Preparation method and application of a pH-responsive self-assembling polypeptide and its hydrogel

Abstract

This invention discloses a pH-responsive self-assembling polypeptide and its preparation method and application for a hydrogel. The pH-responsive self-assembling polypeptide L5 is designed to self-assemble under neutral pH (pH 7.4) conditions, forming a three-dimensional gel network with specific structure and properties, demonstrating its drug-carrying capacity. In an acidic pH (pH 5.5) environment, it can depolymerize and exhibit broad-spectrum antimicrobial activity, showing potential as an antimicrobial wound dressing. Experiments have confirmed that the self-assembling polypeptide hydrogel constructed in this invention, loaded with endolysin molecules, achieves sustained release of the endolysin, effectively killing Staphylococcus aureus (S. aureus). It also improves the stability and bioavailability of the endolysin, exhibits good safety, and exhibits synergistic effects in promoting the healing of S. aureus-infected wounds. This product has promising application prospects in the treatment of pathogen-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, 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 a crucial biological process that occurs after skin injury, encompassing four phases: hemostasis, inflammation, proliferation, and remodeling. Pathogen colonization and biofilm formation at the wound site are key factors that delay healing and increase the risk of pathogen dissemination. Pathogen infection of wounds can lead to excessive production of inflammatory cytokines, hindering the normal progression of the inflammatory phase and promoting the development of chronic wounds before the bacteria are completely eliminated. Furthermore, during infection, pathogens typically produce metabolites such as acetic acid, lactic acid, and malic acid, resulting in an acidic pH at the infected wound site of around 5.5. However, most antimicrobial agents, including antimicrobial peptides, exhibit low activity under acidic conditions. Gauze, polymer bandages, and cotton are commonly used wound dressings, but they are ineffective in preventing bacterial infection. Furthermore, frequent changes of wound dressings can damage regenerating tissue. Therefore, the development of novel wound dressings with excellent antimicrobial activity, anti-inflammatory properties, and the ability to promote wound healing is of great importance.

[0003] Endolysins are phage-encoded peptidoglycan hydrolases 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 less likely to develop resistance 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 the body, 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, constructing a suitable delivery system remains a challenge.

[0004] Hydrogels are widely used in biomedical applications such as tissue engineering, drug delivery, wound dressings, and soft artificial organs. Hydrogels possess a three-dimensional network structure formed by cross-linked polymers, enabling the embedding of a variety of antimicrobial agents within the gel matrix, thereby improving drug bioavailability. Within this field, peptide-based supramolecular hydrogels have attracted increasing attention due to their excellent biocompatibility, high bioactivity, intelligent sensing capabilities, and flexible adjustability. Furthermore, peptides can be prepared using solid-phase peptide synthesizers. Amphiphilic peptides can be constructed into various micro- and nanostructures by adjusting their self-assembly parameters and can respond to environmental changes such as temperature, pH, enzymes, and ionic strength to achieve specific functions. Due to the acidic conditions of pathological environments, pH-responsive peptide hydrogels can not only form gels to encapsulate drugs but also release drugs at the site of infection in response to changes in pH, thereby achieving controlled and targeted drug delivery. 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; and 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:

[0008] A pH-responsive self-assembling polypeptide has an amino acid sequence of Ac-Lys-Pro-Val-Phe-Gln-Phe-Leu-Phe-His-Glu-NH2.

[0009] A method for preparing a pH-responsive self-assembling 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.

[0010] 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.

[0011] The pH-responsive self-assembled drug-loaded hydrogel prepared based on the method.

[0012] In some embodiments of the present invention, the hydrogel self-assembles to form a hydrogel under neutral pH conditions, and disassembles under acidic pH conditions to release endolysin and exhibit antibacterial activity.

[0013] In some embodiments of the present invention, the acidic condition is pH < 6.

[0014] The pH-responsive self-assembling drug-loaded hydrogel is used in the preparation of a skin external dressing for preventing wound infection or promoting wound healing.

