PSM alpha 1 targeting peptide and corresponding application and biomedical material

Abstract

The invention provides a PSM [alpha] 1 targeting polypeptide. The amino acid sequence of the targeting polypeptide is selected from the group consisting of KPTWATG, IDPSKYS, YLTPFQS, GLTTTRY, IVNMSYH and VNVEVTLVN. The invention provides an application of the PSM alpha 1 targeting polypeptide in a biomedical material for resisting bacteria and / or promoting wound healing. The invention also provides a corresponding antibacterial photothermal therapy biomedical material and a photothermal therapy biomedical material for promoting wound healing. The targeting polypeptide specifically bound with the PSM alpha 1 can effectively inhibit the fibrosis process of the PSM alpha 1. According to the nanoparticle applied to the treatment of the MRSA infected biofilm of the diabetes wound surface, the biofilm formation inhibition rate can reach 94.7 + / -1.5%, and the nanoparticle realizes decomposition of the biofilm and killing of deep bacteria by utilizing a photothermal effect.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a PSMα1 targeting peptide and an application thereof in a nanosystem of synergistic antibacterial activity of CuS nanoparticles. Background Art

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is the most common opportunistic pathogen in humans and one of the most difficult bacteria to treat. Compared with common S. aureus, MRSA has an increased expression of PSM, which strengthens the stability of the biofilm. On the one hand, MRSA biofilms help bacteria resist the environment, and on the other hand, they hinder the penetration of drugs and achieve host immune evasion, and contribute to the secretion of bacterial virulence. Studies have shown that the strategy of inhibiting biofilm formation by targeting amyloid proteins is an effective way to inhibit biofilm formation and achieve antibacterial effects. PSMα1 is an amyloid protein secreted by MRSA, which can self-assemble to form amyloid fibers, especially PSMα1 to form strong and irreversible cross-βsheet structure fibers, which play an important role in the stability of biofilms.

[0003] Phage display technology refers to the ability of virus particles to display foreign proteins on their surface. The specific process is to insert exogenously encoded polypeptides or proteins into the appropriate position of the phage coat protein structure, so that the exogenous polypeptides or proteins form fusion proteins on the capsid protein of the phage. With the reassembly of the progeny phage, it is presented on the phage surface, and then a subset of biomolecules carrying specific characteristics and functions is screened from the diverse phage library by affinity. Usually in phage display technology, there is a library of diverse peptide or protein variants on the phage surface (usually 1010-1012). After 3-5 rounds of enrichment, the proportion of phages that can specifically recognize the target molecule is gradually increased, and finally the antibody molecules and their gene sequences that specifically bind to the antigen are screened for further application.

[0004] In order to achieve the anti-biofilm formation strategy mediated by PSMα1, this study screened out the targeting peptide KG7 with the strongest affinity for PSMα1 through phage display technology. KG7 can not only act on the core fragment of PSMα1 to form a beta-sheet structure, inhibit its fibrillation and thus prevent the formation of biofilm. At the same time, KG7 is a typical amphipathic peptide with a positively charged lysine at its N-terminus, which can be adsorbed on the bacterial surface through charge action, thereby destroying the integrity of the cell membrane structure, leading to the leakage of bacterial internal components and bacterial lysis.

[0005] In recent years, copper sulfide (CuS) nanoparticles have been widely used in many biomedical fields such as drug delivery and photothermal therapy. The near-infrared absorption ability of CuS comes from the dd band transition of copper ions. It is often used as an antibacterial agent due to its excellent photothermal performance. When CuS is used as a drug delivery system, hollow CuS has better drug delivery performance and a larger specific surface area for loading drugs. At the same time, CuS accelerates the release of copper ions at high temperatures, causing macrophage M2 polarization, thereby promoting angiogenesis to achieve the purpose of tissue repair.

[0006] In view of the significant effect of KG7 in inhibiting the fibrosis of PSMα1 and thus preventing the formation of biofilm, there are still some defects. KG7 cannot effectively remove mature biofilm, which limits its practical application. In order to further optimize the killing effect of KG7 on mature biofilm, KG7 was combined with photothermal therapy to design a nanosystem with synergistic antibacterial effect. Mature biofilm can be ablated under local photothermal treatment without causing damage to surrounding normal tissues. At the same time, photothermal treatment changes the permeability of bacterial biofilm, allowing targeted peptides to penetrate and exert targeted antibacterial effects. This chapter realizes the degradation of mature biofilm based on the photothermal performance of CuS, and designs the PPDA@CuS system to solve the problem of limited antibacterial effect in MRSA mature biofilm. This paper first synthesizes hollow CuS nanoparticles with a size of about 150nm, which have excellent photothermal performance under near-infrared light. In order to achieve the coupling of CuS and KG7, a polydopamine (PDA) coating was modified on the surface of CuS to prepare PDA@CuS. PDA contains a large number of secondary amines and catechol groups, which can destroy the membrane integrity of bacteria and cause bacterial death, while improving the adhesion and photothermal conversion performance of the substrate material. Finally, the targeted peptide KG7 was grafted on the surface of the PDA coating to form a PPDA@CuS system, which can achieve the following three performance improvements: ① Introducing photothermal therapy to achieve local high-temperature killing of bacteria and the removal of deep bacteria and mature biofilms. ② Photothermal heating promotes the release of copper ions, promotes macrophage M2 polarization, and accelerates tissue repair. ③ Combined with the KG7-mediated EPS component targeted biofilm formation inhibition strategy, the challenge of biofilm regulation in chronic wound sites can be achieved. Summary of the invention

[0007] The first object of the present invention is to provide six PSMα1 targeting polypeptides having strong affinity and binding specificity to PSMα1, wherein the amino acid sequence of the targeting polypeptide is selected from: KPTWATG, IDPSKYS, YLTPFQS, GLTTTRY, IVNMSYH and VNVEVTLVN, and the order of affinity is KG7 (KPTWATG), IS7 (IDPSKYS), YS7 (YLTPFQS), GY7 (GLTTTRY), IH7 (IVNMSYH) and VN7 (VEVTLVN).

[0008] The second object of the present invention is to provide the use of the PSMα1 targeting polypeptide in antibacterial and / or wound healing promoting biomedical materials.

[0009] Preferably, the PSMα1 targeting polypeptide is used in conjunction with CuS nanoparticles.

[0010] Preferably, the PSMα1 targeting polypeptide is grafted onto the surface of the CuS nanoparticles via a dopamine coating.

