A method for preparing a polypeptide hydrogel and its application

Abstract

This invention provides a method for preparing a polypeptide hydrogel and its applications. The structure of the polypeptide hydrogel is shown in the figure below. The basic framework of the hydrogel is constructed from arginine and naphthaleneacetic acid, naproxen, phenylalanine, and glycine as basic units. Naproxen, as an anti-inflammatory drug, is introduced into the hydrogel to increase its bioactivity. Glycine and phenylalanine act as hydrophilic / hydrophobic modulators, while arginine and histidine regulate the hydrogel's properties. This polypeptide can be synthesized via solid-phase synthesis, and the preparation process is mature and readily available. The polypeptide of this invention can self-assemble into a supramolecular hydrogel in water, which can scavenge reactive oxygen species, inhibit the expression of TLR4-related gene pathways, and improve the inflammatory environment by downregulating NF-κB. The polypeptide hydrogel of this invention can also promote macrophage polarization, improve the wound environment through immunomodulation, and promote wound healing, showing broad application prospects in diabetic wound repair.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and novel drug development, and more specifically, to a method for preparing a polypeptide hydrogel and its application. Background Technology

[0002] Diabetic wounds are a common type of chronic wound, with multiple contributing factors including hyperglycemia, vascular disease, and neuropathy, placing a significant burden on patients' lives and the healthcare system. The complexity of these wounds leads to a lack of sufficient wound care materials, making it difficult to meet the treatment needs of diabetic wounds. Hydrogels, due to their porous structure, good swelling properties, ability to maintain a moist environment, and functional modifications, offer significant advantages in wound treatment. Peptide hydrogels exhibit better biocompatibility and biodegradability than ordinary polymer gels. Furthermore, they possess advantages such as ligand receptor recognition, stimulus-responsive self-assembly, and extracellular matrix mimicry. Through rationally designed peptide sequences, various biological functions can be achieved, such as inducing macrophage polarization, improving the microenvironment of diabetic wounds, and enabling spatiotemporal control of the wound. Therefore, peptide hydrogels hold great therapeutic potential in the repair of diabetic wounds.

[0003] In diabetic chronic wounds, the reduced ability of macrophages to clear apoptotic cells due to hyperglycemia and AGEs hinders the transition from a pro-inflammatory to an anti-inflammatory phenotype. This is one reason for the increased M1 / M2 macrophage ratio in chronic wounds. In diabetic chronic wounds, pro-inflammatory cytokines secreted by M1 macrophages, such as monocyte chemoattractant protein 1 (MCP-1) and IL-1β, recruit more macrophages to the wound site and polarize them into the M1 phenotype. This positive feedback loop maintains a long-term high level of inflammation in the wound environment. Therefore, the impediment to the transition from M1 to M2 macrophages is also a significant factor hindering the normal healing of diabetic chronic wounds. NF-κB translocates from the cytoplasm to the nucleus, binds to specific DNA sequences, regulates inflammatory cytokines, and participates in the inflammatory response; it is also a major obstacle to the M1-M2 polarization of macrophages. Studies have shown that silencing the TLR4 gene, an upstream regulatory gene of the NF-κB pathway, reduces TLR4 mRNA expression and significantly decreases the content of free NF-κBp65 in the cell nucleus, thereby reducing the activation of the NF-κB pathway. Therefore, inhibiting TLR4 is of great significance for regulating the inflammatory environment of diabetic wounds.

[0004] Chinese patent CN109157504A, published on August 15, 2018, discloses a polypeptide hydrogel, its preparation method, and its applications. The preparation method employs a solid-phase polypeptide synthesis approach, first sequentially linking non-natural amino acids to a resin, then obtaining a white powdery solid through shearing, purification, and freeze-drying. The white powdery solid is then dispersed, sonicated, heated until the system becomes clear, and finally cooled to obtain a self-assembled polypeptide hydrogel. The polypeptide hydrogel of this invention can effectively regulate and control the kinetics of drug release. The literature (Liping Zhou, Tong Huo, Wenmin Zhang, Na Han, Yongqiang Wen, Peixun Zhang, New techniques and methods for prevention and treatment of symptomatic traumatic neuroma: A systematic review, Frontiers in Neurology, 10.3389 / fneur.2023.1086806, 14, (2023)) describes the preparation of a multifunctional DNA hydrogel dressing through DNA grafting, dynamic cross-linking with polyethyleneimine, and doping with heat-inducing black phosphorus quantum dots. This DNA hydrogel dressing exhibits appropriate mechanical properties, self-healing ability, writability, and tissue adhesion, providing an effective, promising, and practical combination therapy for treating diabetic infected wounds. However, no reports have yet been found regarding the preparation method and application of a polypeptide hydrogel like the one described in this invention. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing and applying a peptide hydrogel containing TLR4-inhibited self-assembling peptides.

