Preparation method and application of pH-responsive self-assembled polypeptide and hydrogel thereof

Abstract

The invention discloses a pH-responsive self-assembled polypeptide and a preparation method and application of a hydrogel thereof, and designs a pH-responsive self-assembled polypeptide L5 which can be self-assembled under a neutral pH (pH 7.4) condition to form a three-dimensional gel network with a specific structure and performance, which shows that the pH-responsive self-assembled polypeptide L5 has drug molecule loading capacity; in an acidic pH (pH 5.5) environment, the polymer can be depolymerized and shows broad-spectrum antibacterial activity, and has the potential of being used as an antibacterial wound dressing. Experiments prove that after endolysin molecules are loaded by adopting the self-assembled polypeptide hydrogel constructed by the invention, continuous release of endolysin can be realized, staphylococcus aureus (staphylococcus aureus) can be effectively killed, and meanwhile, the stability and bioavailability of endolysin are improved; the composition has good safety and a synergistic interaction capability of promoting healing of staphylococcus aureus infected wounds, and has a good application prospect in treatment of pathogenic bacteria infected wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical materials, and specifically relates to a pH-responsive self-assembling polypeptide, and also relates to a method for preparing a pH-responsive self-assembling drug-loaded hydrogel, and the use of the pH-responsive self-assembling drug-loaded hydrogel in the preparation of a skin dressing for preventing wound infection or promoting wound healing. Background Art

[0002] Skin wound healing is an important biological process that occurs after skin injury, including four stages: hemostasis, inflammation, proliferation, and remodeling. Pathogen colonization and biofilm formation at the wound site are key factors that delay the healing process and increase the risk of pathogen dissemination in the body. Pathogen infection of the wound may lead to excessive production of inflammatory cytokines, hinder the normal progress of the inflammatory stage, and promote the formation of chronic wounds before the bacteria are completely cleared. In addition, during the infection, pathogens usually produce metabolites such as acetic acid, lactic acid, and malic acid, resulting in acidic wound infection sites with a pH of about 5.5. However, most antimicrobial preparations, including antimicrobial peptides, show low activity under acidic conditions. Gauze, polymer bandages, and cotton are commonly used wound dressings, but they cannot effectively prevent bacterial infection. Moreover, frequent changes of wound dressings may cause damage to regenerated tissues. Therefore, it is of great significance to develop new wound dressings with good antimicrobial activity, anti-inflammatory function, and promotion of wound healing.

[0003] Endolysins are peptidoglycan hydrolases encoded by bacteriophages that can directly act on the chemical bonds in peptidoglycan, leading to bacterial osmotic death. Compared with bacteriophages, endolysins have the advantages of a wide spectrum of bacterial lysis, fast bactericidal speed, effective removal of biofilms, and bacteria are not easily resistant to endolysins. They are expected to develop into an important means to combat drug-resistant bacterial infections. However, as a protein, endolysins are easily affected by various microenvironments, generally have poor stability, low bioavailability, and are easily inactivated in vivo, which greatly limits the development and application of endolysins. The technology of using carriers to encapsulate endolysins is considered to be a key strategy to improve the application value of endolysins. However, in order to successfully deliver endolysins, it is still a challenge to construct a suitable delivery system.

[0004] Hydrogels are widely used in the biomedical field, such as tissue engineering, drug delivery, wound dressings, and soft artificial organs. Hydrogels have a three-dimensional network structure formed by cross-linking of high molecular polymers, which enables them to embed a variety of antimicrobial agents into the gel matrix, thereby improving the bioavailability of drugs. In the field of hydrogel applications, peptide-based supramolecular hydrogels have attracted more and more attention due to their excellent biocompatibility, high bioactivity, intelligent sensing ability, and flexible adjustability. At the same time, peptides can be prepared by solid-phase peptide synthesizers. Amphiphilic peptides can construct various micro- and nanostructures by adjusting self-assembly parameters, and can respond to various environmental changes, such as temperature, pH, enzymes, and ionic strength, to achieve specific functions. Due to the acidic conditions of the pathological environment, pH-responsive peptide hydrogels can not only form gel-encapsulated drugs, but also release drugs at the site of infection due to pH changes, thereby achieving controllable and targeted delivery of drugs. Therefore, the design and construction of pH-responsive peptide hydrogels has become a research hotspot in the biomedical field.

