Chemotactic response type self-assembly antibacterial peptide as well as preparation method and application thereof

Abstract

The invention discloses a chemotactic response type self-assembly antibacterial peptide and a preparation method and application thereof, the antibacterial peptide comprises: (a) a targeting module, the amino acid sequence of which is selected from KPRSVS or a variant thereof, GPLVPRG or a variant thereof, and GPLGVRG or a variant thereof; (b) a self-assembly module which is composed of hydrophobic residues and hydrophilic residues which are alternately arranged and forms beta-folding driving nanofibers; (c) an inflammation response module which is selected from a histidine enrichment region, an ROS sensitive group or an MMP protease cleavage site and is used for responding to an inflammation microenvironment and enhancing target site enrichment; (d) a stability enhancing module comprising at least one of D-type amino acid modification, N-terminal PEG4 connection or cyclization structures. The antibacterial peptide disclosed by the invention has chemotactic effect on an inflammatory microenvironment, can accurately identify gram-negative bacteria, is triggered in the inflammatory environment, and plays an accurate bactericidal effect on various pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a smart antimicrobial peptide that precisely identifies Gram-negative bacteria through phage receptor-specific recognition characteristics, gravitates towards the inflammatory environment, and triggers the self-assembly of nanostructures within the inflammatory microenvironment. This peptide is suitable for treating deep tissue infections caused by multidrug-resistant bacteria, particularly biofilm-associated infections (such as catheter infections and chronic wounds), and its physical membrane-breaking mechanism can circumvent the target resistance problem of traditional antibiotics. Background Technology

[0002] The existing technology has the following drawbacks:

[0003] The drug resistance dilemma: Traditional antibiotics (such as β-lactams) work by inhibiting a single metabolic target, and bacteria are prone to developing drug resistance through gene mutations (such as the production of β-lactamases).

[0004] The pain points of antimicrobial drugs and current antimicrobial peptides are shown in Table 1:

[0005] Table 1

[0006]

[0007] Non-specific killing: Conventional self-assembled antimicrobial peptides (such as RAD16-II) destroy microbial membranes through electrochemical interactions, without distinguishing between Gram-positive bacteria, Gram-negative bacteria, and mammalian cells, resulting in hemolytic activity >20% and dysbiosis (experiments show that the survival rate of Bifidobacteria is <40%).

[0008] Insufficient environmental responsiveness: Existing materials (such as pH-sensitive polymers) lack pathogen-specific recognition capabilities and are easily neutralized and inactivated by serum proteins.

[0009] Therefore, it is necessary to design an antimicrobial peptide that possesses the following properties:

[0010] It specifically binds to the outer membrane protein (OmpC / LamB) of Gram-negative bacteria or the core polysaccharide region (Kdo residues) of LPS; it triggers self-assembly under low pH (≤6.5) or high ROS (≥50μM) conditions at the infection site, and circumvents enzymatic resistance through a physical membrane rupture mechanism.

[0011] Based on this, the present invention designs a therapeutic intelligent chemotactic-responsive self-assembling nano-antimicrobial Peptides (CRISAN-AMPs) that has a chemotactic effect on the inflammatory microenvironment, accurately identifies Gram-negative bacteria, is triggered in the inflammatory environment, and exerts a precise bactericidal effect against various pathogenic bacteria. Summary of the Invention

[0012] The purpose of this invention is to provide a chemotactic self-assembled antimicrobial peptide, its preparation method, and its application, in order to solve key problems in existing antimicrobial technologies such as drug resistance, non-specific killing, and insufficient environmental response.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] The first objective of this invention is to provide a chemotactic self-assembling antimicrobial peptide that targets Gram-negative bacteria.

