Chimeric polypeptide, self-healing injectable hydrogel containing chimeric polypeptide as well as preparation method and application of self-healing injectable hydrogel

Abstract

The invention relates to a chimeric polypeptide M-IB-367, the amino acid sequence of the chimeric polypeptide is as shown in SEQ ID NO.1, the chimeric polypeptide comprises IB-367 antibacterial peptide, an MMP cleavable sequence and an angiogenesis peptide fragment, the chimeric polypeptide can play an instant antibacterial role in the early stage of infection, the cleavable sequence is recognized by high-concentration MMP enzyme to cut off the cleavable sequence in the repair promoting period, and the chimeric polypeptide M-IB-367 is obtained. Angiogenesis peptide in-situ exposure starts angiogenesis and granulation tissue formation, and wound healing is accelerated; the invention also relates to a self-healing injectable hydrogel and a preparation method thereof, an aldehyde group on an HA-CHO chain segment reacts with a hydrazide group on Gel-ADH to generate an acylhydrazone bond, and primary crosslinking is formed; an aldehyde group of hyaluronic acid reacts with a sulfydryl group of cysteine of the chimeric polypeptide to form secondary crosslinking, and EGF is loaded into the hydrogel through physical embedding and weak current action; experiments prove that the HA / Gel coated M-IB-367 / EGF hydrogel prepared by the preparation method disclosed by the invention has the advantages of high in-vitro antibacterial rate, strong angiogenesis capability, and strong in-vivo anti-inflammatory effect and wound healing promotion capability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a chimeric polypeptide, a self-healing injectable hydrogel containing the same, its preparation method and application. Background Technology

[0002] Under various pathological conditions, tissue damage is often accompanied by persistent inflammatory responses, impaired local blood circulation, and decreased immune function, leading to obstructed repair processes. Burns, large-area skin defects, and chronic wounds caused by metabolic diseases such as diabetes present problems such as long healing cycles, high infection risks, and insufficient regenerative capacity. Among these, diabetic wounds, due to reduced local growth factor secretion, restricted angiogenesis, and insufficient oxygen supply, are considered one of the important pathological factors hindering normal healing. Currently, the clinical treatment of chronic wounds mainly involves mechanical debridement combined with antibiotic irrigation, which is not only costly and causes great pain to patients, but also damages newly formed granulation tissue. Furthermore, its effectiveness in controlling infections caused by multidrug-resistant (MDR) bacteria such as Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) remains limited.

[0003] Besides skin and soft tissue injuries, endometrial injury is another common and difficult-to-repair type of tissue damage. Under the influence of factors such as induced abortion, intrauterine procedures, infection, or chronic inflammation, the basal layer of the endometrium is easily damaged, leading to a decline in endometrial regeneration capacity. When the local inflammatory response persists after injury or the microenvironment is imbalanced, it can easily lead to pathological changes such as incomplete endometrial repair, fibrosis, or intrauterine adhesions, thereby affecting fertility. Existing clinical interventions, such as hormone therapy or physical barrier interventions, are insufficient to actively promote endometrial tissue regeneration and reconstruction while simultaneously suppressing inflammation and infection.

[0004] Epidermal growth factor (EGF), as a crucial regulator in tissue repair, exhibits significant anti-inflammatory properties during skin wound healing, while simultaneously promoting collagen synthesis and re-epithelialization, thus accelerating wound closure. In endometrial repair, EGF has also been shown to promote the proliferation of endometrial epithelial and stromal cells, improve glandular structure development, and support vascular network reconstruction. However, free EGF exhibits poor stability and is easily inactivated in vivo, with a short biological half-life, thus limiting its sustained therapeutic effect. Therefore, controlled release of EGF using various biomaterial delivery systems, such as hydrogels, scaffolds, nanofibers, microspheres, and cationic polymers, has become a significant research direction.

