, while dead cells stained with PI showed red fluorescence.

[0111] Results Analysis

[0112] Compared to the traditional CFU counting method, the live-dead staining method more directly reflects the antibacterial activity of nanomaterials. The blank group almost entirely showed green fluorescence, indicating that the bacteria, without drug treatment, were normal and had a high survival rate. Figure 5 The AP, MnO2, and AP-MnO2@ZIF-8 treatment groups all exhibited abundant green fluorescence and some red fluorescence, which is related to the lethal effects of AP, CDT therapy, and cationic antibacterial drugs on the bacteria. Red fluorescence increased with increasing drug concentration. At a concentration of 150 μg / mL for AP-MnO2@ZIF-8, the field of view was almost entirely red fluorescence, indicating near-complete bacterial death. Figure 5 As shown, the bacterial mortality rate of the AP-MnO2@ZIF-8 group was greater than 95%, indicating that AP-MnO2@ZIF-8 has a significant antibacterial effect and exhibits the superiority of the CDT / AP combination.

[0113] Example 6

[0114] This experimental example provides morphological observation of cells treated with AP-MnO2@ZIF-8.

[0115] To observe the disruption of bacterial structure by AP-MnO2@ZIF-8, bacteria treated with AP-MnO2@ZIF-8 were dehydrated, and their structural condition was observed using SEM. Logarithmic-phase *Escherichia coli* and *Staphylococcus aureus* were treated with different concentrations (50 μg / mL, 100 μg / mL, 150 μg / mL) of AP-MnO2@ZIF-8. AP (100 μg / mL) and AP-MnO2 (100 μg / mL) served as controls. PBS-treated groups served as blanks. Bacteria were collected by centrifugation at 4000 rpm for 10 min, washed twice with PBS, fixed with 2.5% glutaraldehyde at 4℃ for 12 h, and then subjected to a gradient dehydration with different concentrations of ethanol (35%, 50%, 70%, 80%, 95%, 100%) for 20 min each time. Finally, the cells were placed in acetone solution for 20 min, centrifuged, and dried using a critical point dehydrator. Bacterial morphology and structure were observed under SEM.

[0116] Results Analysis

[0117] Morphological changes in bacterial cells were analyzed using scanning electron microscopy. Figure 6 Untreated bacterial cells remained almost intact, while AP-treated cells showed damage, indicating that AP has a destructive effect on bacteria. Cells treated with AP-MnO2@ZIF-8 exhibited significant physical breakage and cell membrane lysis. Particularly at an AP-MnO2@ZIF-8 concentration of 150 μg / mL, cell damage exceeded 90%. This is likely due to the binding of positively charged AP to the negatively charged cell membrane, and the insertion of AP's long carbon chains into the cell membrane, leading to cell lysis. Cell damage results in more manganese dioxide entering the cell and generating ROS, thereby enhancing the effects of AP and CDT. Therefore, the AP-MnO2@ZIF-8 composite nanomaterial exhibits good bactericidal effects.

[0118] Example 7

[0119] This embodiment provides an experiment on the effect of AP-MnO2@ZIF-8 on promoting wound healing in mice.

[0120] Female Kunming mice (approximately 25g; 4-5 weeks old) were fed continuously for 7 days according to the guidelines in the "Guidelines for the Care and Use of Laboratory Animals," and their physical condition was observed. Subsequently, mice in good condition were divided into groups and their hair was removed. Then, under anesthesia, an oval wound approximately 1.5cm-2cm long was made on the back of each mouse using dissecting scissors. For two consecutive days, 100μL of activated Staphylococcus aureus (1×10⁻⁶) was dripped into each wound. 6 (CFU / mL), and follow-up treatment will be carried out after the wound becomes inflamed.

[0121] Mice with wounds infected with Staphylococcus aureus were randomly divided into five groups (n=5-7): control group, AP group, MnO2 group, AP-MnO2 group, and AP-MnO2@ZIF-8 group. The wounds in the control group were treated daily with 100 μL of PBS, the MnO2 group with 100 μL of MnO2 daily, and the AP-MnO2@ZIF-8 group with 100 μL of AP-MnO2@ZIF-8 daily. Wounds were photographed for 8 consecutive days after each treatment. After treatment, the mice were euthanized using standard methods, and the epidermis and other organs were harvested for hematoxylin-eosin (H&E) staining.

[0122] To investigate the toxic effects of near-infrared spectroscopy and AP-MnO2@ZIF-8 on the heart, liver, spleen, lungs, and kidneys of mice, injured mice were divided into four groups: control group, AP group, MnO2 group, AP-MnO2 group, and AP-MnO2@ZIF-8 group. Mice were treated until wound healing and euthanized 30 days after treatment. Organs were collected, and H&E staining was used to observe the staining of cross-sections.

