Modified recombinant III-type collagen hydrogel loaded with herba houttuyniae vesicles and application of modified recombinant III-type collagen hydrogel

Abstract

The invention relates to the field of biological materials, in particular to modified recombinant III-type collagen hydrogel loaded with herba houttuyniae vesicles and application of the modified recombinant III-type collagen hydrogel loaded with the herba houttuyniae vesicles. The hydrogel is prepared from the herba houttuyniae vesicles, modified recombinant human-derived III-type collagen and polyvinyl alcohol, modified recombinant human-derived three-type collagen and polyvinyl alcohol are subjected to physical and chemical double-crosslinking to form collagen hydrogel, the collagen hydrogel is loaded with plant extracellular vesicles prepared from traditional Chinese medicine herba houttuyniae, and when the collagen hydrogel is applied to diabetic wounds, the hydrogel keeps a moist environment for the wounds; due to change of microenvironments such as active oxygen and pH in skin tissues, boric acid ester bonds in the collagen hydrogel can be promoted to be broken, so that the active oxygen is removed, and the wrapped herba houttuyniae vesicles are released into wounds.

Description

Technical Field

[0001] The invention relates to the field of biomaterials, and in particular to a modified recombinant type III collagen hydrogel loaded with houttuynia cordata vesicles and an application thereof. Background Art

[0002] Wound management in patients with diabetes presents significant challenges due to the complexity of diabetic wound healing. These wounds impose a huge socioeconomic burden and severely affect the quality of life of patients. The removal of reactive oxygen species (ROS) is essential for the proper healing of wounds. However, elevated blood glucose levels in diabetic individuals can lead to excessive local production and accumulation of ROS, resulting in cell and tissue damage and increased tissue inflammation. In addition, hyperglycemia can impair the function of endothelial and smooth muscle cells, disrupt the vascular network, reduce local neovascularization, and further impede the wound healing process. These factors together contribute to the poor treatment outcomes observed in diabetic wounds. Therefore, innovative treatments that mitigate oxidative damage, enhance angiogenesis, and inhibit tissue inflammation are urgently needed to improve the outcomes of diabetic wound care.

[0003] Plant-derived extracellular vesicles (PDEVs) have great potential in mitigating oxidative damage, promoting tissue regeneration, and exerting anti-inflammatory effects. These natural vesicles have several key advantages, including high stability, inherent bioactivity, and efficient uptake by recipient cells. PDEVs are promising for diabetic wound therapy due to their antioxidant, anti-inflammatory, and proliferative properties. A particularly promising source of PDEVs is Houttuynia cordata thunb (HC), a perennial herb from the family Trichosanthis that is well-known in traditional Chinese medicine for its wide range of therapeutic effects.

[0004] However, the wound healing process is prolonged and requires continuous tissue growth. The short half-life and rapid clearance of extracellular vesicles (EVs) pose significant challenges, limiting their further application. To overcome these limitations and maximize their therapeutic potential, it is critical to develop a suitable carrier system to effectively protect and deliver PDEVs in the diabetic wound environment.

[0005] Hydrogels are widely used as wound dressings due to their excellent moisture retention, biocompatibility, and customizable physical structure. However, hydrogel drug delivery systems also have certain shortcomings, and many hydrogels lack good controllability in drug release. The release rate of drugs is often affected by environmental changes (such as pH, temperature, etc.) or the structural characteristics of the hydrogel itself, resulting in unstable or too fast or too slow drug release, which is difficult to meet clinical needs. Hydrogels usually have low mechanical strength, which makes them easy to break or unstable in shape in certain practical applications, especially when used in vivo. If hydrogels are used for local drug delivery, higher mechanical strength may be required to maintain structural stability. Summary of the invention

[0006] The purpose of the present invention is to provide a modified recombinant type III collagen hydrogel loaded with houttuynia cordata vesicles and its application. Houttuynia cordata vesicles are combined with modified recombinant human type III collagen to develop a microenvironment-responsive collagen hydrogel made of a physically inert polymer PVA, which can deliver houttuynia cordata vesicles to diabetic wounds.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The hydrogel provided by the present invention is composed of houttuynia cordata vesicles, modified recombinant human type III collagen and polymer PVA;

[0009] The preparation method of Houttuynia cordata vesicles (HC-EVs) is as follows: weigh 200g of Houttuynia cordata slices (purchased from Sinopharm Feng Liaoxing (Foshan) Medicinal Materials Slices Co., Ltd.), add 500ml of high-pressure sterile PBS, squeeze the juice with a juicer, and centrifuge the obtained juice at 500×g for 20 minutes, 2000×g for 30 minutes, and 10000×g for 60 minutes in a 4°C centrifuge (Baiyang, R18, Beijing, China) to remove large plant tissues and cell fragments to obtain Houttuynia cordata vesicles. Subsequently, the Houttuynia cordata vesicles were collected and ultracentrifuged at 100,000×g for 70 minutes and resuspended in PBS. Sterile filtration was performed with 0.22μm, and the obtained liquid was finally stored at -80°C for standby use.

