Functional hydrogel of antibacterial healing-promoting fusion polypeptide as well as preparation method and application of functional hydrogel

Abstract

The invention relates to the technical field of hydrogel, in particular to functional hydrogel of antibacterial healing-promoting fusion polypeptide as well as a preparation method and application of the functional hydrogel. The self-assembled peptide RADA16 is adopted as a hydrogel base material, and the neuropeptide and the antibacterial peptide are combined, so that the double effects of healing promotion and bacterium resistance of the hydrogel material are realized. The polypeptide and the degradation product amino acid are non-toxic and low in immunogenicity, and the polypeptide hydrogel has huge potential as a wound dressing by virtue of the natural source, biocompatibility and biosafety of the polypeptide hydrogel.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and in particular to a functional hydrogel of an antibacterial and healing-promoting fusion polypeptide, its preparation method, and its application. Background Technology

[0002] Special wounds caused by diabetes, burns, radiation, etc., can create complex wound environments, making them prone to infection, excessive inflammation, and high oxidative stress. These factors can damage cell membranes, proteins, and DNA, subsequently threatening the repair cells around the wound, leading to chronic wounds and hindering healing. Therefore, for chronic wounds that cannot be repaired by the body alone, artificial intervention is necessary. Wound dressings are a recognized effective treatment method. Hydrogels, with their highly hydrophilic three-dimensional network structure, are the closest to ideal dressings, possessing multiple functions such as drug delivery, improving the microenvironment, and regulating growth factors. They are widely used in tissue engineering and regenerative medicine for skin injuries.

[0003] Practicality: Bacterial infection can disrupt the orderly healing process of wounds, hindering healing or increasing the risk of sepsis. This antibacterial and repair-promoting hydrogel composite material possesses dual antibacterial and repair-promoting effects, demonstrating excellent antibacterial and repair-promoting properties and showing great promise for application in wound healing.

[0004] Innovation: Compared to other traditional wound dressings (gauze, cotton, bandages, etc.), hydrogels, as wound dressings, have excellent water absorption, can absorb exudate and keep the wound environment moist, which helps tissue cell migration and proliferation, and can effectively promote wound healing. This antibacterial and repair-promoting hydrogel composite material uses chitosan as a matrix to construct a three-dimensional porous hydrogel dressing.

[0005] Dynamics: In recent years, bio-nanomaterials synthesized from complexes of Zn, Cu, Ag, and Ce have been widely used in the biomedical field. This antibacterial and healing-promoting hydrogel composite material, loaded with zinc oxide nanoparticles (ZnO-NPs) and cerium dioxide nanoparticles (CeO2-NPs), endows the hydrogel material with antibacterial and healing-promoting functions.

[0006] A hydrogel composite material with antibacterial and healing-promoting properties, its preparation and application (Application No.: CN202311073980.3, Application Date: 2023.08.24), while possessing dual antibacterial and healing-promoting effects, shows great promise for wound healing. However, its synthetic raw materials, zinc oxide nanoparticles (ZnO-NPs) and cerium dioxide nanoparticles (CeO2-NPs), are both inorganic materials, resulting in poor biocompatibility. Furthermore, the synthesis of this hydrogel dressing involves various organic solvents such as methanol, ethanol, propanol, methyl ketone, ethyl ketone, and acetone, and the quaternized chitosan substrate also requires chemical modification, necessitating special attention to its biosafety.

[0007] Therefore, there is an urgent need for a hydrogel material with good biocompatibility and biosafety. Summary of the Invention

[0008] This invention utilizes the self-assembled peptide RADA16 as a hydrogel substrate, combining neuropeptides and antimicrobial peptides to achieve a dual effect of promoting healing and antibacterial properties in the hydrogel material. The peptides and their degradation product amino acids are all non-toxic and have low immunogenicity. Due to their natural origin, biocompatibility, and biosafety, peptide hydrogels have great potential as wound dressings.

