A phase separation polypeptide based on larks coding and antibacterial applications

Abstract

This invention discloses a phase-separated polypeptide based on LARKS encoding and its antibacterial application. The polypeptide has the structural formula A-B-(G). m -X1GGGS-X2G; where A is the phase separation driving unit, selected from π-electron-rich aromatic hydrophobic segments; B is a cationic amino acid, selected from lysine, arginine, and histidine; (G) m The linker is a flexible linker, m=0,1,2 or3, and G is glycine; X1GGGS is the LARKS core sequence, X1 is selected from phenylalanine, tyrosine, tryptophan, and serine; X2G is the C-terminal flexible tail, and X2 is selected from glycine, serine, threonine, asparagine, glutamine, and glutamic acid; the phase separation driving unit induces liquid-liquid phase separation of the polypeptide through π-π interaction and forms cation-π interaction with its adjacent cationic amino acid B.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a phase-separated polypeptide based on LARKS encoding and its antibacterial application. Background Technology

[0002] The widespread use of antibiotics has led to the evolution of complex drug resistance mechanisms in microorganisms, resulting in the frequent emergence of multidrug-resistant bacteria. Traditional antibiotics often target bacterial metabolic or synthetic pathways, easily inducing resistance, and increasing the dosage often results in severe toxic side effects. Therefore, developing antibacterial strategies that can synergize with traditional drugs, reduce dosage, and are independent of typical biochemical targets is of great significance.

[0003] Antimicrobial peptides are considered ideal candidates due to their broad-spectrum mechanism of disrupting bacterial membranes. Among them, peptides with amyloid-like characteristics can self-assemble into aggregates rich in β-sheet conformations, which are microscopically highly ordered nanofibers with good mechanical strength and stability.

[0004] However, these fibers tend to exhibit high dissociation energies and irreversible aggregation characteristics, making them difficult to degrade under certain physiological conditions and prone to long-term residues. Furthermore, the structural similarity between amyloid peptides and pathological aggregates poses a risk of inducing the aggregation of heterogeneous proteins.

[0005] To balance material function and safety, low-complexity amyloid-like reversible kinked segments (LARKS), which are widely distributed in intrinsically disordered human proteins, have provided inspiration for the construction of controllable antimicrobial materials.

[0006] LARKS undergoes liquid-liquid phase separation through weak intermolecular interactions, forming fluid aggregates. Driven by the environment, these aggregates undergo a phase transition from disorder to order, precipitating rigid nanofibers within them. Ultimately, the cross-linking of these nanofibers leads to the complete transformation of the aggregates into a gel. Compared to typical irreversible amyloid fibers with a β-sheet structure, LARKS-based fibers exhibit significant reversibility and environmental responsiveness, enabling controlled dissociation in response to environmental changes and effectively avoiding the risk of cumulative toxicity.

[0007] However, current research on LARKS largely focuses on its functional characterization or phase transition kinetics regulation. How to utilize the structures formed during the ordered phase transition, such as highly ordered fibers and high-density hydrogen bond networks, to achieve efficient capture and enrichment of environmental antibacterial drugs remains a subject of systematic research. Existing technologies lack strategies that can effectively combine the bacterial membrane-disrupting activity of LARKS materials with the phase transition-induced drug spatial enrichment effect to achieve highly efficient synergistic bactericidal activity, thus limiting the advancement of LARKS-encoded materials towards engineering applications and clinical translation. Summary of the Invention

[0008] To address the aforementioned problems in existing technologies, this invention provides a phase-separated peptide based on LARKS encoding and its antibacterial applications. This peptide, through unique molecular design, forms fluid aggregates with disordered molecular assembly via cation-π and π-π interactions within a specific concentration range and solution environment. As the ambient temperature decreases or the ionic strength changes, the LARKS fragments within the aggregates undergo ordered arrangement through hydrogen bonding and hydrophobic stacking, assembling into nanofibers with a reversible β-sheet conformation. These nanofibers spontaneously arrange themselves within the aggregates and generate polarized light signals while maintaining liquid fluidity; that is, the aggregates transform into a liquid crystal state. Further environmental changes can lead to fiber overlap and microgel formation.

