Polypeptide as well as product, preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide as well as a product, a preparation method and application thereof. According to the invention, a brand new high-efficiency broad-spectrum antibacterial peptide AK15 is screened out on the basis of an artificial intelligence model AMP (Amplified Matrix Protein)-Hydrogel-Designer. The MIC of the antibacterial peptide on standard strains of six clinical common bacteria is less than or equal to 16 mu g / ml, and the antibacterial peptide can inhibit multiple clinical drug-resistant strains and has a relatively strong broad-spectrum antibacterial effect. Furthermore, the antibacterial peptide can be cross-linked with a biological hydrogel material, and the prepared hydrogel material has the effect of promoting wound healing besides resisting bacteria and has huge application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polypeptide and its products, preparation methods and applications. Background Art

[0002] Antibiotic resistance has become a major challenge facing global public health, with a significant increase in global morbidity and mortality. Although various antibacterial strategies have been developed and implemented, including physical, chemical and biological methods, currently, the main reliance is on the combined use of antibiotics with different mechanisms of action to improve efficacy. However, combination therapy also has obvious drawbacks, including the possible generation of unstable synergistic effects or even antagonistic effects; the simultaneous use of antibiotics may lead to an increase in adverse reactions. In addition, it may accelerate the acquisition of multidrug resistance by bacteria, increasing the complexity of clinical treatment. The complexity of antibiotic resistance stems from the diversity and adaptability of bacterial resistance mechanisms, as well as the intricate nature of the bacterial ecosystem. Given this complex situation, it is urgent to develop precise and efficient solutions for drug-resistant bacteria to address serious infection problems.

[0003] Biomaterials have shown unique utility in the field of combating drug-resistant bacterial infections and promoting healing. PEG-4SH has good biocompatibility, low toxicity to cells, tissues, etc. in the body, and is not likely to cause adverse effects such as immune reactions in the body, and can safely serve as a matrix for biomaterials, especially hydrogels. On the other hand, the sulfhydryl groups in its molecules can undergo specific coupling reactions with material components containing specific functional groups, such as sulfhydryl-containing material components, thereby achieving targeted modification of drug molecules or other bioactive molecules, improving the stability and water solubility of bioactive substances, preventing bioactive substances from being rapidly degraded or inactivated in the body, extending the circulation time and action time in the body, and at the same time, it can also reduce the immunogenicity of bioactive substances and reduce the possibility of triggering immune reactions in the body.

[0004] Antimicrobial peptides (AMPs) are an integral part of the innate immune system that is ubiquitously present in the biological world and have received extensive attention due to their therapeutic potential against a variety of bacterial infections. Different from traditional antibiotics, AMPs exhibit many advantageous characteristics such as being less likely to develop resistance, highlighting their potential as alternative therapeutic agents for anti-bacterial infections.

[0005] Although it has been widely reported that antimicrobial peptides are added as antibacterial components to biomaterials, simply mixing them with other components of the material has disadvantages such as fast release, short action time, and unstable antibacterial performance. Therefore, it is necessary to find a highly efficient and broad-spectrum antimicrobial peptide containing sulfhydryl (cysteine) that can undergo complex crosslinking with PEG-4SH, which has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] The purpose of the first aspect of the present invention is to provide polypeptide AK15 or its salt.

[0007] The object of the second aspect of the present invention is to provide a conjugate.

[0008] The object of the third aspect of the present invention is to provide a fusion protein.

[0009] The object of the fourth aspect of the present invention is to provide biomaterials related to the polypeptide AK15 of the first aspect of the present invention, the conjugate of the second aspect of the present invention, and / or the fusion protein of the third aspect of the present invention.

[0010] The object of the fifth aspect of the present invention is to provide a composition.

[0011] The object of the sixth aspect of the present invention is to provide the use of the antibacterial peptide or its salt of the first aspect of the present invention, the conjugate of the second aspect, the fusion protein of the third aspect, the biomaterial of the fourth aspect, and the composition of the fifth aspect.

[0012] The object of the seventh aspect of the present invention is to provide a product.

[0013] The object of the eighth aspect of the present invention is to provide a hydrogel.

[0014] The object of the ninth aspect of the present invention is to provide a method for preparing a hydrogel.

[0015] In order to achieve the above objects of the present invention, the technical solutions adopted by the present invention are as follows:

[0016] The first aspect of the present invention provides a polypeptide AK15 or its salt, and the amino acid sequence of the polypeptide AK15 is:

[0017] a1) SEQ ID NO.1; or

[0018] a2) An amino acid sequence obtained by substituting and / or deleting and / or adding one or several amino acids to SEQ ID NO.1 and having the same function as SEQ ID NO.1; or

[0019] a3) An amino acid sequence having more than 95% homology with SEQ ID NO.1.

[0020] The second aspect of the present invention provides a conjugate, comprising a modification part and the polypeptide AK15 of the first aspect of the present invention, and the modification part is located at the N-terminal and / or C-terminal of the polypeptide AK15.

[0021] In some embodiments of the present invention, the modification part is located at the C-terminal of the polypeptide AK15.

[0022] In some embodiments of the present invention, the modifying moiety includes at least one of chemical modification, targeting moiety, fluorescent dye, and protein tag; further includes chemical modification.

[0023] In some embodiments of the present invention, the chemical modification includes at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation, ubiquitination; further includes amidation.

[0024] In some embodiments of the present invention, the targeting moiety includes at least one of ligand, receptor, antibody.

[0025] In some embodiments of the present invention, the fluorescent dye includes FITC.

[0026] In some embodiments of the present invention, the protein tag includes at least one of His, Flag, GST, MBP, HA, Myc, GFP.

[0027] The third aspect of the present invention provides a fusion protein, comprising the polypeptide of the first aspect of the present invention or a salt thereof, and other polypeptides.

[0028] In some embodiments of the present invention, the other polypeptide is located at the N-terminus and / or C-terminus of the polypeptide AK15.

[0029] In some embodiments of the present invention, the other polypeptide and the polypeptide AK15 of claim 1 are linked to the N-terminus and / or C-terminus of the polypeptide AK15 through a linker.

[0030] The fourth aspect of the present invention provides a biomaterial related to the polypeptide AK15 of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention, and the biomaterial includes at least one of b1) - b8):

[0031] b1) A nucleic acid molecule encoding the polypeptide AK15 of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention;

[0032] b2) An expression cassette containing the nucleic acid molecule of b1);

[0033] b3) A vector containing the nucleic acid molecule of b1);

[0034] b4) A vector containing the expression cassette of b2);

[0035] b5) A transgenic cell line containing the nucleic acid molecule of b1);

[0036] b6) A transgenic cell line containing the expression cassette of b2);

[0037] b7) A transgenic cell line containing the vector described in b3);

[0038] b8) A transgenic cell line containing the vector described in b4).

[0039] In some embodiments of the present invention, the transgenic cell line does not contain propagation materials.

[0040] The fifth aspect of the present invention provides a composition comprising at least one of c1) - c3):

[0041] c1) The polypeptide AK15 of the first aspect of the present invention or a salt thereof;

[0042] c2) The conjugate of the second aspect of the present invention;

[0043] c3) The fusion protein of the third aspect of the present invention.

[0044] In some embodiments of the present invention, the composition further comprises other active antibacterial components.

[0045] In some embodiments of the present invention, the other active antibacterial components comprise at least one of penicillin antibiotics (such as ampicillin, oxacillin, etc.), cephalosporin antibiotics (such as cefradine, cefuroxime, cefoperazone, etc.), carbapenem antibiotics (such as imipenem, meropenem, etc.), lipo(glyco)peptide antibiotics (such as polymyxins and vancomycin, etc.), aminoglycoside antibiotics, tetracycline antibiotics (such as doxycycline, oxytetracycline, etc.), lincosamide antibiotics (lincomycin, clindamycin), macrolide antibiotics (such as azithromycin, roxithromycin, erythromycin, etc.), fluoroquinolone antibiotics (such as ciprofloxacin, lomefloxacin, etc.), and fusidic acid.