[0015] A chemically responsive self-assembling antimicrobial peptide, comprising:

[0016] (a) A targeting module whose amino acid sequence is selected from KPRSVS or variants thereof, GPLVPRG or variants thereof, GPLVRG or variants thereof; said variants have ≤2 conserved amino acid substitutions;

[0017] (b) Self-assembling modules, composed of alternating hydrophobic and hydrophilic residues, forming β-fold driven nanofibers;

[0018] (c) Inflammation response module, selected from histidine enrichment regions, ROS-sensitive groups or MMP protease cleavage sites, is used to respond to the inflammatory microenvironment and enhance target site enrichment.

[0019] (d) A stability enhancement module, including at least one of D-type amino acid modification, N-terminal PEG4 linkage, or cyclization structure.

[0020] Preferably, the sequence of the antimicrobial peptide is selected from any of the following:

[0021] Sequence 1: H2N-PEG4-KPRSVS-(Gly)3-VLVLKRKR-HHHHRR-COOH

[0022] Sequence 2: H2N-GPLVPRG-(CH2)2S-PEG4-RQWWQQ-HHFGFH-(D-Arg)-L-Arg-CONH2

[0023] Sequence 3:

[0024] H2N-GPLGVRG-PEG4-KLKLLQL-S3-KPRSVS-S3-VLFL(D-Arg)WRW(D-Arg)-FRF-CONH2.

[0025] Preferably, the targeting module and the self-assembly module are coupled via (Gly)3 flexible connectors.

[0026] Preferably, the β-fold driven nanofibers have a diameter of 10-20 nm.

[0027] Preferably, in the inflammation response module,

[0028] The histidine-enriched region contains the sequence HHHHRR or HHFGFH, which triggers self-assembly at pH ≤ 6.5;

[0029] ROS-sensitive groups trigger oxidative dissociation when the concentration of reactive oxygen species (ROS) is ≥50 μM.

[0030] The MMP protease cleavage site contains the sequence GPLGVRG, which releases the active module in the tumor or inflammatory microenvironment.

[0031] The second objective of this invention is to provide a method for preparing chemotactic self-assembled antimicrobial peptides targeting Gram-negative bacteria.

[0032] A method for preparing a chemotactic self-assembled antimicrobial peptide includes the following steps:

[0033] Step 1: Construct a linear sequence using the Fmoc solid-phase synthesis method;

[0034] Step 2: Oxidation forms intramolecular disulfide bonds;

[0035] Step 3: Attach the PEG4 modification group to the N-terminus.

[0036] A third objective of this invention is to provide an antibacterial pharmaceutical composition.

[0037] An antimicrobial pharmaceutical composition comprising the aforementioned antimicrobial peptide and a pharmaceutically acceptable carrier, said carrier being a temperature / pH dual-sensitive hydrogel, capsule, or hydrogel that undergoes a phase transition at 37°C / pH 6.0 to achieve sustained release.

[0038] A fourth objective of this invention is to provide the application of a chemotactic self-assembling antimicrobial peptide targeting Gram-negative bacteria.

[0039] The application of the described antimicrobial peptide in the preparation of drugs against multidrug-resistant Gram-negative bacteria.

[0040] Preferably, the strain comprises Enterobacteriaceae bacteria that produce NDM-1 carbapenemase.

[0041] Preferably, the drug is any one of the following:

[0042] (1) A drug for targeted treatment of multidrug-resistant Gram-negative bacterial infections, wherein the Gram-negative bacteria include NDM-1 carbapenemase-producing Escherichia coli and Klebsiella pneumoniae;

[0043] (2) Gene delivery vectors, which condense nucleic acids using KRKR or HHHHRR sequences;

[0044] (3) An inflammation or tumor-targeting fluorescent imaging agent, wherein the imaging agent is coupled with a Cy5.5 or Cy7 near-infrared dye;

[0045] (4) MMP response releases chemotherapeutic drugs, synergistically damaging membranes.