[0009] During this process, the high-density hydrogen bond network formed inside the fiber can accurately anchor external antibacterial components, achieving efficient enrichment; at the same time, the highly ordered rigid fiber physically penetrates the bacterial cell membrane, synergistically with the enriched antibacterial components, achieving highly efficient synergistic bactericidal action against Gram-negative and Gram-positive bacteria.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] In a first aspect, the present invention provides a phase-separating polypeptide based on LARKS encoding, the structural formula of which is: AB-(G) m -X1GGGS-X2G in: A is the phase separation driving unit, selected from aromatic hydrophobic segments rich in π electrons; B is a cationic amino acid, selected from lysine (K), arginine (R), and histidine (H); (G) m For flexible linkers, m = 0, 1, 2 or 3, G is glycine; X1GGGS is the core sequence of LARKS, and X1 is selected from one of phenylalanine (F), tyrosine (Y), tryptophan (W), and serine (S). X2G is a C-terminal flexible tail, and X2 is selected from one of glycine (G), serine (S), threonine (T), asparagine (N), glutamine (Q), and glutamic acid (E). The phase separation driving unit induces liquid-liquid phase separation of the polypeptide through π-π interactions and forms cation-π interactions with its adjacent cationic amino acid B.

[0012] The phase separation drive unit is connected to the N end of the LARKS sequence.

[0013] The phase separation driving unit A is selected from one of tetraphenylethylene, naphthyl, pyrene, fluorenyl, phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0014] The flexible connector (G) m In this case, m = 2.

[0015] The polypeptide is capable of undergoing an ordered phase transition under triggering conditions, including temperature changes, ionic strength changes, pH changes, or changes in the concentration of lactic acid produced by physiological metabolism.

[0016] The peptide concentration range for the triggering conditions is typically between 10 μM and 10 mM.

[0017] The polypeptide undergoes at least three of the following phase transitions under the triggering conditions to form a microgel network: single-phase solution, aggregate, liquid crystal, and microgel.

[0018] The fibrous structure can be specifically stained with thiosulfate T; and the fibrous structure can enrich exogenous antibacterial components. The fibrous structure generated by the ordered phase transition of the polypeptide has a physical-mechanical puncture effect, which can disrupt the integrity of the bacterial cell membrane.

[0019] Directional hydrogen bonds and binding sites can be formed between the fibers. These sites can non-covalently bind with antibacterial drugs through interactions such as hydrogen bonds, π-π, electrostatics, or hydrophobicity, thereby enriching the antibacterial components at the fibers.

[0020] The peptides, under specific concentrations and conditions, form disordered and fluid aggregates through cation-π interactions. As the ambient temperature decreases or the ionic strength changes, the LARKS fragments within the aggregates become ordered through hydrogen bonding and hydrophobic stacking, assembling into ordered fibers with a β-sheet conformation. The aggregates generate polarized light signals while maintaining fluidity, transforming into a liquid crystal state. Further environmental changes can lead to fiber overlap and gel formation. During this process, a high-density hydrogen bond network forms within the fibers. This network anchors external antibacterial components to the fiber backbone through intermolecular interactions, achieving antibacterial enrichment. Simultaneously, the highly ordered rigid fibers physically penetrate bacterial cell membranes and possess their own antibacterial capabilities. Together with the enriched antibacterial components, they achieve synergistic bactericidal activity against both Gram-negative and Gram-positive bacteria.

[0021] The polypeptide can be prepared by chemical synthesis or bioengineering expression, and the chemical synthesis method includes, but is not limited to, solid-phase polypeptide synthesis or liquid-phase polypeptide synthesis.

[0022] Specifically, the preparation method generally includes the following steps: (1) Construction of the main chain: Based on the LARKS sequence and the linking flexible fragment described in the first aspect, the amino acid monomers are sequentially condensed and coupled to complete the directional extension of the peptide backbone; (2) Introduction of phase separation driving unit: The phase separation driving unit is covalently coupled to the end of the above polypeptide backbone by an activating agent; (3) Global deprotection and purification: Remove the protecting groups of each amino acid side chain in the sequence, separate the product from the reaction system, and obtain the composite antibacterial material monomer with the required purity by separation and purification techniques such as liquid chromatography.