[0005] Therefore, in order to better solve practical problems such as the removal of pathogens in infected wounds and wound healing, the present invention develops a method for preparing a pH-responsive self-assembling polypeptide and its hydrogel, and evaluates its application effect. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide a pH-responsive self-assembling polypeptide; a second object of the present invention is to provide a method for preparing a pH-responsive self-assembling drug-loaded hydrogel; a third object of the present invention is to provide a pH-responsive self-assembling drug-loaded hydrogel prepared based on the method; a fourth object of the present invention is to provide the use of the pH-responsive self-assembling drug-loaded hydrogel in the preparation of a skin dressing for preventing wound infection or promoting wound healing.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: A pH-responsive self-assembling polypeptide, the amino acid sequence of the polypeptide is: Ac-Lys-Pro-Val-Phe-Gln-Phe-Leu-Phe-His-Glu-NH2.

[0008] A method for preparing a pH-responsive self-assembled drug-loaded hydrogel comprises the following steps: dissolving the polypeptide according to claim 1 in an endolysin solution, adjusting the pH value to 7.35-7.45, mixing until fully dissolved, and standing at room temperature for more than 30 minutes to form a hydrogel.

[0009] In some embodiments of the present invention, the concentration of the endolysin is 1 mg / mL, and the final concentration of the polypeptide is 10 mg / mL.

[0010] The pH-responsive self-assembled drug-loaded hydrogel prepared based on the method.

[0011] In some embodiments of the present invention, the hydrogel self-assembles to form a hydrogel under neutral pH conditions, and disassembles to release endolysin and exhibit antibacterial activity under acidic pH conditions.

[0012] In some embodiments of the present invention, the pH is an acidic condition of pH < 6.

[0013] The pH-responsive self-assembled drug-loaded hydrogel is used in the preparation of a skin external dressing for preventing wound infection or promoting wound healing.

[0014] In some embodiments of the invention, the infection is caused by Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii or Escherichia coli.

[0015] The beneficial effects of the present invention are as follows: the self-assembling peptide L5 hydrogel prepared by the present invention has pH responsiveness, can self-assemble under neutral (pH 7.4) conditions to form a three-dimensional gel network with specific structure and performance, indicating that it has drug molecule loading capacity; it can depolymerize in an acidic pH (pH 5.5) environment and exhibit broad-spectrum antibacterial activity, and has antibacterial effects on Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii or Escherichia coli, and has the potential to be used as an antibacterial wound dressing. After the self-assembling polypeptide hydrogel constructed by the present invention is loaded with endolysin molecules, the sustained release of endolysin can be achieved, which can effectively kill Staphylococcus aureus (S. aureus), while improving the stability and bioavailability of endolysin, having good safety and synergistic enhancement ability to promote the healing of wounds infected with S. aureus, significantly improving the wound healing rate, and reducing the bacterial load of infected skin. Therefore, pH-responsive self-assembling polypeptide L5 and its drug-loaded hydrogel have good application prospects in the treatment of pathogen-infected wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration: Figure 1 is the chemical structure formula of pH-responsive self-assembling polypeptide L5. Ac-KPVFQFLFHE-NH2 is the abbreviation of Ac-Lys-Pro-Val-Phe-Gln-Phe-Leu-Phe-His-Glu-NH2.

[0017] Figure 2 Figure 2 is the gelling property of the self-assembling polypeptide L5; Figure A is the gelling state of L5 observed at pH 7.4 and pH 5.5 by the vial inversion method; Figure B is the injectable property of L5 hydrogel.

[0018] Figure 3 Circular dichroism spectra of hydrogel formed by polypeptide L5 at pH 7.4 and pH 5.5.

[0019] Figure 4 Figure 3 is the morphology of L5 hydrogel; Figure A is a cryo-electron microscopy image (scale bar: 20 µm); Figure B is a transmission electron microscopy image (scale bar: 1 µm); Figure C is an atomic force microscopy image; Figure D is the fiber diameter of L5 at pH 7.4; Figure E is the fiber diameter of L5 at pH 5.5.

[0020] Figure 5 Figure 2 is the antibacterial activity test results of hydrogel L5 against Staphylococcus aureus; Figure A shows the antibacterial activity of L5 under different pH conditions; Figure B shows the antibacterial activity of L5 under different pH conditions detected by the viable bacteria counting method; Student's t-test was used for statistical test to compare the differences between the L5 treatment group and the PBS control group under different pH conditions, ***P<0.001, ns indicates that the difference is not statistically significant; Figure C shows the effect of ionic strength on the activity of L5.