[0015] In some embodiments of the present invention, the infection is caused by Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, or Escherichia coli.

[0016] The beneficial effects of the present invention include: the self-assembling peptide L5 hydrogel prepared in this invention is pH-responsive and can self-assemble under neutral conditions (pH 7.4) to form a three-dimensional gel network with specific structure and properties, indicating its ability to load drug molecules; it can depolymerize in an acidic pH environment (pH 5.5) and exhibit broad-spectrum antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli, showing potential as an antimicrobial wound dressing. The self-assembling peptide hydrogel constructed in this invention, loaded with endolysin molecules, achieves sustained release of endolysin, effectively killing Staphylococcus aureus (S. aureus). It also improves the stability and bioavailability of endolysin, exhibits good safety, and exhibits synergistic effects in promoting the healing of S. aureus-infected wounds, significantly increasing the wound healing rate and reducing the bacterial load on infected skin. Therefore, the pH-responsive self-assembling peptide L5 and its drug-loaded hydrogel have promising application prospects in the treatment of pathogen-infected wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0018] Figure 1 The chemical structure of pH-responsive self-assembling peptide L5 is shown in Figure 2. Ac-KPVFQFLFHE-NH2 is the abbreviation of Ac-Lys-Pro-Val-Phe-Gln-Phe-Leu-Phe-His-Glu-NH2.

[0019] Figure 2 Figure 3 is the gelling property of the self-assembling polypeptide L5; Figure A shows the gelling state of L5 at pH 7.4 and pH 5.5 observed by the vial inversion method; Figure B shows the injectable properties of L5 hydrogel.

[0020] Figure 3 Circular dichroism spectra of hydrogel formed by polypeptide L5 at pH 7.4 and pH 5.5.

[0021] Figure 4 Figure 3 is the morphological characterization 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.

[0022] Figure 5 Figure 2 shows 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 analysis to compare the differences between the L5-treated group and the PBS control group under different pH conditions, ***P<0.001, ns indicates no statistically significant difference; Figure C shows the effect of ionic strength on L5 activity.

[0023] Figure 6 The results of the broad-spectrum antibacterial activity test of hydrogel L5.

[0024] Figure 7 These are the high-performance liquid chromatography detection results 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.

[0025] Figure 8 The thermal stability of the hydrogel L5@LysSYL was analyzed by one-way ANOVA. **P<0.01, ***P<0.001, and ns indicate no statistically significant difference compared with 4 °C.

[0026] Figure 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.

[0027] Figure 10 The bactericidal activity of hydrogel L5@LysSYL against Staphylococcus aureus was compared using one-way analysis of variance to compare the differences between different treatment groups and the PBS control group, or the differences between specific groups, ***P<0.001.

[0028] Figure 11Figure 2 shows the safety evaluation results of the L5@LysSYL hydrogel. Panel A shows symptom observation; Panel B shows H&E staining analysis. The black arrow indicates hyperkeratosis of the epidermal stratum corneum; the red arrow indicates thickening of the epidermal stratum spinosum; the yellow arrow indicates regularly elongated, clubbed epidermal peduncles; the blue arrow indicates dilated subcutaneous capillaries; and the brown arrow indicates scattered inflammatory cell infiltration in the subcutaneous connective tissue. Scale bar: 200 μm.