[0046] In some embodiments of the present invention, the other active antibacterial components comprise at least one of tetracycline antibiotics (such as doxycycline, minocycline, oxytetracycline, etc.), lincosamide antibiotics (lincomycin, clindamycin), macrolide antibiotics (such as azithromycin, roxithromycin, erythromycin, etc.), and fusidic acid.

[0047] In some embodiments of the present invention, the bacterium comprises at least one of bacteria and fungi; further comprises bacteria; still further comprises at least one of Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Propionibacterium acnes; even further comprises Propionibacterium acnes.

[0048] The sixth aspect of the present invention provides any one of the polypeptide AK15 or its salt according to the first aspect of the present invention, the conjugate according to the second aspect of the present invention, the fusion protein according to the third aspect of the present invention, the biological material according to the fourth aspect of the present invention, and / or the composition according to the fifth aspect of the present invention for any one of the following applications d1) to d2):

[0049] d1) Preparing an antibacterial product;

[0050] d2) Preparing a product for promoting wound healing.

[0051] In some embodiments of the present invention, the product comprises at least one of a drug, a feed, a feed additive, a preservative, a daily chemical product, a fabric, a paper product, a medical material, and a cell culture medium.

[0052] In some embodiments of the present invention, the daily chemical product comprises at least one of a facial cleanser, a hand sanitizer, a body wash, a shampoo, a mouthwash, a toothpaste, a soap, a cosmetic, a feminine care lotion, a laundry soap, a laundry detergent, a washing powder, a dishwashing liquid, a disinfectant, and a toilet cleaner.

[0053] In some embodiments of the present invention, the fabric comprises at least one of clothing, bedding, disinfected tissues, accessories, and bandages.

[0054] In some embodiments of the present invention, the paper product comprises at least one of a sanitary napkin, a panty liner, a diaper, and a urine pad.

[0055] In some embodiments of the present invention, the medical material comprises at least one of a tissue regeneration material, a tissue repair material, a medical hydrogel material, and a medical antibacterial material.

[0056] In some embodiments of the present invention, the bacteria in d1) comprise at least one of bacteria and fungi; further comprising bacteria; still further comprising at least one of Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Propionibacterium acnes; and even further comprising Propionibacterium acnes.

[0057] In some embodiments of the present invention, the product in d2) comprises at least one of a drug, a daily chemical product, a medical device, and a health product.

[0058] In some embodiments of the present invention, the daily chemical product comprises at least one of a facial cleanser, a body wash, a soap, and a cosmetic.

[0059] In some embodiments of the present invention, the dosage form of the drug comprises at least one of an injection, an oral preparation, and an external preparation.

[0060] The seventh aspect of the present invention provides a product comprising at least one of e1) to e4):

[0061] e1) The polypeptide AK15 as claimed in claim 1 or a salt thereof;

[0062] e2) The conjugate as claimed in any one of claims 2 - 3;

[0063] e3) The fusion protein as claimed in any one of claims 4 - 5;

[0064] e4) The composition as claimed in claim 7;

[0065] The product is any one of f1) to f2):

[0066] f1) An antibacterial product;

[0067] f2) A product for promoting wound healing.

[0068] The product comprises at least one of drugs, feeds, feed additives, preservatives, daily chemical products, fabrics, paper products, medical materials, cell culture media.

[0069] In some embodiments of the present invention, the product comprises at least one of drugs, feeds, feed additives, preservatives, daily chemical products, fabrics, paper products, medical materials, cell culture media.

[0070] In some embodiments of the present invention, the daily chemical products comprise at least one of facial cleansers, hand sanitizers, body washes, shampoos, mouthwashes, toothpastes, soaps, cosmetics, feminine care lotions, laundry soaps, laundry detergents, laundry powders, dishwashing liquids, disinfectants, toilet cleaners.

[0071] In some embodiments of the present invention, the fabrics comprise at least one of clothing, bedding, disinfected tissues, accessories, bandages.

[0072] In some embodiments of the present invention, the paper products comprise at least one of sanitary napkins, pantiliners, diapers, urine pads.

[0073] In some embodiments of the present invention, the medical materials comprise at least one of tissue regeneration materials, tissue repair materials, medical hydrogel materials, medical antibacterial materials.

[0074] In some embodiments of the present invention, the bacteria in d1) comprise at least one of bacteria and fungi; further comprising bacteria; still further comprising at least one of Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Propionibacterium acnes; and even further comprising Propionibacterium acnes.

[0075] In some embodiments of the present invention, the product described in d2) comprises at least one of drugs, daily chemical products, medical devices, and health care products.

[0076] In some embodiments of the present invention, the daily chemical products comprise at least one of facial cleanser, body wash, soap, and cosmetics.

[0077] In some embodiments of the present invention, the dosage form of the drug comprises at least one of injections, oral preparations, and topical preparations.

[0078] The eighth aspect of the present invention provides a hydrogel comprising at least one of e1) to e4):

[0079] e1) The polypeptide AK15 or a salt thereof as claimed in claim 1;

[0080] e2) The conjugate as claimed in any one of claims 2-3;

[0081] e3) The fusion protein as claimed in any one of claims 4-5;

[0082] e4) The composition as claimed in claim 7;

[0083] And a colloidal part.

[0084] In some embodiments of the present invention, the colloidal part comprises natural biogels and synthetic biogels.

[0085] In some embodiments of the present invention, the natural biogels comprise at least one of collagen, hyaluronic acid, sodium alginate, chitosan, gelatin, and fibrin.

[0086] In some embodiments of the present invention, the synthetic biogels comprise at least one of polyethylene glycol (PEG)-based hydrogels, polyacrylic acid (PAA), poly-N-isopropylacrylamide (PNIPAM), and polyvinyl alcohol (PVA) hydrogels.

[0087] In some embodiments of the present invention, the hydrogel is a polyethylene glycol (PEG)-based hydrogel; further, it is thiol-modified PEG, such as PEG-4SH.

[0088] In some embodiments of the present invention, the hydrogel further comprises a piezoelectric material, such as BTO.

[0089] In some embodiments of the present invention, the volume-mass ratio of the hydrogel to the polypeptide AK15 or a salt thereof is: 1 to 1000 μg of the polypeptide AK15 or a salt thereof per 1 mL of the hydrogel; preferably, 10 to 500 μg of the polypeptide AK15 or a salt thereof per 1 mL of the hydrogel.

[0090] The ninth aspect of the present invention provides a method for preparing the hydrogel of the eighth aspect of the present invention, comprising the following steps:

[0091] Mix the hydrogel with at least one of e1) to e4) to obtain.

[0092] The beneficial effects of the present invention are:

[0093] Based on the artificial intelligence model AMP-hydrogel-Designer, the present invention screens out a brand-new highly efficient broad-spectrum antibacterial peptide AK15. The MIC of this antibacterial peptide against the standard strains of 6 common clinical bacteria is less than or equal to 16 μg / ml, and it can inhibit multiple clinical drug-resistant strains, showing a strong broad-spectrum antibacterial effect. Further, this antibacterial peptide can be crosslinked with the biological hydrogel material, and the prepared hydrogel material not only has antibacterial properties but also has the effect of promoting wound healing, showing great application potential. Description of the Drawings

[0094] The following further describes the present invention in conjunction with the drawings and embodiments, where:

[0095] Figure 1 is the high-performance liquid chromatography result of AK15.

[0096] Figure 2 is the mass spectrum of AK15.

[0097] Figure 3 is the result of generating and screening AK15 by the AMP-hydrogel-Designer model, where: A) the training data set of AMP-hydrogel-Designer; B) the structure of the AMP-hydrogel-Designer model; C) the experimental verification of antibacterial peptide candidates; D) the detailed generation and screening steps of AK15; E) the evolution of various peptide property scores during RL.