[0021] Figure 6 The results of the broad-spectrum antibacterial activity test of hydrogel L5.

[0022] Figure 7 The results of high performance liquid chromatography detection of endolysin LysSYL in the self-assembling polypeptide L5 loaded endolysin (L5@LysSYL) hydrogel; Figure A is the high performance liquid chromatogram of endolysin LysSYL; Figure B is the standard curve of endolysin LysSYL.

[0023] Figure 8 Thermal stability of hydrogel L5@LysSYL; one-way ANOVA was used, **P<0.01, ***P<0.001 and ns indicate no statistically significant difference compared with 4 °C.

[0024] Fig. 9 Figure 3 is the morphological characterization of the hydrogel L5@LysSYL; Figure A is a cryo-electron microscopy image (scale bar: 20 µm); Figure B is a transmission electron microscopy image; Figure C is an atomic force microscopy image; Figure D is the fiber diameter of L5@LysSYL at pH 7.4.

[0025] Fig.10 The bactericidal activity of hydrogel L5@LysSYL against Staphylococcus aureus; One-way ANOVA was used to compare the differences between different treatment groups and the PBS control group or the differences between specific groups, ***P<0.001.

[0026] Fig.11The safety evaluation results of hydrogel L5@LysSYL; Figure A shows symptom observation; Figure B shows H&E staining analysis. The black arrows in the figure indicate hyperkeratosis of the epidermal stratum corneum; the red arrows indicate thickening of the epidermal stratum spinosum; the yellow arrows indicate regular extension of the epidermal foot in a club-like shape; the blue arrows indicate dilation of subcutaneous capillaries; and the brown arrows indicate scattered infiltration of inflammatory cells in the subcutaneous connective tissue; scale bar: 200 μm.

[0027] Fig.12 The hydrogel L5@LysSYL promotes the healing of wounds in mice infected with Staphylococcus aureus; Figure A is a schematic diagram of the animal experiment design; Figure B is a representative picture of the wound area of ​​mice in different treatment groups. The scale bar is 0.5 cm; Figure C shows the wound healing marks of different treatment groups within 14 days; Figure D shows the wound healing rate of mice in different treatment groups; One-way ANOVA was used to compare the differences between different treatment groups and the PBS control group or the differences between specific groups, *P<0.05, **P<0.01, ***P<0.001, ns indicates no statistically significant difference; Figure E shows the bacterial load of mouse skin tissue after treatment in different treatment groups. One-way ANOVA was used to compare the differences between different treatment groups and the PBS control group, *P<0.05, ***P<0.001, ns indicates no statistically significant difference. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0029] The strains, reagents and materials used in the present invention are: Staphylococcus aureus USA300 (ATCC BAA-1556, purchased from ATCC, USA); Staphylococcus aureus N315 (ATCC 29213, purchased from ATCC, USA); Staphylococcus aureus XN108 was isolated from a burn patient in the burn department of a hospital affiliated to our school and preserved by our laboratory; Escherichia coli O157:H7 (purchased from Beijing Anxinkang Technology Co., Ltd., China); Acinetobacter baumannii ATCC 19606 (purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., China); Pseudomonas aeruginosa PAO1 (purchased from Beijing Anxinkang Technology Co., Ltd., China); yeast extract, agar powder, and trypsin (purchased from Oxoid, UK); BHI medium powder (purchased from Oxoid, UK); endolysin LysSYL (recombinantly expressed in our laboratory, reference: Liu H., et al. LysSYL: a broad-spectrum phage endolysin targeting Staphylococcusspecies and eradicating S. aureus biofilms. Microbial Cell Factories, 2024,23(1):89); mupirocin (Tianjin Sino-US SmithKline, China); circular dichroism spectrometer (JASCO, Japan); transmission electron microscope (JEOL, Japan); cryo-electron microscope (Zeiss, Germany); atomic force microscope (Bruker, Germany); liquid chromatograph (Agilent, USA); chromatographic column model: ZORBAX SB, 300C18 column; other reagents were of biological grade or analytical grade.

[0030] Example 1: Design and synthesis of pH-responsive self-assembling polypeptide L5.