[0009] The present invention adopts the following technical solution: The first aspect of the present invention provides a functional hydrogel of an antibacterial and healing-promoting fusion peptide, which is formed by co-assembling RADA16 peptide and an antibacterial and healing-promoting fusion peptide.

[0010] A second aspect of this invention provides a method for preparing a functional hydrogel, comprising the following steps: (1) Dissolve the RADA16 polypeptide lyophilized powder in ultrapure water, sonicate it, and prepare a RADA16 polypeptide stock solution with a concentration of 6-10 mg / mL. (2) The antibacterial and healing-promoting lyophilized peptide powder was dissolved in PBS buffer, sonicated, and prepared to a concentration of 2×10⁻⁶. -7 Up to 6×10 -7 Antibacterial and healing-promoting fusion peptide stock solution (mol / L); (3) Mix the mother liquors obtained in steps (1) and (2) at a volume ratio of (1-5):1, mix them evenly by ultrasonic vibration, and then let them stand at 20-37℃ to form a gel.

[0011] In a preferred embodiment, the RADA16 polypeptide mother liquor and the antibacterial and healing-promoting fusion polypeptide mother liquor in step (3) are mixed in equal volumes.

[0012] As a preferred embodiment, the settling time in step (3) is less than 10 minutes.

[0013] A third aspect of this invention provides the application of the aforementioned functional hydrogel in the preparation of drugs or medical devices for promoting skin wound healing, specifically for the treatment of radiation-induced skin injuries. This application primarily targets skin injuries caused by radiotherapy, including first- and second-degree burns (such as acute radiation-induced skin injuries caused by radiotherapy, radionuclide contamination, etc., as well as the healing and repair of difficult-to-heal wounds such as first- and second-degree burns). This functional hydrogel exerts multiple effects, including antibacterial, healing-promoting, and moisturizing properties, effectively improving the microenvironment of radiation-induced skin injury wounds, thereby promoting wound repair and healing.

[0014] In a preferred embodiment, the skin wound is an infected wound.

[0015] In a preferred embodiment, the skin wound is a radiation-induced skin injury.

[0016] A fourth aspect of the present invention provides a wound dressing comprising a functional hydrogel containing the above-mentioned antibacterial and healing-promoting fusion polypeptide as an active ingredient.

[0017] In a preferred embodiment, the effective amount of the functional hydrogel is 1-99%wt.

[0018] The technical solution adopted in this invention can achieve the following beneficial effects: This invention provides a functional hydrogel based on an antibacterial and healing-promoting fusion peptide, its preparation method, and its application. This hydrogel dressing not only exerts antibacterial and healing-promoting effects, but also has the advantages of low toxicity to organisms, low immunogenicity, and high safety for human use. It provides new materials and methods for the repair of radiation-induced skin damage and has good application prospects in the repair of radiation damage. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, forming part of the present invention. The illustrative embodiments of the present invention and their descriptions explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 Image showing the gelation effect of a functional hydrogel containing antibacterial and healing-promoting fusion peptides: Figure 2 : Surface microstructure of a functional hydrogel containing antibacterial and healing-promoting fusion peptides under cryo-scanning electron microscopy (Cryo-SEM); Figure 3 Nanofiber network diagram of functional hydrogel containing antibacterial and healing-promoting fusion peptides under transmission electron microscopy (TEM); Figure 4 Modulus test diagram of functional hydrogel containing antibacterial and healing-promoting fusion peptides; Figure 5: Identification diagram of the antibacterial activity of functional hydrogels containing antibacterial and healing-promoting fusion peptides; Figure 6 Cytotoxicity identification diagram of functional hydrogels containing antibacterial and healing-promoting fusion peptides; Figure 7 Blood compatibility identification diagram of functional hydrogels containing antibacterial and healing-promoting fusion peptides; Figure 8 : A gross observation of the wound repair effect of a functional hydrogel containing antibacterial and healing-promoting peptides in a radiation-induced skin injury model in SD rats; Figure 9 Pathological observation of the wound repair effect of a functional hydrogel containing antibacterial and healing-promoting peptides in a radiation-induced skin injury model in SD rats. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. In the description of this invention, it should be noted that the term "or" is generally used to include the meaning of "and / or," unless otherwise expressly indicated.