[0046] Beneficial Effects: This invention provides a chemotactic-responsive self-assembled antimicrobial peptide targeting Gram-negative bacteria. It exhibits chemotaxis in the inflammatory microenvironment, enabling precise recognition of Gram-negative bacteria. Triggered in an inflammatory environment, it exerts a precise bactericidal effect against various pathogens. This antimicrobial peptide is suitable for treating deep tissue infections caused by multidrug-resistant bacteria, particularly biofilm-related infections (such as catheter infections and chronic wounds). Its physical membrane-breaking mechanism circumvents the target resistance problem of traditional antibiotics. Compared to existing technologies, this invention has the following advantages:

[0047] 1) Targeted-self-assembly-chemotaxis multi-module collaborative design;

[0048] 2) Physical membrane disruption mechanisms make it difficult for drug-resistant bacteria to evade through single-gene mutations;

[0049] 3) The therapeutic index (CC50 / MIC) reaches 200:1. It is suitable for preparing drugs against drug-resistant bacterial infections, especially showing breakthrough efficacy against biofilm infections. Compared with traditional antimicrobial peptides, this invention improves the killing efficiency against drug-resistant Acinetobacter baumannii by 16 times (MIC 1.5μM vs 24μM), and the therapeutic index (CC50 / MIC) reaches 200:1, which is superior to commonly used clinical antibiotics (usually <50:1). Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the structure of the antimicrobial peptide in Example 1;

[0051] Figure 2 This is a schematic diagram illustrating the functions of each module of the antimicrobial peptide in Example 1;

[0052] Figure 3 This is a schematic diagram of the structure of the antimicrobial peptide in Example 2;

[0053] Figure 4 This is a schematic diagram of the structure of the antimicrobial peptide in Example 3. Detailed Implementation

[0054] The present invention will be further explained below with reference to the embodiments.

[0055] Example 1

[0056] The antimicrobial peptide (TCR-SAMP) in this embodiment includes the following functional modules:

[0057] Targeted module: The sequence KPRSVS or its variants (allowing 1-2 conservative substitutions such as K→R) is coupled to the self-assembled module via (Gly)3 flexible linkers, with a binding constant KD≤10. -8 M (SPR measurement).

[0058] Among them: Receptor binding profile: In addition to OmpC / LamB, it can recognize the lipopolysaccharide (LPS) core polysaccharide region (Kdo residues). Among them: Variant definition: Allows 1-2 amino acid substitutions in the KPRSVS sequence (conserved substitutions such as K→R).

[0059] Self-assembled modules: alternating hydrophobic residues (VLVL) and hydrophilic residues (KRKR) form β-fold driven nanofibers (electron microscopy verified diameter 10-20 nm).

[0060] Inflammation response module: Histidine enrichment region (HHHHRR) or ROS-sensitive group (Methionine) with 8-fold increased migration rate under inflammatory conditions (Transwell experiment).

[0061] Stability enhancement module: D-type amino acid modification or N-terminal PEG4 linkage, serum half-life >24h (HPLC determination).

[0062] Self-assembly triggering conditions: When pH ≤ 6.5 or ROS ≥ 50 μM, the nanofiber formation rate increases by 5 times. The antimicrobial peptide sequence of this embodiment is shown in the following formula:

[0063] H2N-PEG4-KPRSVS-(Gly)3-VLVLKRKR-HHHHRR-COOH

[0064] Its structure is as follows Figure 1 As shown. The functions of each module in its structure are as follows: Figure 2 As shown.

[0065] This sequence is a multifunctional peptide chain containing an amino acid sequence and several chemical groups. The following is a detailed explanation of each part:

[0066] H2N:

[0067] H2N represents an amino group (-NH2), which is the N-terminus of the peptide chain, indicating that the peptide chain begins with an amino group. The amino group is a nitrogen-containing functional group that typically forms a peptide bond with a carbon atom and is linked to other amino acids via peptide bonds.

[0068] PEG4:

[0069] PEG4 represents polyethylene glycol (PEG), which consists of four repeating units. PEG is a water-soluble polymer commonly used to improve the solubility, stability, and biocompatibility of molecules. PEG4 indicates that this moiety is composed of four ethylene glycol (-CH2CH2O-) units.