[0031] Taking into full consideration the characteristics of amino acids (such as hydrophobicity, hydrophilicity, charge properties, etc.) and the minimum functional motif, a pH-responsive self-assembling polypeptide L5 was designed. Its amino acid sequence is Ac-Lys-Pro-Val-Phe-Gln-Phe-Leu-Phe-His-Glu-NH2, abbreviated as Ac-KPVFQFLFHE-NH2 (SEQ ID NO.1), with N-terminal acetylation (Ac) and C-terminal amidation (NH2). Its chemical structure is shown in Figure 1 The synthesis of peptide L5 was entrusted to Shanghai Qiangyao Biotechnology Co., Ltd. (ChinaPeptides Co., Ltd). It was prepared by Fmoc solid phase synthesis method, and the purity of the peptide was greater than 98%.

[0032] Example 2: Preparation method of pH-responsive self-assembling polypeptide L5 hydrogel.

[0033] Weigh L5 polypeptide powder in a 1.5 mL EP tube, dissolve it in PBS buffer (pH 7.4 or 5.5, 10 mM) (final concentration of L5 is 10 mg / mL), sonicate for 5 min to completely dissolve polypeptide L5, blow it evenly with a pipette, centrifuge at 1,000 × g for 2 min to remove bubbles, let it stand at room temperature for more than 30 min, invert the vial, and observe whether L5 can form a hydrogel. The results show that polypeptide L5 can form a stable hydrogel under neutral (pH 7.4) conditions and an unstable gel under acidic (pH 5.5) conditions, indicating that L5 has pH-responsive gel-forming properties ( Figure 2 , A). Injectable hydrogels show good application potential in the fields of tissue engineering and wound healing due to their ability to retain moisture, absorb wound exudate, permeate oxygen, and adapt to irregular wound shapes. L5 hydrogel (10 mg / mL) can be injected into the disc through a needle with an inner diameter of 0.4 mm, and the word "GEL" is formed, which shows that L5 hydrogel has good injectability ( Figure 2 , B).

[0034] Example 3: Secondary structure characterization of pH-responsive self-assembling polypeptide L5 hydrogel.

[0035] Circular dichroism (CD) spectroscopy was used to further analyze the changes in the secondary structure of L5 at pH 7.4 and pH 5.5. 200 µL of PBS buffer (pH 7.4 or pH 5.5, 10 mM) was added to the L5 hydrogel (50 µL) and incubated at 37°C for 2 h. The PBS was removed, the gel surface was rinsed with ddH2O, and then diluted with ddH2O to a solution concentration of 100 µg / mL. The solution was scanned three times continuously on a JASCO J-1500 circular dichroism spectrometer with a scanning wavelength range of 180–260 nm, and the percentage of secondary structure of L5 under different pH conditions was calculated using Chirascan software. Figure 3 As shown in the figure, L5 can form a stable hydrogel under neutral (pH 7.4) conditions. The sample has a positive peak at 195 nm and a negative peak at 208 nm, indicating that L5 has β-sheet and α-helix conformations. However, the CD chromatogram of L5 changed significantly under pH 5.5 conditions, showing a negative peak at 200 nm, indicating that L5 presents a random coil conformation under pH 5.5 conditions, and the ordered structure of the hydrogel is destroyed.

[0036] Example 4: Morphological characterization of pH-responsive self-assembling polypeptide L5 hydrogel.

[0037] The microstructure of hydrogel L5 under different pH conditions was observed by cryo-electron microscopy (cryo-EM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The results of cryo-EM observation showed that L5 exhibited a uniform and interwoven three-dimensional porous structure under pH 7.4, with an average pore size of 5.6 µm ( Figure 4 , A), which provides a suitable supramolecular network for drug delivery. However, due to the protonation of K and H residues in the L5 sequence, their three-dimensional supramolecular network structure was disassembled and destroyed under acidic conditions (pH 5.5). Transmission electron microscopy ( Figure 4 , B) and atomic force microscopy ( Figure 4 , C) further revealed that L5 formed an intertwined nanofiber network structure under pH 7.4 conditions, while under acidic conditions, the nanofibers became incomplete and broken. The fiber diameter of L5 under pH 7.4 conditions was 9.1 nm ( Figure 4 , D), which is significantly larger than the fiber diameter (6.5 nm) at pH 5.5 ( Figure 4 , E). In conclusion, hydrogel L5 has a pH-responsive nanofibrous structure, and its supramolecular network structure at pH 7.4 suggests that it has potential for application in drug encapsulation and delivery.

