Antibacterial polypeptide high polymer material, product containing antibacterial polypeptide high polymer material, and preparation method and application of antibacterial polypeptide high polymer material

Abstract

The invention discloses an antibacterial polypeptide high polymer material, a product containing the antibacterial polypeptide high polymer material, and a preparation method and application of the antibacterial polypeptide high polymer material, and belongs to the technical fields of high polymer materials, bacterial infection treatment and medicines. The preparation method of the antibacterial polypeptide high polymer material comprises the following steps: carrying out esterification reaction on enol and glutamic acid, and carrying out glutamic acid side group modification to obtain an R2-NCA monomer; taking R1-NH2 as an initiator, and reacting with the R2-NCA monomer to obtain polypeptide; primary amine or quaternary amine is modified to a polypeptide side group through click reaction of sulfydryl and double bonds, and finally the antibacterial polypeptide high polymer material is prepared. The antibacterial polypeptide high polymer material with a specific structure prepared by the method can effectively penetrate through and resist a biological membrane matrix barrier and specifically target bacterial membrane phospholipids; the compound shows excellent in-vivo antibacterial activity, can effectively inhibit biofilm-related infection and promote repair of infected parts, and has good treatment effect and application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of polymer materials, bacterial infection treatment and pharmaceutical technology, and particularly relates to an antimicrobial polypeptide polymer material, products containing the same, preparation methods and applications. Background Technology

[0002] Bacterial biofilm infections pose a significant challenge to clinical treatment. Composed of microbial communities and their secreted extracellular polymeric substances (EPS), these biofilms form physical and chemical barriers, significantly reducing the permeability and effectiveness of antibiotics. Biofilm-associated infections account for more than 60% of human bacterial infections and are commonly found in chronic wound infections, medical device-related infections (such as those involving catheters and artificial joints), and respiratory infections (such as cystic fibrosis). Traditional antibiotics are effective against planktonic bacteria but have limited effectiveness against bacteria in a metabolically dormant state within biofilms and are prone to inducing drug resistance. The World Health Organization (WHO) has listed drug-resistant bacteria as a global public health crisis, necessitating the development of novel antimicrobial strategies.

[0003] Antimicrobial polypeptides (AMPs) are a class of synthetic polymers inspired by natural antimicrobial peptides. They disrupt bacterial membrane integrity by mimicking the amphiphilic structure and cationic properties of these peptides. Compared to natural antimicrobial peptides, AMPs can be optimized for stability, toxicity, and broad-spectrum activity through modular design. Some AMPs can penetrate the biomembrane matrix, targeting negatively charged EPS components via electrostatic interactions and disrupting bacterial membrane potential, even against dormant bacteria. Furthermore, their low tendency to induce drug resistance makes them ideal drug candidates.

[0004] Despite existing research confirming the potential of antimicrobial peptides, their application in biofilm infections still faces challenges: the positively charged amphiphilic structure of antimicrobial peptides is key to their disruption of bacterial cell membranes, enabling non-specific killing and adhesion. However, this structure can also cause them to non-specifically damage normal tissue cell membranes, resulting in strong toxicity and limiting dosage. Furthermore, this structure makes them readily bind to proteins and other substances, thus losing their antimicrobial activity. Studies have shown that the bactericidal effect of antimicrobial peptides is susceptible to environmental factors; for example, many natural antimicrobial peptides lose their antimicrobial activity under physiological conditions (high protein concentration, high salt concentration, divalent cations or polyanions). Moreover, the biofilm matrix contains a large amount of polysaccharides, proteins, nucleic acids, etc., and these negatively charged substances may inhibit the bactericidal activity of antimicrobial peptides, hindering their penetration into the biofilm.

[0005] Therefore, the design of highly efficient and selective antimicrobial peptides against biofilms needs to meet the following requirements: improve the selectivity / targeting of antimicrobial peptides to bacteria through reasonable structural design, reduce the non-specific binding of antimicrobial peptides to normal tissue cells, reduce damage to normal cells, and reduce the inactivation caused by non-specific adhesion of antimicrobial peptides to the biofilm matrix. SUMMARY

[0006] In order to solve the above technical problems, the present application provides an antibacterial poly-peptide macromolecular material, a product containing the same, a preparation method and application thereof.

[0007] To achieve the above object, the present application provides the following technical solutions.

[0008] An antibacterial poly-peptide macromolecular material, comprising an antibacterial poly-peptide, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; wherein the antibacterial poly-peptide has a structural formula as shown in formula (I):

[0009]

[0010] (I)

[0011] In formula (I), R1 is selected from or ; wherein h is 4-10, i is 4-113; R1 is more preferably at least one of , , , ;

[0012] R2 is selected from at least one of , ;

[0013] n is 6-30.

[0014] The antibacterial poly-peptide macromolecular material is used for preparing a medicine for preventing and / or treating bacterial infection.

[0015] The preparation method of the antibacterial poly-peptide macromolecular material comprises the following steps:

[0016] Esterification reaction is performed on an enol and glutamic acid, and then the product is reacted with triphosgene (bis (trichloromethyl) carbonate, chemical formula C3Cl6O3) to perform side group modification of glutamic acid, so as to obtain R2-NCA monomers;

[0017] R1-NH2 is used as an initiator to react with the R2-NCA monomers to obtain a poly-peptide;

[0018] Primary amines or quaternary amines are modified to the side groups of the poly-peptide by using a click reaction of a thiol group and a double bond, so as to finally prepare the antibacterial poly-peptide macromolecular material.

[0019] Further, the preparation method has the following specific steps:

[0020] (1) esterification of L-glutamic acid with 3-buten-1-ol in the presence of concentrated sulfuric acid, removal of alcohol under reduced pressure, neutralization to neutral with saturated sodium carbonate, and extraction by suction filtration to obtain a white solid; dissolving the white solid in a water / isopropanol mixture (v1 / v2 = 1:1) to reflux, cooling and crystallization, extraction by suction filtration and washing with ethyl ether, and drying under reduced pressure and freeze drying to obtain γ-(3-butenyl ester)-L-glutamic acid, which is dissolved in tetrahydrofuran (DMF), and triphosgene is added under ice bath to react, separation and purification to obtain R2-NCA monomer.

[0021] (2) dissolving the R2-NCA monomer in DMF, adding an initiator R1-NH2 to the solution to perform ring-opening polymerization, and performing precipitation and purification to obtain an amino-functionalized poly-peptide;

[0022] (3) mixing the poly-peptide and 2-aminoethanethiol hydrochloride in DMF, and performing thiol-double bond click reaction under deoxygenation, light shielding and ultraviolet light initiation, and performing dialysis purification and freeze drying after the reaction is completed to obtain an amine group-modified antibacterial poly-peptide, i.e., the antibacterial poly-peptide macromolecular material;

[0023] or,

[0024] mixing the poly-peptide and N,N-dimethylthiohexylamine hydrochloride in DMF, and performing thiol-double bond click reaction under deoxygenation, light shielding and ultraviolet light initiation, and performing dialysis purification and freeze drying after the reaction is completed, and reacting the obtained intermediate product with benzyl bromide and sodium bicarbonate in acetonitrile, and performing dialysis impurity removal and freeze drying of the reaction solution to obtain an amine group-modified antibacterial poly-peptide, i.e., the antibacterial poly-peptide macromolecular material.

[0025] Further, in step (1), the mass ratio of L-glutamic acid to 3-buten-1-ol is 10.0 g: 15 mL; and / or,

[0026] The mass ratio of γ-(3-butenyl ester)-L-glutamic acid to triphosgene is 10:6.

[0027] Further, in step (2), the molar ratio of the R2-NCA monomer to the initiator R1-NH2 is 10-20:1. Preferably, the molar ratio is 10:1 or 20:1.

[0028] Further, in step (3), the molar ratio of the poly-peptide to 2-aminoethanethiol hydrochloride, based on the molar amount of the side group double bond of the poly-peptide, is 1:3; and / or,

[0029] The molar ratio of the poly-peptide to N,N-dimethylthiohexylamine hydrochloride, based on the molar amount of the side group double bond of the poly-peptide, is 1:3; and / or,

[0030] The mass ratio of the intermediate product to the benzyl bromide is 50:380.

[0031] A medicine for preventing and / or treating bacterial infection, comprising an active ingredient and pharmaceutically acceptable adjuvants and / or carriers.

[0032] The active ingredient comprises an antibacterial polypeptide macromolecular material.

[0033] Further, the active ingredient further comprises an antibacterial drug, which is used in combination with the antibacterial polypeptide macromolecular material, and is administered alone or in combination.

[0034] Further, the administration mode of the medicine for preventing and / or treating bacterial infection comprises oral, rectal, parenteral.

[0035] Further, the dosage form of the medicine for preventing and / or treating bacterial infection comprises solid dosage form and liquid dosage form.

[0036] The solid dosage form comprises capsules, tablets, pills, powders and granules.

[0037] The liquid dosage form comprises pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.

[0038] Compared with the prior art, the present application has the following advantages and technical effects:

[0039] The antibacterial polypeptide macromolecular material disclosed by the present application has the advantages of being able to effectively penetrate and resist the barrier of biological membrane matrix, and specifically target bacterial membrane phospholipids, and overcomes the limitation that traditional antibacterial agents are difficult to act on bacteria in biological membranes.

[0040] The antibacterial polypeptide macromolecular material disclosed by the present application has high efficient cleaning ability on bacterial biological membranes.