[0011] Preferably, the biomedical material is an antibacterial and / or wound healing promoting material for MRSA infection of diabetic wounds. In order to achieve the first object of the present invention, the present invention provides a targeting polypeptide of PSMα1, the main feature of which is that the amino acid sequence of the targeting polypeptide is selected from: KPTWATG, IDPSKYS, YLTPFQS, GLTTTRY, IVNMSYH and VNVEVTLVN.

[0012] In order to achieve the second purpose of the present invention, the present invention provides the preparation of copper sulfide nanomaterials modified with PSMα1 targeting peptide and the research on their antibacterial properties and wound healing promotion.

[0013] The third object of the present invention is to provide an antibacterial photothermal therapy biomedical material, which mainly comprises CuS nanoparticles and PSMα1 targeting polypeptide, wherein the PSMα1 targeting polypeptide is grafted onto the surface of the CuS nanoparticles through a dopamine coating, and the amino acid sequence of the targeting polypeptide is KPTWATG.

[0014] The fourth object of the present invention is to provide a wound healing-promoting photothermal therapy biomedical material, which mainly comprises CuS nanoparticles and PSMα1 targeting polypeptide, wherein the PSMα1 targeting polypeptide is grafted onto the surface of the CuS nanoparticles through a dopamine coating, and the amino acid sequence of the targeting polypeptide is KPTWATG.

[0015] The advantage of the present invention is that the present invention firstly obtains a targeting polypeptide sequence that specifically binds to PSMα1 by using the technology of phage display library and bio-panning, and then performs molecular simulation and isothermal titration calorimetry analysis on PSMα1 and the targeting polypeptide of PSMα1. At the same time, based on the phenol-soluble regulatory protein PSMα1 in the EPS component of S. aureus biofilm as a target, EPS targeting peptides for inhibiting biofilm formation are screened and obtained, and further grafted to the surface of CuS nanoparticles with high photothermal conversion rate through a dopamine coating, a targeted composite nanosystem for biofilm regulation and promotion of chronic wound healing is successfully constructed. In this system, CuS and PDA achieve the decomposition of mature biofilms and direct killing of bacteria in diabetic chronic wounds under near-infrared laser irradiation. Secondly, with the assistance of the targeting peptide KG7 of PSMα1, PSMα1 fibrosis is inhibited, thereby inhibiting the formation of biofilms and subsequent biofilm skeletons. The key to this regulatory mechanism is that KG7 mainly acts on the core fragment GIIKVIK of PSMα1 fibrosis. More importantly, hollow copper sulfide increases the copper ion content under laser irradiation, prompting macrophages to polarize toward M2, promoting angiogenesis and thus repairing infected tissues. In summary, this study provides a reliable way to prevent bacteria and promote wound healing for chronic diabetic wound infection, and also provides new ideas for biofilm regulation strategies based on EPS component targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The recovery rate of each round of phage screening during the bio-panning process of PSMα1 targeting peptides.

[0017] Figure 2 The sequences, theoretical isoelectric points and molecular weights of the six PSMα1 affinity peptides obtained by bio-panning.

[0018] Figure 3 ELISA validation results of phages containing different PSMα1 targeting peptide sequences.

[0019] Figure 4 The ITC verification results of KG7 (a, c) and IH7 (b, d) on PSMα1.

[0020] Figure 5 The prediction results of the possibility of hot spot sequences forming Beta-sheet structure in PSMα1.

[0021] Figure 6 Analysis of key interacting amino acid sites after docking of PSMα1 and KG7.

[0022] Figure 7 This is the high performance liquid chromatogram of KG7.

[0023] Figure 8 The changes of ThT fluorescence intensity of PSMα1 in different buffers over time.

[0024] Fig. 9 TEM images of various CuS systems and elemental mapping images of PPDA@CuS.

[0025] Fig.10 The coating results and quantification results of the sterilization rate of the MRSA blend liquid after NIR treatment for 10 minutes.

[0026] Fig.11 These are the quantitative results of crystal violet staining of the biofilm formed after co-culture of different materials with MRSA at 100 μg / mL for 48 hours and the corresponding staining pictures.

[0027] Fig.12 The copper ion release content of each CuS nanosystem before and after NIR.

[0028] Fig.13 Laser confocal photography was taken for each CuS system promoting polarization of macrophage M2 cells after NIR irradiation.

[0029] Fig.14 Quantitative results of macrophage M2 polarization by flow cytometry.

[0030] Fig.15 SEM images of the destructive performance tests of various CuS systems on biofilms in vivo.

[0031] Fig.16 The wound healing status of each group is shown for wounds at 1, 4, 7, 10, and 14 days.

[0032] Fig.17 Statistics of wound size of ulcer wounds in diabetic mice treated with various CuS systems within 14 days.

[0033] Fig.18 Plate counts of residual bacteria in the wounds of diabetic mice ulcers 7 days after treatment with each CuS system.

[0034] Fig.19 H&E sections of the wound skin of diabetic mice ulcer wounds 14 days after treatment with various CuS systems, where G represents granulation tissue; NE represents epidermis; ND represents dermis (scale bar: 100 μm).

[0035] Fig. 20 Gimesa sections of the wounds of diabetic mice ulcers 14 days after treatment with various CuS systems (scale bar: 100 μm).

[0036] Fig.21Analysis of macrophage polarization in the wound skin of diabetic mice ulcer wounds 14 days after treatment with various CuS systems (scale bar: 100 μm). DETAILED DESCRIPTION

[0037] The technology of the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following specific embodiments are only used to help those skilled in the art understand the present invention, and are not intended to limit the present invention.

[0038] Example 1. Screening of PSMα1 targeting peptides

[0039] 1.PSMα1 Biopanning of targeted peptides

[0040] ① Target preparation and host bacteria activation:

[0041] a. Inoculate the glycerol-preserved strain ER2738 on an LB-Tet plate and incubate it upside down at 37°C overnight. Seal the plate with sealing film and store it at 4°C in the dark.

[0042] b. Prepare 6 small sterile polystyrene culture dishes and add 1.5 mL of 100 μg / mL streptavidin solution (dissolved in 0.1 M NaHCO3, pH 8.6) to each dish. Swirl thoroughly to wet the surface.

[0043] c. Incubate in a humidified container at 4°C with gentle shaking overnight and store at 4°C until use.

[0044] d. Pick ER2738 monoclone from LB-Tet plate and inoculate into 20 mL LB liquid culture medium. Cultivate in 250 mL Erlenmeyer flask at 37°C with vigorous shaking until early logarithmic phase.