[0038] Example 5: Detection of pH-responsive antibacterial activity of self-assembling polypeptide L5.

[0039] The antibacterial activity of L5 against S. aureus USA300 under different pH conditions was tested. A single colony of S. aureus USA300 was activated overnight and cultured to the logarithmic growth phase (about 2 h of culture). The bacterial suspension was centrifuged at 6,000 × g for 10 min, washed once with PBS (10 mM, pH 7.4), and then resuspended in PBS buffer (10 mM) with different pH values ​​(pH 7.4, pH 6.5, pH 6.0, pH 5.5, pH 5.0, and pH 4.5). The bacterial concentration was adjusted to 5 × 10 6 CFU / mL; 200 µL of bacterial suspension was added to 50 µL of peptide hydrogel (concentration of 10 mg / mL) formed in advance in a 1.5 mL EP tube, and incubated at 37 °C for 8 h; after the incubation, the bacterial solution was serially diluted and plated, cultured at 37 °C overnight, and photographed and counted the number of colonies the next day. The results are as follows Figure 5 As shown in Figure , L5 has no bactericidal activity in the pH range of 7.4–6.0, and its bactericidal activity is significantly enhanced under acidic conditions (pH < 6.0). After 4 h of L5 treatment, the number of bacteria decreased by 1.84, 2.00, and 5.52 orders of magnitude under pH 5.5, pH 5.0, and pH 4.5, respectively, compared with the control group ( Figure 5, B). Therefore, L5 has pH-responsive antibacterial activity.

[0040] In addition, it was found that the ionic strength of PBS buffer had a significant effect on the antibacterial activity of L5. 6 CFU / mL concentration of Staphylococcus aureus USA300, Staphylococcus aureus N315, Staphylococcus aureus XN108, Acinetobacter baumannii ATCC19606, Pseudomonas aeruginosa PAO1 and Escherichia coli O157:H7 suspensions were incubated with 50 µL of hydrogel in a 37°C incubator for 8 h; after the incubation, 50 µL of bacterial solution was evenly spread on the plate, dried, and placed in a 37°C incubator overnight. The plate was taken out and photographed the next day. The results showed that under pH 5.5, 50 mM PBS significantly inhibited the antibacterial activity of L5, while 10 mM PBS had no such inhibitory effect ( Figure 5 , C). Plate coating experiments further demonstrated that L5 had a broad-spectrum antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli at pH 5.5 ( Figure 6 ).

[0041] Example 6: Preparation of self-assembling polypeptide L5 loaded with endolysin LysSYL hydrogel (L5@LysSYL) and detection of encapsulation efficiency.

[0042] Endolysin LysSYL was diluted to 1 mg / mL with PBS (pH 7.4, 10 mM) for later use. 5 mg of polypeptide (L5) powder was weighed with a precision balance and dissolved in 500 µL of the above LysSYL solution to a final concentration of 10 mg / mL. Ultrasonication was performed for 5 min, and the mixture was evenly blown with a pipette. Centrifugation was performed at 1,000 × g for 2 min to remove bubbles, and the mixture was allowed to stand at room temperature for more than 30 min until the hydrogel L5@LysSYL was formed. Subsequently, the encapsulation efficiency of LysSYL in the L5@LysSYL hydrogel was detected by high performance liquid chromatography. The results showed that the peak time of endolysin LysSYL was 12.691 min ( Figure 7 , A), with the concentration of endolysin as the abscissa and the peak area as the ordinate, a standard curve was established, indicating that LysSYL had a good linear relationship in the range of 10–1,000 µg / mL ( Figure 7 , B). The amount of LysSYL in the flushing solution was calculated using the standard curve, and it was found that when the concentration of endolysin was 1 mg / mL, the entrapment efficiency reached 85.7%.

[0043] Example 7: Thermal stability of the self-assembling polypeptide L5-loaded endolysin hydrogel L5@LysSYL.