[0041] The antibacterial polypeptide macromolecular material disclosed by the present application exhibits excellent in vivo antibacterial activity, can effectively inhibit biological membrane related infections, promote the repair of infected sites, and has good treatment effect and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0042] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute improper limitations on the present application. In the drawings:

[0043] Figure 1 is a Cm-CA nuclear magnetic hydrogen spectrum;

[0044] Figure 2 is mPEG x -CA n (x=45 or 113, n=10 or 20) nuclear magnetic hydrogen spectrum;

[0045] Figure 3NMR spectra of C6-MMBen;

[0046] Figure 4 Circular dichroism spectra of antibacterial peptides;

[0047] Figure 5 Statistical analysis of the interaction strength between antibacterial peptides and phospholipids (POPG, POPS);

[0048] Figure 6 Statistical analysis of the selectivity of antibacterial peptides; A, statistical analysis of the MIC values of antibacterial peptides; B, characterization of the hemolytic activity of antibacterial peptides; (C) characterization of the selectivity of antibacterial activity;

[0049] Figure 7 Thermal changes of antibacterial peptides interacting with proteins, DNA, and glycans;

[0050] Figure 8 Statistical analysis of the interaction strength between antibacterial peptides and POPG in the presence of a biofilm matrix or biofilm;

[0051] Figure 9 Characterization of the biofilm removal by antibacterial peptides; A, the inhibitory effect of drugs on the formation of bacterial biofilms; B, the removal of formed biofilms by drugs; C, the removal of biofilm matrix by drugs; D, the killing of bacteria in biofilms by drugs;

[0052] Figure 10 Live / dead staining of bacterial biofilms after drug treatment;

[0053] Figure 11 Investigation of the penetration of drugs into biofilms and targeting of bacteria in biofilms; A, experimental schematic; B, representative photos of the penetration of antibacterial peptides (Cy5-labeled, red) into bacterial biofilms after 3D confocal imaging; C, Z-axis photos of the penetration of antibacterial peptides (Cy5-labeled, red) into bacterial biofilms (GFP expression, green) after 3D confocal imaging; D, Z-axis photos of the penetration of antibacterial peptides (Cy5-labeled, red) into bacterial biofilms (mCherry expression, green) after 3D confocal imaging;

[0054] Figure 12 Mechanism study of the influence of proteins on the bactericidal activity of antibacterial peptides; statistical analysis of the particle size after co-incubation of antibacterial peptides with BSA (A), representative TEM photos (B); influence of BSA on the antibacterial activity of antibacterial peptides (C);

[0055] Figure 13 Study of antibacterial peptides in the treatment of bladder bacterial (E. coli ATCC 25922) infection in mice; A, experimental schematic; B, statistical analysis of the bacterial load after treatment of bladder infection in mice with antibacterial peptides; C, representative photos of H&E staining of tissues after treatment of bladder infection in mice with antibacterial peptides;

[0056] Figure 14 The study of antibacterial peptide treatment of mice with bladder bacterial infection; the statistical analysis of bacterial load in tissues of drug-treated mice with bladder infection of drug-resistant E. coli (A) and MRSA (B); representative photos of H&E staining of tissues of drug-treated mice with bladder infection of drug-resistant E. coli (C) and MRSA (D);

[0057] Figure 15 The study of drug peptide treatment of mice with bladder biofilm infection; A is an experimental schematic diagram; B is the statistical analysis of bacterial load in tissues of drug-treated mice with bladder bacterial biofilm infection;

[0058] Figure 16 The staining of sections of drug-treated mice with bladder biofilm infection; the red arrow points to the biofilm after crystal violet staining;

[0059] Figure 17 The study of alkyl amine-initiated, side group primary amine or quaternary amine functionalized poly-γ-(3-butenolide)-L-glutamate (poly-peptide) for the treatment of mouse periodontitis infection; A is an experimental schematic diagram; B is a representative photo of three-dimensional reconstruction after CT imaging of different groups; C is a representative photo of CT longitudinal section of different groups; the statistical analysis of CEJ-ABC (D), BV / TV (E), and Tp. Sp (F) of different groups;

[0060] Figure 18 The statistical analysis of CEJ-ABC (A), BV / TV (B), and Tp. Sp (C) of alkyl amine-initiated, side group primary amine functionalized poly-γ-(3-butenolide)-L-glutamate (poly-peptide) for the treatment of mouse periodontitis infection;

[0061] Figure 19 Representative photos of H&E staining (A) and Trap staining (B) of tissues of mice treated with alkyl amine-initiated, side group primary amine or quaternary amine functionalized poly-γ-(3-butenolide)-L-glutamate (poly-peptide);

[0062] Figure 20 Statistical analysis of the relative expression of TNF-α (A), iNOS (B), and IL-10 mRNA (C) in gingival tissues of mice treated with alkyl amine-initiated, side group primary amine or quaternary amine functionalized poly-γ-(3-butenolide)-L-glutamate (poly-peptide);

[0063] Figure 21 The study of alkyl amine-initiated, side group primary amine or quaternary amine functionalized poly-γ-(3-butenolide)-L-glutamate (poly-peptide) against biofilm of periodontitis-causing bacteria; A is the inhibitory effect of antibacterial peptide on Fn biofilm formation; B is the study of antibacterial peptide on the removal of Fn biofilm. DETAILED DESCRIPTION

[0064] The embodiment of the present application discloses an antibacterial polypeptide with a structure shown in formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof:

[0065]

[0066] (I)

[0067] In formula (I), R1 is selected from at least one of 、 、 、 、

[0068] R2 is selected from at least one of 、

[0069] In some embodiments, the antibacterial polypeptide is mixed with the salt solution at a ratio of 0.4 mg-30 mg: 4-6 mL.

[0070] The present application provides an application of the above antibacterial polypeptide or a stereoisomer or a pharmaceutically acceptable salt thereof in preparing a medicine for preventing and / or treating bacterial infection.

[0071] The bacterial infection model is bladder infection, pneumonia infection, muscle infection, and abdominal cavity infection.

[0072] The embodiment of the present application provides a medicine for preventing and / or treating bacterial infection, which is prepared from active ingredients and pharmaceutically acceptable adjuvants and / or carriers.

[0073] The active ingredients include the above antibacterial polypeptide or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0074] The embodiment of the present application provides a combined medicine for preventing and / or treating bacterial infection, and the active ingredients of the combined medicine include:

[0075] Component 1: the above antibacterial polypeptide or a stereoisomer or a pharmaceutically acceptable salt thereof; and

[0076] Component 2: an antibacterial medicine other than component 1.

[0077] The component 1 and the component 2 are respectively independent administration units, or the component 1 and the component 2 jointly form a combined administration unit, and specifically:

[0078] ​​The compound of formula (I) disclosed in the present application can be used in combination with other known antibacterial agents. When used in combination, the compound of formula (I) and the known agent can be administered to the patient simultaneously, separately or sequentially. The compound of formula (I) can be administered simultaneously or sequentially with the other known antibacterial agent.

[0079] When the compound of formula (I) is administered with another drug(s), a pharmaceutical composition containing both the compound of formula (I) and the known drug(s) is preferred. Administration of the drug combination also includes administration of the compound of formula (I) and the other known drug(s) within overlapping time periods.

[0080] When the compound of formula (I) is used in combination with other known drug(s), the dosage of the compound of formula (I) or the known drug(s) can be either the same as, or less than, that when administered alone.

[0081] The pharmaceutical composition for preventing and / or treating bacterial infection disclosed in the present application can be used in non-human mammals or humans.

[0082] The pharmaceutically acceptable excipient used in the pharmaceutical composition for preventing and / or treating bacterial infection disclosed in the present application refers to one or more compatible solid or liquid fillers or gel materials which are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0083] "Compatible" herein means that the components of the composition are capable of being commingled with the active ingredient of the present application (e.g., antibacterial polypeptide of formula I) and each other, without any significant degradation or inactivation of the active ingredient.

[0084] The pharmaceutically acceptable excipient used in the pharmaceutical composition for preventing and / or treating bacterial infection disclosed in the present application includes, but is not limited to, one or more of the following: solvents, excipients, fillers, compatibilizers, binders, humectants, disintegrants, retardants, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers, lubricants, wetting agents, suspending agents, flavoring agents and fragrances.

[0085] The pharmaceutically acceptable excipient includes, but is not limited to, cellulose and its derivatives (e.g., sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (e.g., propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween®), wetting agents (e.g., sodium dodecyl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water.

[0086] The mode of administration of the active ingredients or pharmaceutical compositions of the present application is not particularly limited, and representative modes of administration include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous).

[0087] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or with:

[0088] (a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid;

[0089] (b) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia;

[0090] (c) humectants, such as glycerol;

[0091] (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0092] (e) solution retarders, such as paraffin;

[0093] (f) absorption accelerators, such as quaternary ammonium compounds;

[0094] (g) wetting agents, such as cetyl alcohol and glycerol monostearate;

[0095] (h) adsorbents, such as kaolin;

[0096] (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof;

[0097] In capsules, tablets, and pills, the dosage form can also comprise buffering agents.

[0098] The solid dosage forms described above can also be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They can contain opacifying agents, and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the digestive tract. Examples of embedding compositions that can be used are polymeric substances and waxes.

[0099] Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, or elixirs. In addition to the active ingredient, the liquid dosage forms can include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, and the like, together with agents from

[0100] In addition to the active ingredient, suspensions can include suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar-agar, or mixtures of these substances.

[0101] The compositions for parenteral injection can comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

[0102] The present application also discloses a preparation method of the antibacterial polypeptide. The polypeptide is synthesized by initiating polymerization of R2-NCA with R1-NH2, and the amine groups are modified by side chain functionalization of R2, so as to obtain a series of antibacterial polypeptide high molecular materials. The reaction formula and the corresponding abbreviations are as follows:

[0103]

[0104] wherein, R1-NH2 is an initiator.

[0105] The side group modification of glutamic acid is realized by esterification of alcohol and glutamic acid under catalysis of concentrated sulfuric acid. After the reaction of glutamic acid and alcohol under catalysis of concentrated sulfuric acid is completed, the acid liquid is neutralized by saturated sodium carbonate solution, and the crude product is obtained by filtration and washing with ice water. The solid product is obtained by recrystallization of the crude product in a mixed solution of isopropyl alcohol and water (1:1, v / v), and the product is washed with ethyl ether, and the pure product is obtained after freeze-drying.

[0106] In the preparation of the polypeptide, the initiator R1-NH2 and the NCA monomer are fed according to a predetermined molar ratio, and the polypeptide solid is obtained by the poor solvent (ethyl ether: n-hexane = 1:1, v / v) precipitation method after the reaction is completed.