[0098] Figure 4 is the preparation and characterization of the hydrogel; where: A) surface modification of BTO nanoparticles; B) SEM image of Cu-BTO nanoparticles; C) TEM image of a single Cu-BTO nanoparticle and the corresponding selected area electron diffraction (SAED) pattern; D) amplitude and phase images, E) hysteresis loop, F) butterfly loop obtained by PFM analysis; G) tissue adhesion test of pig skin and human hand; H) SEM image of AI-AMP-hydrogel, with the orange arrow indicating Cu-BTO nanoparticles; I) Fourier transform infrared spectroscopy; J) XPS and K) open circuit voltage and short circuit current of AI-AMP-hydrogel under ultrasonic power of 0, 1, and 2 W / cm2.

[0099] Figure 5 In vitro antibacterial properties of AI-AMP-hydrogel; A) Representative colony images of MRSA and Escherichia coli treated with different samples; B) Anti-MRSA biofilm activity of AI-AMP-hydrogel; C) Live / dead staining images of MRSA and Escherichia coli after different treatments; D) TEM images of MRSA and Escherichia coli; E) SEM images of MRSA and Escherichia coli; F) Antibacterial mechanism of AK15; G, H) Colony forming units (CFU) of MRSA and Escherichia coli after different treatments; I) Quantitative analysis of surviving MRSA biofilms; J, K) Survival rates of MRSA and Escherichia coli after different treatments; (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0100] Figure 6 Results of transcriptomics analysis, where: A) Volcano plot of DEGs between the control group and AI-AMP-hydrogel under ultrasound; B) Cluster analysis of the top 15 genes in upregulated and downregulated DEGs; C) GO enrichment analysis of all DEGs; D) KEGG enrichment analysis of all DEGs; E) PPI network of upregulated and downregulated DEGs; F) Schematic diagram of the anti-MRSA mechanism of AI-AMP-hydrogel.

[0101] Figure 7 Results of the biocompatibility and wound healing properties of AI-AMP-hydrogel, where: A) Schematic diagram of the Transwell experiment; B) Schematic diagram of the scratch experiment; C) Representative migration experiment and live / dead staining images of L929 cells after different treatments; D) Bright field (BF) and fluorescence (FL) images of the L929 cell scratch experiment after different treatments; E) Determination of the survival rate of L929 cells after AK15 treatment; F) Survival rates of L929 cells after 1, 2, and 3 days of different treatments; G) Quantitative analysis of L929 cells; H) Quantitative analysis of the cell coverage area at different time points. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0102] Figure 8 Angiogenic ability of AI-AMP-hydrogel in vitro, where: A) Angiogenesis images of HUVECs treated with different samples after 3 or 6 hours; B) Immunofluorescence images of HUVECs after different treatments; C, D) Quantitative analysis of angiogenesis of HUVEC cells treated with different samples after 6 hours; E) Quantitative analysis of the average fluorescence intensity of CD31; (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0103] Figure 9Quantitative analysis of the relative mRNA expression of COL-I (A), COL-III (B) and VEGF (C).

[0104] Figure 10 Results of accelerated wound healing of AI-AMP-hydrogel in a rat neck infected with MRSA wound model, where: A) Schematic diagram of the whole experimental process; B) Wound images at different times; C) Schematic diagram of the change in the area of the infected wound; D) Quantitative analysis of the relative wound closure area; E, F, G) Quantitative analysis of wound healing length, epidermal thickness and collagen area; H) H&E and masson staining results of samples collected on the 6th and 12th days; (*p<0.05, **p<0.01, **p<0.001, ****p<0.0001).

[0105] Figure 11 Results of immunofluorescence staining of wound tissues, where: A) CD31 staining (red) of samples collected on the 6th and 12th days; B, C, D) Quantitative analysis of the average fluorescence intensity of CD31, COL-I and COL-III; E) Schematic diagram of AI-AMP-hydrogel for wound healing; F) COL-I and COL-III staining (red) of samples collected on the 6th and 12th days; (*p<0.05, **p<0.01, **p<0.001, ****p<0.0001).

[0106] Figure 12 Results of DLS analysis of the particle size of Cu-BTO nanoparticles.

[0107] Figure 13 Spectrum of Cu-BTO nanoparticles analyzed by EDS.

[0108] Figure 14 Results of XPS analysis of Cu element in Cu-BTO nanoparticles.

[0109] Figure 15 Spectrum results of XRD analysis of Cu-BTO nanoparticles.

[0110] Figure 16 For D 33 Spectrum results of the analysis of Cu-BTO nanoparticles.

[0111] Figure 17 Results of the self-healing performance of AI-AMP-hydrogel.

[0112] Figure 18 Results of the mechanical properties of AI-AMP-hydrogel measured by a rheometer. Detailed implementation methods

[0113] The concept of the present invention and the resulting technical effects will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts all fall within the scope of protection of the present invention.

[0114] The sources of important reagent materials of the present invention are as follows:

[0115] The Live / Dead Cell Staining Kit, CCK8 reagent, and 0.25% trypsin digestion solution were purchased from Yacoin Biotechnology Co., Ltd. (China). CAMHB and MH media were purchased from Haibo Biotechnology Co., Ltd. (Qingdao). DMEM medium and FBS were purchased from Thermo Fisher Scientific Inc. (USA). Matrigel without growth factors was purchased from Corning Incorporated (USA). All cell culture inserts (PC membrane, pore size 8 μm) were purchased from LABSELECT (China). Primers for COL-I, COL-III, VEGF, and GAPDH were provided by Shanghai Sangon Biotech Co., Ltd. PEG-4SH (purity: 96%, molecular weight: 10 kDa) was purchased from SINOPEG Co. (Shenzhen). G4-PAMAM was provided by Weihai Chenyuan New Materials Co., Ltd., product number 163442-67-9.

[0116] Example 1

[0117] The information on bacterial strains and cell lines used in the present invention is as follows:

[0118] Six standard strains used in this study, including Enterococcus faecalis ATCC 29212, Klebsiella pneumoniae ATCC700603, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, and Escherichia coli ATCC 25922, as well as MRSA ATCC 43300, were all provided by the Microbiology Laboratory of the General Hospital of the Southern Theater Command. Mouse fibroblasts (L929) and human umbilical vein endothelial cells (HUVECs) were purchased from CytoCan (Shanghai) Biotechnology Co., Ltd. All bacterial strains were cultured in CAMHB or CAMHA at a culture temperature of 37°C. L929 and HUVEC grew in complete medium (DMEM, containing 10% fetal bovine serum (FBS)) at 37°C in a 5% CO2 environment.

[0119] The identification and antimicrobial susceptibility testing of the 6 clinical isolates (Klebsiella pneumoniae (327004, 325016, and 418015), Pseudomonas aeruginosa 304238, Acinetobacter baumannii 316039, and Escherichia coli 103231) isolated in this example were completed by the Vitek MS system (bioMérieux, France) and the Vitek-2compact system (bioMérieux, France), respectively. The results are shown in Table 1.

[0120] Table 1 Antimicrobial susceptibility testing of 6 clinical isolates

[0121]

[0122]

[0123] R, resistant; S, sensitive; I, intermediate; -, intrinsically resistant; ND, not determined.

[0124] Example 2 Screening of antimicrobial peptide AK15

[0125] The AMP-GPT model was pre-trained using the GPT model on a large-scale UniProt dataset. Subsequently, the publicly available antimicrobial peptide datasets APD3, CAMP, DBAASP, DBAMP, and DRAMP were used to perform prompt tuning on this model to obtain the AMP-prompt model. The knowledge distillation technique was used to compress AMP-prompt into a more efficient student model AMP-Distillation based on the recurrent neural network (RNN). To better adjust the biological and physicochemical properties of the generated candidate antimicrobial peptides, reinforcement learning (RL) was applied to optimize the model after prompt tuning. During the training process of AMP-GPT and AMP-Distillation, the "CLS" token was appended to the amino acid sequence as input, while the sequence appended with the "SEP" token was used as the label for training the model.