[0005] In recent years, multifunctional hydrogels have attracted widespread attention due to their anti-inflammatory, hemostatic, and drug delivery capabilities. Adding EGF to in-situ molded injectable hydrogels has shown great application potential for treating refractory wounds. However, the degradation by bacterial metabolites and the formation of bacterial biofilms affect the diffusion of growth factors and their binding to receptors. Furthermore, the moist environment of traditional hydrogels promotes bacterial growth and lacks inherent resistance to bacterial infection. To enhance antibacterial properties, inherent antibacterial hydrogels and combination strategies of hydrogels with antibacterial agents have been explored. Antimicrobial peptides (AMPs) are a class of naturally occurring molecules widely distributed in organisms. Compared to broad-spectrum antibiotics, AMPs exert their antibacterial function through non-targeted membrane cleavage mechanisms, thus being unaffected by traditional drug resistance mechanisms. However, AMPs are easily degraded by proteases in vivo, resulting in short local retention times and difficulty in maintaining long-term antibacterial effects. Besides serving as a reservoir for epidermal growth factor, hydrogels are also used as carriers for antimicrobial peptides. This strategy not only achieves the synergistic release of antimicrobial peptides and growth factors, but also effectively reduces the direct contact between antimicrobial peptides and proteases, slowing down the degradation rate and thus improving the controllability of treatment. Natural polymers hyaluronic acid and gelatin have good biocompatibility and biodegradability, making them suitable for the preparation and application of hydrogels. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a chimeric polypeptide, a self-healing injectable hydrogel containing the polypeptide, its preparation method, and its applications.

[0007] The technical solution of this invention is as follows: A chimeric polypeptide M-IB-367, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] A method for preparing a self-healing injectable hydrogel containing the chimeric polypeptide includes the following steps: S1. Preparation of HA-CHO: NaIO4 was added to HA solution, mixed, reacted in the dark, and then freeze-dried to obtain HA-CHO; S2. Preparation of Gel-ADH: Gelatin was dissolved in an acidic buffer solution, activated by a catalyst, coupled with ADH, and lyophilized to obtain Gel-ADH; S3. Preparation of HA / Gel@M-IB-367 / EGF hydrogel: Dissolve HA-CHO powder and add chimeric polypeptide M-IB-367 to obtain solution A. The amino acid sequence of the chimeric polypeptide is shown in SEQ ID NO.1. Dissolve Gel-ADH powder and add EGF to obtain solution B. Mix solutions A and B, react, and let stand to form a gel, thus obtaining HA / Gel@M-IB-367 / EGF hydrogel.

[0009] Furthermore, the mass ratio of HA to NaIO4 in step S1 is 2:(1-2).

[0010] Furthermore, the process parameters for the light-protected reaction in step S1 are: stirring and reacting under light-protected conditions for 20-28 hours.

[0011] Further, the catalyst in step S2 is EDC and HOBT, and the mass ratio of gelatin: EDC: HOBT: ADH is 1:(1-1.5):(0.5-1):(5-6); before the freeze drying in step S2, a dialysis step is also included, and the process parameters of the dialysis step are: molecular weight cutoff of 10kDa, first dialyzing in NaCl solution for 48-72h, and then dialyzing in deionized water for 48-72h.

[0012] Furthermore, the acidic buffer solution mentioned in step S2 is a MES solution with a pH of 6.5.

[0013] Further, in step S3, the final concentration of HA-CHO in the HA / Gel@M-IB-367 / EGF hydrogel is 6-10 wt%, the final concentration of Gel-ADH is 10-15 wt%, the final concentration of the chimeric polypeptide M-IB-367 is 25 μg / mL, and the final concentration of EGF is 25 μg / mL.

[0014] The self-healing injectable hydrogel prepared according to the preparation method described above.

[0015] The application of the chimeric polypeptide M-IB-367 or the self-healing injectable hydrogel in the preparation of drugs or medical dressings that promote wound healing.

[0016] Furthermore, the wound is a chronic, difficult-to-heal diabetic wound, a wound infected with multidrug-resistant bacteria, or a skin defect accompanied by microcirculatory disorders; the multidrug-resistant bacteria include at least one of methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.

