Puerarin-polypeptide co-assembled nano hydrogel as well as preparation method and application thereof

Abstract

The invention discloses puerarin-polypeptide co-assembled nano hydrogel, and belongs to the technical field of biological medicines. In order to solve the problems that puerarin is poor in water solubility and stability and diabetic wounds are difficult to heal, the hydrogel comprises self-assembled polypeptide and puerarin with the mass ratio of 1: (0.5-4), the molecular formula of the self-assembled polypeptide is R-GDFDFDY, D represents amino acid in a D configuration, and R is a non-steroidal anti-inflammatory drug. The self-assembled polypeptide and the puerarin are co-assembled to form the nano hydrogel, so that the solubility and the stability of the puerarin are improved, and the nano hydrogel can be used for preparing the diabetes wound healing medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology. More specifically, this invention relates to a puerarin-peptide co-assembled nanohydrogel, its preparation method, and its applications. Background Technology

[0002] Wounds caused by trauma, burns, surgery, and chronic diseases, especially chronic wounds which are becoming increasingly severe due to the rising incidence of obesity and diabetes, face enormous challenges in treatment. The repair of these wounds involves complex biological processes in multiple stages, such as inflammation regulation, cell proliferation, and angiogenesis. Traditional invasive treatments, such as surgical debridement and skin grafting, not only carry risks of bleeding and infection but also require long-term bed rest for recovery. The cost of a single treatment can reach tens of thousands of dollars, placing a double burden on patients and the healthcare system.

[0003] Non-invasive gel dressings have become a research hotspot in wound treatment due to their three-dimensional network structure similar to soft tissue, injectable in-situ gelation properties, and targeted drug delivery capabilities. Currently, there are many types of biomaterial-based hydrogel dressings, including peptide-based, chitosan-based, and collagen-based systems. Among them, supramolecular hydrogels based on self-assembled peptides, due to their fibrous network structure being highly similar to fibrous proteins in the extracellular matrix (ECM), can provide a biomimetic microenvironment for cell adhesion and migration, exhibiting unique advantages in promoting tissue repair. For example, short peptide self-assembled nanomaterials based on GFFY motifs have been shown to promote antigen presentation and lymph node enrichment by activating humoral and cellular immunity. Their successful applications in tumor immunotherapy and vaccine adjuvants have provided new ideas for wound healing dressing design.

[0004] Puerarin, a natural flavonoid extracted from kudzu root, possesses multiple biological activities, including antioxidant, anti-inflammatory, and regulation of glucose and lipid metabolism. Studies have shown that it can reduce oxidative stress damage by inhibiting NADPH oxidase activity, downregulate the expression of pro-inflammatory factors such as TNF-α and IL-6, reduce the accumulation of advanced glycation end products (AGEs), and improve insulin resistance, demonstrating a clear therapeutic effect in the prevention and treatment of diabetes and its complications. However, puerarin lacks an ideal carrier for wound repair: free puerarin is prone to insufficient local drug concentration due to rapid degradation or metabolism, while traditional formulations such as petrolatum ointment have drawbacks such as uncontrollable drug release, dressing adhesion, and water retention, severely limiting its clinical translation in wound healing.

[0005] While existing single-component hydrogel dressings can partially solve the carrier problem, they still have the limitation of single function: simple peptide hydrogels lack antioxidant and glycemic regulation capabilities, making it difficult to cope with the oxidative stress microenvironment of diabetic wounds; and when puerarin is used alone, its low delivery efficiency prevents it from fully leveraging the advantages of multi-target therapy.

[0006] Therefore, constructing a composite hydrogel system that combines self-assembly properties and drug co-loading function to achieve efficient delivery and functional synergy of puerarin has become a key technical challenge in overcoming the bottleneck of diabetic wound healing treatment. Summary of the Invention

[0007] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0008] To achieve these objectives and other advantages according to the present invention, a puerarin-peptide co-assembled nanohydrogel is provided, comprising a self-assembled peptide and puerarin in a mass ratio of 1:0.5~4, wherein the self-assembled peptide has the molecular formula RG. D F D F D Y, D This indicates that the amino acid is in the D configuration, and R represents a nonsteroidal anti-inflammatory drug.