[0126] In this study, this example utilized the Escherichia coli and Staphylococcus aureus MIC prediction values of AMP-hydrogel Designer, the Marcel AMP classifier, and the presence of individual cysteine residues to provide reward scores.

[0127] This example investigated the variation of different peptide attribute scores during the entire RL process. As Figure 3As shown in Figure E above, the X-axis represents RL iterations and the Y-axis represents the scores of each property. These scores gradually increase with each RL iteration. The overall reward score increases from nearly 1.6 to 3.0, and the probability that the peptide exhibits antibacterial activity increases from 0.5 to approximately 0.75. The predicted MIC values for Escherichia coli and Staphylococcus aureus drop significantly from around 600 to below 100. Figure 3 The property distribution for every 20 to 100 iterations is shown in Figure E below, indicating a shift of the properties towards more optimal values, with the best distribution occurring around the 60th iteration. Throughout the RL process, the system in this example systematically collects the generated polypeptides and sorts them according to their reward values, selecting the top 100 candidate peptides for further screening.

[0128] Subsequently, this example refined the generated polypeptide sequences. As Figure 3 shown in Figure D, screening was performed using CAMP, AMP-Scanner, and Macrel AMP classification probabilities to eliminate non-AMP sequences and only retain sequences containing a single cysteine. After eliminating all unqualified candidate sequences, AK15 (sequence shown in Table 2) was selected for experimental verification based on the average MIC prediction scores for Escherichia coli and Staphylococcus aureus.

[0129] Table 2 Sequence of antibacterial peptide AK15 and alignment with known antibacterial peptide sequences

[0130]

[0131] a Alignment was performed with the APD3 antibacterial peptide database (https: / / aps.unmc.edu / alignment) (alignment time: January 9, 2025).

[0132] Example 3 Synthesis of AK15 polypeptide

[0133] AK15 was custom synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd. Its purity (95.75%) and molecular weight were determined by high performance liquid chromatography (HPLC) and mass spectrometry.

[0134] The high performance liquid chromatography data of the synthesized AK15 is as Figure 1 shown in Table 3, and the mass spectrometry data is as Figure 2 shown.

[0135] Table 3 High performance liquid chromatography data of AK15

[0136] Peak# Ret.Time Area Height Area% 1 5.558 7374 64360 0.6294 2 7.907 5866 35474 0.3469 3 8.938 14477 101706 0.9947 4 9.586 25505 182651 1.786 5 9.854 1059999 9790719 95.75 Total 10225257 1120864 100.000

[0137] Example 4 Determination of the MIC value of AK15

[0138] According to the regulations of the Clinical and Laboratory Standards Institute (CLSI) of the United States, the broth microdilution method was used to determine the MIC value of AK15 against the strains. First, the stock solution of AK15 (5120 μg / mL, dissolved in sterile distilled water) was diluted to 512 μg / mL with CAMHB, and then two-fold serial dilutions were performed in a 96-well plate to form a gradient concentration of AK15 from 0.25 to 512 μg / mL. The concentration of the bacterial suspension in the logarithmic growth phase was adjusted to 0.5 McFarland, diluted 100-fold with fresh CAMHB, and then added to the wells containing AK15 at a concentration of 100 μL per well, with a final bacterial density of approximately 5×10 5 CFU / mL and an AK15 gradient concentration of 0.125 to 256 μg / mL. After incubation at 37 °C for 16 - 20 hours (20 - 24 hours for Acinetobacter baumannii), the MIC value was determined as the lowest concentration of AK15 at which no obvious bacterial growth was observed.

[0139] As shown in Table 4, AK15 has strong broad-spectrum antibacterial activity, and the MIC against the standard strains of 6 common clinical bacteria is less than or equal to 16 μg / ml, with the lowest MIC against Acinetobacter baumannii being 4 μg / ml. The results in Table 5 show that the MIC of AK15 against 4 clinical drug-resistant strains is less than or equal to 16 μg / ml, and the lowest MIC against drug-resistant Acinetobacter baumannii is 4 μg / ml.

[0140] Table 4 MIC values of antibacterial peptide AK15 against the standard strains of six common clinical bacteria

[0141]

[0142] Table 5 MIC values of antibacterial peptide AK15 against six clinical drug-resistant strains

[0143]

[0144] a Pan-drug-resistant (PDR); b Extensively drug-resistant (XDR); c Multidrug-resistant (MDR).

[0145] It can be seen that AK15 has a strong bactericidal effect on clinical drug-resistant bacteria.

[0146] Example 5 Preparation and characterization of AK15 cross-linked hydrogel

[0147] 1. Preparation of hydrogel

[0148] Tetragonal barium titanate (BTO) was ultrasonically dispersed in anhydrous N,N-dimethylformamide (DMF), and the mass ratio of barium titanate to PAMAM was 100:1. A DMF solution dissolving the fourth-generation polyamide-amine dendrimer polymer (G4-PAMAM) was added to the suspension, and then the mixture was refluxed and heated in N2 for 12 hours. The G4-PAMAM-modified BTO was centrifuged at 12,000 rpm for 6 minutes, then washed with water and methanol, and finally dried in vacuo. Then, the G4-PAMAM-coated BTO was dispersed in deionized water, and copper chloride (0.1 M) was added to the mixture. The suspension was stirred at room temperature under N2 conditions for 48 hours. Then, Cu-BTO was separated by centrifugation, washed with water and methanol, and finally dried in vacuo.

[0149] PEG-4SH was dissolved in deionized water to obtain a 100 mg / mL solution, and then various hydrogels were prepared as follows:

[0150] 1) PEG-4SH was mixed with 0.5 mM CuSO4 at a volume ratio of 1:1 to form Cu-hydrogel;

[0151] 2) A 1:1 volume ratio mixture of PEG-4SH and 0.5 mM CuSO4 containing 128 μg / mL AK15 was used to generate AK15-hydrogel;

[0152] 3) A 1:1 volume ratio mixture of PEG-4SH and 0.2 mg / mL Cu-BTO was used to generate BTO-hydrogel;

[0153] 4) A 1:1 volume ratio mixture of PEG-4SH and 0.2 mg / mL Cu-BTO containing 128 μg / mL AK15 was used to generate AI-AMP-hydrogel.

[0154] Each hydrogel was transparent and suitable for injection. All precursor solutions were sterilized through a 0.22 μm sterile filter membrane.

[0155] 2. Characterization of Hydrogels

[0156] The components of the samples were identified using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and nuclear magnetic resonance spectroscopy. Scanning electron microscopy and electron microscopy were used to analyze the morphology of the samples. A rheometer was used to study the mechanical properties of the materials. A piezoresponse force microscope (PFM) was used to characterize the piezoelectric properties of the nanoparticles. Ultrasonic waves were used as the excitation source, and a digital oscilloscope was used to measure the piezoelectric output of the hydrogels.

[0157] Using transmission electron microscopy (TEM, Figure 4In C), scanning electron microscopy (SEM, Figure 4 In B) and dynamic light scattering (DLS, Figure 12 ) were used to analyze the morphology and size of the modified nanoparticles. The results showed that the particle size of the Cu-BTO nanoparticles was uniform and consistent, with an average diameter of about 195 nm. PAMAM was uniformly coated on the surface of BTO with a thickness of about 20 nm. Energy dispersive X-ray spectroscopy (EDS, Figure 13 ) and XPS analysis ( Figure 4 In J) indicated that the N and Cu elements were uniformly distributed on the surface of the nanoparticles, indicating the successful synthesis of the PAMAM / copper composite coating. Further detailed spectral analysis of copper elements in the XPS data ( Figure 14 ) showed that the binding energies of Cu2p3 / 2 and Cu2p1 / 2 decreased from 934.16 eV and 953.44 eV to 932.21 eV and 951.92 eV, respectively. This decrease was attributed to the weakening of the charge density of copper after the lone pair electrons of the amino group in PAMAM coordinated with copper ions, resulting in a slight decrease in the binding energy. To study the piezoelectric properties of Cu-BTO, X-ray diffraction (XRD) was used in this example to characterize its crystal structure ( Figure 15 ). Characteristic diffraction peaks corresponding to the (100), (110), (200), and (211) crystal planes of tetragonal BTO were observed at 2θ angles of 22.2°, 31.5°, 45.3°, and 56.2°, respectively, indicating that Cu-BTO had a piezoelectric tetragonal phase structure and the PAMAM / Cu composite coating had no effect on the crystal phase of BTO. In addition, piezoresponse force microscopy (PFM) was also used to evaluate the piezoelectric properties. Under the reverse electric field, Cu-BTO showed good bright and dark area alignment in the amplitude and phase images ( Figure 4 In D). In addition, the hysteresis and butterfly loops of Cu-BTO showed obvious polarization reversal behavior ( Figure 4 In E,F). The D33 curve ( Figure 16 ) showed that the piezoelectric constant of Cu-BTO was about 16.14 pm / V, indicating its excellent piezoelectric properties.