[0017] Compared with the prior art, the present invention has at least the following advantages: 1. This invention relates to a chimeric polypeptide M-IB-367, the amino acid sequence of which is shown in SEQ ID NO. 1, wherein RGGLCYCRGRFCVCVGR is the IB-367 antimicrobial peptide, GPQGIAGQ is the MMP-cleavable sequence, and KLTQEWQK is the angiogenic peptide. This sequence contains multiple cysteine ​​residues (identified by the letter C), whose side chains contain thiol groups that react with the aldehyde group -SHO in hyaluronic acid, causing the chimeric polypeptide to be covalently anchored to the hydrogel network. The chimeric polypeptide plays different roles in the early stage of infection (stage one) and the second stage of wound healing. In the early stage of wound infection, a large number of inflammatory cells accumulate, and the intact structure of the IB-367 antimicrobial peptide in the chimeric polypeptide provides immediate antimicrobial activity. During the healing phase, as infection is controlled, MMP enzymes accumulate to a certain concentration. High concentrations of MMP enzymes recognize and cleave the cleavable sequence, exposing the angiogenic peptide in situ to the hydrogel surface, initiating angiogenesis and granulation tissue formation, and accelerating wound healing.

[0018] 2. This invention also relates to a self-healing injectable hydrogel and its preparation method. The aldehyde group (-CHO) on the oxidized hyaluronic acid (HA-CHO) chain reacts specifically with the hydrazide group (-CONHNH2) on the hydrazide-modified gelatin (Gel-ADH) to generate a structurally stable hydrazone bond (-C=N-NH-CO-), forming a primary cross-link. The aldehyde group -CHO of hyaluronic acid reacts with the thiol group of cysteine ​​in the chimeric polypeptide to form a secondary cross-link, prolonging the residence time of the polypeptide, and finally obtaining a stable self-healing hydrogel system. Epidermal growth factor (EGF) is loaded into the above dynamic network through physical embedding and weak electrochemical interaction. Experimental verification shows that the HA / Gel@M-IB-367 / EGF hydrogel prepared by the method described in this invention has a high in vitro antibacterial rate, strong angiogenesis ability, and strong in vivo anti-inflammatory effect and wound healing promotion ability. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0020] Figure 1 These are physical images of HA / Gel at different concentrations of HA-CHO in Example 1 of the present invention; Figure 2 The above figures show the in vitro antibacterial performance of the four groups of hydrogels in Example 2 of this invention. Figure 3 These are images showing the cell viability / death staining results of four groups of hydrogels in Example 3 of this invention; Figure 4 This is a graph showing the angiogenesis results of four groups of hydrogels in Example 3 of the present invention; Figure 5These are images showing the wound healing results of four groups of hydrogels in Embodiment 4 of the present invention; Figure 6 The figure shows the results of the four groups of hydrogels in Example 5 of this invention in promoting the recovery of fertility in rats with endometrial damage. Detailed Implementation

[0021] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0022] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0023] The materials and methods described in this invention are as follows.

[0024] Materials, cell lines, and animals: EGF and M-IB-367 were purchased from Sangon Biotech (Shanghai) Co., Ltd. Sodium hyaluronate (HA) and adipic acid dihydrazide (ADH) were purchased from Shandong Focus Freda Biotechnology Co., Ltd. Sodium periodate (NaIO4), morpholine ethanesulfonic acid (MES), amide condensing agent (HOBT), and carbodiimide (EDC) were purchased from Shanghai Titan Technology Co., Ltd. Antibacterial experiments were conducted using Staphylococcus aureus (Gram-positive), Pseudomonas aeruginosa (Gram-negative), and Escherichia coli (Gram-negative). SYTO9-PI, CCK-8, and Calcein-Am / PI kits were purchased from Beyotime Biotechnology Co., Ltd. The human umbilical vein endothelial cells (HUVECs) used in the experiments were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai). Male ICR mice were purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd. Streptozotocin (STZ) was purchased from Shanghai Maclean Biochemical Reagent Co., Ltd.