[0044] To verify whether the stability of LysSYL was improved after being encapsulated into L5 hydrogel, the thermal stability of hydrogel L5@LysSYL was tested. The hydrogel L5@LysSYL (L5 concentration: 10 mg / mL; LysSYL concentration: 1 mg / mL) and LysSYL (concentration: 1 mg / mL) were placed in a metal bath at 80 °C for 10 min, and 200 μL of 5×10 7 CFU / mL of Staphylococcus aureus USA300 suspension was incubated with 50 µL of heated LysSYL or hydrogel L5@LysSYL at 37 °C for 8 h. After the incubation, 10-fold serial dilutions were performed for plate counting, and LysSYL stored at 4 °C was used as a positive control (bactericidal activity was set to 100%). The results showed that compared with the relative activity of free LysSYL (73.5%), the relative activity of L5@LysSYL after LysSYL encapsulation in L5 was significantly increased after 80 °C treatment (95.2%, P<0.01) ( Figure 8 ), that is, L5 encapsulation increased the thermal stability of endolysin LysSYL by 21.7%.

[0045] Example 8: Morphological characterization of the self-assembling polypeptide L5-loaded endolysin hydrogel L5@LysSYL.

[0046] The microstructure of the hydrogel L5@LysSYL under different pH conditions was observed using cryo-electron microscopy, transmission electron microscopy, and atomic force microscopy. The cryo-electron microscopy results showed that L5@LysSYL had a three-dimensional porous network structure similar to that of L5 ( Fig. 9 , A), however, the average pore size of L5@LysSYL (22.5 µm) was significantly larger than that of L5 hydrogel (5.6 µm, P < 0.01, Figure 4 , A). Transmission electron microscopy and atomic force observation results show that under pH 7.4 conditions, L5@LysSYL can also form an intertwined nanofiber network structure ( Fig. 9 , B, C). In addition, the fiber diameter of L5@LysSYL is 13.3 nm ( Fig. 9 , D), larger than L5 at pH 7.4 (9.1 nm) ( Figure 4 , D) and pH 5.5 (6.5 nm) ( Figure 4 , E) of the fiber diameter.

[0047] Example 9: Synergistic antibacterial activity and safety of self-assembling polypeptide L5 loaded with endolysin hydrogel L5@LysSYL.

[0048] The antibacterial activity of hydrogel L5@LysSYL against Staphylococcus aureus USA300 was detected by the viable bacteria counting method. 50 μL of LysSYL (concentration: 1 mg / mL), L5 (concentration: 10 mg / mL) and L5@LysSYL (L5 concentration: 10 mg / mL; LysSYL concentration: 1 mg / mL) were added to three 1.5 mL EP tubes, and then 200 μL of 5×10 7 The suspension of Staphylococcus aureus USA300 with a CFU / mL was placed in a 37 °C incubator for 1 h and 8 h respectively; after the incubation, it was diluted 10 times and plated, and the plate was placed at 37 °C for overnight incubation. The colony count was counted the next day. Fig.10 As shown in the figure, compared with the treatment with L5 alone, the antibacterial activity of L5@LysSYL against S. aureus USA300 was significantly enhanced (P<0.001). After the S. aureus USA300 was treated with L5@LysSYL for 1h and 8h, the number of bacteria decreased by 2.3 and 3.9 orders of magnitude, respectively. This indicates that the hydrogel L5 loaded with LysSYL has a synergistic bactericidal effect.

[0049] In addition, the safety of L5@LysSYL was preliminarily studied. The results showed that after the back skin of healthy mice was treated with 100 μL L5@LysSYL (L5 concentration: 10 mg / mL; LysSYL concentration: 1 mg / mL), LysSYL (concentration: 1 mg / mL) and L5 (concentration: 10 mg / mL), there were no obvious macroscopic symptoms on the surface ( Fig.11 , A), the symptoms of these treatment groups were similar to those of the PBS (10 mM, pH 7.4) control group. However, the skin treated with 30% sodium dodecyl sulfate (SDS) showed epidermal exfoliation and erythema ( Fig.11 , A). H&E staining results showed that the mouse skin tissue treated with 30% SDS showed hyperkeratosis of the stratum corneum, thickening of the epidermal stratum spinosum, dilation of subcutaneous capillaries, and infiltration of inflammatory cells. In contrast, LysSYL, L5, and L5@LysSYL hydrogel treatments did not show obvious pathological features ( Fig.11 , B). The above results indicate that the hydrogel L5@LysSYL is safe when used as wound dressing.