[0045] e. Take a coated plate, pour off the coating solution, and shake the plate vigorously on a clean paper towel to remove as much residual solution as possible. Fill each plate with blocking solution and incubate at 4°C for 1 hour.

[0046] f. Prepare a 5 mg / mL stock solution of biotin-labeled PSMα1 in DMSO; take 3 μL of the stock solution and 10 μL of the original library and mix them in 1.5 mL of TBST and incubate at room temperature for 60 min.

[0047] g. Pour off the blocking solution and quickly wash the plate 6 times with TBST buffer. Spin each time to ensure thorough washing, pour off the buffer, and tap off any remaining solution.

[0048] ② Initial screening of bacteriophages:

[0049] a. Take a cleaned blocking plate, add phage-target protein solution, and incubate at room temperature for 10 minutes.

[0050] b. Add biotin to a final concentration of 0.1 mM and continue incubation for 5 min.

[0051] c. Pour off the unbound phages and discard the remaining solution.

[0052] d. Wash the plate 10 times with TBST.

[0053] e. Use 1 mL of 0.2 M Glycine-HCl buffer (pH 2.2) to separate the bound molecules. Shake gently for more than 10 min, then transfer the eluate to another clean centrifuge tube, and then neutralize the eluate with 150 μL of 1 M Tris-HCl buffer (pH 9.1).

[0054] ③ Titer determination of the eluate:

[0055] a. Inoculate a single colony of ER2738 into 5-10 mL of LB medium and shake until OD 600 About 0.5.

[0056] b. Melt the upper agar in a microwave oven. Add 3 mL of the upper agar to each phage dilution tube in a sterile test tube and store at 45°C for later use.

[0057] c. Pre-warm the LB / IPTG / Xgal plate at 37°C. d. Perform 10-fold serial dilutions of the phages using LB medium. 1 -10 4 dilution.

[0058] e. When the bacterial culture reaches the mid-logarithmic phase, add 200 μL of the culture from each phage dilution tube to a microcentrifuge tube.

[0059] f. Add 10 μL of phage of different dilutions to each tube, shake to mix thoroughly, and incubate at room temperature for 1-5 minutes.

[0060] g. Add the infected cells to the upper agar culture tube (pre-warmed at 45°C), mix quickly and thoroughly, and immediately pour onto the LB / IPTG / Xgal plate (pre-warmed at 37°C) and spread evenly.

[0061] h. After the plate has cooled and solidified, invert and incubate at 37°C overnight.

[0062] i. Count the number of blue plaques on plates with less than 100 blue plaques. Then multiply by the dilution factor to get the titer per 10 μL of phage.

[0063] ④ Phage amplification:

[0064] a. Inoculate the remaining eluate into 20 mL of the pre-logarithmic stage culture of ER2738 and culture at 37°C for 4.5 h with vigorous shaking.

[0065] b. Transfer the culture to a centrifuge tube and centrifuge at 10,000 rpm for 10 min at 4°C. Transfer the supernatant to a new centrifuge tube and centrifuge again.

[0066] c. Use a pipette to remove the upper 80% of the supernatant and place it in a new tube. Add 1 / 6 volume of PEG / NaCl buffer and allow the phage to precipitate overnight at 4°C.

[0067] d. Centrifuge at 4℃ and 10000rpm for 15min. After discarding the supernatant, centrifuge briefly again and remove the remaining supernatant with a pipette.

[0068] e. The pellet was resuspended in 1 mL of TBS buffer, transferred to a new centrifuge tube, and centrifuged at 4°C for 5 min to pellet residual cells.

[0069] f. Transfer the supernatant to a new centrifuge tube and add 1 / 6 volume of PEG / NaCl buffer for precipitation. Keep on ice for 15-60 minutes. Centrifuge at 4°C for 10 minutes, discard the supernatant, centrifuge briefly again, and remove the remaining supernatant.

[0070] g. Resuspend the precipitate in 200 μL TBS. Centrifuge the solution for 1 min to precipitate all insoluble matter. Transfer the supernatant to another new centrifuge tube to obtain the amplified eluate and store at 4°C.

[0071] ⑤ Determination of phage titer after amplification:

[0072] a. Inoculate a single colony of ER2738 into 5-10 mL of LB medium and shake until OD 600 About 0.5.

[0073] b. Melt the upper agar in a microwave oven. Add 3 mL of the upper agar to each phage dilution tube in a sterile test tube and store at 45°C for later use.

[0074] c. Pre-warm LB / IPTG / Xgal plates at 37°C.

[0075] d. Use LB medium to dilute the phage 10-fold. 8 -10 11 dilution.

[0076] e. When the bacterial culture reaches the mid-logarithmic phase, add 200 μL of the culture from each phage dilution tube to a microcentrifuge tube.

[0077] f. Add 10 μL of phage of different dilutions to each tube, shake to mix thoroughly, and incubate at room temperature for 1-5 minutes.

[0078] g. Add the infected cells to the upper agar culture tube (pre-warmed at 45°C), mix quickly and thoroughly, and immediately pour onto the LB / IPTG / Xgal plate (pre-warmed at 37°C) and spread evenly.

[0079] h. After the plate has cooled and solidified, invert and incubate at 37°C overnight.

[0080] i. Count the number of blue plaques on plates with less than 100 blue plaques. Then multiply by the dilution factor to get the titer per 10 μL of phage.

[0081] ⑥ The 2nd and 3rd rounds of selection:

[0082] a. Count the blue plaques on the plate according to step 3 above to calculate the titer. Use this value to estimate the titer corresponding to 1-2×10 11 The amount of pfu added.

[0083] b. Perform the second and third rounds of panning using the same method as above, and gradually increase the concentration of Tween (0.3%-0.5%) during the washing steps to improve the stringency and specificity of the screening.

[0084] ⑦ Amplification and purification of monoclonal clones:

[0085] a. Take the LB-Tet overnight culture of ER2738 and dilute it into LB medium at a ratio of 1:100, and aliquot 2 mL into culture tubes. Select one tube for each clone to be identified. Pick 16 clones from the samples of the third round of panning.

[0086] b. Incubate at 37℃ with shaking for 4.5-5h.

[0087] c. Transfer the culture to a centrifuge tube and centrifuge for 30 seconds. Transfer the supernatant to a new tube and centrifuge again. Use a pipette to remove 80% of the supernatant and transfer it to a new centrifuge tube to obtain the amplified phage stock solution, which can be stored at 4°C for a short period of time.