[0023] As one embodiment of the present invention, the antibacterial material is synthesized using a solid-phase synthesis method, comprising the following steps: (1) Resin pretreatment: Dichlororesin was placed in a synthesis column and dimethylformamide was added to swell it; (2) Elongation of polypeptide chain: Following the sequence from C-terminus to N-terminus, amino acid coupling, ninhydrin detection, and Fmoc protecting group removal are performed cyclically until the synthesis of the entire polypeptide fragment is completed. (3) Coupling of phase separation driving unit: The phase separation driving unit, activating reagent and condensing reagent are dissolved in solvent, added to the synthesis column for reaction, and ninhydrin is detected after the reaction is completed; (4) Material cutting and collection: The resin after reaction is filtered and dried, transferred to a centrifuge tube, the cutting liquid is added and stirred, the filtrate is collected after filtration and separation, the crude product is obtained by precipitation, centrifugation and washing, and finally purified by reversed-phase high-performance liquid chromatography to obtain the target antibacterial material.

[0024] In step (2), the amino acid coupling uses Fmoc-amino acid, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide as the condensation system, and N,N-diisopropylethylamine as the activating reagent. In the ninhydrin detection, if the resin and solution remain yellow and there is no color reaction, it is judged as negative, indicating that the coupling is complete. If a dark blue or purple color appears, it indicates that the coupling is incomplete and the reaction time needs to be extended or the coupling needs to be repeated. The Fmoc protecting group is removed by treating with piperidine DMF solution. Only when the ninhydrin detection is positive after deprotection can the coupling of the next amino acid proceed.

[0025] The phase separation driving unit in step (3) is TPE-COOH, the activating agent is N,N-diisopropylethylamine, and the condensing agent is 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide.

[0026] The cutting fluid in step (4) is a mixture of trifluoroacetic acid / water / triisopropylsilane, the precipitate is made of ice-cold diethyl ether, and the centrifugation conditions are 3000-5000 g, 10-20 min, and 4-25°C.

[0027] The antibacterial material can be synthesized using solid-phase synthesis or conventional techniques in this field.

[0028] Secondly, the present invention also provides a thermally reversible antibacterial fiber material based on LARKS-encoded peptides, comprising: The polypeptides described above; and Exogenous antibacterial components are enriched on the fibrous structure formed by the polypeptide through non-covalent interactions.

[0029] The exogenous antibacterial component is an antibacterial drug with a hydrogen bond donor-receptor or aromatic structure, including at least one of antimicrobial peptides, glycopeptide antibiotics, aminoglycoside antibiotics, quinolone antibiotics, or small molecule chemical drugs containing aromatic rings.

[0030] Directional hydrogen bonds and binding sites can be formed between the fibers. These sites can non-covalently bind with antibacterial drugs through interactions such as hydrogen bonds, π-π, electrostatics, or hydrophobicity, thereby enriching the antibacterial components at the fibers.

[0031] Thirdly, the present invention provides an application of the aforementioned antimicrobial material, the application of which includes the prevention and treatment of bacterial infections, the inhibition and removal of bacterial biofilms, the preparation of antimicrobial coatings on the surfaces of medical implants and devices, wound repair and anti-infection dressings, synergistic drug delivery systems, and antimicrobial applications in the fields of public health and personal protection.

[0032] Compared with the prior art, the present invention has the following beneficial effects: (1) Highly efficient enrichment dominated by fibrosis: This invention uses the directional hydrogen bond network and interfacial binding sites formed during the fibrosis stage to efficiently enrich antibacterial components, significantly increasing the local concentration of antibacterial components at the interface and reducing the action time.

[0033] (2) Synergistic enhancement of sterilization: The rigid nanofibers exert a direct physical puncture effect on the bacterial membrane, while being enriched with antibacterial components to exert a sterilization or penetration enhancement effect. The synergistic effect of the two can significantly improve the killing efficiency of Gram-negative and Gram-positive bacteria.

[0034] (3) Controllable phase transition and material safety: Based on the low binding energy of LARKS, by controlling the fibrosis conditions and sequence design, the controllable formation and reversible dissociation of hydrogen bond network can be achieved, so that the material can be controlled to dissociate into oligomer or monomer state after completing the sterilization function, which is beneficial to reduce the long-term residue and potential accumulation toxicity risk of the material in the body.