[0009] A method for preparing the puerarin-peptide co-assembled nanohydrogel is provided, comprising the following steps: The self-assembled peptide and puerarin were added to phosphate buffer and mixed. The mixture was heated and the pH of the mixture was adjusted to 7-8 using sodium carbonate solution until it was completely dissolved. After cooling at room temperature, a puerarin-peptide co-assembled nanohydrogel was formed, wherein the mass ratio of the self-assembled peptide to puerarin was 1-4:0.75-7.5.

[0010] Preferably, the self-assembled polypeptide includes a nonsteroidal anti-inflammatory drug, short peptide G, etc. D F D F D Y.

[0011] Preferably, the method for preparing the self-assembled polypeptide includes the following steps: S1. Dissolve N-(9-fluorenylmethoxycarbonyl)-O-tert-butyl-D-tyrosine in dichloromethane, then add diisopropylethylamine and mix to obtain reaction solution I; S2. After fully swelling the 2-Cl-Trt resin with anhydrous dichloromethane, remove the dichloromethane, add reaction solution I and react it in a solid-phase synthesizer on a shaker at room temperature. After the reaction is completed, mixture 1 is obtained. S3. Remove the liquid from mixture 1, wash several times with anhydrous dichloromethane, add the blocking solution, and react on a shaker at room temperature. After the reaction is complete, mixture 2 is obtained, wherein the blocking solution includes dichloromethane:N,N-diisopropylethylamine:methanol in a volume ratio of 17:1:2. S4. Remove the liquid from mixture 2 by washing it several times with anhydrous dichloromethane and N,N-dimethylformamide, respectively. Then add piperidine solution and react at room temperature. After the reaction is complete, remove the liquid and wash it several times with N,N-dimethylformamide. The solvent of piperidine solution is N,N-dimethylformamide. S5. Add O-benzotriazole-tetramethylurea hexafluorophosphate, diisopropylethylamine, and N,N-dimethylformamide to N-(9-fluorenylmethoxycarbonyl)-D-phenylalanine to obtain reaction solution II. Then add reaction solution II to the solid phase washed in step four and react in a solid phase synthesizer until the reaction is completed. S6. Repeat steps S4 and S5, adding N-(9-fluorenylmethoxycarbonyl)-L-phenylalanine and N-(9-fluorenylmethoxycarbonyl)-glycine sequentially. After the last amino acid is added, wash several times with N,N-dimethylformamide, add piperidine solution and react at room temperature. After the reaction is complete, remove the liquid, and then wash several times with N,N-dimethylformamide to obtain the short peptide G. D F D F D Y; S7. Add peptide coupling reagent HBTU, diisopropylethylamine, and N,N-dimethylformamide to a nonsteroidal anti-inflammatory drug to obtain reaction solution III. Then, react reaction solution III with short peptide G. D F D F D Y was reacted in a solid-phase synthesizer. After the reaction was completed, the liquid was removed, and then the product was washed several times with N,N-dimethylformamide and anhydrous dichloromethane to obtain the intermediate product. S8. Add a cutting fluid to the intermediate product of the solid-phase synthesizer to cut RG from the 2-Cl-Trt resin. D F D F D Y was concentrated and dried to obtain a crude product, which was then purified to obtain the self-assembled polypeptide.

[0012] Preferably, the volume fraction of the piperidine solution in steps S4 and S6 is 20-40%, and the solvent of the piperidine solution is N,N-dimethylformamide.

[0013] Preferably, the cutting fluid comprises trifluoroacetic acid:triisopropylsilane:water in a volume ratio of 95:2.5:2.5.

[0014] Preferably, the nonsteroidal anti-inflammatory drug includes naproxen, flurbiprofen, and ketoprofen.

[0015] This invention provides the application of the aforementioned puerarin-peptide co-assembled nanohydrogel in the preparation of a drug for healing diabetic wounds.