[0088] d. Transfer 500 μL of phage supernatant to another new tube.

[0089] e. Add 200 μL of PEG / NaCl solution, mix thoroughly and incubate at room temperature for 10-20 minutes.

[0090] f. Centrifuge at 14000 rpm for 10 min and discard the supernatant.

[0091] g. After brief centrifugation, remove the remaining supernatant with a pipette.

[0092] h. Thoroughly resuspend the precipitate in 100 μL iodide buffer and tap the tube vigorously to ensure that the precipitate is dissolved. Add 250 μL ethanol and incubate at room temperature for 10 minutes.

[0093] i. Centrifuge at 14000 rpm for 10 min and discard the supernatant. Wash the precipitate with 0.5 mL of 70% ethanol, centrifuge and discard the supernatant, then briefly vacuum dry.

[0094] j. Resuspend the pellet in 30 μL TE buffer and store at -20°C.

[0095] ⑧Phage titer determination.

[0096] ⑨DNA extraction and sequencing of combined clones.

[0097] ⑩ Determination of affinity polypeptide sequence.

[0098] like Figure 1 As shown in the figure, the recovery rate data of each round of phage screening after three rounds of bio-panning. The six affinity peptides KG7 (KPTWATG), IS7 (IDPSKYS), YS7 (YLTPFQS), GY7 (GLTTTRY), IH7 (IVNMSYH) and VN7 (VEVTLVN) were obtained after three rounds of bio-panning. In each round of panning, the unbound phages were removed by vigorous washing, and the remaining phages were amplified and purified before being used as input for the next round. The recovery efficiency of each round is constantly improving, and the number of recovery in the third round is 3.2 times. The output / input phage ratio of each round can be used as the phage recovery efficiency. The results show that the recovery rate of the target phage ranges from 2.75×10 -9 Increased to 1.78×10 -7 .

[0099] In order to obtain the final target sequence, we sequenced the phages obtained after multiple rounds of in vitro selection and subtraction. The final results showed that a total of 11 different phages were obtained, and a total of six target sequences were found after sequencing. Figure 2 As shown, the frequency of occurrence, theoretical isoelectric point and molecular weight of each sequence are listed. The frequency of occurrence of phages with target sequences can reflect the affinity between the target sequence and the target. Among them, KG7 (KPTWATG) has the highest frequency of occurrence (4 / 11), which indicates that it is more likely to bind to the PSMα1 monomer. IS7 (IDPSKYS) and YS7 (YLTPFQS) have a frequency of 2 / 11, and the remaining polypeptide sequences GY7 (GLTTTRY), IH7 (IVNMSYH), and VN7 (VEVTLVN) have only appeared once. At the same time, the molecular weight of the polypeptide sequence was calculated, and the relative molecular mass of KG7 was 744.85, which is convenient for the subsequent confirmation of the relative molecular mass of mass spectrometry and subsequent experiments.

[0100] 2. ELISA / ITC Identification of Affinity Peptides

[0101] Select the primers synthesized by the following sequencing companies:

[0102] ELISA method: The target phage containing the above six polypeptides is amplified and cultured to verify their affinity and selectivity for the target molecule. The specific experimental process can be referred to the instructions of the above kit. The main experimental steps include (1) amplification of binding clones; (2) phage precipitation; (3) phage suspension and titer determination; (4) coating of target molecules; (5) blocking and washing of 96-well plates; (6) phage binding to target molecules; (7) addition of enzyme-labeled antibodies; (8) preparation of HRP substrate solution; (9) enzyme-substrate reaction; (10) result detection, and finally recording the absorbance value at 405nm with an ELISA reader.

[0103] ITC method: IH7 was used as a control to verify the strength of KG7's interaction with the target molecule PSMα1. The specific operation process is as follows: PSMα1 was dissolved in hexafluoroisopropanol and then dried in a vacuum at 40°C for 4 hours. After complete drying, ultrapure water was used to dissolve it, and the final concentrations were PSMα1 (50μm), KG7 (1000μm) and IH7 (1000μm). After ultrasonication for 5 minutes, centrifugation was performed at 12500rpm and the supernatant was analyzed using a Micro Cal PEAQ-ITC instrument.

[0104] like Figure 3 As shown in the above ELISA results, KG7 and IH7 have relatively strong binding ability to PSMα1. In order to further explore the interaction strength between them and PSMα1, the present invention uses KG7 and IH7 (purity greater than 95%) for ITC test, in which IH7 is used as a control. The results are shown in Figure 4 As shown in the figure, as the titration time increases, the reactions of IH7 and KG7 continue to release heat, and the Gibbs free energy (ΔG) and molar binding enthalpy (ΔH) of the reaction of both with PSMα1 are negative values, -80 kcal / mol and -1.6 kcal / mol, respectively, indicating that IH7 and KG7 can spontaneously bind to PSMα1, and the main binding forces between the two polypeptides and PSMα1 are hydrogen bonds and van der Waals forces. Among them, the ΔTS value of KG7 is -5.57 kcal / mol, which indicates that hydrophobic interactions occur. In addition, the smaller the binding constant value (Kd), the stronger the affinity between the two. The affinity Kd value of KG7 and PSMα1 is 5.26×10 -6 , which is much smaller than the Kd value of IH7 and PSMα1, 3.48×10 -4 , which indicates that KG7 has a stronger affinity for PSMα1.

[0105] 3. Prediction of KG7 and PSMα1 binding sites

[0106] PSMα1 (MGIIAGIIKVIKSLIEQFTGK) is a polypeptide molecule containing only 21 amino acids. The present invention first uses AGGRESCAN to predict its core amino acid sequence of fibrillation. AGGRESCAN is an online computational biology tool provided by the Autonomous University of Barcelona, ​​which can predict the polymerization hotspot area in the polypeptide, that is, the area in the protein that is most likely to form fibers. Its calculation is based on the aggregation propensity value (amino-acidaggregation-propensity value, a3v) of each amino acid obtained in the experiment. Subsequently, the present invention used the Pepsitefinder algorithm of the RPBS online server and the CASB docker server to predict the binding site between KG7 and PSMα1 respectively. Among them, PSMα1 (5KHB) comes from the PDB database, and the predicted structure of KG7 comes from the PEP-FOLD algorithm of the RPBS online server. CABS-dock is an online molecular docking tool provided by the Computational Biology Laboratory of the University of Warsaw, which is suitable for the interaction between proteins and polypeptides. CABS-dock uses the CABS coarse-grained protein model for docking simulation and is a molecular docking program using a flexible docking mode. In the CABS model, each amino acid is simplified into four interaction centers, the force field is based on the statistical potential of the studied proteins, and the molecular dynamics follows the Monte Carlo method. Figure 5 As shown, the probability of GIIKVIK sequence at positions 6-12 in PSMα1 forming a β-sheet is more than 95%. Figure 6 As shown, isoleucine at position 7 and lysine at position 12 of PSMα1 are the most critical amino acid sites in the interaction with KG7.