[0006] On the one hand, a self-assembled polypeptide is provided, the polypeptide sequence being (Naproxen)-FGGRGGHG GGG-(3-Aminobenzeneboronic acid) (Remark: Naproxen is naproxen, 3-Aminobenzeneboronic acid is 3-aminobenzeneboronic acid).

[0007] In a second aspect, a polypeptide hydrogel is provided, the polypeptide hydrogel comprising the aforementioned self-assembled polypeptide.

[0008] Thirdly, a method for preparing the aforementioned polypeptide hydrogel is provided, comprising the following steps:

[0009] S1 Weigh the self-assembled freeze-dried powder into a container;

[0010] Add S2 to the deionized water solution and mix well;

[0011] S3 dissolves the mixed solution under ultrasonic conditions;

[0012] S4 can form an adhesive hydrogel by letting the dissolved solution stand at room temperature for more than 10 minutes.

[0013] As a preferred example, in step S1, a polypeptide lyophilized powder with a purity of ≥95% is used.

[0014] More preferably, in step S2, deionized water is used.

[0015] More preferably, in step S3, the temperature used is room temperature, and the ultrasonic conditions are 100W for 30s.

[0016] More preferably, in step S4, the concentration of the polypeptide used is 30 mg / mL.

[0017] Fourthly, the invention provides an application of the aforementioned polypeptide hydrogel in the preparation of a wound healing drug.

[0018] As a preferred example, the wound is a diabetic chronic wound.

[0019] More preferably, the polypeptide hydrogel promotes the repair of diabetic wounds by inhibiting TLR4 to promote macrophage polarization.

[0020] The advantages of this invention are:

[0021] The polypeptide hydrogel of this invention has been tested for various indicators. The hydrogel is a fibrous, intertwined structure. In vitro experiments show that the polypeptide hydrogel has good biocompatibility, such as cell compatibility and blood compatibility, and can promote the migration of human umbilical vein endothelial cells (HUVECs). It also improves the inflammatory response by regulating the TLR4 pathway, reducing the inflammatory expression of the NF-κB pathway. In vivo experiments show that, compared with ordinary excipients, this polypeptide hydrogel can significantly promote the healing of chronic wounds in diabetic rats. The hydrogel possesses certain extensibility and adhesiveness, and inhibits TLR4 to promote macrophage polarization, thus improving anti-inflammatory effects. These characteristics make this hydrogel a promising clinical translational material for diabetic wounds. Attached Figure Description

[0022] Figure 1 This is an inverted experimental photograph of a polypeptide hydrogel.

[0023] Figure 2 This is a transmission electron microscope image of a polypeptide hydrogel.

[0024] Figure 3 The images show the infrared spectra of the polypeptide hydrogel before and after gelation.

[0025] Figure 4-6 This is a diagram of a cytotoxicity experiment using a polypeptide hydrogel.

[0026] Figure 7 This is a diagram of the blood compatibility experiment of the polypeptide hydrogel.

[0027] Figure 8 This is a diagram illustrating how peptide hydrogels promote cell migration.

[0028] Figure 9 This is a statistical graph showing the migration rate of peptide hydrogels.

[0029] Figure 10 Images show the staining of live and dead cells and the scavenging of reactive oxygen species in the polypeptide hydrogel.

[0030] Figure 11 This is a diagram showing how peptide hydrogels promote macrophage polarization.

[0031] Figure 12 Immunoblotting of polypeptide hydrogels.

[0032] Figure 13 Flow cytometry was used to examine the polarization of macrophage raw264.7.

[0033] Figure 14 Image showing how peptide hydrogel promotes wound healing on the back of diabetic rats.

[0034] Figure 15 Statistical chart showing the rate of peptide hydrogel promoting wound healing in diabetic patients. Detailed Implementation

[0035] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and are intended to limit the scope of application of the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of application of the invention. Methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional conditions or according to the conditions provided in the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. The specific embodiments of the present invention will be described in detail below with reference to the technical solutions and accompanying drawings.

[0036] The structure of the polypeptide hydrogel in the examples is shown in the figure below. The basic framework of the hydrogel is constructed from arginine and naphthaleneacetic acid, naproxen, phenylalanine, and glycine as basic units.

[0037]

[0038] Example 1: Preparation and Morphological Characterization of Peptide Hydrogels

[0039] Preparation of peptide hydrogel: Weigh 5 mg of lyophilized peptide powder (Naproxen)-FGGRGGHG GGG-(3-Aminobenzeneboronic acid) (Remark: Naproxen is naproxen, 3-Aminobenzeneboronic acid is 3-aminobenzeneboronic acid), add 0.5 mg of ultrapure water, sonicate to dissolve, and let stand for 10 minutes to obtain peptide hydrogel. Figure 1 As shown, the inverted vial demonstrates the formation of the gel.