[0107] The method of side chain modification is as follows: in order to obtain the side group primary amine functionalized antibacterial polypeptide, the primary amine is modified to the side group of the polypeptide through the click reaction of thiol and double bond, after the modification is completed, the reaction solution is transferred to a dialysis bag with a molecular weight cut-off of 1 kDa, dialysis in deionized water for 24 h, water is changed every 2 h, and freeze-drying is performed, thereby obtaining the side group primary amine functionalized antibacterial polypeptide. In order to obtain the side group quaternary amine functionalized antibacterial polypeptide, the tertiary amine is modified to the side group of the polypeptide through the click reaction of thiol and double bond, and the method is consistent with that of the primary amine functionalization; further, the tertiary amine is modified to obtain the side group quaternary amine functionalized antibacterial polypeptide through the reaction of the tertiary amine and benzyl bromide.

[0108] The "room temperature" described in the present application refers to 20-30℃, unless otherwise specified.

[0109] The "parts" described in the present application refer to mass parts, unless otherwise specified.

[0110] The raw materials used in the present application are obtained by purchase on the market.

[0111] The technical solutions of the present application are further described through the following examples.

[0112] Example 1: Alkyl amine initiation, side group primary amine functionalized poly γ-(3-butenyl ester)-L-glutamic acid (polypeptide)

[0113] When n=10, ButLG-NCA is initiated with Cm-NH2 to obtain Cm-ButLG 10 The primary amine group is modified in the side group through the reaction of thiol and double bond to obtain the primary amine functionalized Cm-CA. The reaction equation is as follows:

[0114]

[0115] The specific preparation steps are as follows:

[0116] (1) Take 10.0 g of L-glutamic acid (L-Glu) and place it in a 100 mL round-bottom flask. Add 15 mL of 3-buten-1-ol and stir until it is evenly dispersed. Place the round-bottom flask in an ice water bath and slowly add 4 mL of concentrated sulfuric acid as a catalyst and dehydrating agent for the esterification reaction. Stir while adding the acid, taking about 20 minutes. Be careful not to add too quickly, as this can cause carbonization. After the addition is complete, place the round-bottom flask in a room temperature environment for 24 hours. The reaction liquid will become a clear, transparent, and viscous liquid. After the reaction is complete, remove the excess 3-buten-1-ol under reduced pressure until no more bubbles are produced. Add a small amount of saturated sodium carbonate solution to the reaction liquid and stir constantly to neutralize the concentrated sulfuric acid in the system. Monitor the pH of the reaction system with pH paper until the solution is neutral, then stop adding the saturated sodium carbonate solution. Use a Buchner funnel to filter and remove the solvent, resulting in a white solid. Wash the solid with a small amount of deionized water that has been treated at 4°C to remove residual salt. Transfer the white solid obtained by filtration to a round-bottom flask and add a mixture of deionized water and isopropyl alcohol (v1 / v2 = 1:1) to disperse the solid. Heat the dispersion in an oil bath (80°C) to reflux, and after the solid is completely dissolved, turn off the reflux system and allow the product to cool and crystallize naturally. Use a suction filter funnel to separate the solid product and wash it with ethyl ether to remove isopropyl alcohol and a small amount of organic impurities, resulting in a shiny, white, flaky solid. Place the solid in a 100 mL round-bottom flask and remove the volatile solvents such as ether under reduced pressure. Then, place the solid in a sample bottle, cool it in liquid nitrogen, and place it in a freeze dryer to freeze and dry the residual small amount of water, resulting in a flaky white solid, which is γ-(3-butenyl ester)-L-glutamic acid (ButLG).

[0117] (2) Take 10 g of ButLG and pump it overnight into a glove box. Add 250 mL of tetrahydrofuran (THF) and transfer it to an ice bath for stirring. Add 6 g of triphosgene and connect a condenser tube. Stir for about 10 minutes, then transfer it to an oil bath and react at 50 °C for about 2.5 hours. After drying, transfer it to a glove box. Remove impurities by column chromatography: first wash the impurities with 1 L of n-hexane, then separate the product using 500 mL of n-hexane: ethyl acetate = 1:1 (v / v) as the eluent. After the product solution is dried, ButLG-NCA is obtained and ready for use.

[0118] (3) Take 1.0 g of ButLG-NCA and dissolve it in 10 mL of N,N-dimethylformamide. Take one-tenth of the molar equivalent of Cm-NH2 of ButLG-NCA and dissolve it in 2 mL of N,N-dimethylformamide. Add the Cm-NH2 solution to the DMF solution in one go using a syringe, and react for 24 hours. Characterize the reaction system by Fourier infrared spectroscopy at 1789 cm -1The disappearance of the absorption peak indicates that the reaction is complete. The reaction liquid is drained, dissolved in 2 mL of dichloromethane, precipitated by adding dropwise into n-hexane, the supernatant is removed, and the polymer Cm-PButLG is obtained by draining. 10 , standby.

[0119] (4) The polymer Cm-PButLG prepared in step (3) is weighed. 10 5.0 g, completely dissolved in 3 mL of DMF. The mass of 2-aminoethanethiol hydrochloride is calculated according to three times the molar amount of the double bond of the side group of the poly-peptide, and after being weighed, it is dissolved in 2 mL of DMF and added to the reaction system, and stirred and dissolved. At the same time, the round-bottom flask is purged with nitrogen for 15 min to remove oxygen in the system to prevent the free radicals from being quenched. 10 mg of 2,2-dimethoxyphenyl phenylacetophenone is dissolved in 1 mL of DMF and added to the reaction system, and the reaction system is wrapped with tin foil for light shielding treatment. Continue to purge the reaction system with nitrogen for 15 min. After the purge is completed, turn on the ultraviolet point light source (wavelength of 365 nm) and insert it into the round-bottom flask to initiate the click reaction. After 1 h of reaction, turn off the point light source, and transfer the reaction liquid to a dialysis bag (molecular weight cut-off of 1 kDa) to remove impurities by dialysis in pure water, and change the water every 2 h, for a total of 24 h. After dialysis is completed, transfer the poly-peptide solution to a 50 mL centrifuge tube, freeze in liquid nitrogen, and then place the sample in a freeze dryer for 48 h to obtain a white solid. The structure of the antibacterial poly-peptide is verified by nuclear magnetic hydrogen spectrum, as shown in Figure 1 .

[0120] Example 2: Polyethylene glycol amine-initiated, side group primary amine functionalized poly-gamma-(3-butenyl lactate)-L-glutamic acid (poly-peptide)

[0121] When n = 10 or 20. ButLG-NCA is initiated with mPEG x -NH2 (x = 45 or 113) to obtain mPEG x -ButLG n (x = 45 or 113, n = 10 or 20), the primary amine group is modified on the side group by the reaction of thiol and double bond to obtain primary amine functionalized mPEG x -CA n (x = 45 or 113, n = 10 or 20). The reaction equation is as follows:

[0122]

[0123] The specific steps are as follows:

[0124] (1) The synthesis method of ButLG-NCA is consistent with that in Example 1.

[0125] (2) Take 1.0 g of ButLG-NCA and dissolve it in 10 mL of N,N-dimethylformamide. Take one-tenth or one-twentieth molar equivalent of mPEG x -NH2(x = 45 or 113) and dissolve it in 2 mL of N,N-dimethylformamide. Add the mPEG x -NH2(x = 45 or 113) solution to the DMF solution, and react for 24 h. The reaction system is characterized by Fourier infrared spectroscopy. The disappearance of the absorption peak at 1789 cm -1 indicates that the reaction is complete. The reaction liquid is drawn off, 2 mL of dichloromethane is added to dissolve it, and it is added dropwise to n-hexane to precipitate it. The supernatant is removed, and the polymer mPEG x -ButLG n (x = 45 or 113, n = 10 or 20) is prepared.

[0126] (3) Take the polymer mPEG x -ButLG n (x = 45 or 113, n = 10 or 20) prepared in step (3). 5.0 g, and add 3 mL of DMF to completely dissolve it. Calculate the mass of 2-aminoethanethiol hydrochloride according to three times the molar amount of the double bonds of the side groups of the poly-peptide, dissolve it in 2 mL of DMF, and add it to the reaction system, and stir to dissolve it. At the same time, the round-bottom flask is purged with nitrogen for 15 min to remove oxygen in the system to prevent the free radicals from being quenched. Take 10 mg of 2,2-dimethoxyphenyl phenylacetophenone and dissolve it in 1 mL of DMF, and add it to the reaction system, and wrap the reaction system with tin foil for light protection. Continue to purge the reaction system with nitrogen for 15 min. After the purging is complete, turn on the ultraviolet point light source (wavelength of 365 nm) and insert it into the round-bottom flask to initiate the click reaction. After 1 h of reaction, turn off the point light source, and transfer the reaction liquid to a dialysis bag (molecular weight cut-off of 1 kDa) to remove impurities by dialysis in pure water, and change the water every 2 h, for a total of 24 h. After the dialysis is complete, transfer the poly-peptide solution to a 50 mL centrifuge tube, freeze it in liquid nitrogen, and then place the sample in a freeze dryer for 48 h to obtain a white solid. The structure of the antibacterial poly-peptide is verified by nuclear magnetic resonance hydrogen spectrum, as shown in Figure 2 .

[0127] Example 3: Alkyl amine initiation, side group quaternary amine functionalized poly-γ-(3-butenol ester)-L-glutamic acid (poly-peptide)

[0128] When n = 10. ButLG-NCA is initiated with C6-NH2 to obtain C6-ButLG 10, the tertiary amine group is modified by the reaction of thiol and double bond, and further the tertiary amine is reacted with benzyl bromide to obtain the side group quaternary amine functionalized antibacterial poly peptide:

[0129]

[0130] The specific steps are as follows:

[0131] (1) The synthesis method of C6-ButLG is consistent with that in Example 1.