[0010] Modified recombinant human type III collagen was prepared by reacting recombinant human type III collagen with 3-amino-4-methoxybenzoic acid (AMB) by oxidative polymerization, and then reacting with 3-aminophenylboronic acid (APBA) in 2-morpholineethanesulfonic acid (MES) buffer in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) crosslinkers to form BA-rhCOL3 (BC).

[0011] The method for preparing the modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles is as follows: 250 mg BC is dissolved in 1 ml of 10 10 Particles / mL of HC-EVs solution. After the mixture was allowed to stand for 30 minutes, it was mixed with 10wt% PVA to prepare HC-EVs@BC hydrogel. The volume ratio of the mixture to the PVA solution was 1:1.

[0012] The microenvironment-responsive collagen hydrogel loaded with houttuynia cordata vesicles of the present invention is composed of houttuynia cordata vesicles, modified recombinant human type III collagen and polymer PVA. When applied to diabetic wounds, the hydrogel maintains a moist environment for the wounds. Due to the presence of active oxygen in the environment and the influence of the pH environment of the skin, the borate bond in the collagen hydrogel is broken, thereby removing active oxygen and releasing the encapsulated houttuynia cordata vesicles into the diabetic wounds. An effective potential treatment method for diabetic wounds is provided.

[0013] Compared with other treatment methods, the present invention has the following beneficial effects:

[0014] (1) The microenvironment-responsive collagen hydrogel loaded with Houttuynia cordata vesicles of the present invention is formed by borate bonds. The borate bonds are broken when they encounter reactive oxygen species (ROS) in the environment and the influence of the pH environment of the skin, thereby removing the reactive oxygen species and releasing the encapsulated Houttuynia cordata vesicles to the diabetic wound.

[0015] (2) The hydrogel of the present invention is simple to prepare and has low cost, which lays a foundation for large-scale preparation.

[0016] (3) The hydrogel is composed of modified recombinant human type III collagen. The structure of the hydrogel can control the slow release of Houttuynia cordata vesicles, ensuring that its active ingredients (such as anti-inflammatory factors, antioxidant factors, etc.) act on the wound continuously and stably, reducing the volatility and peak concentration of the drug, avoiding the rapid disappearance of the drug effect, and thus prolonging the efficacy time. When the hydrogel slowly releases the active ingredients in Houttuynia cordata, combined with the repair function of type III collagen, an optimized microenvironment can be formed in the local area. Houttuynia cordata reduces inflammation and oxidative stress and enhances the repair and support effect of the hydrogel on cells, making the effects of the two complement and promote each other. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Synthesis of BC and characterization of hydrogels (a) Schematic diagram of BC synthesis process. (b) 1HNMR spectra. (c) FTIR spectra of rhCOL3 and BC. (d) Photographs of the transition from solution to hydrogel by mixing PVA solution and BC solution and representative diagrams of the injectability of the hydrogel. (e) The behavior of the hydrogel sample in a dilute state with increasing shear rate. (f) Changes in G' and G" of the hydrogel sample with increasing strain from 1% to 100%.

[0018] Figure 2 Characterization of hydrogels and HC-EVs. (a) SEM images of B-CH and HC-EVs@B-CH. (b) TEM image of HC-EVs. (c) Particle size distribution of HC-EVs. (d) Schematic diagram of the microenvironmental response mechanism of boronate bonds. (e) In vitro release characteristics of hydrogel samples under different environmental conditions. (f) Quantitative analysis of in vitro release data.

[0019] Figure 3 In vitro release characteristics of hydrogel samples under different glucose environmental conditions and quantitative analysis of in vitro release data.