[0016] Preferably, the puerarin-peptide co-assembled nanohydrogel promotes wound healing by regulating macrophage polarization, reducing AGEs accumulation at the wound site, and restoring blood circulation.

[0017] The present invention has at least the following beneficial effects: First, the self-assembly of the present invention forms a nano-hydrogel by co-assembling peptides and puerarin, which significantly improves the water solubility and stability of puerarin and solves the problem of low delivery efficiency caused by its poor water solubility and low gastrointestinal stability.

[0018] Secondly, the water content of the puerarin-peptide co-assembled nanohydrogel prepared by this invention decreases over time but still exhibits excellent performance, with a water content as high as 48% at 170 hours. This excellent water retention is crucial for its use as a wound dressing, as it can provide a moist environment for the wound and promote wound healing.

[0019] Secondly, the present invention prepares puerarin-peptide co-assembled nanohydrogels, which regulate macrophage polarization, reduce AGEs accumulation at wound sites, and restore blood regeneration, thus effectively promoting wound healing in diabetic patients. Animal experiments show that the puerarin-peptide co-assembled nanohydrogels are significantly better than the mixture of puerarin-petrolatum and peptide hydrogels in promoting wound healing, and show the best effect on day 14.

[0020] Fourth, this invention breaks through the functional limitations of single-component hydrogels, combining self-assembly properties and drug co-loading capabilities to achieve efficient delivery and functional synergy of puerarin. It solves the problems of simple polypeptide hydrogels lacking antioxidant and glycemic regulation capabilities, and the low delivery efficiency of puerarin when used alone.