[0132] (2) Take C6-PButLG 10 Polymer 5.0 g, add 3 mL DMF to completely dissolve. Calculate the mass of N, N-dimethyl thioamine hydrochloride according to the three times molar amount of the side group double bond of the poly peptide, weigh it after dissolving in 2 mL DMF and adding it to the reaction system, and stir to dissolve. At the same time, the round bottom flask is purged with nitrogen for 15 min to remove oxygen molecules in the system to prevent free radicals from being quenched. Take 10 mg 2, 2-dimethoxyphenyl phenylacetophenone and dissolve it in 1 mL DMF, add it to the reaction system, and wrap the reaction system with tin foil for light protection. Continue to purge the reaction system with nitrogen for 15 min. After purging is completed, turn on the ultraviolet point light source (wavelength is 365 nm) and insert it into the round bottom flask to initiate the click reaction. After 1 h of reaction, turn off the point light source, transfer the reaction solution to a dialysis bag (molecular weight cut-off is 1 kDa), and dialyze in pure water to remove impurities, change water every 2 h, and dialyze for 24 h. After dialysis is completed, transfer the poly peptide solution to a 50 mL centrifuge tube, freeze in liquid nitrogen, and then place the sample in a freeze dryer for 48 h to obtain a white solid.

[0133] (3) Weigh 50 mg of the side group tertiary amine functionalized antibacterial poly peptide and dissolve it in 3 mL acetonitrile. Take 380 mg of benzyl bromide, dissolve it in 2 mL acetonitrile and add it to the reaction system. Add 1.5 times the molar amount of sodium bicarbonate to the side group of the tertiary amine functionalized poly peptide, and react at 80 ℃ for 24 h. After the reaction is completed, transfer the reaction solution to a dialysis bag (molecular weight cut-off is 1 kDa), and dialyze in pure water to remove impurities, change water every 2 h, and dialyze for 24 h. After dialysis is completed, transfer the antibacterial poly peptide solution to a 50 mL centrifuge tube, freeze in liquid nitrogen, and then place the sample in a freeze dryer for 48 h to obtain a white flocculent product, which is characterized by nuclear magnetic hydrogen spectrum to verify the structure of the antibacterial poly peptide (C6-MMBen), as shown in Figure 3

[0134] Effect verification:

[0135] I. Circular dichroism characterization of the helical structure of the antibacterial poly peptide

[0136] ​The helicity of the antimicrobial peptide was characterized using circular dichroism spectroscopy as follows: 5 mg of the antimicrobial peptide was dissolved in physiological saline to prepare a concentration of 10 mg / mL. -1 The antimicrobial peptide stock solution was prepared. 10 µL of the stock solution was added to 990 µL of physiological saline to obtain an antimicrobial peptide concentration of 0.1 mg / mL. -1 The solution was prepared using a 200 µL solution in a quartz cuvette with a 0.5 mm optical path length for circular dichroism spectroscopy. The measurement range was set to 200–260 nm, and the test time was 1 min. Each sample was tested twice at a temperature of 25 °C. After each sample was tested, the cuvette was rinsed once with physiological saline, then once with protein cleaning solution, and finally once with physiological saline to ensure it was thoroughly cleaned and would not affect subsequent tests. After the tests, the data were processed using Chirascan software. The data were averaged twice, and the physiological saline test results were used as background subtraction. The antimicrobial peptide helicity was calculated using CDNN software (Milli-Degrees was used for data simulation analysis). The experimental results are shown below. Figure 4 As shown in the figure, the synthesized antimicrobial peptides all have strong absorption peaks at 208 nm and 222 nm, proving that the series of antimicrobial peptides are all helical structures.

[0137] II. Isothermal titration quantitative calorimetry characterization of the interaction between antimicrobial peptides and phospholipids

[0138] Take 5 mg of antimicrobial peptide and dissolve it in PBS to prepare a 1.25 mM solution. Take POPG and POPS separately and dissolve them in PBS to prepare 1 mg / mL solutions. -1 A 1.25 mM solution was prepared, and the process required ultrasonic assistance (65 Hz, 30 s sonication, 5 s pause, total 2 min sonication) for dissolution. The interaction strength between the antimicrobial peptide and different phospholipids was characterized using ITC. The phospholipid solution was titrated with the antimicrobial peptide solution. ~200 μL of phospholipid solution was added to the sample cell, and ~70 μL of antimicrobial peptide solution was injected using a syringe to titrate the phospholipids. The test conditions were: stirring speed 750 rpm, reference power 10 μcal / s, test temperature 25 ℃, a total of 19 drops were titrated, with each drop equilibrated at 150 s intervals. The heat of dilution was subtracted by using the antimicrobial peptide solution to titrate PBS solution as background. The experimental results are as follows: Figure 5 As shown, C16-CA and C12-CA have a stronger effect on POPS than on POPG, while C6-CA and C6-MMBen have a stronger effect on POPG than on POPG. This demonstrates that the antimicrobial peptides C6-CA and C6-MMBen can selectively bind to POPG.

[0139] III. Characterization of the antimicrobial selectivity of antimicrobial peptides

[0140] (1) Antimicrobial activity characterization: The antimicrobial activity of the antimicrobial peptide was characterized by detecting the minimum inhibitory concentration (MIC) value of the antimicrobial peptide in the M9 medium without protein. After obtaining the bacteria in the stationary phase (E. coli ATCC 25922), a certain concentration of bacterial suspension was prepared with the medium (the concentration was 2 × 10 6 CFU mL -1 ). The antimicrobial peptide solution was prepared as follows: 10 mg of the antimicrobial peptide was dissolved with 0.9 % NaCl solution to obtain a mother liquor of 10 mg mL -1 , which was stored in a 4 ℃ refrigerator for later use. 12.8 μL of the mother liquor was added to 487 μL of M9 to obtain an antimicrobial peptide solution with a concentration of 256 μg mL -1 , and 100 μL of the solution was added to a 2 mL centrifuge tube, which was then diluted with M9 in gradient to obtain antimicrobial peptide concentrations of 256, 128, 64, 32, 16, 8, 4 and 0 μg mL -1 . A sterile 96-well plate was taken, 50 μL of the drug solution and 50 μL of the bacterial solution with corresponding concentrations were added to each well, the final concentration of the drug was 128, 64, 32, 16, 8, 4, 2 and 0 μg mL -1 , and the final concentration of the bacteria was 1 × 10 6 CFU mL -1 . The plate was placed in a 37 ℃ constant temperature incubator for incubation for 24 h, after the incubation was completed, the plate was taken out, the incubation wells were blown and mixed uniformly using a pipette gun, and the optical density (OD) value at 600 nm was measured using a microplate reader, 100 μL of the medium was used as a negative control, and the data was processed according to formula 1:

[0141] (Formula 1)

[0142] Wherein, OD Sample is the OD value of the experimental well, and OD Control is the OD value of the medium. The drug concentration when the OD 600 nm is about 0 is defined as the MIC value of the drug to the bacteria. The experimental results are shown in A of Figure 6

[0143] ​(2) Characterization of hemolytic activity: 1 mL of fresh sheep blood was added to 24 mL of PBS solution and centrifuged at 4 ℃ (3000 rpm, 5 min). After removing the supernatant, sheep blood red blood cells were collected. The red blood cells were resuspended in PBS and washed until the supernatant was colorless. After washing, the sheep blood was diluted with 25 mL of PBS solution to obtain a 4% sheep blood dilution, which was stored at 4 ℃. Approximately 5 mg of antimicrobial peptide was dissolved in sterile PBS to prepare 10 mg / mL solution. -1 The mother liquor was prepared at concentrations of 1024, 512, 256, 128, 64, 32, 16, 8, and 4 µg / mL. -1 Prepare gradient concentrations of antimicrobial peptide solutions. Add PBS solution to the negative control and 0.2% Triton X to the positive control. Take 100 µL of each solution from different groups into 1.5 mL centrifuge tubes (three replicates per concentration), and add an equal volume of 4% sheep blood diluent. Incubate the mixture at 37 ℃ for 1 h, then centrifuge at 2500 rpm for 5 min to separate intact red blood cells. Take 100 µL of the supernatant from each well and transfer it to a 96-well plate. Measure the absorbance (OD) of the supernatant at 576 nm using a microplate reader. 576 nm The hemolysis rate is calculated using Formula 2:

[0144] (Formula 2)

[0145] Among them, OD Sample This is the absorbance value of the experimental group samples, OD. Negative control This is the absorbance value of the negative control group, OD. Positive control This is the absorbance value of the positive control group sample. The experimental results are as follows: Figure 6 As shown in B.

[0146] (3) Characterization of antimicrobial selectivity: The concentration of antimicrobial peptides at a hemolysis rate of 10% was determined. 10 HC 10 ) and MIC The ratio of n) is defined as the selectivity value of the antimicrobial polypeptide, and the selectivity results are as follows: Figure 6 As shown in C; the selective calculation formula is as follows:

[0147] (Formula 3)

[0148] IV. Characterization of the interaction strength between antimicrobial peptides and biomembrane matrix

[0149] BSA was used to simulate the protein in the biological membrane matrix, DNA from bovine thymus was used to simulate the DNA in the biological membrane matrix, and hyaluronic acid was used to simulate the glycan in the biological membrane matrix. The interaction intensity of the antibacterial peptide with these substances was studied, respectively. In the experiment, the antibacterial peptide concentration was 1.25 mM, the BSA concentration was 12.5 mg mL -1 , the DNA concentration was 0.1 mg mL -1 , and the glycan concentration was 0.5 mg mL -1 . ITC was used to characterize the interaction intensity of the antibacterial peptide with different biological membrane components. The antibacterial peptide solution was used to titrate the biological membrane component solution, 200 μL of the biological membrane component solution was added to the sample cell, and 70 μL of the antibacterial peptide solution was used to titrate the biological membrane component with the sample needle. The test conditions were as follows: stirring speed 750 rpm, reference power 10 μcal / s, test temperature 25 ℃, a total of 19 drops were titrated, and the equilibrium was performed every 150 s. Among them, the antibacterial peptide solution was used to titrate the PBS solution as the background to deduct the dilution heat. The experimental results are shown in Figure 7 , and it can be seen from Figure 7 that C6-CA has weak effect on BSA protein, and C6-MMBen has strong effect on BSA; C6-CA and C6-MMBen have weak interaction with DNA and glycan.