[0029] Figure 12 Figure 4: The hydrogel L5@LysSYL promotes wound healing in mice infected with Staphylococcus aureus. Panel A shows the animal experimental design. Panel B shows representative images of wound areas in mice from different treatment groups. Scale bar, 0.5 cm. Panel C shows wound healing traces in different treatment groups over 14 days. Panel D shows the wound healing rate in mice from different treatment groups. One-way ANOVA was used to compare the differences between the different treatment groups and the PBS control group, or between specific groups. *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no statistically significant difference. Panel E shows the bacterial load in mouse skin tissue after treatment in different treatment groups. One-way ANOVA was used to compare the differences between the different treatment groups and the PBS control group. *P < 0.05, ***P < 0.001, ns indicates no statistically significant difference. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0031] The strains, reagents and materials used in the present invention are:

[0032] 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 an affiliated hospital of our school and is maintained in 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); and endolysin LysSYL (recombinantly expressed in our laboratory, reference: Liu H., et al. LysSYL: a broad-spectrum phage endolysin targeting Staphylococcus species 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 chromatography (Agilent, USA); chromatographic column model: ZORBAX SB, 300C18 column; all other reagents were of biological grade or analytical grade.

[0033] Example 1: Design and synthesis of pH-responsive self-assembling polypeptide L5.

[0034] Taking into full consideration the characteristics of amino acids (such as hydrophobicity, hydrophilicity, charge, etc.) and the minimum functional motif, a pH-responsive self-assembling peptide 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 commissioned to Shanghai Qiangyao Biotechnology Co., Ltd. (ChinaPeptides Co., Ltd.). It was prepared using the Fmoc solid-phase synthesis method, and the peptide purity was greater than 98%.

[0035] Example 2: Preparation method of pH-responsive self-assembling polypeptide L5 hydrogel.

[0036] L5 peptide powder was weighed into a 1.5 mL EP tube and dissolved in PBS buffer (pH 7.4 or 5.5, 10 mM) (final L5 concentration was 10 mg / mL). The solution was sonicated for 5 minutes to completely dissolve the peptide L5. After pipetting evenly, the solution was centrifuged at 1,000 × g for 2 minutes to remove bubbles. The solution was allowed to stand at room temperature for at least 30 minutes. The vial was inverted to observe whether L5 could form a hydrogel. The results showed that peptide L5 formed a stable hydrogel under neutral conditions (pH 7.4) and an unstable gel under acidic conditions (pH 5.5), indicating that L5 has pH-responsive gel-forming properties ( Figure 2 , A). Injectable hydrogels have shown good application potential in tissue engineering and wound healing due to their ability to retain water, 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, forming the word "GEL", which shows that L5 hydrogel has good injectability ( Figure 2 , B).

[0037] Example 3: Secondary structure characterization of pH-responsive self-assembling polypeptide L5 hydrogel.

[0038] 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 Chirascan software was used to calculate the secondary structure percentage of L5 under different pH conditions. Figure 3 As shown in the figure, L5 can form a stable hydrogel under neutral conditions (pH 7.4). This sample has a positive peak at 195 nm and a negative peak at 208 nm, indicating that L5 has β-sheet and α-helical conformations. However, the CD chromatogram of L5 changes significantly at pH 5.5, showing a negative peak at 200 nm, indicating that L5 adopts a random coil conformation at pH 5.5, and the ordered structure of the hydrogel is destroyed.

[0039] Example 4: Morphological characterization of pH-responsive self-assembling polypeptide L5 hydrogel.

[0040] 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). Cryo-EM observations showed that L5 exhibited a uniform and interwoven three-dimensional porous structure at 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 at pH 7.4 was 9.1 nm ( Figure 4 , D), which is significantly larger than the fiber diameter (6.5 nm) at pH 5.5 ( Figure 4 In summary, hydrogel L5 possesses a pH-responsive nanofibrous structure, and its supramolecular network structure at pH 7.4 suggests its potential application in drug encapsulation and delivery.

[0041] Example 5: Detection of pH-responsive antibacterial activity of self-assembling polypeptide L5.