[0021] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0022] Figure 1 An optical photograph of the polypeptide self-assembled nanohydrogel according to one of the technical solutions of the present invention; Figure 2 An optical photograph of the puerarin-peptide co-assembled nanohydrogel according to one of the technical solutions of the present invention; Figure 3 An electron micrograph of the polypeptide self-assembled nanohydrogel according to one of the technical solutions of the present invention; Figure 4 This is an electron microscope image of the puerarin-peptide co-assembled nanohydrogel according to one of the technical solutions of the present invention; Figure 5The fluorescence spectrum of a polypeptide self-assembled nanohydrogel, puerarin, and puerarin-polypeptide co-assembled nanohydrogel at an excitation wavelength of 272 nm is shown in one of the technical solutions of the present invention. Figure 6 The water retention properties of a polypeptide self-assembled nanohydrogel and a puerarin-polypeptide co-assembled nanohydrogel, which are one of the technical solutions of the present invention, are shown in the figure. Figure 7 The mechanical properties of a polypeptide self-assembled nanohydrogel, representing one of the technical solutions of this invention, are shown in the diagram. Figure 8 The mechanical properties of the puerarin-peptide co-assembled nanohydrogel, according to one of the technical solutions of the present invention, are shown in the figure. Figure 9 The hemolysis rate diagram shows the hemolysis rate of peptide self-assembled nanohydrogel, puerarin, and puerarin-peptide co-assembled nanohydrogel, which are one of the technical solutions of the present invention. Figure 10 This is a representative wound diagram illustrating the peptide self-assembled nanohydrogel, puerarin, and puerarin-peptide co-assembled nanohydrogel that promotes diabetic wound healing, representing one of the technical solutions of this invention. Figure 11 This is a statistical chart showing the effect of peptide self-assembled nanohydrogel, puerarin, and puerarin-peptide co-assembled nanohydrogel on promoting the wound area of ​​diabetic wounds, according to one of the technical solutions of the present invention. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0024] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified. In the description of this invention, the orientation or positional relationship indicated by the terms is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing this invention and simplifying the description. It does not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0025] <Example 1> The preparation method of self-assembled peptides includes the following steps: S1. 0.5 mmol of N-(9-fluorenylmethoxycarbonyl)-O-tert-butyl-D-tyrosine (Fmoc-D-Tyr(tbu)-OH) with D configuration was dissolved in 10 mL of dichloromethane, and then 1 mmol of diisopropylethylamine was added and mixed to obtain reaction solution I. S2: 0.5 mmol 2-Cl-Trt resin was swollen with 15 mL of anhydrous dichloromethane for 15 min (fully swollen), then the dichloromethane was removed, reaction solution I was added, and the mixture was reacted in a solid-phase synthesizer on a shaker at room temperature for 1 h to obtain mixture 1; S3: Remove the liquid from mixture 1, wash 5 times with 10 mL of anhydrous dichloromethane, add 20 mL of blocking solution, and react on a shaker at room temperature for 20 min to obtain mixture 2, wherein the blocking solution includes dichloromethane:N,N-diisopropylethylamine:methanol in a volume ratio of 17:1:2. S4: Remove the liquid from mixture 2, wash 5 times each with anhydrous dichloromethane and N,N-dimethylformamide, then add 10 mL of 20% piperidine solution, react at room temperature for 35 min, remove the liquid, and wash 5 times with N,N-dimethylformamide, wherein the solvent of the piperidine solution is N,N-dimethylformamide; S5: 1.5 mmol of O-benzotriazole-tetramethylurea hexafluorophosphate, 2 mmol of diisopropylethylamine, and 10 mL of N,N-dimethylformamide were added to 1 mmol of N-(9-fluorenylmethoxycarbonyl)-D-phenylalanine (Fmoc-D-Phe-OH) with the D configuration to obtain reaction solution II. Then, reaction solution II was added to the solid phase washed in step four and reacted in a solid phase synthesizer for 2 h. S6: Repeat steps S4 and S5 above, adding N-(9-fluorenylmethoxycarbonyl)-D-phenylalanine (Fmoc-D-Phe-OH) and N-(9-fluorenylmethoxycarbonyl)-glycine (Fmoc-Gly-OH) in sequence. After the last amino acid is added, wash five times with N,N-dimethylformamide, add 20% piperidine solution, react at room temperature for 35 min, remove the liquid, and then wash five more times with N,N-dimethylformamide to obtain the short peptide G. D F D F D Y; S7: Add 1.5 mmol of peptide coupling reagent HBTU, 2 mmol of diisopropylethylamine, and 10 mL of N,N-dimethylformamide to 1 mmol of nonsteroidal anti-inflammatory drugs (naproxen, flurbiprofen, and ketoprofen, respectively), dissolve to obtain reaction solution III, and then react reaction solution III with short peptide G. D F D F D Y was reacted in a solid-phase synthesizer for 2 h, and then washed 5 times each with N,N-dimethylformamide and anhydrous dichloromethane, respectively. S8: Add 10 mL of cutting fluid (volume ratio of trifluoroacetic acid: triisopropylsilane: water = 95:2.5:2.5) to the solid-phase synthesizer, and add RG.D F D F D Y was cleaved from 2-Cl-Trt resin, concentrated, and dried to obtain a crude product. The crude product was purified by HPLC (high performance liquid chromatography) to obtain the self-assembled polypeptide. When the nonsteroidal anti-inflammatory drug is naproxen, the structural formula of the resulting self-assembled polypeptide is: When the nonsteroidal anti-inflammatory drug is flurbiprofen, the structural formula of the resulting self-assembled polypeptide is: When the nonsteroidal anti-inflammatory drug is ketoprofen, the structural formula of the resulting self-assembled polypeptide is: .

[0026] <Example 2> The preparation method of polypeptide hydrogels includes the following steps: Weigh 1-4 mg of the self-assembled polypeptide powder (naproxen-G). D F D F D Y) Add 0.9 mL of phosphate buffer to a glass vial, heat with an alcohol lamp while adjusting the pH of the solution with 1 M sodium carbonate solution. After the compound is completely dissolved, adjust the final pH to 7-8, with a final volume of 1 mL. The preferred final pH of the solution is 7.4. After standing and cooling to room temperature, a peptide hydrogel is obtained. Figure 1 As shown.

[0027] <Example 3> The preparation method of puerarin-peptide co-assembled nanohydrogels includes the following steps: 1–4 mg of the self-assembled peptide (naproxen-G) D F D F D Y) is mixed with 0.75~7.5 mg of puerarin in 1~2 mL of phosphate buffer, heated, and the pH of the mixture is adjusted to 7~8 using 1 M sodium carbonate solution, preferably with a final pH of 7.4, until completely dissolved. After cooling at room temperature, a puerarin-peptide co-assembled nanohydrogel is formed. Figure 2 As shown.