[0107] 4. Solid Phase Synthesis of Peptides and Liquid Chromatography-Mass Spectrometry Analysis of KG7

[0108] The peptides were synthesized by Xinhao Peptide Biotechnology Co., Ltd. The purity and relative molecular mass were subsequently analyzed using liquid chromatography-mass spectrometry. The specific operation was: the supernatant was analyzed using a triple quadrupole mass spectrometer Shimadzu LCMS-8045. All peptides were analyzed using ESI in positive ionization mode, and two transitions of the peptides were collected by multiple reaction monitoring mode. The results are shown in Figure 2. Figure 7 As shown, the relative molecular mass of KG7 is 759.70 and the purity is 95.216% (>95%), which meets the requirements for subsequent use.

[0109] Subsequently, before verifying the efficiency of KG7 in inhibiting PSMα1 fibrosis, the present invention first characterized the conditions and process of PSMα1 fibrosis. In order to explore the fibrosis conditions of PSMα1, the present invention used three buffers (phosphate buffer, phosphate buffer and Tris-HCl buffer) to incubate PSMα1 monomers, and used ThT fluorescence staining intensity to evaluate the β-sheet content during fibrosis. The results are as follows: Figure 8 As shown in the figure, the degree of fibrosis of PSMα1 in PB and PBS was very weak even after 120 h. The ThT fluorescence of PSMα1 in Tris-HCl buffer was significantly enhanced after 120 h, indicating that PSMα1 can form fibrosis and form a beta-sheet structure under this condition.

[0110] 5. Pretreatment of Peptides

[0111] First, the PSMα1 lyophilized peptide powder (purity>95%) was dissolved in hexafluoroisopropanol to prepare a mother solution with a final concentration of 1 mg / mL, and then the mother solution was ultrasonicated in an ice water bath for 5 minutes, and it was divided into different centrifuge tubes at 65 μL / tube, and the constant temperature vacuum treatment was carried out for about 12 hours until the organic solvent was completely volatilized. The treated polypeptide was stored in a refrigerator at -20°C before use. During the experiment, the pretreated polypeptide was redissolved in 100 μL ultrapure water, then ultrasonicated in an ice bath for 5 minutes, and finally diluted to a final concentration (50 μM) with 10 mM phosphate buffer (PBS, pH=7.4) and phosphate buffer (PB, pH=7.4), respectively, and cultured at 37°C.

[0112] Example 2. Preparation of CuS

[0113] Weigh 17.1 mg of copper chloride dihydrate and dissolve it completely in 60 mL of isopropanol by ultrasonication. After 20 minutes, add 200 mg of polyvinyl pyrrolidone (PVP K30), stir magnetically at room temperature until it is completely dissolved, and then add 1 mmol of sodium hydroxide solution. At this time, the solution is blue-green. Add 0.4 mL of 40% hydrazine hydrate solution to the above solution, stir at 25 ° C for 15 minutes until the solution turns orange, and cuprous oxide nanoparticles are generated. After collecting the nanoparticles, wash them repeatedly with ethanol and deionized water for at least 3 times and resuspend them with deionized water. Then add 35 μL of 35% ammonium sulfide solution by mass, and stir magnetically at room temperature for 2 hours to generate hollow CuS nanoparticles. Collect by centrifugation at 10000 rpm for 15 minutes, wash them three times with deionized water and ethanol, and dry the product in a vacuum drying oven at 60 ° C for use.

[0114] Example 3. Preparation of PDA@CuS

[0115] Based on the CuS prepared in Example 2, 5 mg of dopamine was weighed and dissolved in 10 mL of Tris-HCl (pH=8.5) buffer, and then 10 mg of CuS nanoparticles were added. After stirring at room temperature for 4 hours, the mixture was collected at 8000 rpm. The excess components were washed repeatedly with deionized water and ethanol solution for 3 times, and the product was dried in a vacuum drying oven at 60°C to obtain PDA@CuS nanoparticles.

[0116] Example 4. Preparation of PSMα1 targeting peptide modified copper sulfide nanomaterials

[0117] Molecular simulation predicts that the binding site of PSMα1 and KG7 contains a hotspot sequence for PSMα1 aggregation to form a β-sheet, and isothermal calorimetry experiments show that PSMα1 has a strong affinity with KG7. In view of the fact that KG7 binds to PSMα1 to inhibit its fibrosis and thus prevent the formation of biofilms, it is very effective, but there are still certain defects. KG7 cannot effectively remove mature biofilms, which limits its practical application. In order to further optimize the killing effect of KG7 on mature biofilms, KG7 is combined with photothermal therapy to design a nanosystem with synergistic antibacterial properties. Based on the PDA@CuS prepared in Example 4, the targeting peptide KG7 was grafted. 10 mg of KG7 polypeptide was weighed and dissolved in 10 mL of Tris-HCl (pH = 8.5) buffer, and 5 mg of PDA@CuS nanoparticles were added. After stirring at room temperature for 24 hours, the mixture was centrifuged and collected. After washing with deionized water three times, the mixture was centrifuged at 8000 rpm and dried in a vacuum oven at 60 ° C to obtain PPDA@CuS nanoparticles.

[0118] Example 5. Morphology of PPDA@CuS

[0119] Copper sulfide nanoparticles and other samples were fully dispersed in anhydrous ethanol. 10 μL of the above sample was directly dropped on a 200-mesh copper grid, dried overnight, then stained with 2% phosphotungstic acid for 30 seconds, and washed with ultrapure water before testing. The test instrument was a JEM-2100 transmission electron microscope (TEM). Fig. 9 As shown in the figure, a uniform coating of about 10 nm appears on the CuS with a diameter of about 150 nm. The existence of C, N, O, Cu, and S elements in the mapping image proves the success of the construction and gradual modification of the nanosystem, among which the Cu and S elements are relatively close to the inner area and appear hollow, which further confirms that the modification is carried out with hollow CuS as the base material.