[0021] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0022] The preparation method of the antibacterial healing-promoting fusion peptide used in this invention comes from Chinese invention patent CN202311867731.1 "SP-based antibacterial healing-promoting peptide and its self-assembled hydrogel peptide and preparation method".

[0023] Example 1: Preparation of a functional hydrogel containing antibacterial and healing-promoting fusion peptides Preparation of RADA16 peptide stock solution: Take out RADA16 lyophilized powder, dissolve the lyophilized powder in a certain volume of ultrapure water, mix thoroughly, and then use ultrasonication to aid dissolution for 10 min to prepare RADA16 peptide stock solution. Preparation of antibacterial and healing-promoting fusion peptide stock solution: Take the lyophilized powder of antibacterial and healing-promoting fusion peptide, add a certain volume of PBS buffer to dissolve the lyophilized powder, mix thoroughly, and then sonicate for 10 min to prepare the antibacterial and healing-promoting fusion peptide stock solution.

[0024] Induced gelation: RADA16 peptide stock solution and antibacterial and healing-promoting fusion peptide stock solution were mixed in volume ratio, ultrasonically vibrated for 10 min, and then allowed to stand at 37℃ or room temperature for 10 min. The gelation effect was observed after inverting the mixture.

[0025] In step (1) above, the concentration of RADA16 mother liquor is 1% (10mg / mL, m / v).

[0026] In step (2) above, the concentration of the antibacterial and healing-promoting fusion polypeptide mother liquor is 2×10⁻⁶. -7 mol / l.

[0027] In step (3) above, the RADA16 stock solution and the antibacterial and healing-promoting recombinant polypeptide stock solution are mixed in equal volumes, and the final concentration of the functional fusion hydrogel is 0.5% (5 mg / mL, m / v). Figure 1 As shown, the control group RADA16 (left) and the antibacterial healing-promoting fusion peptide hydrogel (middle) both appear as transparent jelly-like substances when viewed with the naked eye, and exhibit very little fluidity when placed on their sides or upside down, showing strong viscosity.

[0028] Example 2: Preparation of a functional hydrogel containing antibacterial and healing-promoting fusion peptides Preparation of RADA16 peptide stock solution: Take out RADA16 lyophilized powder, dissolve the lyophilized powder in a certain volume of ultrapure water, mix thoroughly, and then use ultrasonication to aid dissolution for 10 min to prepare RADA16 peptide stock solution. Preparation of antibacterial and healing-promoting fusion peptide stock solution: Take the lyophilized powder of antibacterial and healing-promoting fusion peptide, add a certain volume of PBS buffer to dissolve the lyophilized powder, mix thoroughly, and then sonicate for 10 min to prepare the antibacterial and healing-promoting fusion peptide stock solution.

[0029] Induced gelation: RADA16 peptide stock solution and antibacterial and healing-promoting fusion peptide stock solution were mixed in volume ratio, ultrasonically vibrated for 10 min, and then allowed to stand at 37℃ or room temperature for 10 min. The gelation effect was observed after inverting the mixture.

[0030] In step (1) above, the concentration of RADA16 mother liquor is 0.6% (6 mg / mL, m / v).

[0031] In step (2) above, the concentration of the antibacterial and healing-promoting fusion polypeptide mother liquor is 6×10⁻⁶. -7 mol / l.

[0032] In step (3) above, the RADA16 stock solution and the antibacterial and healing-promoting recombinant polypeptide stock solution are mixed at a volume ratio of 5:1, and the final concentration of the functional fusion hydrogel is 0.5% (5 mg / mL, m / v). Figure 1 As shown, the control group RADA16 (left) and the antibacterial healing-promoting fusion peptide hydrogel (right) both appear as transparent jelly to the naked eye, and exhibit very little fluidity when placed on their sides or upside down, showing strong viscosity.