[0123] Results Analysis

[0124] A wound healing model was established to evaluate the in vivo efficacy of AP-MnO2@ZIF-8 composite nanomaterials in treating wound infections. Mice were treated with AP, MnO2, AP-MnO2, and AP-MnO2@ZIF-8, respectively, while the control group was treated with PBS. After 9 days of treatment, the body weight of mice in all five groups reached dynamic equilibrium with fluctuations of less than 2g, indicating that the nanomaterials had no significant toxic side effects on mice. Figure 7 A). Wound image of a mouse ( Figure 7 C) reflects the antibacterial and wound-healing effects of nanomaterials. The change in the wound area curve is shown in... Figure 7 As shown in Figure B, after 9 days of drug treatment, the wound area in the AP-MnO2@ZIF-8 group mice decreased by 91.5%, approaching healing. In the control group, the wound color intensified at the end of the treatment period, possibly due to persistent inflammation hindering spontaneous healing. The AP, MnO2, and AP-MnO2 groups showed signs of healing, but residual wound areas remained at the end of the treatment cycle. The wound color in the manganese dioxide group darkened, possibly due to the deposition of black manganese dioxide particles on the wound surface caused by continuous administration. It was also observed that the treated mice exhibited better wound crusting and healing, with the AP-MnO2@ZIF-8 group showing the most significant effect. H&E staining results of mouse wound skin (…) Figure 7D) shows an abnormal bluish-purple area on the epidermis of the control group, indicating an inflammatory response. The AP, MnO2, and AP-MnO2 groups showed reduced inflammation, but a large number of inflammatory cells remained. The AP-MnO2@ZIF-8 group exhibited the least inflammation, indicating the best wound healing. Therefore, the efficacy of AP or CDT alone against MDR bacterial wound infection is limited, while the combined treatment of AP / CDT is significantly effective. These results demonstrate that the AP-MnO2@ZIF-8 composite nanomaterial possesses strong anti-infection and wound-healing properties.

Claims

1. An acid-sensitive antibacterial material AP-MnO2@ZIF-8, characterized in that, The material comprises a zeolite-like imidazole framework ZIF-8, hollow mesoporous manganese dioxide nanospheres, and an antimicrobial peptide. The manganese dioxide nanospheres have a hollow mesoporous core-shell structure, and the antimicrobial peptide is connected to the hollow mesoporous manganese dioxide nanospheres through coordination bonds and / or electrostatic adsorption. ZIF-8 encapsulates the hollow mesoporous manganese dioxide nanospheres modified with the antimicrobial peptide. The amino acid sequence of the antimicrobial peptide is RRRWWWWV.

2. The acid-sensitive antibacterial material AP-MnO2@ZIF-8 according to claim 1, characterized in that, The weight ratio of the manganese dioxide nanospheres to the antimicrobial peptides is 2:

1.

3. The preparation method of the acid-sensitive antibacterial material AP-MnO2@ZIF-8 according to claim 1 or 2, characterized in that, Includes the following steps: S1: Synthesis of AP-MnO2: The product AP-MnO2 was prepared by mixing and stirring hollow mesoporous manganese dioxide nanospheres with antimicrobial peptide AP. S2: Synthesis of AP-MnO2@ZIF-8. In S1, AP-MnO2 microspheres were prepared and mixed with zinc nitrate hexahydrate. After stirring, 2-methylimidazole solution was added and stirred to obtain ZIF-8-encapsulated manganese dioxide composite nanoparticles loaded with antimicrobial peptides.

4. The preparation method of the acid-sensitive antibacterial material AP-MnO2@ZIF-8 according to claim 3, characterized in that, The mass ratio of the hollow mesoporous manganese dioxide nanospheres to the antimicrobial peptides is 2:

1.

5. The preparation method of the acid-sensitive antibacterial material AP-MnO2@ZIF-8 according to claim 3 or 4, characterized in that, The method for preparing the hollow mesoporous manganese dioxide nanospheres is as follows: manganese dioxide is generated on the surface of silica nanospheres, and then the silica is etched to obtain hollow mesoporous manganese dioxide nanospheres, specifically including the following steps: The silica precipitate was dispersed in water; sodium hydroxide solution and hexadecyltrimethylammonium bromide were added to the mixture and gently stirred; potassium permanganate was then slowly added to the mixture to form a dispersion, followed by the addition of ethanol; the dispersion was stirred at 35 °C for 12 h, and then the temperature was increased to 50 °C and stirred for another 6 h; the mixture was centrifuged, and the supernatant was collected, yielding a brown SiO2 / MnO2 aqueous dispersion; the precipitate was redispersed in sodium carbonate solution, and air was removed from the flask; the mixture was stirred at 60 °C for 20 h. Centrifugation and washing with water yielded hollow mesoporous manganese dioxide nanospheres.

6. The preparation method of the acid-sensitive antibacterial material AP-MnO2@ZIF-8 according to claim 5, characterized in that, The mass ratio of hexadecyltrimethylammonium bromide to sodium carbonate in the dispersion system is 1:(10.5~11).

7. The preparation method of the acid-sensitive antibacterial material AP-MnO2@ZIF-8 according to claim 5, characterized in that, The synthesis of the antimicrobial peptide includes: selecting Wang resin as the carrier for amino acid binding, using a mixture of N,N-diisopropylethylamine, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide as the condensation reagent, and using the Fomc solid-phase synthesis method to synthesize the peptide from the carboxyl terminus to the amino terminus according to the amino acid sequence of RRRWWWWV.

8. The use of the acid-sensitive antibacterial material AP-MnO2@ZIF-8 as described in claim 1 or 2 and / or the acid-sensitive antibacterial material AP-MnO2@ZIF-8 prepared by any of the preparation methods described in claims 3-7 in the preparation of drugs for treating drug-resistant bacterial infections and / or promoting wound healing.

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