[0035] (4) Versatility and engineering convenience: The antibacterial material fixes different types of drug molecules through short-range interactions, can be compatible with multiple antibacterial modules and can be combined as needed, and the phase change behavior and enrichment efficiency can be adjusted by conventional parameters, which facilitates process scale-up and application transformation. Attached Figure Description

[0036] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is an optical characterization of the phase transition process of the polypeptide sequence with temperature change in Example 1; Figure 2 Optical characterization of the polypeptide sequence after phase transition in Example 2; Figure 3 The images show the fluorescence microscopy characterization of the enriched vancomycin after the ordered phase transition of the polypeptide sequence in Example 1; where A is the fluorescence microscopy characterization of the polypeptide TPE; and B is the fluorescence microscopy characterization of the enriched vancomycin. Figure 4 The scanning electron microscope characterization of the polypeptide sequence ordered by phase transition and enriched with vancomycin in Example 1, followed by co-incubation with bacteria. Figure 5 This refers to the degradation of the polypeptide sequence in Example 1 after an ordered phase transition in a simulated physiological environment. Detailed Implementation

[0037] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0038] Example 1: Peptide TPE Synthesis of HGGYGGGSDG Target peptide TPE The synthesis of HGGYGGGSDG (SEQ ID NO.1), wherein tetraphenylethylene (TPE) is the phase separation driving unit, histidine H is a cationic amino acid, GG is a flexible linker, YGGGS (SEQ ID NO.2) is the LARKS core sequence, and DG is the C-terminal flexible tail.

[0039] The solid-state synthesis method was used for synthesis, and the specific steps are as follows: (1) Resin pretreatment: Dichloro resin (degree of substitution 1 mmol / g) was placed in the synthesis column and dimethylformamide (DMF) was added to swell for 30 minutes.

[0040] (2) Elongation of the polypeptide chain: Following the sequence from the C-terminus to the N-terminus, the following operations are performed cyclically: a. Amino acid coupling: Four molar amounts of Fmoc-amino acid, four molar amounts of 1-hydroxybenzotriazole (HOBt), and four molar amounts of N,N'-diisopropylcarbodiimide (DIC) were dissolved in DMF, followed by the addition of eight molar amounts of N,N-diisopropylethylamine (DIEA) for activation. The mixed solution was added to a synthesis column, and nitrogen gas was injected and bubbled for 1 h.

[0041] b. Ninhydrin detection after coupling: Take 20-30 resin particles after reaction into a 1.5 mL centrifuge tube, and add 5% ninhydrin ethanol solution, 80% phenol ethanol solution, and anhydrous pyridine in a 2:1:1 ratio. Heat at 110°C for 2 min. If the resin and solution retain their original yellow color and no color reaction occurs, it is considered negative, indicating that the coupling step is complete. If the resin particles or solution show a deep blue or purple color, it indicates that unreacted free amino groups still exist in the system, and the coupling is incomplete. In this case, the reaction time needs to be extended, or the amino acids and condensation reagent should be weighed again for a second coupling until the result is negative.

[0042] c. After complete coupling, treat with 20% piperidine in DMF solution for 30 minutes to remove Fmoc protection. After deprotection, confirm a positive result again by ninhydrin detection; the resin particles or solution should show a deep blue or purple color before proceeding with the coupling of the next amino acid.

[0043] Repeat the above steps until the synthesis of the entire polypeptide fragment HGGYGGGSDG is complete.

[0044] (3) Coupling of TPE: Weigh 4 molar amounts of TPE-COOH, 4 molar amounts of HOBt, and 4 molar amounts of N,N'-diisopropylcarbodiimide DIC and dissolve them in DMF. Then add 8 molar amounts of N,N-diisopropylethylamine (DIEA) for activation. Add the mixed solution to the synthesis column and inject nitrogen gas for bubbling reaction for 2 h. After the reaction is completed, perform ninhydrin detection again until the result is negative.

[0045] (4) Material cutting and collection: The reacted resin was vacuum filtered and dried, then transferred to a centrifuge tube. A cutting solution of trifluoroacetic acid / water / triisopropylsilane with a volume ratio of 95:2.5:2.5 was added and stirred for 8 hours. Subsequently, the resin-peptide mixture was separated by vacuum filtration, and the filtrate was collected. The peptide was precipitated with ice-cold ether, and the crude product was collected by low-temperature centrifugation (5000 g, 10 min, 4°C). The peptide was washed three times with ice-cold ether. Finally, the crude peptide was dissolved in water, and the precipitated peptide was further purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain TPE-HGGYGGGSDG.

[0046] Example 2 The following polypeptide was synthesized in this embodiment: Nap-HGGYGGGSDG (SEQ ID NO.1), wherein 2-naphthylNap is the phase separation driving unit, histidine H is a cationic amino acid, GG is a flexible linker, YGGGS (SEQ ID NO.2) is the LARKS core sequence, and DG is the C-terminal flexible tail.