[0120] Example 6. Analysis of in vitro antibacterial properties of PPDA@CuS

[0121] The Gram-positive bacteria MRSA43300 was selected for in vitro antibacterial effect evaluation. Before the experiment, the Gram-positive bacteria MRSA43300 was placed on a sheep blood agar (SBA) plate and cultured overnight at 37°C. Single colonies of each strain were collected and cultured overnight at 37°C in 4 mL of liquid TSB medium. After the above bacteria were centrifuged and washed three times with sterile PBS (10mM, pH 7.4), the bacteria were diluted to 106CFU / mL for subsequent experiments. First, 500 μL of the above bacterial suspension (1×106CFU / mL) was added to each well, and then copper sulfide nanoparticles (final concentration of 100 μg / mL) were added and incubated at 37°C for 1h, 3h, 6h and 8h respectively. Then, the gradient dilution method was used for coating, and the plate count method was used to evaluate the antibacterial effect of each sample. According to the operating procedures of the Live / DeadBacLight Bacteria Survival Kit, the bacterial suspension treated with nanoparticles was gently rinsed three times with PBS, and then stained with 500 μL of mixed staining reagent for 30 minutes under light-proof conditions. Finally, the bacteria were observed under a fluorescence microscope. Among them, green fluorescence represents live bacteria, while red fluorescence represents dead bacteria.

[0122] like Fig.10 As shown in the figure, KG7 and CuS nanoparticles can indeed partially inhibit the growth and proliferation of MRSA, with the inhibition rates reaching 73% and 67% respectively compared with the control group. PDA@CuS and PPDA@CuS nanoparticles can reduce the number of bacterial survival by 2-3 orders of magnitude. After NIR treatment, the number of bacterial survival in the CuS+NIR treatment group dropped to 2×103CFU / mL, while the number of bacterial survival in the other two groups (PDA@CuS+NIR and PPDA@CuS+NIR) was 0, achieving 100% killing and inhibition of MRSA. Similarly, the PPDA@CuS nanosystem also has a good killing and inhibition effect on the survival of E.coli, and the antibacterial effect of the PPDA@CuS+NIR group reaches 100%.

[0123] Example 7. Analysis of the anti-biofilm performance of PPDA@CuS in vitro

[0124] First, add 500 μL of MRSA suspension (1×10 6CFU / mL), and then different systems (PBS, PEP, CuS, PDA@CuS, PPDA@CuS, CuS+NIR, PDA@CuS+NIR and PPDA@CuS+NIR, final concentration of 100μg / mL) were added for different treatments. Incubate at 37°C for 1 day, then gently rinse 3 times with PBS. Add 500μL of methanol to each well to fix the biofilm for 15 minutes, and then add 500μL of 0.4% crystal violet ethanol solution to react for 15 minutes. The crystal violet stained plate was photographed with a camera, and the absorbance of the solution at 590nm was measured. Fig.11 As shown in the figure, the nanomaterials treated without NIR all showed anti-biofilm activity, of which KG7 (300 μM) showed 51.3% anti-biofilm activity, and the biofilm inhibition activity of the PPDA@CuS system was significantly higher than that of CuS and PDA@CuS. The NIR treated groups all showed the highest biofilm inhibition activity of 94.7%. Obviously, the PPDA@CuS system had the highest anti-biofilm activity, which was due to the inhibitory effect of KG7 on PSMα1 assembly and the synergistic antibacterial effect of KG7 and PDA@CuS system.

[0125] Example 8. Analysis of Copper Ion-Induced Macrophage Polarization of PPDA@CuS

[0126] Mouse macrophage Raw246.7 cells were used to test the effect of copper ions in nanoparticle extract on macrophage M2 polarization. Specifically, 1x10 5 Cells / well were cultured in DMEM medium, including 10% fetal bovine serum and 1% penicillin / streptomycin, in a 96-well plate at 37°C in a humidified atmosphere of 5% CO2. After 12 h, different types of copper sulfide nanoparticles were added to serum-free medium (CuS, PDA@CuS and PPDA@CuS, final concentration of 100 μg / mL), laser irradiated for 10 min, and the nanoparticles were removed using a 0.22 μm filter after cooling. The copper ion extract was then added to the above culture dish and incubated for 1 day. The old medium was discarded to collect the cells, and 80% methanol solution was added to each well for fixation at 4°C for 40 min. After centrifugation, the excess solution was removed and fresh DMEM containing 5 μL CD206 polyclonal antibody was added. After the samples were incubated at 37°C for 60 min, the excess dye was removed and macrophage polarization analysis was performed under flow cytometry. For confocal fluorescence imaging, the cells were first fixed at 4°C for 30 min by adding 80% methanol solution. After that, the excess solution was removed and fresh DMEM containing 5 μL CD206 polyclonal antibody was added for staining for 60 min. After removing the excess dye, the cell status was observed and recorded using a confocal microscope. At least 3 replicates were measured in each group.

[0127] To verify the wound healing performance of the PPDA@CuS system, the copper ion release content of each nanosystem under laser irradiation and without laser irradiation was first characterized. Fig.12 As shown, each nanosystem releases copper ions with or without NIR. CuS alone releases copper ions up to 1.3 mg / L, but the release of copper ions is delayed with the modification of PDA and KG7. After laser irradiation, the content of copper ions shows a clear upward trend, and the PPDA@CuS group before illumination increases from 0.7 mg / L to 1.0 mg / L, indicating that the increase in copper ion content after illumination may aggravate the M2 polarization of macrophages.

[0128] To verify the ability of copper ions released by each nanosystem to promote macrophage M2 polarization, this paper used CD206 fluorescent antibody labeling for CLSM photography and flow cytometry analysis. The increase in CD206 expression is a significant feature of macrophage M2 polarization. The CLSM results are as follows Fig.13 As shown in the figure, the IL-4 group was used as a positive control, and its red fluorescence was the strongest. The fluorescence intensity of PPDA@CuS+NIR, PDA@CuS+NIR and CuS+NIR increased successively, because the increasing content of released copper ions led to an increase in the polarization of macrophage M2, which further indicated that the modification of CuS surface by PDA and KG7 delayed the release of copper ions. The fluorescence intensity was further quantified by flow cytometry, and the specific results are shown in the figure. Fig.14 As shown, the M2 macrophage content in the PBS group was 12.3%, while the macrophage polarization rate increased from 12.3% to 28.1% after PPDA@CuS+NIR treatment, indicating that the copper ions released by the system effectively promoted the polarization of macrophage M2 and achieved the effect of promoting tissue repair, which corresponds to the CLSM results.