[0158] FTIR analysis determined different peaks related to PAMAM, BTO, disulfide bonds, and PEG peaks, confirming the binding of Cu-BTO nanoparticles in the hydrogel ( Figure 4 In I). The Cu-S coordination bonds in the hydrogel were dynamically self-formed, endowing the hydrogel with self-healing properties. Tissue adhesion tests showed that AI-AMP-hydrogel had excellent adhesion and could still firmly adhere to tissues after deformation tests on pig skin and muscle. This enabled it to adhere well to wounds in active areas and would not fall off due to frequent movement ( Figure 4In (G). Hydrogels for wound healing must have good hygroscopicity and degradability, and the porous structure of AI-AMP-hydrogel can ensure effective moisture absorption. When soaked in PBS solution, the hydrogel swells by more than 2500%. After cutting, the hydrogel gradually self-heals within ten minutes ( Figure 17 ). In addition, under the action of high shear force, the Cu-S coordination bond will break, making the hydrogel fluid and facilitating injection. A rheometer is used to measure the mechanical properties of the hydrogel ( Figure 18 ). At low shear strain, the storage modulus exceeds the loss modulus, presenting a solid-like state. As the shear strain increases, the loss modulus gradually increases; when the shear strain exceeds 300%, due to the loss modulus being higher than the storage modulus, the hydrogel presents a fluid state. This is because the Cu-S coordination bond in the hydrogel weakens with the increase of shear stress; under the action of high shear force, the coordination bond dissociates reversibly, causing the hydrogel to transform into a fluid state. When the shear force disappears, the coordination bond reforms, making the hydrogel return to a solid-like state, thus showing injectability.

[0159] Example 6 Antibacterial Properties of AI-AMP-hydrogel

[0160] 1. Experimental Methods

[0161] Colony Counting Test: Add 200 μL of hydrogel to a 1.5 mL EP tube and let the gel solidify. Adjust the bacterial suspension of MRSA (ATCC 43300) or Escherichia coli (ATCC 25922) in the logarithmic growth phase to 1×107 CFU / mL with fresh sterile CAMHB, and then add 10 μL of the suspension to the gel surface. After static incubation at 37°C for 2 hours, resuspend the bacteria on the hydrogel surface with 1 mL of sterile PBS. Dilute the resuspended solution 10-fold with PBS, and then spread 100 μL of the diluted suspension on a sterile MH agar plate. After static incubation at 37°C for 18 hours, photograph the results and use ImageJ software to count the colonies formed on the agar plate.

[0162] Bacterial Live / Dead Staining Test: Centrifuge the bacterial suspension in the logarithmic growth phase at 10,000 rpm for 5 minutes. Wash once with physiological saline and adjust to 1×10 8 CFU / mL, then incubate 200 μL of the bacterial suspension with 200 μL of the hydrogel for 2 hours. Then, use a live / dead bacterial staining kit (Beyotime) to stain the co-cultured bacterial suspension in the dark at 37°C for 15 minutes. Observe with an inverted fluorescence microscope and calculate the bactericidal rate. The formula for calculating the bactericidal rate (%) is (N red / N0)×100, where N redis the number of bacteria emitting red fluorescence, and N0 is the total number of bacteria emitting both red and green fluorescence. After staining, viable bacteria with intact membranes emit green fluorescence, while dead bacteria with damaged membranes emit red fluorescence.

[0163] Crystal violet staining of biofilms: Adjust the MRSA suspension to 0.5 McFarland with CAMHB medium and then add 1 mL to each well of a 24-well plate. Replace the medium with fresh medium after 24 hours and discard the old medium after 48 hours. Carefully wash the wells three times with PBS to remove unattached bacteria, thus allowing the MRSA biofilm to adhere to the 24-well plate. Place a chamber containing 200 μL of hydrogel into the well with the biofilm, and then add 600 μL of PBS. Incubate the samples at 37 °C for 6 hours, then remove the hydrogel and gently wash it three times with PBS to remove floating bacteria. After drying at room temperature, add 500 μL of absolute ethanol and fix at 4 °C for 15 minutes. Then remove the ethanol and stain the biofilm with 500 μL of 1% crystal violet for 30 minutes. After gently washing three times with PBS and drying at room temperature, take pictures of the samples. Subsequently, add 500 μL of ethanol to each well and incubate for 20 minutes to dissolve the crystal violet. Transfer the solution to a 96-well plate and measure the OD using a microplate reader 595 。

[0164] As Figure 5 shown, compared with the control group, AI-AMP-hydrogel showed strong antibacterial activity against both E. coli and MRSA, with an inhibition rate as high as 99.99%, while Cu-hydrogel and BTO-hydrogel had no inhibitory effect on these bacterial strains. Bacterial live / dead staining detection ( Figure 5 C) confirmed these findings, and the number of viable bacteria in the AI-AMP-hydrogel treatment group was significantly reduced compared with the growth control group, Cu-hydrogel group, and BTO-hydrogel group ( Figure 5 J, K). In addition, AI-AMP-hydrogel also had the ability to disrupt the MRSA biofilm, so the survival rate of the biofilm treated with AI-AMP-hydrogel was significantly lower than that of the other three groups ( Figure 5 B, I). In summary, these results indicate that AI-AMP-hydrogel has potent and broad-spectrum in vitro antibacterial activity.

[0165] Exploration of the antibacterial mechanism of Example 7 AK15

[0166] 1. Experimental method

[0167] TEM (Transmission Electron Microscopy) Characterization: Cell Harvest and Treatment: Centrifuge (4000 rpm, 5 minutes) at room temperature to harvest 10 mL of log-phase MRSA (ATCC 43300) or Escherichia coli (ATCC 25922) suspension. Incubate the bacterial suspension with an equal volume of gel at 37 °C and 150 rpm for 5 hours, then centrifuge the mixture (4000 rpm, 5 minutes) at 4 °C. After discarding the supernatant, resuspend the pellet in PBS, transfer an appropriate volume of the pellet to a 1.5 mL tube, centrifuge (8000 rpm, 10 minutes) at 4 °C, remove the supernatant, slowly add pre-cooled glutaraldehyde (1 mL), and fix overnight at 4 °C. Rinse twice with PBS within 10 minutes, fix with 1% osmium tetroxide at room temperature for 2 hours, and rinse twice with PBS within 5 minutes. Dehydrate the samples through an ethanol series (50% and 70%, 10 minutes each) and an acetone series (80%, 90%, and 100%, 10 minutes each).

[0168] Embedding and Sectioning: Immerse the samples in a 1:1 mixture of acetone and Epon 812 epoxy resin for 40 minutes, then immerse in pure Epon 812 at 37 °C overnight. Finally, embed the samples in Epon 812 in a cylindrical mold at 60 °C for 48 hours. Carefully cut the embedded samples into 40 nm ultra-thin sections using a Leica ultramicrotome, then transfer the sections to copper grids and let them dry overnight. Stain with saturated uranyl acetate solution (dissolved in 70% ethanol) for 3 minutes, then stain with lead citrate for 3 minutes, and then let the copper grids dry completely. Acquire images using TEM (HITACHI H-7650).