[0025] Inverted vial experiment: The final concentration of Gel-ADH was set to 12 wt%, and the concentrations of HA-CHO were adjusted to 6 wt%, 8 wt%, and 10 wt%. Then, equal volumes of the two solutions were mixed, inverted, and the vials were observed and photographed to capture the gelation process.

[0026] Antibacterial / bactericidal activity assay: Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were selected as experimental strains to investigate the antibacterial activity of the hydrogel in vitro. 300 μL of the hydrogel was placed in a 1.5 mL centrifuge tube, followed by the addition of an equal volume of 1×10⁻⁶ micrograms of hydrogel. 5 A bacterial suspension of CFU / mL was placed on top of the hydrogel. The mixture was incubated with shaking at 120 rpm and 37°C. After 18 hours of incubation, appropriate amounts of the bacterial suspension were taken out, diluted, and evenly spread on plates. The plates were inverted for incubation to evaluate the bactericidal effect of the hydrogel, and photographs were taken for recording.

[0027] Live / dead cell staining: HUVECs were seeded in 24-well culture plates and cultured overnight to ensure cell adhesion. Subsequently, the cells were washed twice with PBS to thoroughly remove residual culture medium. After 24 hours of hydrogel treatment, the cells were stained using Calcein-AM (excitation / emission wavelengths: 485 / 530 nm) and PI (excitation / emission: 535 / 617 nm). Specifically, 1 μL of PI and 1 μL of AM were added to 1 mL of cells and incubated in the dark for 25 minutes. After incubation, the cells were washed three times with PBS, and finally, photographs were taken using a fluorescence inverted microscope.

[0028] Cell formation: After pre-cooling the 24-well plates to 4°C, add an appropriate amount of matrix gel to each well and incubate at 37°C for 1 hour to allow it to solidify completely. Then, seed each well with 500 μL of HUVEC cell suspension (2 × 10⁻⁶ cells / well). 4 Cells were treated with PBS, HA / Gel, M-IB-367 / EGF, and HA / Gel@M-IB-367 / EGF, respectively, and incubated at 37°C in a 5% CO2 incubator. After incubation, angiogenesis was observed using an optical microscope, and the number of branches and total length of the vascular-like structures were quantitatively analyzed using ImageJ software.

[0029] Effect of hydrogel on wound healing in diabetic mice with MRSA infection: Male ICR mice were fed a high-fat diet for 4 weeks and then injected intraperitoneally with streptozotocin (STZ) 50 mg / kg / day. -1 Blood samples were collected from the tail vein of mice for four consecutive days to determine random blood glucose levels. Mice with successfully established blood glucose levels were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital, followed by the creation of a 6 mm diameter circular wound in the shaved skin area. A 1×10⁻⁶ sodium pentobarbital solution was then used. 850 μL of MRSA bacterial suspension (CFU / mL) was inoculated at the wound site. The established diabetic wound infection model was randomly divided into four groups of 10 mice each, named Control (PBS), HA / Gel, M-IB-367 / EGF, and HA / Gel@M-IB-367 / EGF, respectively. The Control group received 200 µL of physiological saline daily, while the other groups received 200 µL of the corresponding hydrogel. Mice were euthanized on days 3, 7, and 14, and wound tissue was collected, homogenized, incubated at 37°C for 18 hours, and colony counted on agar plates. Simultaneously, the wound was photographed and the wound area was calculated.