[0129] Example 9. Analysis of implant biofilm infection of PPDA@CuS

[0130] Balb / c mice were divided into groups, with no less than 5 mice in each group. After anesthesia with 1% sodium pentobarbital solution, the back skin was shaved and disinfected with 75% alcohol. A disposable sterile scalpel was used to make an incision of about 1 cm in the back skin, and a sterilized titanium sheet (1 cm in diameter) was inserted subcutaneously, and the wound was carefully sutured. MRSA suspension (108 CFU / mL) was injected on the surface of the implanted titanium sheet. One day after infection, the mice were treated with different groups for 7 days, and then the mice were killed, the titanium sheet was obtained and immediately washed twice with PBS (pH = 7.4), fixed with pentanediol at 4°C for 12 hours, and then dehydrated with graded ethanol (50, 60, 70, 80, 90, 100% v / v) for 10 minutes, freeze-dried and coated with platinum, and the biofilm on the surface of the titanium sheet was observed using a scanning electron microscope.

[0131] In order to observe the structure and residual bacteria status of implant biofilms in vivo, implants (titanium sheets) were collected on the 7th day after different treatments and characterized using Bio-SEM. Fig.15 As shown in the figure, after the treatment, obvious fiber structures can be observed in the control group (as shown by the yellow arrow), which is the manifestation of MRSA forming a dense biofilm. At the same time, a large number of intact bacteria (as shown by the red arrow) are covered under the biofilm, which is the existence form of bacteria in normal biofilm. In the treatment groups of each single component (CuS, PDA@CuS, PEP and PPDA@CuS), a small amount of loose and porous biofilm and some dead bacteria (as shown by the red arrow) can be seen, and there is still an obvious fiber structure, which may be due to the poor biofilm regulation performance of the single component. After laser irradiation, a large number of dead bacteria exist in the biofilm of CuS, PDA@CuS and PPDA@CuS. In the PPDA@CuS+NIR system, there are a large number of nanoparticles on the surface of the bacteria, the bacterial morphology is incomplete and the contents flow out, all of which indicate the death of bacteria. More importantly, the fiber structure of the biofilm is significantly eliminated, which is the result of the synergistic effect of KG7 and photothermal. These data suggest that PPDA@CuS+NIR can disrupt the biofilm structure and prevent biofilm regeneration, making the bacteria more easily eliminated by the host.

[0132] Example 10. In vivo wound healing evaluation of PPDA@CuS

[0133] Balb / c mice were fasted for 12h the night before, and then freshly prepared STZ solution (10mg / mL dissolved in sodium citrate solution at 4℃) was intraperitoneally injected, with an injection volume of 150mg / kg per mouse. After that, they were raised normally, and blood glucose was tested one week later. Non-fasting blood glucose greater than 16.7 indicated that diabetic mice were successfully constructed. Then they were randomly divided into groups, with at least five mice in each group, and the backs were shaved. After that, 1% sodium pentobarbital solution was used for anesthesia, and a 1cm diameter wound was created on the back of the mouse using a skin punch. Immediately after that, MRSA suspension (108CFU / mL) was inoculated for infection for 12h. Different materials were used for diabetic infected wound sites. During the two weeks of treatment, the wound healing was observed and the wound size was recorded by taking photos every day. After the treatment, H&E and Giemsa sections were analyzed for wound tissue, and macrophage polarization staining was performed to evaluate the wound recovery at the infected site. Major organs and blood were collected for biosafety evaluation. All animal experiments were conducted in accordance with the Law on the Use and Care of Research Animals (Document No. 55 of 2001).

[0134] Paraffin embedding and sectioning: After 14 days of treatment, the mice were killed, and their heart, liver, spleen, lung, kidney and wound tissues were embedded in paraffin. The collected tissue samples were fixed with 4% paraformaldehyde solution, and the tissue samples were completely immersed in the fixative. The fixed tissue samples were dehydrated, and the tissue samples were immersed in ethanol solutions of different concentrations (70%, 80%, 90%, 100% v / v) in turn, and stayed in each concentration of ethanol for 10 minutes. After dehydration, the tissue samples were gradually immersed in pentadiene for 1 hour. The infiltrated tissue samples were placed in a 60°C paraffin solution to allow them to be fully immersed. Transfer the impregnated tissue samples to the paraffin mold, and then pour in the paraffin. Make sure that the tissue sample is upright in the mold, and try to avoid the formation of gaps and bubbles. After condensation at room temperature for 10 minutes, the paraffin is cooled and solidified, and then stored in a refrigerator at 4°C.

[0135] H&E staining: Prepare thin slices of the embedded tissue samples on a freezing microtome, and then place them on a baking machine for 5 hours. After the slices are placed in xylene to remove the paraffin on the slices, and the slices are immersed in different concentrations of ethanol solutions (100%, 90%, 80%, 70% v / v) for 10 minutes, and the tissues are restored to moisture and washed twice with ultrapure water. Immerse the slices in hematoxylin dye to stain the nuclei for 30 seconds, then rinse the slices in clean water for 10 minutes to remove excess stain. Then use 0.7% hydrochloric acid ethanol for differentiation, and wash twice with ultrapure water after the end. Immerse in 0.5% eosin ethanol for 10 seconds again, repeat the above cleaning process, and then immerse the slices in different concentrations of ethanol solutions (60%, 70%, 80%, 90%, 100% v / v) for dehydration, and finally use paraffin to seal the slices for observation and photography.

[0136] Giemsa staining: Prepare thin sections of the embedded tissue samples on a freezing microtome, then remove the paraffin on the sections with xylene, and soak the sections in different concentrations of ethanol solutions (100%, 90%, 80%, 70% v / v) for 10 minutes, and wash them twice with ultrapure water after restoring the moisture of the tissue. Immerse the smear in methyl acetate for 10-15 minutes. Then immerse the sections in Giemsa staining solution for 20 minutes. Then gently rinse the excess dye with ultrapure water, and then dry the sections and seal them with paraffin for observation and photography.

[0137] Macrophage polarization staining: Remove the paraffin on the slices with xylene, and soak the slices in different concentrations of ethanol solution (100%, 90%, 80%, 70% v / v) for 10 minutes in turn. After restoring the moisture of the tissue, wash it twice with ultrapure water. Use 0.1% Triton X-100 to permeabilize the smear so that the dye can enter the cell. Then use 5% bovine serum albumin or 10% sheep serum for blocking to block nonspecific protein binding sites and reduce false positive signals. After the end, use CD206 to mark for 1 hour, rinse with PBS for 5 minutes, and then dry the slices and seal them with paraffin for observation and photography.