[0150] V. Characterization of the influence of the biological membrane matrix on the interaction between the antibacterial peptide and PG phospholipid

[0151] BSA was used to simulate the protein in the biological membrane matrix, DNA from bovine thymus was used to simulate the DNA in the biological membrane matrix, and hyaluronic acid was used to simulate the glycan in the biological membrane matrix. The interaction intensity of the antibacterial peptide with these substances was studied, respectively. In the experiment, the antibacterial peptide concentration was 1.25 mM, the BSA concentration was 12.5 mg mL -1 , the DNA concentration was 0.1 mg mL -1 , and the glycan concentration was 0.5 mg mL -1 . ITC was used to characterize the interaction intensity of the antibacterial peptide with different biological membrane components. The antibacterial peptide solution was used to titrate the biological membrane component solution, 200 μL of the biological membrane component solution was added to the sample cell, and 70 μL of the antibacterial peptide solution was used to titrate the biological membrane component with the sample needle. The test conditions were as follows: stirring speed 750 rpm, reference power 10 μcal / s, test temperature 25 ℃, a total of 19 drops were titrated, and the equilibrium was performed every 150 s. Among them, the antibacterial peptide solution was used to titrate the PBS solution as the background to deduct the dilution heat. The experimental results are shown in Figure 8As shown in the figure, the protein (BSA) has no effect on the action of C6-CA and PG, and can significantly inhibit the action of C6-MMBen and PG; DNA and polysaccharide can weaken the action intensity of the antibacterial peptide and PG, and the weakening effect on C6-CA is smaller, while the weakening effect on C6-MMBen is larger; the bacterial biofilm matrix can also weaken the action intensity of the antibacterial peptide and PG, and the weakening effect on C6-CA is smaller, while the weakening effect on C6-MMBen is larger. The numbers above the column chart in the data represent the weakening multiple.

[0152] Six, Study on the biofilm resistance activity of antibacterial peptide

[0153] (1) Inhibition of antibacterial peptide on biofilm formation: After obtaining the bacteria in the platform phase (E. coli ATCC 25922), a bacterial suspension (concentration of 2 × 10 6 CFU / mL) was prepared using MHB medium containing 1% glucose. The antibacterial peptide was diluted with MHB medium containing 1% glucose to obtain a series of solutions with concentrations of 256, 128, 64, 32, 16 and 8 μg / mL. At the same time, ampicillin, a clinically used drug, was used as a control drug, and ampicillin was diluted twice in gradient according to the dilution method of the antibacterial peptide to obtain solutions with concentrations of 256 to 8 μg / mL. In a sterile 96-well plate, 50 μL of bacterial suspension and 50 μL of drug solution with corresponding concentration were added to each well, and the final concentration of the drug was 128, 64, 32, 16, 8, 4 and 0 μg / mL, and the final concentration of the bacteria was 1 × 10 6 CFU / mL, and the plate was incubated in a 37 ℃ constant temperature incubator for 24 h. After incubation, the supernatant was removed with a insulin syringe, and after washing with sterile PBS, 100 μL of thiazolyl blue solution (0.5 mg / mL, dissolved in MHB medium containing 1% glucose) was added to each well, and incubated in a 37 ℃ constant temperature incubator for 6 h. After incubation, the supernatant was removed with a insulin syringe, 100 μL of DMSO was added to each well to dissolve methylene blue, and the OD 500 nm was tested.

[0154] (Formula 4)

[0155] Wherein, OD Sample represents the absorption value of the experimental well at 500 nm, OD DMSO represents the absorption value of the solvent DMSO at 500 nm, and OD Control represents the absorption value of the 0 μg / mL treatment well at 500 nm, and the experimental results are shown in Figure 9 A.

[0156] (2) The removal effect of antibacterial peptide on the formed biofilm: The removal ability of the formed biofilm is an important index for evaluating the resistance of drugs to biofilm. In order to study the removal effect of antibacterial peptide on the formed biofilm, the biofilm sample was prepared: after obtaining the bacteria in the stationary phase (E. coli ATCC 25922), the bacterial suspension (concentration of 1 × 10 6 CFU / mL) was prepared using MHB medium containing 1% glucose. In a sterile 96-well plate, 100 μL of bacterial suspension was added to each well, and the plate was incubated in a 37 ℃ constant temperature incubator for 48 h. After the incubation, the supernatant was removed with an insulin injection needle, and clear biofilm samples were formed at the bottom of the wells. The drug was diluted with MHB medium containing 1% glucose to obtain a series of solutions with concentrations of 128, 64, 32, 16, 8, 4 and 0 μg / mL, and 100 μL of the drug was added to the wells containing the biofilm and incubated in a 37 ℃ constant temperature incubator for 24 h. After the drug incubation was completed, the supernatant was removed with an insulin needle, and 100 μL of thiazolyl blue solution (0.5 mg / mL, dissolved in MHB medium containing 1% glucose) was added to each well after washing with sterile PBS, and the plate was incubated in a 37 ℃ constant temperature incubator for 6 h. After the incubation was completed, the supernatant was removed with an insulin syringe, 100 μL of DMSO was added to each well to dissolve the MTT, and the OD 500 nm was measured. The biofilm removal rate was calculated using formula 5.

[0157] (Formula 5)

[0158] Wherein, OD Sample represents the absorbance of the experimental well at 500 nm, OD DMSO represents the absorbance of the solvent DMSO at 500 nm, and OD Control represents the absorbance of the 0 μg / mL treatment well at 500 nm. The experimental results are shown in B of Figure 9 , it can be seen that C6-CA has good antibiofilm activity, and the antibiofilm activity is comparable to that of ampicillin; and the antibiofilm activity of C6-MMBen is weak.

[0159] (3) To determine the biomass of biofilm after different treatments, mature biofilm of E. coli ATCC 25922 was exposed to antibacterial peptides or ampicillin at a concentration of 128 pg / mL for 24 hours. Subsequently, the treated biofilm was then fixed with formalin for 1 hour and air-dried. The biofilm was then stained with 0.02% crystal violet solution for 30 minutes and then washed with normal saline for three times. After staining, the biofilm was dissolved with 95% ethanol for 30 minutes. The OD of the dissolved solution was measured at 590 nm by using a microplate reader (PowerWave XS2, BioTek) to quantify the relative biomass of the biofilm. The experimental results are shown in Figure 9 As shown in FIG. 6C, it can be seen that C6-CA has a good biofilm matrix removal effect, and its removal activity is comparable to that of ampicillin; while C6-MMBen has a weak biofilm matrix removal effect.

[0160] (4) To evaluate the bactericidal activity of RAPs against bacteria in biofilm, mature biofilm of E. coli ATCC 25922 was exposed to RAPs or ampicillin at a concentration of 128 pg / mL for 12 hours. After treatment, the biofilm was collected in PBS and was destroyed by vigorous vortex. The viable cell count was then determined by the dilution plating method. The experimental results are shown in Figure 9 As shown in FIG. 6D, it can be seen that C6-CA has stronger resistance to biofilm activity.

[0161] Seven, Live-Dead Staining of Bacteria after Incubation of Antibacterial Peptides with Bacterial Biofilm

[0162] The method of live-dead staining was used to stain the bacteria after drug treatment of the biofilm to explore the survival of bacteria after the antibacterial peptides removed the biofilm. After obtaining the logarithmic phase bacteria (E. coli ATCC 25922), a bacterial suspension (concentration of 1 × 10 6 CFU / mL) was prepared using MHB medium containing 1% glucose, 1 mL of which was taken into a confocal culture dish and cultured for 48 h. After the culture was completed, the upper culture medium was sucked off with a pipette gun, and washed with sterile PBS, 1 mL of antibacterial peptide and ampicillin solution (the drug was prepared with MHB medium containing 1% glucose, and the concentration was 128 pg / mL) was added, and 1 mL of MHB medium containing 1% glucose was added as a negative control. After incubation in a 37 °C constant temperature incubator for 6 h, the supernatant was sucked off with a pipette gun, washed with sterile PBS, and physiological saline was used to prepare SYTO 9 / PI dye solution (the preparation method is described in the reagent instruction manual), 700 pL of dye solution was added to each dish, and the staining was carried out in the dark for 15 min. After the staining was completed, the dye was sucked off and 1% low-melting-point agarose solution (~ 50 °C) was added to fix the bacteria. The sample was observed using an Olympus rotating disk confocal microscope and recorded, and the experimental results are shown in Figure 10As shown, C6-CA has stronger biofilm clearance activity, and its clearance effect is stronger than that of ampicillin at the same concentration; C6-MMBen has relatively weak biofilm clearance effect.