[0047] TPE-RGGYGGGSDG (SEQ ID NO.3), wherein tetraphenylethylene (TPE) is the phase separation driving unit, arginine (R) is a cationic amino acid, GG is a flexible linker, YGGGS (SEQ ID NO.2) is the LARKS core sequence, and DG is the C-terminal flexible tail.

[0048] TPE-HGGFGGGSDG (SEQ ID NO.4), wherein tetraphenylethylene TPE is the phase separation driving unit, histidine H is a cationic amino acid, GG is a flexible linker, FGGGS (SEQ ID NO.5) is the LARKS core sequence, and DG is a C-terminal flexible tail.

[0049] TPE-HGGYGGGSQG (SEQ NO.6), wherein tetraphenylethylene TPE is the phase separation driving unit, histidine H is a cationic amino acid, GG is a flexible linker, YGGGS (SEQ ID NO.2) is the LARKS core sequence, and QG is a C-terminal flexible tail.

[0050] The synthesis method is the same as in Example 1.

[0051] Example 3 Characterization of multi-stage phase transitions in materials The material TPE-HGGYGGGSDG synthesized in Example 1 was added to physiological saline and heated to 80°C to fully dissolve it, preparing a solution with a concentration of 2 mg / mL. The solution was transferred to a 1 mm thick glass slide groove and sealed. The slide was placed on a high-precision cooling stage and cooled from 80°C to 25°C at a cooling rate of 0.1°C / min. The evolution of the material's phase state during the cooling process was observed using a polarizing microscope. Figure 1 ).

[0052] like Figure 1 As shown, the material exhibits significant multi-stage phase transition characteristics in physiological saline as the temperature decreases. The system successively undergoes a single-phase solution state, a condensate state generated by liquid-liquid phase separation, a highly ordered liquid crystal state, and finally evolves into a stable microgel network.

[0053] This multi-stage phase transition process demonstrates that the material has good environmental responsiveness and controllable self-assembly capability.

[0054] Example 4: Characterization of multi-stage phase transitions in materials The materials Nap-HGGYGGGSDG, TPE-RGGYGGGSDG, TPE-HSGGFGGGSDG, and TPE-HGGYGGGSQG synthesized in Example 2 were added to physiological saline and heated to 80°C to fully dissolve them, preparing a solution with a concentration of 2 mg / mL. The solution was transferred to a 1 mm thick glass slide groove and sealed. The slide was placed on a high-precision heating and cooling stage and cooled from 80°C to 25°C at a cooling rate of 0.1°C / min. The results were observed using a polarizing microscope. Figure 2 ).

[0055] like Figure 2 As shown, each sequence precipitated a second phase in physiological saline, and formed a birefringent liquid crystal and gel in the later stage of cooling, proving that different sequences all have the ability of liquid-liquid phase separation and ordered phase transition.

[0056] Example 5: Highly efficient enrichment of antibacterial components The material TPE-HGGYGGGSDG synthesized in Example 1 was added to physiological saline and heated to 80°C to fully dissolve it, preparing a solution with a concentration of 2 mg / mL. 100 μL of the above solution was added to a 96-well plate, and the temperature was lowered from 80°C to 25°C at a cooling rate of 0.5°C / min to induce an ordered phase transition. After the system gelled, 50 μg / mL of FITC-labeled vancomycin was added to the system. After standing for 1 h, the distribution of vancomycin within the material was observed using a fluorescence microscope. Figure 3 (where A represents the fluorescence microscopy characterization of the peptide TPE; B represents the fluorescence microscopy characterization of vancomycin enrichment).

[0057] like Figure 3 As shown, the green fluorescence intensity inside the gel is significantly higher than the background fluorescence intensity of the external solution environment, indicating that the nanofiber network formed during the ordered phase transition of the material can achieve efficient enrichment of external antibacterial components.

[0058] Example 6 Synergistic bactericidal effect of antibacterial materials Take the TPE material synthesized in Example 1 HGGYGGGSDG was added to physiological saline and heated to 80°C to prepare a 2 mg / mL solution. 400 μL of this solution was added to a 24-well plate containing sterile silica discs, and the peptide solution was cooled from 80°C to 25°C at a cooling rate of 0.5°C / min. 50 μg / mL FITC-labeled vancomycin was added to the system, and the mixture was allowed to stand for 1 h to allow for complete enrichment. Subsequently, 10 μg / mL FITC-labeled vancomycin was added to the system. 6 CFU / mL Escherichia coli and Staphylococcus aureus suspension were co-incubated for 2 h.