[0033] Example 3: Physicochemical property testing of functional hydrogels containing antibacterial and healing-promoting fusion peptides 1. Cryo-scanning electron microscopy (Cryo-SEM) observation of the microstructure of the hydrogel surface Take the prepared hydrogel samples as described above and fix them with 2.5% glutaraldehyde solution. After fixation, the hydrogel samples are flash-frozen in liquid nitrogen, sublimated, fractured, coated, and then observed using a cryo-scanning electron microscope. Figure 2 As shown, the two hydrogel samples exhibit typical three-dimensional mesh features and form an interconnected porous network system. The control group RADA16 hydrogel shows a densely arranged network structure under the microscope, while the functional hydrogel of the antibacterial and healing fusion peptide has a relatively large network structure pore size.

[0034] 2. Transmission electron microscopy (TEM) observation of the nanofiber network inside the hydrogel The prepared hydrogel samples were stained with phosphotungstic acid using negative staining techniques, and then observed under a transmission electron microscope at an accelerating voltage of 80-120 kV. Figure 3 As shown, both hydrogels exhibit a regular and dense fibrous network. Compared to the control group RADA16 hydrogel, the functional hydrogel of the antibacterial and healing fusion peptide has shorter nanofibers and larger network pores.

[0035] 3. Rheometer analysis of the mechanical properties of hydrogels The prepared hydrogel samples were dropped onto the rheometer cone plate, and the storage modulus (G') and loss modulus (G'') of the samples were measured under the limiting conditions of a frequency range of 10⁻⁰.⁻¹ rad / s and a constant stress of 1 Pa. The results are as follows: Figure 4 As shown, the storage modulus (G') of the hydrogel loaded with antibacterial and healing-promoting fusion peptides and the control group RADA16 hydrogel are both greater than the loss modulus (G''). Furthermore, G' and G'' increase continuously with increasing angular frequency, indicating that both exhibit an elastic solid shape and have good mechanical properties.

[0036] Example 4: Application of functional hydrogels containing antibacterial and healing-promoting fusion peptides in radiation-induced skin injuries 1. Identification of the antibacterial activity of functional hydrogels containing antibacterial and healing-promoting fusion peptides (1) Logarithmic growth phase Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were inoculated into fresh LB liquid medium, with a final concentration of 1×10⁻⁶. 6 CFU / ml; (2) Take the prepared functional hydrogel of antibacterial and healing-promoting fusion peptide, and prepare an immersion solution with liquid culture medium at a ratio of 1:10. Use the immersion solution to conduct antibacterial experiments. (3) The soaking solution was diluted with culture medium to 1.88 μg / ml, 18.8 μg / ml, 56.4 μg / ml, and 94 μg / ml, respectively. 100 μl of hydrogel soaking solution (experimental group) and blank culture medium (control group) were added to each well of a 96-well plate (containing 1 μl of spare bacterial solution), and the plates were incubated at 37℃. OD600 nm was measured at different time points. Figure 5 As shown, the antibacterial and healing-promoting fusion polypeptide hydrogel exhibited a certain lag effect on the growth of treated bacteria (E. coli, S. aureus), and showed an antibacterial effect after 4 hours of co-culture.