[0163] Eight, penetration of antibacterial peptides into bacterial biofilm and bacterial targeting effect

[0164] To study the penetration of RAPs in biofilm, a mature biofilm was established using an E. coli strain expressing GFP in the cytoplasm. The biofilm was co-incubated with Cy5-labeled antibacterial peptides for 2 hours, and then washed with sterile PBS to remove unbound RAPs. The biofilm was then fixed with 0.1% agarose gel, and imaged in the GFP channel and Cy5 channel using a spinning disk confocal microscope (Olympus, Japan). The schematic diagram is shown in FIG. 6A. The obtained images were reconstructed into 3D stacked images using Imaris software, so as to analyze the penetration of RAPs in the biofilm. The experimental results are shown in FIG. 6B and C. Figure 11 Figure 11 A mature biofilm was established on the cell membrane using an E. coli strain expressing mCherry protein, so as to detect the targeting effect of RAPs on bacteria in the biofilm. The biofilm was co-incubated with cy5-labeled rap for 2 hours, and then washed with sterile PBS to remove unbound rap. The biofilm was then fixed with 0.1% agarose gel, and imaged in the mCherry channel and Cy5 channel using a spinning disk confocal microscope. The obtained images were reconstructed into 3D stacked images using Imaris software, so as to analyze the penetration and bacterial targeting of RAPs in the biofilm. The experimental results are shown in FIG. 7D. It can be seen from the above experimental results that C6-CA can efficiently penetrate the bacterial biofilm and efficiently target the bacteria in the biofilm. Figure 11

[0165] Nine, mechanism study of the effect of proteins on antibacterial activity of antibacterial peptides

[0166] ​​(1) Characterization of aggregate morphology after protein-antimicrobial peptide binding: To study the morphology of aggregates after antimicrobial peptide binding with BSA, dynamic light scattering (DLS) nanoparticle size analyzer was used to characterize the particle size of antimicrobial peptide after co-incubation with BSA, and TEM was used to characterize the particle morphology. The experimental method is as follows: Antimicrobial peptide was dissolved in pure water to obtain a solution with a concentration of 256 μg / mL, and BSA was dissolved in pure water to obtain a solution with a concentration of 25 mg / mL. 500 μL of antimicrobial peptide solution and 500 μL of BSA solution were added to a 2 mL centrifuge tube and co-incubated at room temperature for 1 h. After incubation, DLS test samples and TEM test samples were prepared respectively. For DLS samples: 100 μL of the mixture was added to 900 μL of pure water, and the particle size of the aggregates was tested by dynamic light scattering. Test conditions: The temperature was 25 ℃, and a total of 3 tests were conducted, with 5 cycles each time, and each cycle lasting 5 s. The experimental results are as follows. Figure 12 As shown in Figure A. For TEM samples: In a clean bench, four copper meshes were placed in 6 cm cell culture dishes and labeled. 10 μL of the mixed solution was dropped onto the copper meshes. After 10 seconds, the liquid was aspirated from the meshes. The culture dishes were then placed in a clean environment and allowed to air dry for approximately 20 minutes. The samples were then characterized, and the experimental results are shown below. Figure 12 As shown in Figure B, the experimental results above show that C6-CA does not form a clear assembly structure after co-incubation with BSA, while C6-MMBen forms a large aggregate after co-incubation with BSA. This indicates that C6-CA does not readily react with BSA and assemble, while C6-MMBen readily forms an aggregated assembly structure after reacting with BSA.

[0167] (2) Effect of protein on the antimicrobial activity of antimicrobial peptides: M9 medium was used as the solvent to prepare a medium with a BSA concentration of 12.5 mg / mL, resulting in BSA-containing M9 medium. Minimum inhibitory concentration (MIC) experiments were conducted on C6-CA and C6-MMBen against E. coli ATCC 25922 in both M9 medium and M9 medium containing BSA (12.5 mg / mL). The MIC values ​​of the antimicrobial peptides in media without BSA were compared to investigate the effect of BSA protein on the antimicrobial activity of the peptides. The experimental results are shown below. Figure 12 As shown in Figure C, it can be seen that BSA has a relatively small inhibitory effect on the antibacterial activity of C6-CA, but a strong inhibitory effect on the antibacterial activity of C6-MMBen.

[0168] 10. Research on the use of antimicrobial polypeptides in the treatment of bacterial bladder infections in mice.

[0169] To investigate the therapeutic effect of the antibacterial peptide on bacterial infection at the animal level, the infection models of mice bladder by standard strain E. coli ATCC 25922, clinically isolated pandrug-resistant E. coli (XDR-E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) were established respectively, and the treatment was carried out by in situ administration. The bacterial load of the bladder tissue after treatment was counted, and the tissue damage and inflammatory infiltration were analyzed by staining the section of the fixed bladder tissue to evaluate the therapeutic effect of the antibacterial peptide drug on infection.

[0170] (1) Research on antibacterial peptide for treating bacterial infection of mouse bladder by standard strain (E. coli ATCC 25922): The mice were deprived of water 18 h before the experiment, and the urine was emptied by repeatedly grabbing the mice in the middle. When the experiment was carried out, the bacteria (E. coli ATCC 259522) in the plateau were obtained, and the bacterial suspension with a concentration of 7.1 × 10 8 CFU / mL was prepared with sterile PBS. The mice were intraperitoneally injected with 160 ~ 180 μL of anesthetic (1% sodium pentobarbital solution), and the mice were completely anesthetized after about 8 ~ 10 min. The mice were placed on a constant temperature heating pad, and the urinary catheter was inserted into the mouse bladder with the aid of lubricating oil, and the residual urine was pressed out of the bladder. The mouse bladder was perfused with 70 μL of bacterial solution (5 × 10 7 CFU / bladder) using a urinary catheter, and the bacterial solution was retained in the bladder for 2 h. Treatment was carried out 6 h after infection: the mice were anesthetized by intraperitoneal injection of ~ 140 μL of anesthetic (1% sodium pentobarbital solution), and the mice were placed on a constant temperature heating pad. The mouse bladder was perfused with 70 μL of sterile PBS (control) or drug solution (C6-MMBen or C6-CA) using a urinary catheter, and was retained for 2 h, wherein the treatment dose of C6-MMBen was 1 mg / mL (70 μg / bladder), and the treatment dose of C6-CA was 0.5 mg / mL (35 μg / bladder) and 1 mg / mL (70 μg / bladder), with 7 mice in each group. After 24 h of bacterial infection, the mice were sacrificed by cervical dislocation, and the mouse bladder was dissected for tissue homogenization grinding (300 μL of sterile water, 4 steel balls with a diameter of 3 mm and 1 steel ball with a diameter of 4 mm were added to the homogenizer; the homogenization program was 80 Hz for 120 s and 60 s for 3 times). The tissue solution was diluted in sterile water (10, 100, 1000, 10000 times), and the above dilutions and the original tissue solution were plated (10 μL of volume) for colony counting. The agar plates were incubated in a 37 ℃ constant temperature incubator for 12 ~ 20 h, and the results were as follows: Figure 13As shown in FIG. 6, it can be seen that the bacterial load in the bladder of mice was significantly reduced after treatment with antibacterial peptides, but the average bacterial load in the 70 μg / bladder C6-CA treatment group (C6-CA_70) was lower than that in the 70 μg / bladder C6-MMBen treatment group (C6-MMBen), indicating that C6-CA has better therapeutic effect.

[0171] (2) Study on antibacterial peptides for treating bladder bacterial infection in mice with drug-resistant strains (E. coli ATCC 25922): The research method of antibacterial peptides for treating bladder infection in mice infected with XDR-E. coli is consistent with that in (1), wherein colistin, a clinically used drug, is used as a control drug, and the dose of colistin is consistent with that of antibacterial peptides. The research method of antibacterial peptides for treating bladder infection in mice infected with MRSA is also consistent with the above method, wherein vancomycin, a clinically used drug, is used as a control drug, and the dose of vancomycin is consistent with that of antibacterial peptides. The experimental results are shown in FIG. 7. Figure 14 As shown in FIG. 7, it can be seen that the bacterial load in the bladder of mice was significantly reduced in the C6-CA treatment group and the antibiotic treatment group, and there was no significant difference between the C6-CA treatment group and the antibiotic treatment group, indicating that C6-CA has a therapeutic effect comparable to that of antibiotics.

[0172] Eleven, Study on antibacterial peptides for treating bladder biofilm infection in mice

[0173] In order to study the therapeutic effect of antibacterial peptides on bacterial biofilm infection at the animal level, a biofilm infection model of standard strain E. coli ATCC 25922 in the bladder of mice was established, and treatment was performed by in situ administration. The bacterial load in the bladder tissue after treatment was counted, and the biofilm clearance effect and inflammatory infiltration were analyzed by staining the fixed bladder tissue sections to evaluate the therapeutic effect of antibacterial peptides on infection.

[0174] Firstly, 7-week-old C57BL / 6j female mice were given 0.1% (volume ratio) Tween-80 by bladder perfusion (50 μl per mouse). After 12 hours of perfusion, the mice were infected by bladder perfusion, and 5x10 8 CFU of E. coli ATCC 25922 strain was injected into each bladder. After 24 hours of infection, PBS, C6-CA (70 μg per bladder) or ampicillin (70 μg per bladder) was given by bladder perfusion, respectively. The mice were sacrificed after 12 hours of treatment. The bladder tissue was taken out, homogenized with sterile water, and then gradient diluted for colony counting on LB agar plates. The bacterial load in the bladder tissue of each mouse (CFU / tissue) was calculated, and the experimental results are shown in FIG. 6. Figure 15Histological analysis, the bladder was collected and fixed with 4% paraformaldehyde in PBS. Healthy mouse bladder as negative control was also fixed with 4% paraformaldehyde in PBS. Then the bladders were paraffin-embedded, sectioned and stained with H&E and crystal violet (0.1%) staining. The results are shown in FIG. 6. Figure 16 As shown in FIG. 6, the results of the above experiments show that both the C6-CA treatment group and the ampicillin treatment group can significantly reduce the bacterial load in the bladder of the mice after biofilm infection, and there is no significant difference between the C6-CA treatment group and the ampicillin treatment group, indicating that C6-CA has a therapeutic effect comparable to that of ampicillin. At the same time, after treatment with C6-CA and ampicillin, the thickness of the bladder biofilm in the mice is reduced, indicating that both C6-CA and ampicillin can eliminate the bladder biofilm.

[0175] Twelfth, research on the use of antibacterial peptide for the treatment of mouse periodontitis

[0176] Periodontitis is a chronic inflammatory disease caused by bacterial microorganisms, affecting the health and life of about 700 million people worldwide. The present application establishes a primary periodontitis infection model to evaluate the therapeutic effect of antibacterial peptide on periodontitis.