[0042] The antibacterial activity of L5 against Staphylococcus aureus USA300 was tested under different pH conditions. A single colony of S. aureus USA300 was activated overnight and cultured to the logarithmic growth phase (approximately 2 h). The bacterial suspension was centrifuged at 6,000 × g for 10 min, washed once with PBS (10 mM, pH 7.4), and resuspended in PBS buffer (10 mM) at 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 suspension was serially diluted and plated, and cultured at 37 °C overnight. The next day, the colonies were photographed and the number of colonies was counted. Figure 5 As shown in Figure 3, L5 has no bactericidal activity in the pH range of 7.4–6.0, but its bactericidal activity is significantly enhanced under acidic conditions (pH < 6.0). After 4 h of L5 treatment, the bacterial counts at pH 5.5, pH 5.0, and pH 4.5 decreased by 1.84, 2.00, and 5.52 orders of magnitude, respectively, compared to the control group ( Figure 5, B). Therefore, L5 has pH-responsive antibacterial activity.

[0043] In addition, it was found that the ionic strength of PBS buffer had a significant effect on the antibacterial activity of L5. 6 Suspensions of S. aureus USA300, S. aureus N315, S. aureus XN108, Acinetobacter baumannii ATCC19606, Pseudomonas aeruginosa PAO1, and Escherichia coli O157:H7 at CFU / mL concentrations were incubated with 50 µL of hydrogel in a 37°C incubator for 8 hours. After incubation, 50 µL of the bacterial suspension was evenly spread on a plate, allowed to dry, and then incubated overnight at 37°C. The plates were then removed and photographed the next day. The results showed that at 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 broad-spectrum antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli at pH 5.5 ( Figure 6 ).

[0044] Example 6: Preparation of self-assembling polypeptide L5 loaded endolysin LysSYL hydrogel (L5@LysSYL) and entrapment efficiency detection.

[0045] Endolysin LysSYL was diluted to 1 mg / mL with PBS (pH 7.4, 10 mM) for later use. 5 mg of peptide (L5) powder was weighed using 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 minutes, and the mixture was then pipetted evenly. The mixture was centrifuged at 1,000 × g for 2 minutes to remove bubbles and allowed to stand at room temperature for more than 30 minutes until the hydrogel L5@LysSYL was formed. Subsequently, high-performance liquid chromatography was used to detect the encapsulation efficiency of LysSYL in the L5@LysSYL hydrogel. 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 rinsate was calculated using a standard curve, and it was found that when the concentration of endolysin was 1 mg / mL, the entrapment efficiency reached 85.7%.

[0046] Example 7: Thermal stability of the self-assembling polypeptide L5-loaded endolysin hydrogel L5@LysSYL.

[0047] To verify whether the stability of LysSYL was improved after being encapsulated into L5 hydrogel, the thermal stability of the 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 heated in a metal bath at 80°C for 10 min, and 200 μL of 5×10 7 A suspension of S. aureus USA300 (CFU / mL) was incubated with 50 µL of heated LysSYL or L5@LysSYL hydrogel at 37°C for 8 hours. Following incubation, the suspension was serially diluted 10-fold and plate counted. LysSYL stored at 4°C served as a positive control (bactericidal activity was set at 100%). The results showed that, compared to the relative activity of free LysSYL (73.5%), the relative activity of L5@LysSYL encapsulated in L5 was significantly increased (95.2%, P < 0.01) after treatment at 80°C ( Figure 8 ), that is, L5 encapsulation increased the thermal stability of endolysin LysSYL by 21.7%.

[0048] Example 8: Morphological characterization of the self-assembling polypeptide L5-loaded endolysin hydrogel L5@LysSYL.

[0049] Cryo-electron microscopy, transmission electron microscopy, and atomic force microscopy were used to observe the microscopic morphology of the hydrogel L5@LysSYL under different pH conditions. Cryo-electron microscopy results showed that L5@LysSYL has a three-dimensional porous network structure similar to L5 ( Figure 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 at pH 7.4, L5@LysSYL can also form an intertwined nanofiber network structure ( Figure 9 , B, C). In addition, the fiber diameter of L5@LysSYL is 13.3 nm ( Figure 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.