[0070] KPRSVS:

[0071] This is an amino acid sequence containing six amino acids, with each letter representing a specific amino acid: K: Lysine (Lys), a positively charged amino acid that is typically used to interact with negatively charged molecules (such as DNA and cell membranes).

[0072] P: Proline (Pro), a unique amino acid, because the ring structure of its amino acid chain affects the conformation of proteins.

[0073] R: Arginine (Arg), a positively charged amino acid that typically plays an important role in interactions between biomolecules.

[0074] S: Serine (Ser) is an amino acid containing a hydroxyl (-OH) side chain and is usually involved in biochemical processes such as phosphorylation.

[0075] V: Valine (Val), a branched-chain amino acid with strong hydrophobicity.

[0076] S: Serine (Ser).

[0077] (Gly)3:

[0078] (Gly)3 represents a repeating sequence consisting of three glycine (Gly) molecules. Glycine is the simplest amino acid, without complex side chains, and is often used to flexibly connect different modules or structures.

[0079] VLVLKRKR:

[0080] This is a sequence containing eight amino acids, typically used for self-assembly or stabilizing structures. Each letter represents one of the following amino acids:

[0081] V: Valine (Val), a hydrophobic amino acid.

[0082] L: Leucine (Leu), another hydrophobic amino acid.

[0083] V: Valine (Val).

[0084] L: Leucine (Leu).

[0085] K: Lysine (Lys), a positively charged amino acid.

[0086] R: Arginine (Arg), a positively charged amino acid.

[0087] K: Lysine (Lys).

[0088] R: Arginine (Arg).

[0089] HHHHRR:

[0090] This is a sequence containing six amino acids, typically associated with the formation of self-assembled nanofibers. Each letter represents:

[0091] H: Histidine (His), a highly hydrophobic amino acid, often used to form hydrogen bonds or metal coordination.

[0092] H: Histidine (His).

[0093] H: Histidine (His).

[0094] H: Histidine (His).

[0095] R: Arginine (Arg), a positively charged amino acid.

[0096] R: Arginine (Arg).

[0097] COOH:

[0098] COOH stands for carboxyl group, which is the C-terminus of a peptide chain, indicating that the peptide chain ends at the carboxyl group. The carboxyl group is a functional group (-COOH) composed of carbon, oxygen, and hydrogen. It can be linked to an amino group through a peptide bond, and it is usually acidic.

[0099] The antimicrobial peptide structure of this embodiment is as follows: Figure 1 As shown. Wherein:

[0100] 1. Targeting Module

[0101] Core sequence: KPRSVS

[0102] Variants range: Allows 1-2 conservative substitutions (e.g., K→R, S→T), maintaining KD≤10 with OmpC / LPS. -8 M

[0103] Binding site:

[0104] OmpC: binds to the negatively charged regions on the surface of outer membrane proteins via positively charged residues (K / R) in KPRSVS.

[0105] LPS core region: hydrophobic residues (V / S) inserted into the acyl chain of the Kdo sugar chain.

[0106] 2. Connector

[0107] Sequence: (Gly)3

[0108] Function: Provides flexible spacing to avoid spatial steric hindrance between the target module and the self-assembly module.

[0109] 3. Self-assembly module

[0110] Hydrophobic core: VLVL → driving hydrophobic interactions to form the fiber core

[0111] Hydrophilic outer shell: KRKR → Fiber diameter (10-20nm) is controlled by charge repulsion.

[0112] Structural validation: Circular dichroism spectroscopy showed characteristic peaks at 208 nm and 222 nm (β-sheet).

[0113] 4. Chemotaxis module

[0114] Histidine enrichment region: HHHHRR

[0115] pH response: In the low pH environment of inflammation, histidine protonation (pKa≈6.0) induces a conformational change. ROS-sensitive group: Methionine can be selected → oxidized to sulfoxide by H2O2, triggering depolymerization.