[0028] <Micromorphology> 10 μL of the polypeptide hydrogel prepared in Example 2 and the puerarin-polypeptide co-assembled nanohydrogel prepared in Example 3 were respectively dropped onto a copper grid and left to stand for one minute. Excess solution was removed using filter paper. The copper grid was then placed in a drying oven and completely dried. The microstructure was observed and recorded using an electron transmission microscope, yielding the following results: Figure 3and Figure 4 The electron microscopy images shown, along with transmission electron microscopy results, reveal that the peptides self-assemble to form nanofibers, which then intertwine to form a three-dimensional network. The puerarin-peptide co-assembled hydrogel exhibits a clear nanofiber network structure, with interwoven fibers. At the edges of the nanofibers, granular material can be observed adhering to the fibers; the particles are regularly shaped and show no obvious free dispersion.

[0029] <Fluorescence Spectroscopy Measurement> The fluorescence emission of peptide hydrogels, puerarin, and puerarin-peptide co-assembled nanohydrogels was detected using a fluorophotometer at an excitation wavelength of 272 nm (the emission wavelength of self-assembled peptides). The obtained fluorescence spectra are shown below. Figure 5 As shown; from Figure 5 As can be seen, the peptide hydrogel exhibits maximum emission at 360 nm, while the puerarin-peptide co-assembled hydrogel exhibits maximum emission at around 460 nm, similar to the maximum emission of puerarin alone. This indicates that intermolecular interactions occur during the co-assembly of peptides and puerarin, ultimately altering the self-assembly state of the peptides.

[0030] <Water retention performance> The polypeptide hydrogel prepared in Example 2 and the puerarin-peptide co-assembled into a nanohydrogel were then allowed to stabilize and weighed using a balance, with the weight recorded as 0 hours. The gel was then left exposed at room temperature, and the weight was recorded at different time points to plot the water retention curve. The results are shown below. Figure 6 As shown, the puerarin-peptide co-assembled nanohydrogel has similar water retention properties to the peptide hydrogel. Although the water content of the hydrogel decreases over time, it is still as high as 48% at 170 hours, showing excellent water retention, which is very important for its use as a wound dressing.

[0031] <Mechanical Properties> The mechanical properties of peptide hydrogels and puerarin-peptide co-assembled nanohydrogels were tested using a rheometer. A 25 mm diameter rheology testing plate was used, with a sample volume of 600 μL. The gap between the plate and the stage was set to 500 μm, and the temperature was set to 25 °C. The results are as follows: Figure 7 and Figure 8 As shown, during dynamic frequency scanning, the strain was set to 0.1%. When the scanning frequency ranged from 0.1 Hz to 10 Hz, the elastic modulus (G') of both hydrogels was greater than their viscous modulus (G''). These results indicate that both hydrogels have excellent mechanical properties.

[0032] <Safety Features> 500 μL of blood was collected from SD rats and centrifuged at 3500 rpm for 5 minutes at 4℃. The supernatant was discarded, and the lower layer of red blood cells was resuspended in 5 mL of physiological saline and centrifuged three times. 250 mg of polypeptide hydrogel, puerarin-polypeptide co-assembled nanohydrogel, and puerarin solution were respectively placed in test tubes with 1 mL of red blood cells and incubated at room temperature for 4 h. A negative control (PBS buffer) and a positive control (Triton X-100) were provided. All samples were centrifuged at 3500 rpm for 5 minutes. The absorbance of the supernatant was measured at 545 nm using a microplate reader. The results are as follows: Figure 9 As shown, the hemolysis rates of puerarin-peptide co-assembled hydrogel, peptide-assembled hydrogel, and puerarin solution were all found to be lower than the internationally recognized hemolysis standard of 5%, indicating that the peptide-puerarin co-assembled hydrogel in this invention has good biocompatibility.