[0050] Example 9: Synergistic antibacterial activity and safety of self-assembling polypeptide L5-loaded endolysin hydrogel L5@LysSYL.

[0051] The antibacterial activity of the hydrogel L5@LysSYL against Staphylococcus aureus USA300 was tested by the viable bacterial 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 200 μL of 5×10 7 A suspension of Staphylococcus aureus USA300 with a CFU / mL was placed in a 37 °C incubator and incubated for 1 h and 8 h respectively. After the incubation, 10-fold serial dilutions were made and plated. The plates were incubated at 37 °C overnight and the colonies were counted the next day. Figure 10 As shown, compared with L5 alone, L5@LysSYL significantly enhanced the antibacterial activity against S. aureus USA300 (P < 0.001). After 1 and 8 hours of L5@LysSYL treatment, the bacterial count of S. aureus USA300 decreased by 2.3 and 3.9 orders of magnitude, respectively. This indicates that the L5-loaded hydrogel exhibits a synergistic bactericidal effect.

[0052] 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 ( Figure 11 , A), the symptoms of these treatment groups were similar to those of the PBS (10 mM, pH 7.4) control group. However, skin treated with 30% sodium dodecyl sulfate (SDS) showed exfoliation and erythema ( Figure 11 , A). H&E staining results showed that the skin tissue of mice treated with 30% SDS showed hyperkeratosis of the stratum corneum, thickening of the epidermal spinous layer, dilation of subcutaneous capillaries, and infiltration of inflammatory cells. In contrast, LysSYL, L5, and L5@LysSYL hydrogels did not show obvious pathological features ( Figure 11 , B). The above results indicate that the hydrogel L5@LysSYL is safe when used as a wound dressing.

[0053] Example 10: Study on the promotion of wound healing by self-assembling peptide L5 loaded with endolysin hydrogel L5@LysSYL

[0054] 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. Figure 12 , A), evaluating the potential of hydrogel L5@LysSYL to promote wound healing. PBS (10 mM, pH 7.4), LysSYL (concentration: 1 mg / mL), L5 (concentration: 10 mg / mL), and L5@LysSYL (L5 concentration: 10 mg / mL; LysSYL concentration: 1 mg / mL) were all applied in 100 µL doses each time; mupirocin (Mup) ointment (concentration: 20 mg / g) was applied with a cotton swab each time; treatment lasted for 14 days, with treatment twice daily for the first 7 days and once daily for the next 7 days. Physically, compared with the other treatment groups, mice in the L5@LysSYL hydrogel treatment group showed superior wound healing effects on both days 7 and 14 ( Figure 12 , B), the wound area continued to decrease with the extension of treatment time ( Figure 12 , C). Quantitative detection showed that the wound healing rate of mice in the L5@LysSYL hydrogel treatment group was the fastest ( Figure 12 , D). On the 7th day, the percentages 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 the hydrogel L5@LysSYL was significantly reduced (P<0.001) ( Figure 12 , E). After 14 days of treatment with the hydrogel L5@LysSYL, the bacterial count in the wound tissue decreased by 6 orders of magnitude, significantly lower than that in the LysSYL-treated group (bacterial count decreased by 2.3 orders of magnitude) and the mupirocin-treated group (bacterial count decreased by 1.5 orders of magnitude). These results demonstrate that the hydrogel L5@LysSYL possesses stronger antibacterial and wound-healing properties than the medical dressing Mup.

[0055] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection 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-assembling drug-loaded hydrogel, characterized by: 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 until fully dissolved, and standing at room temperature for more than 30 minutes to form a hydrogel.

3. The method for preparing the pH-responsive self-assembling 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-assembling drug-loaded hydrogel prepared by the method according to any one of claims 2 to 3.

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

6. The pH-responsive self-assembling drug-loaded hydrogel according to claim 5, characterized in that: The acidic condition is pH < 6.

7. Use of the pH-responsive self-assembling 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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