[0040] TEM morphology characterization of the peptide hydrogel: The peptide hydrogel prepared as described above was shaken well, and 10 μL was dropped onto a 200-mesh carbon support membrane. After drying, it was stained with 1% phosphotungstic acid solution for approximately 30 seconds, washed three times with ultrapure water, and dried before TEM testing. Figure 2 The polypeptide hydrogel is formed by dense nanofibers intertwined.

[0041] Infrared spectroscopy determination of peptide hydrogel: The hydrogel prepared by the above method was freeze-dried at -50℃ to obtain a lyophilized hydrogel powder. Potassium bromide was added at a ratio of 1:20, and the powder was compressed into a pellet. The infrared spectrum was measured and compared with that of the lyophilized peptide powder. Figure 3 The infrared spectrum shows a red shift in the absorption wavelength in the gel state, indicating that hydrogen bonds are formed between molecules in the gel state.

[0042] Example 2: The effect of hydrogels on cell growth

[0043] Peptide hydrogel solutions with concentrations of 20 μg / mL, 15 μg / mL, 10 μg / mL, and 5 μg / mL were prepared according to the hydrogel preparation method in Example 1. The above hydrogels of different concentrations were added to 96-well plates and subjected to overnight UV irradiation to determine cytotoxicity.

[0044] Cytotoxicity of the hydrogel: Mouse epithelial-like fibroblast L929 cells and human umbilical vein endothelial cells (HUVECs) were used to investigate the cytotoxicity of the hydrogel and cell proliferation. The prepared hydrogel solution was sterilized at 60°C for 12 h, mixed, and poured into sterile culture dishes. The cells were cultured in a CO2 incubator using 10% fetal bovine serum and DMEM (containing antibiotics) as the complete growth medium. L929 cells were seeded at a density of 20,000 cells / well in 96-well plates and cultured for 24 h before the hydrogel dish was added to the wells. After culturing for 1, 3, and 5 days, the cell culture medium was replaced with 100 μL of fresh DMEM medium, and 10% (v / v) CCK-8 reagent (Beyotime, China) was added. After culturing at 37°C for 2 hours, cell viability was quantified using a 450 nm microplate reader (TECAN, Switzerland). After 24 hours, cell proliferation and viability under hydrogel conditions were assessed using the LIVE / DEAD live / dead cell assay. Figure 4-6 In addition, the Live / Dead kit, consisting of calcein (green fluorescence) and propidium iodide (red fluorescence), is used to determine the viability of L929 cells. Furthermore, fluorescence microscopy is used to measure fluorescence images (e.g., Figure 10 ).

[0045] The effect of peptide hydrogel on cell migration was determined using a scratch assay: First, cells were seeded in 24-well plates. When cell confluence was ideal, a scratch was made in the center of each well using a 200 μL needle tip, followed by rinsing with PBS. Cells were then incubated with peptide hydrogel extraction buffer, and wound closure was observed periodically for up to 12 hours. The wound area was quantified using ImageJ software, and migration patterns were analyzed (…). Figure 8 To calculate mobility () Figure 9 ).

[0046] Blood compatibility of the hydrogel: Red blood cells were separated from mouse blood by centrifugation (1000 rpm) for 10 minutes. The obtained red blood cells were washed three times with Tris buffer and diluted to a final concentration of 5% (v / v). 500 μL of hydrogel and 500 μL of stock solution of red blood cells were added to 24-well microplates and incubated at 37°C with shaking at 150 rpm for 1 hour. Subsequently, the contents of the microplate wells were centrifuged (1000 rpm) for 10 minutes, and 100 μL of the supernatant was transferred to 96-well microplates. The absorbance of the supernatant at 540 nm was measured using a molecular device. A positive control was Triton X-100, and a negative control was Tris buffer. The hemolysis rate is calculated using the formula: Hemolysis (%) = [(Ap-Ab) / (At-Ab)] × 100%, where Ap is the absorbance value of the experimental group, At is the absorbance value of the Triton x-100 positive control, and Ab is the absorbance value of the Tris buffer solution. Figure 7 ).

[0047] Immunoblot: RAW264.7 protein was separated using RIPA lysis buffer, quantified using a BCA protein assay kit, separated by SDS-PAGE, and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was then incubated overnight at 4°C with primary antibody, anti-TLR4, anti-NF-κB, and anti-Actin. The PVDF membrane was then immersed in 5% skim milk for 1 hour, followed by incubation with secondary antibody at room temperature for 2 hours. Finally, the membrane was washed three times with TBST, and the blot was observed using an enhanced chemiluminescence detection system. Figure 12 ).