[0138] The specific method is to treat the chronic wounds of infected mice with PBS, CuS, PDA@CuS, PEP, PPDA@CuS, CuS+NIR, PDA@CuS+NIR and PPDA@CuS+NIR on the first day of treatment, and record the changes in the size of the wounds of the mice on the 1st, 4th, 7th, 10th and 14th days. The results are as follows: Fig.16 As shown. The wounds of mice in the illumination groups (CuS+NIR, PDA@CuS+NIR and PPDA@CuS+NIR) had obvious antibacterial and wound healing effects. As can be seen from the figure, there was basically no yellow exudate produced in all NIR groups during the treatment period, which indicates that the infection at the wound site was suppressed. At the same time, compared with the group without NIR, all wounds in the NIR group healed better at the same time point, indicating that photothermal therapy inhibited the growth of MRSA and promoted the wound healing process.

[0139] Wound healing rate specific data such as Fig.17 As shown in the figure, first, the overall wound size tends to heal over time. Among them, the PEP group had a poor treatment effect, and the wound size was basically the same as that of the PBS group at the same time point. This is because the mature biofilm will excrete or degrade small molecule agents.

[0140] In order to evaluate the bacterial killing of each system in vivo, the bacteria from the wound tissue on the seventh day of incubation were plated, such as Fig.18 As shown. The results show that MRSA in the PPDA@CuS+NIR group was basically completely eliminated. Compared with the PBS group, CuS, PDA@CuS and PPDA@CuS all had certain bactericidal effects, with bacterial survival rates of 77.0%, 65.9% and 63.7%, respectively. At the same time, the amount of MRSA residues in the wounds of mice in the NIR treatment group was significantly reduced, and the bacterial survival rate of the PPDA@CuS+NIR group was 1.2%. The above results all indicate that the PPDA@CuS system has excellent antibacterial / wound healing capabilities.

[0141] like Fig.19As shown, granulation tissue and epidermis formation reflect the wound healing to a certain extent. Compared with most of the groups without NIR (CuS, PDA@CuS, PEP), the NIR groups formed the epidermis, and even the dermis and new hair follicles were formed at the wound site in the PPDA@CuS+NIR group. The degree of wound recovery is consistent with the above-mentioned wound size statistics, among which the PPDA@CuS+NIR group has the best healing effect, and the wound size is 1133μm after 14 days of treatment. The above results show that PPDA@CuS+NIR can achieve the effect of accelerating wound healing. Giemsa section is a sign of bacterial residues (indicated by the red arrow), such as Fig. 20 As shown, the number of bacteria in the PPDA@CuS+NIR group was the least among all the treatment groups. At the same time, the presence of bacteria can cause wounds to be difficult to heal, and the results of the above skin H&E sections and Gimesa sections are consistent. After 14 days of treatment in mice, iNOS and CD206 staining were performed on the wound skin to evaluate the polarization of macrophages in the wound site. CD206 is a marker for M2 macrophages, which has the function of controlling inflammation and promoting wound tissue healing. The results are shown in Fig.21 As shown, the control PBS group showed the strongest fluorescence signal, while the fluorescence of each treatment group decreased, which is a manifestation of weakened inflammatory response. In addition, the fluorescence signal of the PPDA@CuS+NIR group was almost non-existent, indicating that the bacteria were effectively eliminated. Furthermore, the CD206 fluorescence of the PPDA@CuS+NIR group was the strongest, which was caused by the expression of anti-inflammatory factors caused by the copper ion-mediated macrophage M2 polarization.

[0142] The present invention uses PSMα1, the main component protein of MRSA biofilm, as the target, and uses phage display and bio-panning technology to finally obtain six targeted peptides that specifically bind to PSMα1. Among them, KG7 (KPTWATG) has the strongest targeting binding performance to PSMα1. It can effectively inhibit the fibrosis process of PSMα1 by binding to the core fibrosis sequence GIIKVIK of the PSMα1 monomer. This inhibitory effect has a significant concentration dependence. When the molar ratio of KG7 to PSMα1 reaches 10:1, its fibrosis inhibition rate can reach 61.5±2.6%, and the inhibition efficiency of MRSA biofilm can also reach 69.1±0.9%. Based on this, the present invention further develops a nanoparticle used in the treatment of MRSA infection biofilm on diabetic wounds. The nanoparticle is mainly composed of copper sulfide as the core, and KG7 is grafted on the surface through a PDA coating. The biofilm formation inhibition rate can reach 94.7±1.5%. Moreover, for mature biofilms, the nanoparticle uses photothermal effect to achieve the decomposition of biofilms and the killing of deep bacteria. The biofilm removal rate can reach 56.8±1.1%, providing a nanomedicine with potential transformation prospects for biofilm infections caused by MRSA and the healing of chronic wounds in diabetes.

[0143] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A PSMα1 targeting polypeptide, characterized in that: The amino acid sequence of the targeting polypeptide is selected from the group consisting of: KPTWATG, IDPSKYS, YLTPFQS, GLTTTRY, IVNMSYH and VNVEVTLVN.

2. Use of the PSMα1 targeting polypeptide according to claim 1 in antibacterial and / or wound healing promoting biomedical materials.

3. The use according to claim 2, characterized in that: The PSMα1 targeting polypeptide is used in coordination with CuS nanoparticles.

4. The use according to claim 3, characterized in that: The PSMα1 targeting polypeptide is grafted onto the surface of the CuS nanoparticles via a dopamine coating.

5. The use according to claim 2, characterized in that: The biomedical material is used for antibacterial and / or wound healing promotion of MRSA infection on diabetic wound surfaces.

6. An antibacterial photothermal therapy biomedical material, characterized in that: The invention comprises CuS nanoparticles and PSMα1 targeting polypeptide. The PSMα1 targeting polypeptide is grafted onto the surface of the CuS nanoparticles through a dopamine coating, and the amino acid sequence of the targeting polypeptide is KPTWATG.

7. A biomedical material for promoting wound healing photothermal therapy, characterized in that: The invention comprises CuS nanoparticles and PSMα1 targeting polypeptide. The PSMα1 targeting polypeptide is grafted onto the surface of the CuS nanoparticles through a dopamine coating. The amino acid sequence of the targeting polypeptide is selected from the group consisting of KPTWATG, IDPSKYS, YLTPFQS, GLTTTRY, IVNMSYH and VNVEVTLVN.

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