[0033] <Experiment on Diabetic Wound Healing> 1. Construction of a diabetic rat model STZ solution preparation: Weigh STZ (streptozotocin) and dissolve it in 0.1 M citrate buffer (pH 4.5) to prepare a 10 mg / mL solution. Then sterilize it with a 0.22 μm filter membrane and store it in the dark. Modeling procedure: Male SD rats (6-8 weeks old) were fasted for 12 hours, and then injected intraperitoneally with STZ solution 65 mg / kg. After STZ injection, the drinking and eating of all experimental rats were closely observed. When the abdominal blood glucose measured by a blood glucose meter was greater than or equal to 11.1 mmol / L, or the non-fasting blood glucose value was greater than 16.7 mmol / L, it was considered that the rats were successfully induced to have diabetes, i.e., the diabetes model was successful.

[0034] 2. Full-thickness skin defect model: After successfully establishing a diabetic rat model, a skin lesion model was created. The rats, after successful modeling, were anesthetized and placed on an operating table with their backs facing upwards. The fur on their backs was removed to fully expose the skin. Following routine surgical disinfection, a 1cm diameter full-thickness layer of skin was removed from the rat's back.

[0035] 3. Grouping and Treatment Rats with successful modeling and defect formation were randomly divided into two groups. The control group had their skin wounds on the back of the rats covered with sterile gauze and fixed, without any other treatment. The treatment groups had their skin wounds on the back of the rats covered with puerarin-peptide co-assembled hydrogel, peptide hydrogel, and control group puerarin-petrolatum mixture samples, respectively, and covered with sterile gauze and fixed. All animals were housed individually. The wound dressings were changed daily, and the wound area was photographed and recorded. The animals' activity and diet were observed daily, and the bedding was changed in a timely manner. The wounds were photographed with a digital camera, and the wound area was calculated using ImageJ software. The healing rate (%) = (initial area - remaining area) / initial area × 100%.

[0036] 4. Indicator Testing Record the wound contraction and scab shedding time over 14 days, and the results are as follows: Figure 10 and Figure 11 As shown, on days 3 and 7, the peptide hydrogel group, puerarin-peptide co-assembled hydrogel group, and puerarin group all exhibited wound-healing effects compared to the control group. Among them, the puerarin-peptide co-assembled hydrogel showed a significantly better wound-healing effect than the mixture of puerarin and petrolatum. On day 10, both the peptide hydrogel group and the peptide-puerarin co-assembled hydrogel group showed wound-healing effects. The wound healing rate of the control group (puerarin-petrolatum mixture) was similar to that of the control group. On day 14, the peptide hydrogel could promote the healing of diabetic wounds, but the puerarin-peptide co-assembled hydrogel group showed the best wound-healing effect, significantly better than the peptide hydrogel, and has good application prospects.

[0037] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

[0038] like Figure 5 As shown, the English explanations are as follows: Wavelength (nm) represents wavelength, and RFU represents fluorescence intensity; like Figure 6 As shown, the English explanations are as follows: Time (h) represents time (hours), and Water Retention (%) represents water retention rate (%). like Figure 7 and Figure 8 As shown, the English explanation is as follows: Frequency (Hz) indicates frequency (Hertz); like Figure 9As shown, the English explanations are as follows: Hemolysis (%) indicates the hemolysis rate (%), PBS is PBS buffer, and Triton is a positive control.

[0039] like Figure 11 As shown, the English explanation is as follows: Wound Area (%) represents the area of ​​the wound (%).

Claims

1. A puerarin-peptide co-assembled nanohydrogel, characterized in that, It includes self-assembled peptides and puerarin in a mass ratio of 1:0.5~4, wherein the molecular formula of the self-assembled peptide is RG. D F D F D Y, D This indicates that the amino acid is in the D configuration, and R represents a nonsteroidal anti-inflammatory drug.