[0030] Evaluation of fertility and live birth in rats with endometrial damage caused by hydrogel: A 25G syringe needle, bent at approximately 0.5 cm at a 45° angle, was used as a scraping tool to damage the endometrium. Female rats were anesthetized by intraperitoneal injection of sodium pentobarbital (120 mg / kg). The skin and abdominal wall were incised along the midline of the rectus abdominis muscle in the lower abdomen to expose the Y-shaped uterine structure, and the right endometrium was mechanically damaged. In the sham surgery group (Sham), only an abdominal incision was performed, without causing any damage to the uterus. In the control group (Control), the right uterus was punctured approximately 1 cm from the ovary using a scraping tool, and the endometrium was repeatedly scraped until the endometrial surface became rough and significant bleeding occurred, while the left uterus remained intact. After three estrous cycles, the damaged endometrium was treated. The injured rats were randomly assigned to Control, HA / Gel, M-IB-367 / EGF, and HA / Gel@M-IB-367 / EGF groups. Fertility in rats was assessed after three consecutive estrous cycles. The day when a vaginal plug was observed in the female rat's vagina was recorded as day 0.5 of gestation. Subsequently, the number of implanted embryos was recorded on day 16.5 of gestation.

[0031] Statistical analysis: Data were evaluated using GraphPad Prism 8.0. The results were analyzed using one-way ANOVA and Dunnett's multiple comparison test. All experiments were biologically replicated (n=3–5). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0032] Example 1: Preparation and Characterization of the Hydrogel of the Present Invention The preparation process of the hydrogel is as follows.

[0033] S1. Synthesis of oxidized hyaluronic acid (HA-CHO) and hydrazide-grafted gelatin (Gel-ADH): 2 g of HA was dissolved in 200 mL of deionized water, followed by the addition of 1.2 g of NaIO4. The mixture was stirred for 24 hours in the dark, then dialyzed (MW: ~10 kDa) for 72 hours, and finally freeze-dried to obtain HA-CHO. To prepare Gel-ADH, 1 g of MES was first dissolved in 200 mL of deionized water, and the pH was adjusted to 6.5. Then, 1 g of gelatin was completely dissolved in the solution, followed by the addition of 1.25 g of EDC and 0.69 g of HOBT. After reacting for 45 minutes, an appropriate amount of ADH was added, and the reaction was continued at room temperature for 24 hours. The resulting solution was dialyzed in NaCl (MW: ~10 kDa) for 72 hours, then dialyzed in deionized water for 48 hours. Finally, the solution was freeze-dried to obtain purified hydrazide-grafted gelatin (Gel-ADH). Preparation of S2.HA / Gel@M-IB-367 / EGF hydrogel: Using a dual syringe, equal volumes of HA-CHO were mixed with Gel-ADH dissolved in phosphate buffered solution (PBS) to prepare an injectable hydrogel. To prepare injectable hydrogels of different concentrations, the final concentration of Gel-ADH was set at 12 wt%, and the concentration of HA-CHO in the hydrogel was adjusted accordingly to 6 wt%, 8 wt%, and 10 wt%. Subsequently, the chimeric peptide M-IB-367 with a final concentration of 25 μg / mL was added to HA-CHO and mixed rapidly. EGF with a final concentration of 25 μg / mL was added to Gel-ADH. The two solutions were mixed in equal volumes to observe the gelation process, thus preparing HA / Gel@M-IB-367 / EGF hydrogel. The amino acid sequence of the chimeric polypeptide M-IB-367 is: RGGLCYCRGRFCVCVGRGPQGIAGQKLTQEWQKS (SEQ ID NO.1), where RGGLCYCRGRFCVCVGR is the IB-367 antimicrobial peptide (SEQ ID NO.2), GPQGIAGQ is the MMP cleavable sequence (SEQ ID NO.3), and KLTQEWQK is the angiogenic peptide (SEQ ID NO.4). This sequence contains multiple cysteine ​​residues (identified by the letter C). The thiol groups on the side chains of these residues can undergo an "aldehyde-thiol" click chemical reaction with the aldehyde groups on oxidized hyaluronic acid (HA-CHO), thereby covalently anchoring the entire chimeric polypeptide to the hydrogel network.

[0034] To obtain the optimal hydrogel ratio for the experiment, the performance of the hydrogel at different concentrations of HA-CHO was first tested. Therefore, the concentration of Gel-ADH in the hydrogel was kept constant, and the final concentration of HA-CHO in the hydrogel was adjusted to 6 wt%, 8 wt%, and 10 wt%. When the HA-CHO concentration reached 10%, the hydrazide groups of Gel-ADH were saturated with the aldehyde groups of HA-CHO. Further increases in the HA-CHO concentration did not change the degree of crosslinking in the hydrogel. Figure 1 Images of HA / Gel at different concentrations of HA-CHO.