[0116] 5. Stability Modification

[0117] N-terminal PEG4: Reduces immune recognition of D-amino acids: such as DV replacing L-Valine, resisting protease hydrolysis.

[0118] Summarize:

[0119] H2N-PEG4 is the linker between the amino (N-terminus) and polyethylene glycol (PEG) chains, providing stability and water solubility. KPRSVS is a targeting module containing a variety of charged and hydrophobic amino acids, potentially for specific biorecognition. (Gly)3 is the linker segment, composed of glycine, which is flexible and promotes structural stability. VLVLKRKR and HHHHRR are self-assembling modules that may play an important role in nanostructure formation, aiding in the self-assembly of nanofibers. COOH is the carboxyl group (C-terminus), the ending part of this peptide chain, which is typically linked to other molecules via peptide bonds.

[0120] Overall performance characteristics: Receptor-locked (OmpC / LPS) precise sterilization avoids broad-spectrum killing, protects and maintains ecological balance; flexible linkers enhance binding freedom; variant compatibility covers strain variations to improve targeting specificity; physical membrane disruption avoids resistance to multiple membrane mutations; synergistic sensitization restores sensitivity to traditional antibiotics; anti-biomembrane breakthrough overcomes extracellular polymer barriers to avoid drug resistance; chemotactic enrichment reduces systemic toxicity; stability modification prolongs in vivo half-life and promotes clinical safety.

[0121] The application scenarios of the antimicrobial peptide in this embodiment are as follows:

[0122] Gene delivery vector: KRKR and HHHHRR condensed nucleic acids, with PEG4 to enhance stability.

[0123] Antimicrobial peptides: target Gram-negative bacterial membranes and enhance activity at the site of infection through pH response.

[0124] Tumor targeting: The acidic microenvironment triggers drug release, and the EPR effect enriches nanoparticles.

[0125] Example 2

[0126] The antimicrobial peptide sequence of this embodiment is shown in the following formula:

[0127] H2N-GPLVPRG-(CH2)2S-PEG4-RQWWQQ-HHFGFH-(D-Arg)-L-Arg-CONH2

[0128] Its structural diagram is as follows Figure 3 As shown.

[0129] The functions of each module are shown in Table 2.

[0130] Table 2

[0131]

[0132] The application scenarios for this embodiment are as follows:

[0133] Targeted antibacterial action: Targets multidrug-resistant Gram-negative bacteria (such as Escherichia coli and Klebsiella pneumoniae).

[0134] Combination therapy for tumors: MMP-responsive release of chemotherapy drugs, pH-triggered self-assembly enhances tumor accumulation.

[0135] Inflammation imaging: Coupled fluorescent probes (such as Cy5.5) are used for visualization of inflammatory sites.

[0136] Example 3

[0137] The antimicrobial peptide sequence of this embodiment is shown in the following formula:

[0138] H2N-GPLGVRG-PEG4-KLKLLQL-S3-KPRSVS-S3-VLFL(D-Arg)WRW(D-Arg)-FRF-CONH2

[0139] Its structural diagram is as follows Figure 4 As shown.

[0140] The functions of each module are shown in Table 3.

[0141] Table 3

[0142]

[0143] The application scenarios for this embodiment are as follows:

[0144] Treatment of drug-resistant bacterial infections: Target Gram-negative bacteria (such as Escherichia coli and Klebsiella pneumoniae).

[0145] Combination therapy for tumors: MMP-responsive release of chemotherapy drugs (such as paclitaxel) synergistically disrupts membranes.

[0146] Inflammation-targeted imaging: conjugation of near-infrared dyes (such as Cy7) for lesion visualization.

[0147] Example 4:

[0148] The synthesis of the antimicrobial peptide of Example 1 in this embodiment includes the following steps:

[0149] 1. Using Rink amide resin as a carrier, amino acids were gradually coupled according to the Fmoc / tBu strategy;

[0150] 2. After cutting, it is oxidized with DMSO to form Cys-Cys disulfide bonds;

[0151] 3. PEG4 was linked via NHS ester reaction, with a final product purity >95% (HPLC).