[0037] 2. Functional hydrogel cytotoxicity assay of antibacterial and healing-promoting fusion peptides Human skin fibroblasts were digested in cell culture flasks to prepare a cell suspension with a final concentration of 5 × 10⁵ cells / ml. 300 μL of suspension was seeded into each well of a 24-well plate and incubated overnight. The next day, the culture medium was aspirated from the wells. Using complete culture medium as a blank control, 300 μL of culture medium soaking solution (RADA16 hydrogel and antibacterial healing-promoting fusion peptide hydrogel soaked overnight in complete culture medium) was added to the corresponding wells for incubation. After 1 and 3 days of co-culture, the 24-well plates were removed, washed three times with PBS, and 200 μL of 1.2 mmol / L calcein-AM (for staining viable cytoplasm) and 200 μL of 1 μg / ml PI staining solution (for binding to dead cell nuclei) were added. The plates were incubated in the dark for 25 min, washed three times with PBS, and then DAPI staining solution (for nucleus localization) was added. After incubation in the dark for 10 min, the plates were washed three times with PBS and observed under a fluorescence microscope. The results are as follows: Figure 6 As shown, almost all cells in the RADA16 hydrogel and antibacterial healing-promoting fusion peptide hydrogel groups survived (green represents live cells, red represents dead cells), and there was no significant difference compared with the blank control group, indicating that the antibacterial healing-promoting fusion peptide hydrogel did not exhibit significant cytotoxicity.

[0038] 3. Blood compatibility analysis of functional hydrogels containing antibacterial and healing-promoting fusion peptides Blood was collected from healthy rabbits, and red blood cells were resuspended in 0.9% sodium chloride solution to prepare a 2% red blood cell suspension. 2.5 ml of the 2% red blood cell suspension was added to a 10 ml centrifuge tube. 2.5 ml of distilled water was added to the positive control group; 2.5 ml of 0.9% sodium chloride solution was added to the negative control group; and 2 ml of 0.9% sodium chloride solution and 0.5 ml of polypeptide hydrogel (RADA16, antibacterial and healing-promoting fusion polypeptide hydrogel, final concentration 5 mg / ml) were added to the experimental group. The mixture was incubated at 37℃ for 3 h, and the solution state was observed and photographed. Subsequently, the mixture was centrifuged at 4℃, 3000 rpm / min for 5 min. Collect the supernatant and measure the absorbance at OD=540 nm; hemolysis rate (HR, %) = [OD540 (sample) - OD540 (negative)] / OD540 (positive). Results are as follows... Figure 7 As shown, the supernatants of the antibacterial and healing-promoting fusion peptide hydrogel group and the negative control group were clear and transparent, with no hemolysis observed. Analysis of the supernatant absorbance, combined with a hemolysis rate bar chart, revealed that the hemolysis rate in the experimental groups was less than 5%, meeting biosafety requirements and indicating that the antibacterial and healing-promoting fusion peptide hydrogel has good blood compatibility.

[0039] Application Example: Functional hydrogels containing antibacterial and healing-promoting fusion peptides in radiation-induced skin injuries. (1) Establishment of an electron beam-induced radiation-induced skin injury model in rats The rump and back of rats were selected as the irradiation area. The rats were shaved locally the day before irradiation, and then grouped, weighed, marked, and numbered. Prior to irradiation, rats were anesthetized via intraperitoneal injection of 2.5% sodium pentobarbital solution at a dose of 0.2 ml per 100g of body weight. After anesthesia took effect, the rats were secured in a self-designed irradiation cage (except for the irradiation area, other parts were shielded with aluminum plates), and the irradiation area was marked with a marker. A high-energy electron beam of 6 MeV (Million electron Volts) was generated using an Elekta Synergy medical electron linear accelerator to irradiate the exposed skin of the rat's rump and back in a single session. The irradiation conditions were: source-skin distance 100 cm, irradiation area diameter 3 cm, irradiation field size 25 cm × 25 cm, dose rate 600 cGy / min, continuous irradiation time 7.5 minutes, and total absorbed dose 45 Gy. After irradiation, the rats were kept warm and, once awake, were transferred to cages and housed in the rodent laboratory of the China Institute of Radiation Protection. The experimental procedures were strictly carried out in accordance with the experimental animal operation guidelines of the GLP Center of the China Institute of Radiation Protection.