[0169] SEM (Scanning Electron Microscopy) Characterization: The cell collection and treatment procedures are the same as those for the TEM procedure. Fix the bacteria with glutaraldehyde overnight at 4 °C, then discard the glutaraldehyde solution. Then rinse the samples three times with 0.1 M PBS for 15 minutes each. Then, fix the samples with 1% osmium tetroxide solution for 2 hours. Pour out the osmium tetroxide solution and rinse the samples three times with 0.1 M PBS for 15 minutes each. Dehydrate in ethanol solutions of increasing concentration (30%, 50%, 70%, 80%, 90%, and 95%) for 15 minutes each, then treat with 100% ethanol for 20 minutes. Then transfer the samples to fresh 100% ethanol for storage. After drying with a dryer (Quorom k850), mount the samples on a sample holder with an ion sputter (Hitachi MC1000) for about 120 seconds. Finally, examine the samples and acquire images using a scanning electron microscope (Hitachi Regulus 8100).

[0170] Transcriptome Analysis:

[0171] Suspend the logarithmic-phase MRSA bacterial suspension with AI-AMP-hydrogel and sonicate it together (1 W / cm 2 , 1 MHz, 5 minutes), and then culture it at 37 °C (150 rpm, 2 hours). Untreated MRSA serves as the control group. Collect MRSA bacterial cells and extract total bacterial RNA using the HiPure Bacterial RNA Kit (Guangzhou Meiji). Sequence the RNA-seq transcriptome in the PE150 sequencing mode using the Illumina NovaSeq6000. After sequencing, perform differential analysis using the DESeq2 software (version 1.20.0), and perform GO and KEGG analyses on the DEGs using the clusterProfiler software package (version 3.8.1).

[0172] 2. Experimental results

[0173] The TEM and SEM results (Figs. 5D, E) show that the biofilms of E. coli and MRSA in the growth control group are smooth and structurally intact, while the biofilms in the AI-AMP-hydrogel group are damaged or even ruptured, resulting in the leakage of cell contents. This indicates that AK15 exerts its antibacterial effect by disrupting the integrity of the bacterial membrane structure.

[0174] To further clarify the anti-MRSA mechanism of AI-AMP-hydrogel, this example analyzed the transcriptome profiles of MRSA from the growth control group and the AI-AMP-hydrogel group. The volcano plot ( Figure 6 A) shows that compared with the control group, MRSA treated with AI-AMP-hydrogel exhibits 233 differentially expressed genes (DEGs), of which 176 are upregulated and 57 are downregulated. Subsequently, this example performed cluster analysis on the top 15 upregulated and downregulated DEGs and plotted a circular heatmap ( Figure 6In B), red represents upregulated DEGs, while blue represents downregulated DEGs. After treatment with AI-AMP-hydrogel, the upregulated DEGs in MRSA were mainly related to cell wall teichoic acid modification (dltA, dltB, dltC, and dltD), regulation of positive charge on the membrane surface (mprF), and copper ion transport (copB, copZ). In contrast, the downregulated DEGs were mainly involved in cell wall damage repair (cwrA, vraR, vraX), membrane stress response (liaF), metabolic activities (argH, uhpT), and hemolytic activity (hlgA, hlgB). These results indicate that AI-AMP-hydrogel not only effectively weakens the cell wall repair ability of MRSA and enhances the destructive effect of AK 15 on the cell wall, but also further improves the antibacterial activity by inhibiting the membrane stress response and interfering with metabolic activities. Although MRSA attempts to protect itself by altering the membrane charge through upregulating the expression of genes such as dltA and mprF, this is completely insufficient to resist the effective bactericidal effect of AI-AMP-hydrogel. Notably, the treatment with AI-AMP-hydrogel downregulates the expression of hlgA and hlgB, indicating a decrease in hemolytic activity. In contrast, the upregulation of copB and copZ expression means that the copper ions released from Cu-BTO in AI-AMP-hydrogel disrupt the copper homeostasis regulatory system of MRSA. GO enrichment analysis of DEGs ( Figure 6 In C) shows that the effects of AI-AMP-hydrogel on MRSA are concentrated on key biological processes such as protein translation, protein-RNA complex assembly, and tRNA modification. Ribosome-related cellular components are significantly enriched, indicating that bacteria may respond to antibacterial stress by enhancing ribosome function and promoting protein synthesis. In addition, the pathways involved in metal ion transmembrane transport are significantly enriched, indicating that AI-AMP-hydrogel may disrupt the metabolism and cell function of MRSA by interfering with metal ion homeostasis and transmembrane transport processes. KEGG enrichment analysis of DEGs ( Figure 6 In D) shows that the effects of AI-AMP-hydrogel on MRSA are concentrated on key biological processes such as ribosome biosynthesis, metabolic pathways, and antimicrobial peptide resistance. Notably, the infection pathway is significantly downregulated, which means that AI-AMP-hydrogel not only precisely kills MRSA, but also has multi-faceted effects on infection by reducing the virulence and infectivity of MRSA. Protein-protein interaction (PPI) network ( Figure 6 In E) shows that top hub genes such as dltC, fusA, rplC, mnh, and saeR are also involved in bacterial membrane alteration, metabolic processes, and virulence factors. Figure 6Figure F shows a schematic diagram illustrating the anti-MRSA mechanism of AI-AMP-hydrogel based on transcriptome sequencing results. Overall, these results indicate that AI-AMP-hydrogel exerts its antibacterial activity by disrupting the bacterial membrane, interfering with metabolism, and reducing bacterial virulence.

[0175] Example 8 Biocompatibility and Cell Proliferation Tests of Hydrogels

[0176] 1. Experimental Methods

[0177] Preparation of hydrogel extract: Immerse 1 mL of AI-AMP-hydrogel in 30 mL of CAMHB, then culture it at 37 °C and 150 rpm for 3 days. The extract is passed through a 0.22 μm sterile filter membrane and stored at 4 °C. Add 10% FBS before use.

[0178] CCK-8 assay: Aliquot 100 μL of the L929 cell suspension into each well of a 96-well plate, with approximately 5000 cells per well, and culture it in an environment of 37 °C and 5% CO2 for 24 hours. Then, add 10 μL of AK15 with gradient concentrations to each well, such that the final concentration of AK15 ranges from 8 μg / mL to 512 μg / mL. After co-culturing for 24 hours, remove the old medium, and add 100 μL of DMEM medium containing 10% (v / v) CCK-8. After incubating the cells in a 5% CO2 environment at 37 °C for 2 hours, measure the OD 450 of the supernatant and calculate the cell viability using Formula 1.

[0179]

[0180] Live / Dead cell staining detection: Seed L929 cells into a 24-well plate, with approximately 50,000 cells per well, to allow them to attach to the surface. Add 200 μL of the hydrogel to the chamber. The ultrasonic group is sonicated at a power of 0.8 W / cm 2 and a frequency of 1 MHz for 1 minute, and then co-culture it in a 5% CO2 environment at 37 °C for 24 hours. Then remove the old medium, add 200 μL of the live / dead staining solution to each well and incubate for 30 minutes. After rinsing twice with PBS, record the images under an inverted fluorescence microscope.

[0181] Cell proliferation assay: Resuspend L929 cells in the hydrogel extract and seed them into a 96-well plate, with approximately 5000 cells per well. After culturing for 1, 2, or 3 days in an environment of 37 °C and 5% CO2, discard the old medium, and add CCK-8 in DMEM (100 μL, 10% (v / v)) to each well. After culturing for 2 hours, measure the OD 450 of the supernatant to calculate the cell viability.