[0059] After incubation, samples were fixed with 5% glutaraldehyde for 1 h, followed by washing three times with 0.5% glutaraldehyde aqueous solution. Dehydration was then performed sequentially using ethanol in volume increments of 25%, 50%, 75%, and 100%. After dehydration, the samples were dried using supercritical carbon dioxide drying technology, and bacterial morphology was observed using scanning electron microscopy. Figure 3 ).

[0060] like Figure 4 As shown, the surface of Escherichia coli and Staphylococcus aureus cells showed obvious shrinkage or structural damage, indicating that the nanofibers enriched with vancomycin exerted a significant synergistic bactericidal effect through the physical and mechanical puncture of the rigid structure and the chemical killing effect of the local high concentration of drug.

[0061] Example 7: Dynamic reversible degradation of materials under simulated physiological conditions The material TPE-HGGYGGGSDG synthesized in Example 1 was added to physiological saline and heated to 80°C to fully dissolve it, preparing a solution with a concentration of 2 mg / mL. 100 μL of the above solution was added to a 96-well plate, and the plate was cooled from 80°C to 25°C at a cooling rate of 0.5°C / min to prepare a microgel network. The supernatant was replaced with DMEM cell culture medium containing 10% fetal bovine serum, and the plate was placed in a 37°C incubator. The morphology of the material was monitored after 48 h. Figure 4 ).

[0062] like Figure 5 As shown, the antibacterial material formed by ordered phase transition can undergo spontaneous slow dissociation and degradation in a culture medium simulating a physiological environment, indicating that the antibacterial material constructed based on LARKS ordered phase transition has excellent dynamic reversibility and can be effectively metabolized after use, avoiding long-term accumulation of toxicity in organisms.

[0063] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that these are merely illustrative examples, and any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A phase-separated polypeptide encoded by LARKS, characterized in that, The structural formula of the polypeptide is: A-B-(G) m -X1GGGS-X2G in: A is the phase separation driving unit, selected from aromatic hydrophobic segments rich in π electrons; B is a cationic amino acid, selected from lysine, arginine, and histidine. (G) m For flexible linkers, m = 0, 1, 2 or 3, G is glycine; X1GGGS is the core sequence of LARKS, and X1 is selected from one of phenylalanine, tyrosine, tryptophan, and serine. X2G is a C-terminal flexible tail, and X2 is selected from one of glycine, serine, threonine, asparagine, glutamine, and glutamic acid. The phase separation driving unit induces liquid-liquid phase separation of the polypeptide through π-π interactions and forms cation-π interactions with its adjacent cationic amino acid B.

2. The polypeptide according to claim 1, characterized in that, The phase separation driving unit A is selected from one of tetraphenylethylene, naphthyl, pyrene, fluorenyl, phenylalanine, tyrosine, and tryptophan.

3. The polypeptide according to claim 1, characterized in that, The flexible connector (G) m In this case, m = 2.

4. The polypeptide according to claim 1, characterized in that, The polypeptide is capable of undergoing fibrillation assembly and reversible phase transition under triggered conditions, including temperature changes, ionic strength changes, pH changes, or lactic acid concentration changes.

5. The polypeptide according to claim 4, characterized in that, The polypeptide undergoes at least three or more of the following phase transitions under the triggering conditions to form a microgel network: single-phase solution, aggregate, liquid crystal, microgel.

6. A thermally reversible antibacterial fiber material based on LARKS-encoded phase-separated peptides, characterized in that, include: The polypeptide according to any one of claims 1-5; as well as, Exogenous antibacterial components are enriched on the fibrous structure formed by the polypeptide through non-covalent interactions.

7. The antibacterial fiber material according to claim 6, characterized in that, The exogenous antibacterial component is an antibacterial drug with a hydrogen bond donor-receptor or aromatic structure, including at least one of antimicrobial peptides, glycopeptide antibiotics, aminoglycoside antibiotics, quinolone antibiotics, and small molecule chemical drugs containing aromatic rings.

8. An application of the thermally reversible antibacterial fiber material as described in claim 6 or 7, characterized in that, The applications include the preparation of materials for the prevention or treatment of bacterial infections, the inhibition and removal of bacterial biofilms, the preparation of antimicrobial coatings for the surfaces of medical implants and devices, wound repair and anti-infection dressings, synergistic drug delivery systems, and antimicrobial applications in the fields of public health and personal protective equipment.

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