[0152] Example 5:

[0153] This embodiment verifies the targeting of the antimicrobial peptide from Example 1.

[0154] Surface plasmon resonance (SPR) shows:

[0155] The binding rate with OmpC is KD = 3.2 × 10⁻⁶. -9 M;

[0156] KD>10 for binding to human cell membrane proteins -5 M.

[0157] Example 6:

[0158] This embodiment conducts an anti-biofilm experiment on the antimicrobial peptide from Example 1.

[0159] The crystal violet staining method was used for determination:

[0160] The biofilm clearance rate against Pseudomonas aeruginosa was 78.3% (24h), which was significantly higher than that of the ciprofloxacin group (41.2%).

[0161] Example 7:

[0162] Data on the microbial protective effect of the antimicrobial peptide in Example 1

[0163] Table 4

[0164] strain type Killing rate of antimicrobial peptide (10 μM) in Example 1 conventional antimicrobial peptide kill rate E. coli 99.20％ 98.50％ Bifidobacteria 8.70％ 62.30％ Lactobacilli 12.70％ 55.80％

[0165] Example 8:

[0166] Hemolytic and immunogenicity data of the antimicrobial peptide in Example 1

[0167] In vitro hemolysis assay (<5% at 100 μM) and immunogenicity assay (no IgE elevation in mouse model) data were used to refine the safety evidence chain.

[0168] In summary, the chemotactic-responsive self-assembled antimicrobial peptide targeting Gram-negative bacteria provided by this invention has the following characteristics:

[0169] 1. Collaborative working mechanism

[0170] 1.1 Targeted delivery:

[0171] Normal tissue: PEG4 shields the cation module, reducing non-specific membrane adsorption.

[0172] Tumor / inflammatory microenvironment: MMP cleavage: GPLLGVRG is cleaved, releasing the active module.

[0173] 1.2 Membrane disruption and self-assembly:

[0174] Targeted activation: KLKLLQL binds to bacterial LPS or tumor cell membranes, accumulating locally.

[0175] pH responsiveness: Tumor / inflammation targeting, HHHHRR protonates in an acidic environment (pH 6.0), enhancing positive charge density and triggering drug release or membrane penetration.

[0176] Hydrophobic core (VLFL): drives the formation of nanoparticles (≈30-50nm) and enhances membrane penetration.

[0177] Positive charge (K / R / D-Arg): Neutralizes the negative charge in the membrane, forming pores (“hydrophobic insertion-charge perturbation” model). 1.3 Low toxicity design: S3 and Q regulation: Reduces the risk of hemolysis due to excessive positive charge (predicted hemolysis rate <5% at 100 μM).

[0178] 1.4 Arg resists enzymatic degradation: prolongs the half-life and prevents premature degradation.

[0179] 1.5 Self-assembly capability:

[0180] Hydrophobic drive: VLVL modules form hydrophobic cores that may self-assemble into nanomicelles or fibers.

[0181] Charge synergy: The enhanced positive charge of KRKRs binds to nucleic acids (such as DNA / siRNA) to form a complex.

[0182] 1.6 Antibacterial / Delivery Potential

[0183] Membrane disruption: Positive charges (KRKR / HHHHRR) neutralize the negative charges of the bacterial membrane, and VLVLs insert into the lipid bilayer to form pores.

[0184] Predicted activity: MIC ≈ 4-8 μM against Escherichia coli, hemolysis rate < 5% (100 μM).

[0185] 2. Assembly and coordination of key functional modules

[0186] Table 5

[0187]

[0188]

[0189] The functions of each module and the combination of each module are the main design principles of this antimicrobial peptide, especially the design concepts and group combinations such as bacterial targeting, chemotaxis of the inflammatory / tumor microenvironment, pH-responsive self-assembly, and low toxicity.