[0020] Figure 4 Antioxidant properties and biocompatibility of HC-EVs and hydrogels (a) Quantitative analysis of ABTS scavenging activity of HC-EVs. (b) Quantitative analysis of hydroxyl radical scavenging by HC-EVs. (c) Quantitative comparison of DPPH radical scavenging activity of HC-EVs and hydrogels. (d) Representative images showing intracellular ROS scavenging (scale bar = 200 μm). (e) Fluorescence intensity spectrum of DCFH-DA during intracellular ROS scavenging. (f) Quantitative fluorescence intensity of DCFH-DA. (g) Hemolysis rate of blood samples treated with HC-EVs and various hydrogels.

[0021] Figure 5 The effects of EVs and hydrogels on cytotoxicity and cell migration were evaluated. (a) Schematic illustration of cell scratch and MTT cytotoxicity assays. (b) Representative images of MTT assays for cytotoxicity of different sample groups on L929 cells at 0 and 24 h (scale bar = 200 μm). (c) Representative images of scratch healing assays of L929 cells at 0 and 36 h, showing the migration ability of different treatment groups (scale bar = 200 μm). (d) Statistical analysis of L929 cell viability in different sample groups in cytotoxicity assays. (e) Statistical analysis of L929 wound closure area in different sample groups.

[0022] Figure 6Figure 3 Evaluation of wound healing by EVs and hydrogels in vivo. (a) Schematic diagram of the experimental timeline and design. (b) Wound closure area measured at different time points in each group. (c) Representative images of wound healing progression in the control group and three treatment groups at days 0, 3, 7, 10, and 14. (d) Quantitative analysis of wound closure rates over a 14-day period in all groups. (e) Blood glucose measurement results in mice at days 0, 7, and 14.

[0023] Figure 7 HC-EVs@B-CH promotes wound healing in vivo. (a) Representative hematoxylin-eosin staining images of wound tissue sections of each group on day 14 (scale bar = 200 μm and 50 μm). (b) Quantitative analysis of wound thickness of each group on day 14. (c) Representative Masson trichrome images of wound tissue sections of each group on day 14 (scale bar = 200 μm and 20 μm). (d) Quantification of collagen deposition density of each group on day 14. (e) Sirius red staining images showing wound tissues of different groups on day 14 (scale bar = 500 μm and 100 μm). (f) Ratio of type III to type I collagen in wound tissues of each group on day 14.

[0024] Figure 8 HC-EVs@B-CH have anti-inflammatory properties and promote angiogenesis (a) Representative immunofluorescence images of CD31 staining in wound tissues of each group on day 14 (scale bar = 50 μm and 20 μm). (b) Quantitative analysis of CD31+ cells. (c) Representative immunofluorescence images of CD86 staining in wound tissues of each group on day 14 (scale bar = 50 μm and 20 μm). (d) Quantitative analysis of CD86+ cells. (e) Representative immunofluorescence images of CD206 staining in wound tissues of each group on day 14 (scale bar = 50 μm and 20 μm). (f) Quantitative analysis of CD206+ cells.

[0025] Fig. 9 Mechanistic analysis of HC-EVs@B-CH in the treatment of diabetic wounds (a) Volcano plot showing transcriptome analysis of differentially expressed genes (n = 3 biological replicates). (b) Heat map of expression levels of differentially expressed genes. (c) and (d) Gene Ontology (GO) analysis of up-regulated and down-regulated genes, respectively. (e) and (f) KEGG pathway enrichment analysis comparing HC-EVs@B-CH and control groups.

[0026] Fig.10 是In vitro analysis of the regulatory effect of HC-EVs@B-CH on the NF-κB / YAP signaling pathway (a) The protein expression levels of p65, p-p65, IκBα, p-IκBα, YAP, and p-YAP in the control group, HC-EVs group, B-CH group, and HC-EVs@B-CH group were detected by western blot. (b) Relative expression level of p65 protein. (c) Relative expression level of p-p65 protein. (d) Relative expression level of IκBα protein. (e) Relative expression level of p-IκBα protein. (f) Relative expression level of YAP protein. (g) Relative expression level of p-YAP protein.

[0027] Fig.11 is a representative picture of hydrogel adhesion.

[0028] Fig.12 is the potential diagram of Houttuynia cordata vesicles (HC-EVs).