[0050] Example 10: Study on the promotion of wound healing by self-assembling peptide L5 loaded with endolysin hydrogel L5@LysSYL The full-thickness skin of mice was inoculated with Staphylococcus aureus USA300 (10 8CFU) wound infection model was used to evaluate the antibacterial efficacy and wound healing promoting ability of the hydrogel L5@LysSYL. Observations and photographs were taken on days 0, 3, 7, and 14 of hydrogel treatment, and skin tissues were collected for testing on days 7 and 14. Fig.12 , A), evaluate the potential of hydrogel L5@LysSYL to promote wound healing. The dosage of PBS (10 mM, pH7.4), LysSYL (concentration: 1 mg / mL), L5 (concentration: 10 mg / mL), and L5@LysSYL (L5 concentration: 10 mg / mL; LysSYL concentration: 1 mg / mL) was 100 µL each time; medical mupirocin (Mup) ointment (concentration: 20 mg / g) was applied with a cotton swab each time; the treatment lasted for 14 days in total, with treatment twice a day for the first 7 days and once a day for the next 7 days. In terms of physical signs, compared with other treatment groups, mice in the L5@LysSYL hydrogel treatment group showed better wound healing effects on days 7 and 14 ( Fig.12 , B), the wound area decreases with the extension of treatment time ( Fig.12 , C). Quantitative detection showed that the wound healing rate of mice in the L5@LysSYL hydrogel treatment group was the fastest ( Fig.12 , D). On the 7th day, the percentage of unhealed wound area of ​​mice in the PBS, LysSYL, L5 and mupirocin (Mup) treatment groups were 92.1%, 75.1%, 69.9% and 77.2%, respectively, while the percentage of unhealed wound area of ​​mice in the hydrogel L5@LysSYL treatment group was 38.4%. On the 14th day, the percentage of unhealed wound area of ​​mice in the hydrogel L5@LysSYL treatment group was only 4.9%, which was significantly lower than that in the PBS (35.0%, P<0.001), LysSYL (27.2%, P<0.01), L5 (24.3%, P<0.001) and Mup (21.1%, P<0.05) treatment groups. In addition, compared with the PBS treatment group, the bacterial load of infected skin after 7 days of treatment with hydrogel L5@LysSYL was significantly reduced (P<0.001) ( Fig.12 , E). After 14 days of treatment with hydrogel L5@LysSYL, the number of bacteria in the wound tissue decreased by 6 orders of magnitude, which was significantly lower than that in the LysSYL treatment group (the number of bacteria decreased by 2.3 orders of magnitude) and the mupirocin treatment group (the number of bacteria decreased by 1.5 orders of magnitude). The above results show that hydrogel L5@LysSYL has stronger antibacterial and wound healing abilities than medical dressing Mup.

[0051] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A pH-responsive self-assembling polypeptide, characterized in that: The amino acid sequence of the polypeptide is: Ac-Lys-Pro-Val-Phe-Gln-Phe-Leu-Phe-His-Glu-NH2.

2. A method for preparing a pH-responsive self-assembled drug-loaded hydrogel, characterized in that: The method comprises the following steps: dissolving the polypeptide according to claim 1 in an endolysin solution, adjusting the pH value to 7.35-7.45, mixing evenly to fully dissolve, and standing at room temperature for more than 30 minutes to form a hydrogel.

3. The method for preparing the pH-responsive self-assembled drug-loaded hydrogel according to claim 2, characterized in that: The concentration of the endolysin was 1 mg / mL, and the final concentration of the polypeptide was 10 mg / mL.

4. A pH-responsive self-assembled drug-loaded hydrogel prepared by the method according to any one of claims 2 to 3.

5. The pH-responsive self-assembled drug-loaded hydrogel according to claim 4, characterized in that: The hydrogel self-assembles to form a hydrogel under neutral pH conditions, and de-assembles to release endolysin and exhibit antibacterial activity under acidic pH conditions.

6. The pH-responsive self-assembled drug-loaded hydrogel according to claim 4, characterized in that: The acidic condition is pH﹤6.

7. Use of the pH-responsive self-assembled drug-loaded hydrogel according to any one of claims 4 to 6 in the preparation of a skin dressing for preventing wound infection or promoting wound healing.

8. The use according to claim 7, characterized in that: The infection is caused by Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii or Escherichia coli.

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