[0035] Example 2: In vitro antibacterial / bactericidal performance testing of hydrogels The wound microenvironment in diabetic patients is complex and susceptible to bacterial infection. Timely anti-infection treatment in the early stages of inflammation is crucial for wound healing. Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative Pseudomonas aeruginosa, and Escherichia coli were selected as representative strains. The bactericidal effect of the hydrogel was assessed using a plate spread assay. Figure 2 As shown in Figure A, the HA / Gel@M-IB-367 / EGF hydrogel exhibits excellent bactericidal effects against both Gram-negative and Gram-positive bacteria, killing all bacteria incubated on its surface within 2 hours. A small number of bacteria remained in the HA / Gel group, while the bacterial survival rate in the M-IB-367 / EGF group was less than 1%. Figure 2 B).

[0036] Example 3: Biological Evaluation of Hydrogels In the field of clinical wound dressing applications, ensuring good biocompatibility is an indispensable prerequisite for hydrogels. Live / dead cell staining using AM / PI showed that HUVEC cells treated with HA / Gel, M-IB-367 / EGF, and HA / Gel@M-IB-367 / EGF exhibited better growth. After 72 hours of culture, the cell density in the hydrogel group was significantly increased compared to the control group. Figure 3 ).

[0037] Angiogenesis is crucial for wound healing. To evaluate the angiogenesis-promoting ability of hydrogels, an angiogenesis experiment was conducted using HUVECs. Statistical analysis of the number and total length of vessels was performed using ImageJ. The results showed that, compared with the control group, both the HA / Gel group and the M-IB-367 / EGF group significantly increased the vessel length and branch number of HUVECs. Notably, while M-IB-367 itself exhibits a certain pro-angiogenic ability, the pro-angiogenic effect was further enhanced when it was co-loaded with EGF in HA / Gel to form the HA / Gel@M-IB-367 / EGF composite hydrogel. This strong evidence suggests that the HA / Gel@M-IB-367 / EGF composite hydrogel can more effectively induce angiogenesis. Figure 4 This provides the necessary vascularization basis for wound repair.

[0038] Example 4: Healing and Anti-inflammatory Effects of Hydrogels on MRSA-Infected Diabetic Wounds This study investigated the anti-inflammatory and wound-healing effects of hydrogels by constructing a MRSA-infected diabetic mouse wound model. Type 1 diabetic mice were induced with STZ (streptozotocin). Random blood glucose levels were measured to observe successful model establishment. After applying MRSA to the wound, mice were randomly divided into four groups: Control, HA / Gel, M-IB-367 / EGF, and HA / Gel@M-IB-367 / EGF, respectively. Wounds from mice receiving different treatments were photographed at specified time intervals (0, 3, 7, and 14 days). Figure 5 As shown in Figures AB, after 14 days of treatment, the HA / Gel@M-IB-367 / EGF group exhibited significant healing effects, significantly superior to the other three groups. Analysis of wound healing rates across groups revealed that the HA / Gel@M-IB-367 / EGF group consistently demonstrated superior healing-promoting capabilities compared to other groups at different time points. Compared to the control group, wounds in all treatment groups rapidly closed from all sides on day 3, maintaining a near-circular shape throughout the healing process. Furthermore, statistical analysis of bacterial colonies in the wounds of mice in each group showed a significant decrease in bacterial count compared to the control group in all treatment groups. Due to its excellent physicochemical properties, the HA / Gel@M-IB-367 / EGF hydrogel demonstrated good anti-inflammatory effects and wound-healing capabilities in treating diabetic wounds infected with MRSA.