[0048] In vitro macrophage regulation: The inflammatory response of cells was stimulated by LPS agonists, followed by gel incubation. The anti-inflammatory effect was assessed by immunofluorescence and flow cytometry. RAW264.7 was stimulated with 100 ng / mL LPS and 20 ng / mL IFN-γ for 24 h to induce M1 polarization, and stimulated with 20 ng / mL IL-4 for 24 h to induce M2 polarization.

[0049] Immunofluorescence and flow cytometry were used to examine macrophage polarization: 100 ng / mL LPS and 20 ng / mL IFN-γ were added to the culture medium for 24 h to induce a systemic inflammatory microenvironment.

[0050] Immunofluorescence staining: Macrophages were fixed / infiltrated / blocked with 4% paraformaldehyde / 0.5% Triton / 10% BSA; incubated with primary antibody CD206 polyclonal antibody (1:2000, Abcam), followed by secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (1:2000). Cell nuclei were then reverse-stained with DAPI and observed using a confocal microscope. Figure 11The number of CD206-positive cells was counted using ImageJ software. CD86 (1:2000) was stained using the same method. Furthermore, flow cytometry was used to assess the effect of the peptide hydrogel on macrophage polarization; 2×10⁻⁶ cells / cells were counted. 5 RAW 264.7 cells were seeded in 6-well plates. One day after seeding, the LPS group received 200 ng / mL LPS in the culture medium, while the experimental group received peptide hydrogel. After two days of culture, RAW 264.7 cells were collected using a scraper and centrifuged at 1000 rpm for 5 minutes. After resuspending and blocking with 1% BSA, the cells were incubated with CD86 (M1 marker) and CD206 (M2 marker) for 30 minutes. The cell suspension was then tested using a BD FACS-Calibur cytometer. Data were analyzed using FlowJo X 10.2 software. Figure 13 ).

[0051] Example 3: Detection of the therapeutic effect of hydrogel on wound healing in diabetic rats in vivo

[0052] Methods to promote chronic wound healing in diabetic rats: The animals used in this study were SPF-grade male SD rats (190±10g). Type 1 diabetic rats were induced by a single intraperitoneal injection of streptozocin (STZ) at 65 mg / kg. One week after administration, rats with a blood glucose level exceeding 16.7 mmol / L were diagnosed with diabetes. STZ is a DNA alkylating agent that selectively accumulates in pancreatic β-cells via the GLUT2 glucose transporter. Its nitrosourea moiety can destroy pancreatic β-cells in a short time, thereby inducing diabetes. A diabetic wound model was established in a selected number of rats 3 weeks after STZ-induced hyperglycemia. Full-thickness defects with a diameter of 10 mm were prepared on the skin of the rat's back using a perforated biopsy instrument. The wounds were rinsed with physiological saline, each covered with sterile gauze, and fixed with elastic adhesive tape. Hydrogel was injected in situ into the wound site. The wounds were recorded using a digital camera at 1, 3, 7, and 11 days, and the wound area was calculated using ImageJ software. The healing rate of the wound was used to evaluate the repair-promoting effect of the hydrogel. Figure 14-15 ).

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A self-assembling polypeptide, characterized in that, The polypeptide sequence is (naproxen)-FGGRGGHGGGG-(3-aminophenylboronic acid).

2. A polypeptide hydrogel, characterized in that, The polypeptide hydrogel comprises the self-assembling polypeptide described in claim 1.

3. The method for preparing the polypeptide gel according to claim 2, characterized in that, Includes the following steps: S1 Weigh the lyophilized powder of the self-assembled peptide into a container; Add S2 to the deionized water solution and mix well; S3 dissolves the mixed solution under ultrasonic conditions; S4 can form an adhesive hydrogel by letting the dissolved solution stand at room temperature for more than 10 minutes.

4. The method of preparation according to claim 3, characterized in that, In step S1, the polypeptide lyophilized powder with a purity of ≥95% is used.

5. The method of preparation according to claim 3, characterized in that, In step S3, the temperature used is room temperature, and the ultrasonic conditions are 100W for 30 seconds.

6. The preparation method according to claim 3, characterized in that, In step S4, the concentration of the polypeptide used is 30 mg / mL.

7. The use of the polypeptide hydrogel according to claim 2 in the preparation of a wound healing drug, characterized in that, The wound is a chronic diabetic wound.

8. The application according to claim 7, characterized in that, The polypeptide hydrogel promotes the repair of diabetic wounds by inhibiting TLR4 and promoting macrophage polarization.

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