[0177] (1) Establishment of periodontitis model and experimental grouping: The animals used in this experiment were purchased from Zhuhai Baisitong Biotechnology Co., Ltd. The animal experiment was approved by the Lingjutopu Ethics Committee. The mice were anesthetized by intraperitoneal injection of 1.25% tri- bromoethanol at 0.2 mL / 10 g, and the limbs were spread and fixed on the mouse plate with adhesive tape. A 5-0 silk thread was used to pull the upper and lower incisors to keep the mouth open, and a microvessel clamp was used to place the silk thread in the interproximal space between the first and second molars and between the second and third molars on both sides of the maxilla. After triple ligation on the palate, the excess thread was cut off. Twenty C57BL / 6 mice (6-8 weeks old, female, SPF level) weighing about 20 g were selected. After 7 days of adaptive feeding in the barrier system animal house, the mice were randomly divided into 4 groups: healthy control group (Control group), periodontitis group (PD group), C6-CA administration group (C6-CA group), and C6-MMBen administration group (C6-MMBen group). From the 3rd day after the establishment of the periodontitis model, local administration was started, with 2 mg / mL gingival local injection and 0.08 mg / mL periodontal local irrigation alternately. When C6-CA and C6-MMBen were administered, microsyringes were used for gingival local injection (10 μL per mouse) on the 3rd, 5th, 7th, and 9th days, and insulin syringes were used for periodontal local irrigation (25 μL per mouse) on the 4th, 6th, and 8th days. 45 -CA 10 , mPEG 45 -CA 20 and mPEG113 -CA 10 The administration mode was 2 mg / mL gingival local injection and 0.08 mg / mL periodontal local irrigation alternately, the local injection volume was 10 μL, and the local irrigation volume was 25 μL. Each group of mice was administered once a day, and all animals were euthanized on the 10th day of the experiment. The animal experiment process is specifically shown in Figure 17 A.

[0178] (2) Alveolar bone imaging and three-dimensional reconstruction: After the mice were sacrificed, the right maxilla was quickly separated, the silk thread was removed, the gingiva was peeled off, and it was fixed in 4% paraformaldehyde solution. After 48 h, it was soaked in 75% alcohol. The right maxilla sample was scanned by Quantum GX2-2-E animal Micro-CT scanning image system, and the specific parameter settings were voltage 90 kV, current 88 μA, slice thickness 10 μm, and exposure time 4 min. Then the collected data were reconstructed into three-dimensional images by Quantum GX2 software. The experimental results are shown in Figs. 2B and 2C, it can be seen that the alveolar bone absorption of the mice after the treatment of the antibacterial peptide is improved, and the C6-CA treatment group has a better improvement effect. Figure 17

[0179] (3) Measurement and analysis of the distance between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC): Using the SkyScan DataViewer software, six specific positions of the right maxillary second molar of the mice were measured on the sagittal and coronal planes, including the buccal mesial, buccal central, buccal distal, and palatal mesial, palatal central, and palatal distal. The vertical distance between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC) was determined to evaluate the differences in alveolar bone absorption between the groups. To ensure data accuracy, two researchers independently completed the measurement work under double-blind conditions, and each sample was measured twice to eliminate possible measurement bias. The experimental results are shown in Fig. 2D. The above results show that the CEJ-ABC of the periodontitis mice is significantly reduced after the treatment of the antibacterial peptide, indicating that the periodontitis of the mice is significantly improved after the treatment of the antibacterial peptide. The C6-CA treatment group has a better improvement effect. Figure 17

[0180] ​​(4) Calculation and analysis of bone volume fraction (BV / TV) and trabecular separation (Tb.Sp): The three-dimensional reconstruction images were saved as sagittal views using SkyScan DataViewer software, and then the buccal mesial root, buccal distal root and bifurcation area of the second maxillary molar were selected for alveolar bone analysis using CT analysis software (CTAn). Fifteen consecutive sections were selected on the sagittal plane, and parameters such as the vertical distance of the cement-enamel junction-alveolar bone crest (CEJ-ABC), bone volume fraction (BV / TV) and trabecular separation (Tb.Sp) were analyzed. The experimental results are shown in Figs. 16A-16C. Figure 17 E and F and Figure 18 It can be seen that after C6-CA or C6-MMBen treatment, the BV / TV of the administration group increased, and the Tb.Sp decreased, indicating that the bone resorption of periodontitis was inhibited, and the alveolar bone microstructure was partially repaired, and C6-CA had better treatment effect; after the administration of the polyethylene glycol amine-initiated, side group primary amine functionalized antibacterial peptide, the CEJ-ABC distance of the administration group was significantly shortened, the BV / TV increased, and the Tb.Sp decreased, indicating that the bone resorption of periodontitis was inhibited, and the alveolar bone microstructure was partially repaired.

[0181] Thirteen, histological section staining of mice after antibacterial peptide treatment of periodontitis infection

[0182] Histological section preparation: After collecting the fresh left maxillary bone specimen, the surface blood stains and hair impurities were removed using physiological saline, and then the specimen was immersed in 4% paraformaldehyde solution for 48 h and transferred to 75% ethanol for storage. Then the sample was placed under flowing tap water for continuous flushing for 6 h to remove residual substances, and then the tissue was placed in 10% EDTA decalcification solution for 4 weeks of decalcification treatment in a 37°C constant temperature oscillator, until the bone tissue became soft and could be easily pierced by a needle tip. After decalcification, the target area was trimmed using a special knife in a fume hood, and the trimmed tissue was placed in a labeled dehydration box. The dehydration program was to use ethanol with increasing concentration (75% to absolute ethanol) for dehydration, then use xylene to make the sample transparent, and finally immerse in paraffin three times, each time for 1 h. After completing the paraffin immersion, the embedding machine was used for embedding, and the melted paraffin was injected into the embedding frame, and the tissue was taken out from the dehydration box before the wax solidified, and placed in the embedding frame with the sagittal plane facing down, and placed in a-20°C cooling table for cooling. After demolding, the excess paraffin around the embedding box was removed using a wax trimming instrument, and the tissue sample was stored at 4°C. Before sectioning, the wax block was frozen overnight in a-20°C environment. Continuous sections were made using a microtome, with a section thickness of 3 μm. After sectioning, the wax sheet was placed in a 60°C oven for baking, and then transferred to 4°C for storage.

[0183] (1) Hematoxylin-eosin staining: First, the sections were dewaxed by placing them in xylene three times for 5 min each. Then, the sections were dehydrated by placing them in absolute ethanol twice for 5 min each and 95% ethanol twice for 5 min each. For staining, the sections were first stained with hematoxylin for 5 min, rinsed with tap water for 1 min, treated with differentiation solution for 2 s, and rinsed with running water for 8 min. After further dehydration, the sections were stained with eosin solution for 2 min. Before mounting, the sections were passed through 95% ethanol twice for 2 min each, absolute ethanol twice for 2 min each, and xylene three times for 3 min each, and finally mounted. The changes in alveolar bone height of the left maxillary second molar were observed using a microscope, and the images were scanned using Aperio Image Scope. The experimental results are shown in FIG. 2A. Figure 19 As shown in FIG. 2A, it can be seen that after treatment with antibacterial peptides, the apical migration of epithelial cells, dense inflammatory cell infiltration, vascular congestion, epithelial ulceration, and the increase in the CEJ-ABC distance were all improved, and the C6-CA treatment group showed better improvement.

[0184] (2) Tartrate-resistant acid phosphatase staining: The embedded tissue sections were preheated in a 37°C incubator to bring the sample to the appropriate temperature. Then, the sample was dewaxed by placing it in xylene solutions I and II for 10 min each to remove the paraffin components. Next, the sample was treated with absolute ethanol for 5 min and then with 75% ethanol solution for 5 min to complete the dehydration process. After that, the sample was transferred to distilled water and left to stand for 2 min to ensure that the tissue was fully hydrated. The pre-treated sections were placed in freshly prepared TRAP incubation solution and incubated at 37°C in the dark for 50 min. After incubation, the residual reagents were immediately removed by rinsing with running water for 1 min. The sample was stained with hematoxylin solution for 2 min and then rinsed with running water to remove excess stain. After staining, the sections were transparentized by placing them in xylene for 5 min. After the sections were slightly dried, they were mounted with neutral resin in preparation for microscopic examination. The distribution of TRAP-positive cells in the alveolar bone region between the first and second molars of the mouse maxilla was observed under a microscope, and images were obtained using an EasyScan6 scanner. Quantitative analysis was performed using Image J software. The experimental results are shown in FIG. 3B. Figure 19 As shown in FIG. 3B, it can be seen that after treatment with antibacterial peptides, the number of osteoclasts (indicated by arrows) decreased, and the C6-CA treatment group showed a more significant decrease.

[0185] Fourteen, Detection of Inflammatory Factors after Treatment of Mouse Periodontitis with Antibacterial Peptides

[0186] After the mice were euthanized, the gingival tissue around the right maxillary second molar was quickly isolated. The isolated sample was gently rinsed with physiological saline to remove surface impurities, and then the treated tissue was quickly frozen and stored in liquid nitrogen.

[0187] The total RNA was isolated from the right maxillary gingival tissue according to the operation manual of the RNA extraction kit. First, the sample to be tested was placed in a mortar and ground to fine particles after adding liquid nitrogen. Then, lysis buffer was added and the tissue was lysed at room temperature for 5 min. The lysate was transferred to a 1.5 ml centrifuge tube, 150 μL Buffer A solution was added, and after vigorous shaking for 15 s, it was left to stand at room temperature for 5 min. Next, it was centrifuged at 15000 g at 4°C for 5 min, and 200 μL supernatant was transferred to a new centrifuge tube. After mixing well with an equal volume of anhydrous ethanol, the mixture was transferred to a centrifugal column, which was centrifuged at 4°C and 4000 g for 1 min. Then, 500 μL Wash Buffer 1 and Wash Buffer 2 were used to wash the centrifugal column in turn, and after each washing, it was centrifuged at 4°C and 12000 g for 1 min. After washing was completed, the centrifugal column was empty at the same conditions for 1 min to remove residual liquid. The centrifugal column was transferred to a new EP tube, and the cap was opened to dry for 2 min. 30 μL Elution Buffer was added to the center of the centrifugal column membrane, which was left to stand at room temperature for 2 min, and then centrifuged at 4°C and 12000 g for 1 min. The centrifugal column was discarded, and the collected liquid was the total RNA of the tissue. The obtained RNA was immediately placed on ice, and after determining the concentration using Nano Drop 2000, it was stored in a -80°C ultra-low temperature refrigerator for standby.