2. The method for preparing puerarin-peptide co-assembled nanohydrogels as described in claim 1, characterized in that, Includes the following steps: The self-assembled peptide and puerarin were added to phosphate buffer and mixed. The mixture was heated and the pH of the mixture was adjusted to 7-8 using sodium carbonate solution until it was completely dissolved. After cooling at room temperature, a puerarin-peptide co-assembled nanohydrogel was formed, wherein the mass ratio of the self-assembled peptide to puerarin was 1-4:0.75-7.

5.

3. The method for preparing puerarin-peptide co-assembled nanohydrogels as described in claim 2, characterized in that, The self-assembled polypeptide includes nonsteroidal anti-inflammatory drugs and short peptide G. D F D F D Y.

4. The method for preparing puerarin-peptide co-assembled nanohydrogels as described in claim 3, characterized in that, The method for preparing the self-assembled polypeptide includes the following steps: S1. Dissolve N-(9-fluorenylmethoxycarbonyl)-O-tert-butyl-D-tyrosine in dichloromethane, then add diisopropylethylamine and mix to obtain reaction solution I; S2. After fully swelling the 2-Cl-Trt resin with anhydrous dichloromethane, remove the dichloromethane, add reaction solution I and react it in a solid-phase synthesizer on a shaker at room temperature. After the reaction is completed, mixture 1 is obtained. S3. Remove the liquid from mixture 1, wash several times with anhydrous dichloromethane, add the blocking solution, and react on a shaker at room temperature. After the reaction is complete, mixture 2 is obtained, wherein the blocking solution includes dichloromethane:N,N-diisopropylethylamine:methanol in a volume ratio of 17:1:

2. S4. Remove the liquid from mixture 2 by washing it several times with anhydrous dichloromethane and N,N-dimethylformamide, respectively. Then add piperidine solution and react at room temperature. After the reaction is complete, remove the liquid and wash it several times with N,N-dimethylformamide. The solvent of piperidine solution is N,N-dimethylformamide. S5. Add O-benzotriazole-tetramethylurea hexafluorophosphate, diisopropylethylamine, and N,N-dimethylformamide to N-(9-fluorenylmethoxycarbonyl)-D-phenylalanine to obtain reaction solution II. Then add reaction solution II to the solid phase washed in step four and react in a solid phase synthesizer until the reaction is completed. S6. Repeat steps S4 and S5, adding N-(9-fluorenylmethoxycarbonyl)-L-phenylalanine and N-(9-fluorenylmethoxycarbonyl)-glycine sequentially. After the last amino acid is added, wash several times with N,N-dimethylformamide, add piperidine solution and react at room temperature. After the reaction is complete, remove the liquid, and then wash several times with N,N-dimethylformamide to obtain the short peptide G. D F D F D Y; S7. Add peptide coupling reagent HBTU, diisopropylethylamine, and N,N-dimethylformamide to a nonsteroidal anti-inflammatory drug, dissolve to obtain reaction solution III, and then react reaction solution III with short peptide G. D F D F D Y was reacted in a solid-phase synthesizer. After the reaction was completed, the liquid was removed, and then the product was washed several times with N,N-dimethylformamide and anhydrous dichloromethane to obtain the intermediate product. S8. Add a cutting fluid to the intermediate product of the solid-phase synthesizer to cut RG from the 2-Cl-Trt resin. D F D F D Y was concentrated and dried to obtain a crude product, which was then purified to obtain the self-assembled polypeptide.

5. The method for preparing puerarin-peptide co-assembled nanohydrogels as described in claim 4, characterized in that, The volume fraction of the piperidine solution in steps S4 and S6 is 20-40%, and the solvent for the piperidine solution is N,N-dimethylformamide.

6. The method for preparing puerarin-peptide co-assembled nanohydrogels as described in claim 4, characterized in that, The cutting fluid comprises trifluoroacetic acid:triisopropylsilane:water in a volume ratio of 95:2.5:2.

5.

7. The application of the puerarin-peptide co-assembled nanohydrogel as described in claim 1 in the preparation of a drug for healing diabetic wounds.

8. The application as described in claim 7, characterized in that, The puerarin-peptide co-assembled nanohydrogel promotes wound healing by regulating macrophage polarization, reducing AGEs accumulation at the wound site, and restoring blood circulation.

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