[0182] 2. Experimental Results

[0183] The cytotoxicity of AK15 against L929 cells is shown in Figure 7 Figure E. Even at a concentration of 128 μg / mL, the survival rate of L929 cells remained at 87.2%. In contrast, the MIC of AK15 against Escherichia coli and Staphylococcus aureus was only 8 μg / mL. This indicates that AK15 has good biocompatibility. Subsequently, in this example, the extracts of AI-AMP-hydrogel were cultured with L929 cells for 1 day, 2 days, and 3 days respectively to measure cell viability ( Figure 7 Figure F). The results showed that there was no significant difference in the cell proliferation rate at all time points compared with the control group, which means that AI-AMP-hydrogel does not affect cell proliferation and has good biocompatibility. The live / dead staining assay was used to further analyze the survival of L929 cells treated with AI-AMP-hydrogel after 24 hours of ultrasonic activation (0.8 W / cm 2 , 1 MHz, 1 minute). As shown in Figure 7 Figure C, whether in contact with ultrasonic waves or not, AI-AMP-hydrogel does not affect the survival of L929 cells.

[0184] Example 9 Evaluation of Cell Migration of Hydrogel

[0185] 1. Experimental Method

[0186] Scratch test: L929 cells were seeded into 6-well plates, with about 1×10 6 cells per well, and cultured in an incubator (37 °C, 5% CO2) until about 90% confluence. A sterile 200 μL pipette tip was used to scratch the cell monolayer, and then the cells were rinsed three times with PBS to remove the detached cells. Then 2 mL of serum-free DMEM was added to each well, and a transwell chamber containing 400 μL of hydrogel was placed into the well. The ultrasonic group was treated at a frequency of 0.8 W / cm 2 and 1 MHz for 1 minute. After culturing for 12 and 24 hours in an environment of 37 °C and 5% CO2, the cells were stained with calcein-AM and observed under an inverted fluorescence microscope. The ImageJ software was used to measure the change in the scratch area to compare the cell migration rate.

[0187] Transwell test: L929 cells were cultured in serum-free DMEM for 12 hours and then co-cultured with the hydrogel. The ultrasonic group was treated at 0.8 W / cm 2 , 1 MHz for 1 minute. After culturing, the cells were seeded into the Transwell wells of a 24-well plate, with about 5×104 One cell, with 200 μL of serum-free DMEM in the upper chamber and 600 μL of complete medium in the lower chamber. After co-culturing for 24 hours, the transwell chamber was taken out, and the cells at the bottom of the lower chamber were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution for 20 minutes. The stained cells were observed under a microscope, and cell counting was performed using ImageJ software.

[0188] 2. Experimental results

[0189] The experimental procedures of the Transwell and scratch assays are shown in Figure 7 A and B respectively.

[0190] The Transwell results are shown in Figure 7 C and Figure 7 G as shown. The cell migration rates of BTO-hydrogel and AI-AMP-hydrogel containing BTO nanoparticles after ultrasound exposure were significantly higher than those of the control group, and no significant difference was found between the non-ultrasound-treated groups, indicating that the bioelectric energy released by the piezoelectric material after ultrasound activation effectively promoted cell migration.

[0191] The scratch assay evaluated the migration and healing potential of cells between different groups by detecting the closure of the scratch wound. As shown in Figure 7 D and Figure 7 H as shown, the application of low-intensity ultrasound (0.8 W / cm 2 , 1 MHz, 1 minute) significantly enhanced the migration of L929 cells in the BTO-hydrogel group and the AI-AMP-hydrogel group.

[0192] Evaluation of cell migration of the hydrogel in Example 10

[0193] 1. Experimental method

[0194] Thaw Matrigel without growth factors overnight at 4 °C. Then, add 20 μL of Matrigel to each well of a pre-cooled 24-well plate and spread the gel evenly with a 200-μL sterile pipette tip. Then incubate at 37 °C for 30 minutes to allow the Matrigel gel to solidify. Resuspend HUVECs that have been starved for 12 hours in complete medium and seed them onto the 24-well plate with the hydrogel-filled transwell chambers at a density of 1×10 5 cells per well. The ultrasound-treated group received 1 minute of 0.8 W / cm 2and 1 MHz ultrasound. After culturing for 3 hours and 6 hours at 37°C under 5% CO2 conditions, the cells were stained with AbFluorTM 594-Phalloidin (Yakeyin) and DAPI (Solarbio), and observed under an inverted fluorescence microscope. The angiogenesis data were analyzed using ImageJ software.

[0195] Immunohistochemistry: HUVECs co-cultured with the hydrogel (ultrasound conditions: 0.8 W / cm 2 and 1 MHz, for 1 minute) were resuspended in complete medium and then seeded onto the cell coverslips in 12-well plates, approximately 1×10 5 cells per well. After culturing for 24 hours in a 5% CO2 environment at 37°C, the cells were fixed with 4% paraformaldehyde for 10 minutes and blocked with 10% goat serum for 45 minutes. The cells were incubated overnight with a primary antibody against CD31 (Affinity) at 4°C, and then incubated with a secondary antibody (Fluor488-conjugated goat anti-rabbit IgG(H+L), Affinity) at room temperature in the dark for 1 hour. Then, the cells were permeabilized with 0.1% Triton X-100 for 5 minutes and stained with AbFluor TM 594-Phalloidin (Yakeyin) in the dark for 30 minutes, and then stained with DAPI (Solarbio) in the dark for 10 minutes. After sealing the coverslips with nail polish, they were observed under a confocal microscope (Nikon), and the CD31 fluorescence data were recorded using ImageJ software.

[0196] 2. Experimental Results

[0197] In this example, the angiogenesis assay ( Figure 8 A therein) and CD31 immunofluorescence staining ( Figure 8 B therein) were used to evaluate the effect of AI-AMP-hydrogel on the angiogenic ability of human umbilical vein endothelial cells (HUVECs) under ultrasound-activated and non-activated conditions. As Figure 8 shown in C, after co-culturing for 6 hours, obvious tube formation was observed in each group, and the number of tubes in the ultrasound-treated BTO-hydrogel group and AI-AMP-hydrogel group was significantly higher than that in other groups. Immunofluorescence detection of HUVECs showed that the fluorescence intensity in the ultrasound-treated BTO-hydrogel group and AI-AMP-hydrogel group was significantly higher than that in other groups. The research results indicate that the bioelectric energy released by the piezoelectric material during ultrasound activation effectively enhances the expression of CD31 and the formation of HUVECs blood vessels.

[0198] Example 11 Expression of Genes Related to Hydrogel Wound Healing

[0199] L929 cells were seeded into 6-well plates at approximately 1×106 cells per well and cultured overnight. Then, chambers containing hydrogels were added, and ultrasound (0.8W / cm 2 , 1 MHz, 1 minute) was applied to the ultrasound group. After culturing for 24 hours in an environment of 37°C and 5% CO2, total RNA was extracted according to the method described in the AFTSpin Animal Tissue / Cell Rapid RNA Extraction Kit (Aibotech), and quantified using a micro-spectrophotometer. RNA was reverse-transcribed into cDNA using the ABScript III RT Master Mix for qPCR with gDNARemover (Aibotech). The qPCR reaction system was prepared according to the BrightCycle Universal SYBR Green qPCRMix with UDG (Aibotech), and real-time quantitative PCR analysis was performed using PCR (lepgen-96).

[0200] The relevant primer sequences are shown in Table 6.

[0201] Table 6 Primer sequences of COL-I, COL-III, and VEGF

[0202]

[0203] As Figure 9 shown in A, B, and C, under the activation of low-intensity ultrasound (US), the expression levels of VEGF, COL-I, and COL-III genes in L929 cells cultured with BTO-hydrogel and AI-AMP-hydrogel were significantly increased compared with the control group and the group without ultrasound treatment.