[0029] Fig.13 Schematic diagram of microenvironment-responsive smart recombinant human type III collagen hydrogel loaded with fishtail vesicles, designed for the treatment of diabetic wounds. (a) Schematic diagram of BC and HC-EVs@B-CH. (b) HC-EVs@B-CH accelerates diabetic wound healing through multiple mechanisms, including enhancing angiogenesis, reducing oxidative stress, stimulating cell proliferation, reducing inflammation, and regulating the NF-κB / YAP signaling pathway. DETAILED DESCRIPTION

[0030] The technical scheme of the present invention is further explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0031] Example 1 Preparation of modified recombinant human type III collagen

[0032] 1 g of recombinant human type III collagen and 100 mg of 3-amino-4-methoxybenzoic acid (AMB) were dissolved in 20 mL of deionized water, and 150 mg of ammonium persulfate (APS) was added as an oxidative polymerization catalyst. After 24 hours of reaction, the solution was dialyzed in deionized water for 3 days and then freeze-dried to obtain AC.

[0033] Next, 2 g of AC was dissolved in 20 mL of 2-morpholineethanesulfonic acid buffer (MES) (pH = 5.0). At the same time, 0.52 g of 3-aminophenylboronic acid (APBA), 0.32 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and 0.12 g of N-hydroxysuccinimide (NHS) were fully dissolved in the buffer. The mixture was stirred in an ice bath for 2 hours. Finally, BC was obtained by freeze drying.

[0034] Example 2 Preparation of modified recombinant human type III collagen hydrogel

[0035] Before preparing the hydrogel, BC and PVA were completely dissolved in deionized water to form solutions with concentrations of 25 wt% and 10 wt%, respectively. Then, the two solutions were quickly mixed in a volume ratio of 1:1 to prepare BC hydrogel (B-CH), and the mixture was placed in a -20 °C refrigerator for three freeze-thaw cycles.

[0036] Example 3 Preparation of Houttuynia cordata vesicles

[0037] Houttuynia cordata slices were purchased from Sinopharm Fengliaoxing (Foshan) Medicinal Materials Slices Co., Ltd. Weigh 200g of Houttuynia cordata slices, add 500ml of high-pressure sterile PBS buffer (1X, pH 7.2-7.4, purchased from Adamas life), squeeze the juice with a juicer, and centrifuge the obtained juice at 500×g for 20 minutes, 2000×g for 30 minutes, and 10000×g for 60 minutes in a 4℃ centrifuge (Baiyang, R18, Beijing, China) to remove large plant tissues and cell fragments to obtain Houttuynia cordata vesicles. Subsequently, the vesicles were collected and ultracentrifuged at 100,000×g for 70 minutes and resuspended in 50mL PBS. The vesicles obtained by the above resuspended vesicles were sterilized and filtered with 0.22μm to obtain Houttuynia cordata vesicles (HC-EVs), and finally the obtained liquid was stored at -80℃ for standby use.

[0038] Example 4 Encapsulation of Houttuynia cordata Vesicles

[0039] Dissolve 250 mg BC in 1 mL of 10 10 The particles / mL of Example 3 were resuspended in the filtered HC-EVs solution. After the mixture was allowed to stand for 30 minutes, it was quickly mixed with 10 wt % PVA at a volume ratio of 1:1 to prepare HC-EVs@BC hydrogel (HC-EVs@B-CH), and then the mixture was placed in a -20°C refrigerator for three freeze-thaw cycles.

[0040] Example 5 Characterization of HC-EVs

[0041] The particle size of HC-EVs was measured using a laser particle size analyzer (Zetasizer Nano S90, Malvern Instruments, UK). Figure 2 (c)).

[0042] HC-EVs were fixed with 2.5% glutaraldehyde and 1% tannic acid for 2 h and then air-dried for morphological analysis. The samples were gold-plated by sputter coating and their morphology was observed using a scanning electron microscope (SEM, ZEISS SUPRA 55, Germany). HC-EVs were negatively stained with phosphotungstic acid on copper wires and their morphology was observed by transmission electron microscopy (TEM, JEOL JEM-2100, Japan). Figure 2 (ab)).

[0043] Example 6 FT-IR spectrum test

[0044] The Fourier transform infrared (FT-IR) spectra of rhCOL3, AC and BC were measured using an FT-IR spectrometer (Nicolet iS50, Thermo Scientific, USA). Figure 1 (c)), wavelength range 4000-400cm 1 .

[0045] Example 7 1 H NMR spectroscopy test

[0046] use 1 H NMR spectrometer (AVANCE III 400M, Bruker, Germany) was used to measure the NMR of rhCOL3, AC and BC. 1 H NMR spectroscopy ( Figure 1 (b)).