[0188] According to the operation manual of the reverse transcription kit, 1 μg of total RNA sample was weighed and transferred to an enzyme-free EP tube. Then, 2 μL of gDNA removal agent was added, mixed gently by blowing and sucking, and reacted at room temperature for 5 min. After the reaction was completed, it was immediately placed on ice, 5 μL of 4×RT Master Mix was added, and mixed well. The reaction system was adjusted to 20 μL with ddH2O, and mixed again. The reverse transcription program was set as follows: 42°C for 15 min, followed by 95°C for 30 s. After the reaction was completed, the obtained cDNA product was stored in a -80°C ultra-low temperature refrigerator for subsequent experiments.

[0189] The mixture required for qRT-PCR reaction was prepared on ice according to the instructions of EZB ® 2x Color SYBR Green gPCR Master Mix (ROX2 plus). The reaction conditions were set as follows: 95°C pre-denaturation for 5 min, followed by 40 cycles of 95°C denaturation for 10 s and 60°C annealing / elongation for 30 s. The obtained cycle threshold (Ct) was detected by a fluorescent quantitative PCR instrument, and the relative expression amount of GAPDH, TNF-α, iNOS and IL-10 mRNA was calculated by 2 -△△CT The experimental results are shown in Table 2 and Figure 1.Figure 20 As shown in the figure, compared with the periodontitis group, the levels of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) were reduced after antibacterial peptide treatment, and the level of interleukin-10 (IL-10) was increased. Among them, the TNF-α reduction in the C6-CA treatment group was greater, which proved that C6-CA had stronger anti-inflammatory effect.

[0190] Table 1 Primer sequence

[0191]

[0192] Fifteen, Study on antibacterial peptides against periodontitis pathogenic bacteria biofilm

[0193] Fusobacterium nucleatum (Fn) is a gram-negative obligate anaerobic bacterium and one of the key oral symbionts. Fn plays a crucial bridge role in the formation of plaque biofilm and promotes the development of periodontitis together with other periodontal disease pathogens. Taking Fn as a model, we evaluated the efficacy of antibacterial peptides in inhibiting Fn biofilm formation and eliminating established biofilm.

[0194] (1) Inhibition effect of antibacterial peptides on Fn bacterial biofilm formation: First, bacterial film-forming medium was configured, 0.5 g of glucose was added to 50 ml of bacterial liquid medium, and after mixing evenly, it was filtered to obtain bacterial film-forming culture medium containing 1% glucose. Then, the Fn bacterial stock solution was diluted to 2×10 6 CFU / mL with the culture medium. Subsequently, C6-CA and C6-MMBen were diluted with normal saline to prepare gradient concentrations of antibacterial peptide solutions, with concentrations of 256 µg / mL, 128 µg / mL, 64 µg / mL, 32 µg / mL, 16 µg / mL, 8 µg / mL. In the 96-well plate, Fn bacterial suspension and corresponding concentration of antibacterial peptide solution were added in a ratio of 1:1 per well, with a total volume of 100 µL per well, so that the final concentration of antibacterial peptides in each well was 128 µg / mL, 64 µg / mL, 32 µg / mL, 16 µg / mL, 8 µg / mL, 4 µg / mL, and the bacterial concentration was 1×10 6CFU / mL, the control group was added with 50 μL bacterial suspension and 50 μL normal saline, and finally the 96-well plate was placed in a 37℃ anaerobic incubator for incubation. After 24 hours, the 96-well plate was tilted, and the bacterial supernatant was carefully sucked along the bottom edge of one side of the plate with an insulin syringe. After the supernatant was completely sucked, a thin film of turbidity was formed at the bottom of the 96-well plate, and then 100 μL of 0.5 mg / ml thiazolyl blue (MTT) solution was added to each well, and incubated in the dark for 4 hours. After the incubation was completed, the MTT liquid was completely sucked with an insulin syringe, and the biofilm at the bottom of the plate was blue-violet in color, and a photograph was taken. 100 μL of DMSO liquid was added to each well to dissolve the methylene blue, and the absorbance value was measured at a wavelength of 500 nm. The biofilm formation rate was calculated using the formula: Figure 21 As shown in FIG. 2A, it can be seen that the antibacterial peptide has an inhibitory effect on the formation of Fn biofilm, and C6-CA has a stronger inhibitory effect.

[0195] (2) Study on the clearing effect of antibacterial peptide on Fn bacteria biofilm: prepare bacterial biofilm culture medium, bacterial suspension and antibacterial peptide solution of C6-CA and C6-MMBen series concentration. In the 96-well plate, add the diluted bacterial suspension, the total amount of each well is 100 μL, and the concentration of bacteria is 1×10 6 CFU / mL, and then the 96-well plate was placed in a 37℃ anaerobic incubator for incubation for 24 hours. After 24 hours, the 96-well plate was tilted, and the bacterial supernatant was carefully sucked along the bottom edge of one side of the plate with an insulin syringe. After the supernatant was completely sucked, the bacterial biofilm culture medium and the corresponding concentration of antibacterial peptide solution were added to the corresponding wells in a 1:1 ratio, and the control group was added with the same volume of bacterial biofilm culture medium and normal saline. The total amount of each well was 100 μL, and after being placed in a 37℃ anaerobic incubator, it was incubated for 24 hours. After 24 hours, MTT staining was performed and the biofilm survival rate was calculated, and the experimental results are shown in FIG. 2B. It can be seen that the antibacterial peptide has a clearing effect on the mature Fn biofilm, and C6-CA has a stronger clearing effect. Figure 21

[0196] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.​

Claims

1. An antibacterial polypeptide polymer material, characterized in that, This includes antimicrobial peptides, or their stereoisomers, or their pharmaceutically acceptable salts; The structural formula of the antimicrobial polypeptide is shown in formula (I): （I） In equation (I), R1 is selected from Or #imgpt2#; where h is 4-10 and i is 4-113; R2 is n is 6-30.

2. The use of the antimicrobial polypeptide polymer material as described in claim 1 in the preparation of medicaments for the prevention and / or treatment of bacterial infections.

3. A method for preparing the antibacterial polypeptide polymer material as described in claim 1, characterized in that, Includes the following steps: Enol undergoes esterification with glutamic acid, and the product is then reacted with triphosgene to obtain the R2-NCA monomer. A polypeptide was obtained by reacting R1-NH2 with the R2-NCA monomer using R2-NH2 as an initiator. The antibacterial polypeptide polymer material was finally prepared by modifying the primary amine onto the side group of the polypeptide using a click reaction between a thiol group and a double bond.

4. The method for preparing the antibacterial polypeptide polymer material according to claim 3, characterized in that, The specific steps are as follows: (1) L-glutamic acid was esterified with 3-buten-1-ol under concentrated sulfuric acid catalysis. After removing the alcohol under reduced pressure, the solid was neutralized with saturated sodium carbonate and filtered to obtain a white solid. The solid was dissolved in a water / isopropanol mixture and refluxed. After cooling and crystallization, the solid was filtered and washed with ice-cold ether. After drying under reduced pressure and freeze drying, γ-(3-butenol ester)-L-glutamic acid was obtained. The solid was dissolved in DMF and reacted with triphosgene under ice bath. After separation and purification, R2-NCA monomer was obtained. (2) The R2-NCA monomer was dissolved in DMF and initiator R1-NH2 was added to it for ring-opening polymerization, followed by precipitation and purification to obtain amino-terminated functionalized peptides. (3) The polypeptide and 2-aminoethanethiol hydrochloride are mixed in DMF and subjected to a thiol-double bond click reaction. After the reaction is completed, the mixture is purified by dialysis and lyophilized to obtain an antimicrobial polypeptide modified with side chain amine groups, which is the antimicrobial polypeptide polymer material. or, The peptide and N,N-dimethylthiohexylamine hydrochloride were mixed in DMF and subjected to a thiol-double bond click reaction. After the reaction was completed, the mixture was purified by dialyzing and lyophilized. The intermediate product was then reacted with benzyl bromide and sodium bicarbonate in acetonitrile. The reaction solution was dialyzed to remove impurities and then lyophilized to obtain the antimicrobial peptide with side chain amine modification, which is the antimicrobial peptide polymer material.

5. The method for preparing the antibacterial polypeptide polymer material according to claim 4, characterized in that, In step (1), the ratio of L-glutamic acid to 3-buten-1-ol is 10.0 g : 15 mL; and / or, The mass ratio of γ-(3-butenol ester)-L-glutamic acid to triphosgene is 10:

6.

6. The method for preparing the antibacterial polypeptide polymer material according to claim 4, characterized in that, In step (2), the molar ratio of the R2-NCA monomer to the initiator R1-NH2 is 10-20:

1.

7. The method for preparing the antibacterial polypeptide polymer material according to claim 4, characterized in that, In step (3), the molar ratio of the polypeptide to 2-aminoethanethiol hydrochloride is 1:3, based on the molar amount of the double bond in the polypeptide side group; and / or, Based on the molar amount of double bonds in the side groups of the peptide, the molar ratio of the peptide to N,N-dimethylmercaptohexylamine hydrochloride is 1:3; and / or, The mass ratio of the intermediate product to the benzyl bromide is 50:

380.

8. A drug for the prevention and / or treatment of bacterial infections, characterized in that, This includes the active ingredient and pharmaceutically acceptable excipients and / or carriers; The active ingredient includes the antimicrobial polypeptide polymer material as described in claim 1.

9. A medicament for preventing and / or treating bacterial infections according to claim 8, characterized in that, The active ingredient also includes an antibacterial drug, which is used in combination with the antibacterial polypeptide polymer material for administration alone or in combination.

10. A medicament for preventing and / or treating bacterial infections according to claim 8, characterized in that, The dosage forms of the medicines used for the prevention and / or treatment of bacterial infections include solid dosage forms and liquid dosage forms; The solid dosage forms include capsules, tablets, pills, powders, and granules; The liquid dosage forms include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures.

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