[0039] Example 5: Hydrogel promotes the recovery of fertility in rats with endometrial damage This invention further investigated the effect of combined treatment with HA / Gel@M-IB-367 / EGF hydrogels on fertility recovery in a rat model of endometrial injury. Results showed that the embryo implantation rate in the right uterine horn of the model group was only 10%, significantly lower than the 49% in the sham-operated group. In contrast, the embryo implantation rate in the HA / Gel@M-IB-367 / EGF combined treatment group increased to 45%, comparable to the sham-operated group. Furthermore, treatment with HA / Gel or M-IB-367 / EGF alone improved endometrial receptivity to embryos to some extent, but its effect still did not reach the level of the sham-operated group. Figure 6 A). To more comprehensively evaluate the in vivo safety of this hydrogel system, this study established a rat model of bilateral uterine horn injury, administered corresponding treatments, and observed the offspring born after treatment. The results showed that no obvious structural malformations were observed in the offspring rats of each treatment group at birth, and their development was good within 14 days after birth. Furthermore, the pups in each treatment group showed good development on day 1 after birth (…). Figure 6 There was no statistically significant difference in weight on day B and day 14. Figure 6 C).

[0040] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A chimeric polypeptide M-IB-367, characterized in that, The amino acid sequence of the chimeric polypeptide is shown in SEQ ID NO.

1.

2. A method for preparing a self-healing injectable hydrogel comprising the chimeric polypeptide of claim 1, characterized in that, Includes the following steps: S1. Preparation of HA-CHO: NaIO4 was added to HA solution, mixed, reacted in the dark, and then freeze-dried to obtain HA-CHO; S2. Preparation of Gel-ADH: Gelatin was dissolved in an acidic buffer solution, activated by a catalyst, coupled with ADH, and lyophilized to obtain Gel-ADH; S3. Preparation of HA / Gel@M-IB-367 / EGF hydrogel: Dissolve HA-CHO powder and add chimeric polypeptide M-IB-367 to obtain solution A. The amino acid sequence of the chimeric polypeptide is shown in SEQ ID NO.

1. Gel-ADH powder was dissolved and EGF was added to obtain solution B; solutions A and B were mixed, reacted, and allowed to stand to form a gel, thus obtaining HA / Gel@M-IB-367 / EGF hydrogel.

3. The preparation method according to claim 2, characterized in that, The mass ratio of HA to NaIO4 in step S1 is 2:(1-2).

4. The preparation method according to claim 2, characterized in that, The process parameters for the light-protected reaction in step S1 are: stirring and reacting under light-protected conditions for 20-28 hours.

5. The preparation method according to claim 2, characterized in that, The catalysts in step S2 are EDC and HOBT, and the mass ratio of gelatin: EDC: HOBT: ADH is 1:(1-1.5):(0.5-1):(5-6). Before the freeze drying in step S2, a dialysis step is also included. The process parameters for the dialysis step are: molecular weight cutoff of 10 kDa, first dialyzing in NaCl solution for 48-72 h, and then dialyzing in deionized water for 48-72 h.

6. The preparation method according to claim 2, characterized in that, The acidic buffer solution mentioned in step S2 is a MES solution with a pH of 6.

5.

7. The preparation method according to claim 2, characterized in that, In step S3, the final concentration of HA-CHO in the HA / Gel@M-IB-367 / EGF hydrogel is 6-10 wt%, the final concentration of Gel-ADH is 10-15 wt%, the final concentration of the chimeric polypeptide M-IB-367 is 25 μg / mL, and the final concentration of EGF is 25 μg / mL.

8. The self-healing injectable hydrogel prepared by the preparation method according to any one of claims 1-7.

9. The use of the chimeric polypeptide M-IB-367 of claim 1 or the self-healing injectable hydrogel of claim 8 in the preparation of a medicament or medical dressing that promotes wound healing.

10. The application according to claim 9, characterized in that, The wound is a chronic, difficult-to-heal wound caused by diabetes, or a wound infected with multidrug-resistant bacteria, or a skin defect accompanied by microcirculatory disorders; the multidrug-resistant bacteria include at least one of methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.

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