[0047] Example 8 In vitro drug release

[0048] To investigate the microenvironmental responsiveness of the hydrogels, protein nanoparticles with a particle size similar to that of HC-EVs (ovalbumin nanoparticles with a particle size of 100 nm) were used to simulate the release of HC-EVs in different environments. These protein nanoparticles were labeled with RhB and their release was detected. An in vivo imaging system (ABL-X5, Tanon, China) was used to measure the emission concentration of the protein nanoparticles (excitation wavelength: 546 nm, emission wavelength: 610 nm). The hydrogels containing fluorescently labeled nanoparticles were placed in dialysis bags and immersed in different PBS solutions (10 mL per group): group 1 (pH 7.4), group 2 (pH 5.0), group 3 (pH 7.4 + 0.2 mM H2O2), group 4 (pH 5.0 + 0.2 mM H2O2), group 5 (pH 7.4 + 4 g / L glucose), group 6 (pH 7.4 + 4 g / L glucose) ( Figure 2 (ef), Figure 3 ).

[0049] Example 9 In vitro anti-hemolytic activity

[0050] 100 μL HC-EVs sample (1×10 10 particles mL -1 ), 100 μL of 100 mg B-CH extract and 100 μL of 100 mg HC-EVs@B-CH extract (extract preparation method: soak the hydrogel in pH 7.4 PBS for 72 h and take the supernatant) were incubated with 2% fresh mouse red blood cells (rbc) for 2 h. PBS and Triton X-100 were used as control groups. The released hemoglobin was quantified by measuring the absorbance at 540 nm using a Spark microplate reader (Tecan, USA) ( Figure 4 (g)).

[0051] Example 10 Cytotoxicity

[0052] The MTT method was used to detect the effect of hydrogel on L929 cells; 1 g of hydrogel was immersed in 5 mL of complete cell culture medium (pH 5.0, containing 0.2 mM H2O2) and extracted for 72 h ( Figure 5 (bd)).

[0053] Example 11 Scratch Test

[0054] L929 cells were seeded in 6-well plates and cultured for 24 h. Subsequently, a scratch was created in the cell monolayer using a pipette tip to simulate a wound. The cells were washed twice with PBS and then incubated with extracts of HC-EVs, B-CH, and HC-EVs@B-CH. Images of the scratch area were taken every 0, 12, 24, and 36 h to monitor the healing of the scratch. The wound healing rate ( Figure 5 (ce)).

[0055] Example 12 In vivo wound healing

[0056] Female ICR mice (6 weeks old, about 20 g) were fasted for 24 h after acclimation for 1 week. 10 mg / mL STZ solution was prepared with 0.1 mol / L sodium citrate buffer (pH 4.0). 100 mg / kg STZ was injected intraperitoneally to induce hyperglycemia in mice, and the blood glucose level of mice was measured using an Accu-ChekActive blood glucose meter; mice with blood glucose levels exceeding 15 mmol / L were selected for study, and their blood glucose levels were monitored during the entire treatment period. Dorsal tissues of the selected mice were obtained and anesthetized with isoflurane. A disposable biopsy punch was used to make an elliptical full-thickness skin wound with a length of 1 cm along the long axis on the upper back to establish a chronic diabetic wound model. According to the treatment method, the diabetic mice were divided into 4 groups: (I) control group (administered with normal saline), (II) HC-EVs group, (III) B-CH group, and (IV) HC-EVs@B-CH group. The wound closure was recorded using a digital camera, and the wound area was analyzed using ImageJ software (NIH, ImageJ2.1.1, USA). The animals were killed on the 7th and 14th days after surgery, and wound samples were collected for analysis ( Figure 6 ).

[0057] Example 13 Histological evaluation of skin samples

[0058] The samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 6 μm thick slices. The wound surface was histologically analyzed by hematoxylin-eosin (HE) staining and Masson's trichrome staining. After staining, images were captured with a microscope (ECLIPSE Ti2, Nikon, Japan) and analyzed with ImageJ software. In immunofluorescence staining, the samples were incubated with anti-CD31, anti-CD86, and anti-CD206 primary antibodies at 4°C overnight, followed by incubation with Cy3-conjugated secondary antibodies at 37°C for 1 hour. DAPI staining was then used for 10 minutes. Finally, images were captured with a microscope (ECLIPSE Ti2, Nikon, Japan) and processed with ImageJ software ( Figure 7 , 8).