[0204] Example 12 Wound repair at the active site

[0205] All animal experiments were approved by the Animal Ethics Committee of Guangdong Provincial People's Hospital (Approval No.: KY2023-054-01). All experimental procedures were strictly carried out in accordance with ethical standards. Male SD rats (8 weeks old, n = 6) were randomly divided into 5 groups (model control group, Cu-hydrogel group, BTO-hydrogel group, AK15-hydrogel group, AI-AMP-hydrogel group), and neck wounds were established. The rats were anesthetized with Zoletil 50 (25 mg / kg), and the neck hair was shaved. A full-thickness skin wound with a diameter of 10 mm was established on the back of the neck of each rat, and then 5 μL of a solution with a concentration of 4×10 9MRSA suspension of CFU / mL, and the hydrogel was applied to the wound. During the entire experiment, rats had unrestricted access to food and water and were housed in micro-isolation cages with controlled humidity, temperature, and a 12-hour light / dark cycle. The wound condition was recorded daily, and the wound area was measured using ImageJ software.

[0206] Microbiological and histological analysis: Rats in each group were euthanized on the 6th and 12th days after infection. The infected tissues were collected and homogenized in 5 mL of PBS to evaluate the bacterial load. In addition, the wound and surrounding tissues were collected for histological evaluation by H&E staining and Masson staining. Immunofluorescence staining was performed to evaluate the expression of CD31, COL I, and COL III.

[0207] In a full-thickness dorsal skin wound model of MRSA-infected rats, the mechanical energy generated by neck movement replaced ultrasound, activated BTO in the AI-AMP-hydrogel, generated bioelectricity, and exerted a potential therapeutic effect. The treatment of MRSA-infected wounds started 2 hours after infection. The treatment day was designated as day 0, and skin samples were collected on the 6th and 12th days for histological and immunofluorescence staining ( Figure 10 in A). The healing results at different time points are shown in Figure 10 in B - D. The wounds in the model control group and the Cu-hydrogel group showed infection and suppuration on the 2nd day, and the healing progress was slow from day 0 to day 12. In contrast, the wounds in the BTO-hydrogel group healed faster, while the AK15-hydrogel group and the AI-AMP-hydrogel group showed significant anti-infection and wound healing abilities, and the wounds in all three groups could be rapidly closed (the wound closure rates were 87.3%, 89.6%, and 99.5% respectively). The wound healing effect of the AI-AMP-hydrogel group was better than that of the BTO-hydrogel group and the AK15-hydrogel group, which may be due to the synergistic effect of AK15 further promoting the bioelectricity released by the piezoelectric material to exert the wound healing effect.

[0208] To further confirm the in vivo healing effect, the wound healing characteristics between different groups were compared by detecting the histological changes of skin tissues on the 6th and 12th days. The results of H&E staining ( Figure 10 in H, E, F) were consistent with the wound closure rate. Compared with other groups, the wound area in the AI-AMP-hydrogel group decreased, and the epidermal thickness increased significantly. To explore the biological mechanism of wound repair, Masson staining was performed in this example to analyze the collagen distribution at the wound site. As Figure 10As shown in H and G, the collagen fiber density of the BTO-hydrogel, AK15-hydrogel, and AI-AMP-hydrogel groups was significantly higher than that of the model control group and the Cu-hydrogel group. CD31 is a marker of vascular endothelial cells, and angiogenesis at the wound site was evaluated by immunofluorescence staining ( Figure 11 in A and B). The results showed that the CD31 expression was higher in the groups containing BTO, and the CD31 expression further increased after adding AK15, indicating that the AI-AMP-hydrogel group had more superior angiogenesis characteristics. The expression levels of COL-I and COL-III at the wound site were further quantified using immunofluorescence ( Figure 11 in F). As Figure 11 shown in C and D, the quantitative results showed that the expression levels of COL-I and COL-III in the BTO-hydrogel group and the AI-AMP-hydrogel group were significantly higher than those in the model control group and the Cu-hydrogel group. Although the expression levels of COL-I and COL-III in the AK15-hydrogel group increased compared with the model control group, they were slightly lower than those in the AI-AMP-hydrogel group. These research results indicate that BTO converting mechanical energy into bioelectricity can effectively promote the expression of collagen at the wound site, and this effect will be further amplified when the infection at the wound site is controlled ( Figure 11 in E).

Claims

1. A polypeptide AK15 or a salt thereof, characterized in that: The amino acid sequence of the polypeptide is shown as any one of a1) to a3); a1) SEQ ID NO.1; or a2) An amino acid sequence obtained by substituting and / or deleting and / or adding one or several amino acids to SEQ ID NO.1 and having the same function as SEQ ID NO.1; or a3) An amino acid sequence having at least 95% homology with SEQ ID NO.

1.

2. A conjugate, characterized in that: The conjugate comprises a modification part and the polypeptide AK15 according to claim 1.

3. The conjugate according to claim 2, characterized in that: The modification part is located at the N-terminus and / or C-terminus of the polypeptide AK15; Preferably, the modification part comprises at least one of chemical modification, targeting part, fluorescent dye and protein tag; Preferably, the chemical modification comprises at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation, ubiquitination; Preferably, the targeting part comprises at least one of ligand, receptor, antibody; Preferably, the fluorescent dye comprises FITC; Preferably, the protein tag comprises at least one of His, Flag, GST, MBP, HA, Myc, GFP.

4. A fusion protein, characterized in that: The fusion protein comprises the polypeptide or a salt thereof according to any one of claims 1 to 3; And other polypeptides.

5. The fusion protein according to claim 4, characterized in that: The other polypeptide is located at the N-terminus and / or C-terminus of the polypeptide AK15; Preferably, the other polypeptide and the polypeptide AK15 according to claim 1 are linked to the N-terminus and / or C-terminus of the polypeptide AK15 through a linker.

6. A biological material related to the polypeptide AK15 described in claim 1, the conjugate according to any one of claims 2-3, or the fusion protein according to any one of claims 4-5, the biological material comprising any one of b1) to b8): b1) A nucleic acid molecule encoding the polypeptide AK15 described in claim 1, the conjugate according to any one of claims 2-3, or the fusion protein according to any one of claims 4-5; b2) An expression cassette comprising the nucleic acid molecule of b1); b3) A vector comprising the nucleic acid molecule of b1); b4) A vector comprising the expression cassette of b2); b5) A transgenic cell line comprising the nucleic acid molecule of b1); b6) A transgenic cell line comprising the expression cassette of b2); b7) A transgenic cell line comprising the vector of b3); b8) A transgenic cell line comprising the vector of b4).

7. A composition comprising at least one of c1) to c3): c1) The polypeptide AK15 or a salt thereof according to claim 1; c2) The conjugate according to any one of claims 2-3; c3) The fusion protein according to any one of claims 4-5; Preferably, the composition further comprises other active antibacterial components; Preferably, the other active antibacterial components include at least one of penicillin antibiotics, cephalosporin antibiotics, carbapenem antibiotics, lipo(glyco)peptide antibiotics, aminoglycoside antibiotics, tetracycline antibiotics, lincosamide antibiotics, macrolide antibiotics, and fluoroquinolone antibiotics.

8. Use of the polypeptide AK15 or its salt according to claim 1, the conjugate according to any one of claims 2-3, the fusion protein according to any one of claims 4-5, the biomaterial according to claim 6, and / or the composition according to claim 7 in any one of d1) to d2): d1) Preparing an antibacterial product; d2) Preparing a product for promoting wound healing.

9. A product comprising at least one of e1) to e4): e1) The polypeptide AK15 or its salt according to claim 1; e2) The conjugate according to any one of claims 2-3; e3) The fusion protein according to any one of claims 4-5; e4) The composition according to claim 7; The product is any one of f1) to f2): f1) An antibacterial product; f2) A product for promoting wound healing; The product comprises at least one of a drug, a feed, a feed additive, a preservative, a daily chemical product, a fabric, a paper product, a medical material, and a cell culture medium.

10. The product according to claim 9, wherein: The product includes a hydrogel; The hydrogel comprises at least one of e1) to e4), and a colloidal part; The colloidal part includes a natural biogel and a synthetic biogel; Preferably, the natural biogel includes at least one of collagen, hyaluronic acid, sodium alginate, chitosan, gelatin, and fibrin; Preferably, the synthetic biogel includes at least one of polyethylene glycol, polyacrylic acid, poly N-isopropylacrylamide, and polyvinyl alcohol.

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