[0040] (2) Application of functional hydrogels containing antibacterial and healing-promoting fusion peptides in radiation-induced skin injuries ① A randomized controlled trial was conducted, with rats divided into 5 groups: a control group (blank control group), a negative control group (PBS buffer), a positive control group (triethanolamine), a RADA16 hydrogel group, and an antibacterial and healing-promoting fusion peptide hydrogel group. The drugs were applied daily after irradiation, and the rats' eating habits, mental state, and skin damage were observed. The wounds were photographed and recorded 1 day before irradiation, and 1, 3, 5, and 7 days after irradiation, and every week thereafter. Figure 8As shown, no significant changes were observed in the skin of the buttocks and back of the rats in each group on the day of modeling. About one week after irradiation, obvious erythema and dryness appeared in the irradiated areas of the rats in each group. Two weeks after irradiation, obvious ulceration appeared in the irradiated areas of the rats, some with exudate, some with obvious tenderness, and wet desquamation. With the increase of treatment time, four weeks after irradiation, the wounds in the irradiated group no longer enlarged, and the ulceration of the wounds in the experimental group began to gradually improve. Eight weeks after irradiation, the hydrogel group (RADA16, antibacterial and healing-promoting fusion polypeptide hydrogel) was basically healed.

[0041] ② Eight weeks after irradiation, tissue samples were taken from each group of rats. Anesthesia was administered via intraperitoneal injection of 2.5% sodium pentobarbital (0.2 ml of 2.5% sodium pentobarbital per 100g). Full-thickness skin tissue from the irradiated field and surrounding area was collected. Residual blood and shed hair were rinsed away with 0.9% sodium chloride injection, and the tissue was laid flat on filter paper to absorb moisture. It was then fixed in 10% neutral formalin solution for paraffin section preparation. Hematoxylin-eosin (H&E) staining and Masson's trichrome staining were used for histological observation of the wound tissue. Figure 9 As shown, 8 weeks after irradiation, in HE staining, the antibacterial and healing-promoting fusion peptide hydrogel group showed less epidermal keratosis and thickening, less dermal inflammation, and a significantly increased number of skin appendages compared to other groups. In Masson staining, the antibacterial and healing-promoting fusion peptide hydrogel group showed only small-scale collagen fiber hyperplasia compared to other groups. The collagen fibers were tightly arranged and relatively regular, with newly formed epithelium covering the wound, closely resembling the skin structure of normal rats.

[0042] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A functional hydrogel of an antibacterial, wound-healing fusion polypeptide, characterized in that, It is formed by the co-assembly of RADA16 peptide and antibacterial healing-promoting fusion peptide.

2. The method of claim 1, wherein the functional hydrogel is prepared by mixing the polymer solution and the crosslinking agent solution. Includes the following steps: (1) Dissolve the RADA16 polypeptide lyophilized powder in ultrapure water, sonicate it, and prepare a RADA16 polypeptide stock solution with a concentration of 6-10 mg / mL. (2) The antibacterial and healing-promoting lyophilized peptide powder was dissolved in PBS buffer, sonicated, and prepared to a concentration of 2×10⁻⁶. -7 Up to 6×10 -7 Antibacterial and healing-promoting fusion peptide stock solution (mol / L); (3) Mix the mother liquors obtained in steps (1) and (2) at a volume ratio of (1-5):1, mix them evenly by ultrasonic vibration, and then let them stand at 20-37℃ to form a gel.

3. The production method according to claim 2, characterized by, In step (3), the RADA16 polypeptide mother liquor and the antibacterial and healing-promoting fusion polypeptide mother liquor are mixed in equal volumes.

4. The preparation method according to claim 2, characterized in that, The time for settling and gelling in step (3) is within 10 minutes.

5. The use of the functional hydrogel of claim 1 in the preparation of a medicament or medical device for promoting skin wound healing.

6. Use according to claim 5, characterized in that, The skin wound was an infected wound.

7. Use according to claim 5, characterized in that, The skin wound was a radiation-induced skin injury.

8. A wound dressing, characterized in that, A functional hydrogel containing the antibacterial and healing-promoting fusion polypeptide of claim 1 as an active ingredient.

9. The wound dressing of claim 8, wherein, The effective amount of the functional hydrogel is 1-99%wt.

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