[0059] Example 14 mRNA Sequencing

[0060] Total RNA was extracted using Trizol reagent, and its quality and concentration were assessed using a spectrophotometer (NanoDrop 2000, Thermo Scientific, USA) and a bioanalyzer (Agilent 2100, Agilent Technologies, Santa Clara, USA). The library was prepared using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, USA), and the sequencing was performed using the Illumina HiSeq X Ten platform (Shanghai Aoyi Biotechnology Co., Ltd., China). The raw reads were processed using Trimmomatic to remove low-quality reads, and the clean reads were aligned to the mouse reference genome (GRCm38) using HISAT2. Gene expression levels were quantified by calculating FPKM values ​​using Cufflinks, and gene read counts were obtained using HTSeq-count. Differential expression analysis was performed using the DESeq R package (2012), with P < 0.05 and |log2 fold-change| ≥ 1.0 as thresholds. The DAVID (Database for annotation, Visualization, and Integrated Discovery) bioinformatics resource (version 6.8, https: / / david.ncifcrf.gov / summary.jsp) was used to perform functional annotation and pathway analysis on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations. Three animals were included in each experimental group ( Fig. 9 ).

[0061] Example 15 Western Blot

[0062] Animal tissues were lysed with RIPA lysis buffer, and protein concentration was determined using a BCA kit. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin for 1 hour at 37°C and then incubated with primary antibodies overnight at 4°C. The primary antibodies used included anti-NF-κB p65 Rabbit pAb (1:300), anti-phospho-NF-κB p65 (S536) Rabbit pAb (1:600), anti-IκBα Rabbit pAb (1:1000), recombinant anti-phospho-IκBα Rabbit mAb (S32) Rabbit pAb (1:1000), anti-yap1 Rabbit pAb (1:1000), and anti-phospho-yap1 (S127) Rabbit pAb (1:500). After washing with TBST, the cells were incubated with HRP-labeled goat anti-rabbit IgG (H+L) (1:10,000). The signal was detected by chemiluminescence (Millipore, Bedford, MA, USA) and quantified using ImageJ software ( Fig.10 ).

[0063] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A modified recombinant type III collagen hydrogel loaded with houttuynia cordata vesicles, characterized in that: The hydrogel consists of houttuynia cordata vesicles, modified recombinant human type III collagen and polyvinyl alcohol; wherein the houttuynia cordata vesicles are extracted from houttuynia cordata.

2. The modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 1, characterized in that: The steps of the preparation method of the Houttuynia cordata vesicle are as follows: (1) Weigh Houttuynia cordata slices, add high-pressure sterile PBS, squeeze the juice with a juicer, and centrifuge the obtained juice in a centrifuge at 4°C to remove large plant tissues and cell debris to obtain Houttuynia cordata vesicles HC-EVs; (2) The Houttuynia cordata vesicles were collected by ultracentrifugation and resuspended in PBS, sterilized by 0.22 μm filtration, and finally stored in separate devices at -80°C for future use.

3. The modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 2, characterized in that: In step (1), the mass volume ratio of Houttuynia cordata slices to sterile PBS is: 2g:5ml; centrifugation refers to: 500×g for 20 minutes, 2000×g for 30 minutes, and 10000×g for 60 minutes.

4. The modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 2, characterized in that: In step (2), ultracentrifugation refers to ultracentrifugation at 100,000×g for 70 minutes.

5. The modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 1, characterized in that: The modified recombinant human type III collagen is prepared by reacting recombinant human type III collagen with 3-amino-4-methoxybenzoic acid (AMB) by oxidative polymerization, and then reacting the recombinant human type III collagen with 3-aminophenylboronic acid (APBA) in a 2-morpholineethanesulfonic acid (MES) buffer under the action of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) crosslinking agents to form BA-rhCOL3 (BC).

6. The modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 1, characterized in that: The preparation method of the modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles is as follows: BC is dissolved in the HC-EVs solution, the mixture is allowed to stand for 30 minutes, and then mixed with PVA, and freeze-thawed to prepare the HC-EVs@BC hydrogel.

7. The modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 6, characterized in that: The mass volume ratio of BC to HC-EVs solution is 250 mg:1 mL; the volume ratio of the mixture to PVA solution is 1:

1.

8. The modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 7, characterized in that: The concentration of HC-EVs solution was 10 10 Particles / mL, the mass concentration of PVA solution is 10%.

9. An application of the modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 1, characterized in that: The modified recombinant type III collagen hydrogel loaded with houttuynia cordata vesicles is used for preparing a medicament for promoting diabetic wound repair.

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