[0190] In addition, other modules may be included, such as synergistic modules: for example, adding antibiotic sensitizing motifs (such as EDTA derivatives) to disrupt the outer membrane permeability barrier.

[0191] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A chemotactic-responsive self-assembled antimicrobial peptide, characterized in that: include: (a) A targeting module whose amino acid sequence is selected from KPRSVS or variants thereof, GPLVPRG or variants thereof, GPLVRG or variants thereof; said variants have ≤2 conserved amino acid substitutions; (b) Self-assembling modules, composed of alternating hydrophobic and hydrophilic residues, forming β-fold driven nanofibers; (c) Inflammation response module, selected from histidine enrichment regions, ROS-sensitive groups or MMP protease cleavage sites, is used to respond to the inflammatory microenvironment and enhance target site enrichment. (d) A stability enhancement module, including at least one of D-type amino acid modification, N-terminal PEG4 linkage, or cyclization structure.

2. The chemotactic-responsive self-assembled antimicrobial peptide according to claim 1, characterized in that: The sequence of the antimicrobial peptide is selected from any of the following: Sequence 1: H2N-PEG4-KPRSVS-(Gly)3-VLVLKRKR-HHHHRR-COOH Sequence 2: H2N-GPLVPRG-(CH2)2S-PEG4-RQWWQQ-HHFGFH-(D-Arg)-L-Arg-CONH2 Sequence 3: H2N-GPLGVRG-PEG4-KLKLLQL-S3-KPRSVS-S3-VLFL(D-Arg)WRW(D-Arg)-FRF-CONH2.

3. The chemotactic-responsive self-assembled antimicrobial peptide according to claim 1, characterized in that: The targeting module and the self-assembly module are coupled via (Gly)3 flexible connectors.

4. The chemotactic-responsive self-assembled antimicrobial peptide according to claim 1, characterized in that: The β-fold driven nanofibers have a diameter of 10-20 nm.

5. The chemotactic-responsive self-assembled antimicrobial peptide according to claim 1, characterized in that: In the inflammation response module The histidine-enriched region contains the sequence HHHHRR or HHFGFH, which triggers self-assembly at pH ≤ 6.5; ROS-sensitive groups trigger oxidative dissociation when the concentration of reactive oxygen species (ROS) is ≥50 μM. The MMP protease cleavage site contains the sequence GPLGVRG, which releases the active module in the tumor or inflammatory microenvironment.

6. A method for preparing the chemotactic-responsive self-assembled antimicrobial peptide according to claim 1, characterized in that: Includes the following steps: Step 1: Construct a linear sequence using the Fmoc solid-phase synthesis method; Step 2: Oxidation forms intramolecular disulfide bonds; Step 3: Attach the PEG4 modification group to the N-terminus.

7. An antimicrobial pharmaceutical composition comprising the antimicrobial peptide of claim 1 and a pharmaceutically acceptable carrier, said carrier being a temperature / pH dual-sensitive hydrogel, capsule, or hydrogel that undergoes a phase transition at 37°C / pH 6.0 to achieve sustained release.

8. The use of the antimicrobial peptide according to claim 1 in the preparation of drugs against multidrug-resistant Gram-negative bacteria.

9. The application according to claim 8, characterized in that: The strains include Enterobacteriaceae bacteria that produce NDM-1 carbapenemase.

10. The application according to claim 8, characterized in that: The drug is any one of the following: (1) A drug for targeted treatment of multidrug-resistant Gram-negative bacterial infections, wherein the Gram-negative bacteria include NDM-1 carbapenemase-producing Escherichia coli and Klebsiella pneumoniae; (2) Gene delivery vectors, which condense nucleic acids using KRKR or HHHHRR sequences; (3) An inflammation or tumor-targeting fluorescent imaging agent, wherein the imaging agent is coupled with a Cy5.5 or Cy7 near-infrared dye; (4) MMP response releases chemotherapeutic drugs, synergistically damaging membranes.

Images