Modified recombinant type iii collagen hydrogel loaded with houttuynia cordata thunb vesicles and application thereof

Abstract

The present application relates to the field of biological materials, and in particular to a modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles and applications thereof, the hydrogel being composed of Houttuynia cordata vesicles, modified recombinant human type III collagen and polyvinyl alcohol, and the modified recombinant human type III collagen and polyvinyl alcohol are crosslinked by physical and chemical methods to form a collagen hydrogel, and then the collagen hydrogel is loaded with plant extracellular vesicles prepared from traditional Chinese medicine Houttuynia cordata, and when the hydrogel is applied to a diabetic wound, the hydrogel keeps the wound in a moist environment, and due to the change of the microenvironment such as active oxygen and pH in the skin tissue, the borate ester bond in the collagen hydrogel can be broken, thereby removing active oxygen and releasing the wrapped Houttuynia cordata vesicles into the wound.

Description

Technical Field

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

[0002] Due to the complexity of diabetic wound healing, wound management in diabetic patients presents significant challenges. These wounds impose a significant socioeconomic burden and severely impact patients' quality of life. The scavenging of reactive oxygen species (ROS) is crucial for proper wound healing. However, elevated blood glucose levels in diabetic individuals can lead to excessive local production and accumulation of ROS, resulting in cellular and tissue damage and exacerbated tissue inflammation. Furthermore, hyperglycemia impairs endothelial and smooth muscle cell function, disrupts vascular networks, reduces local neovascularization, and further impedes the wound healing process. These factors collectively 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 outcomes in diabetic wound care.

[0003] Plant-derived extracellular vesicles (PDEVs) have significant potential in mitigating oxidative damage, promoting tissue regeneration, and exerting anti-inflammatory effects. These natural vesicles possess several key advantages, including high stability, inherent bioactivity, and efficient uptake by recipient cells. Due to their antioxidant, anti-inflammatory, and proliferative properties, PDEVs hold promise for diabetic wound therapy. A particularly promising source of PDEVs is Houttuynia cordata thunb (HC), a perennial herb from the family Trichosanthes, renowned 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 crucial 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. 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 administration, 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 solutions:

[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: 200g of Houttuynia cordata slices (purchased from Sinopharm Feng Liaoxing (Foshan) Medicinal Materials Pieces Co., Ltd.) were weighed, added to 500ml of sterile autoclaved PBS, and juiced with a juicer. The resulting juice was centrifuged in a 4°C centrifuge (Baiyang, R18, Beijing, China) at 500×g for 20 minutes, 2000×g for 30 minutes, and 10,000×g for 60 minutes to remove large plant tissue and cell debris 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. The solution was sterilized by 0.22μm filtration and the resulting liquid was stored in aliquots at -80°C until use.

[0010] Modified recombinant human type III collagen was prepared by oxidative polymerization of recombinant human type III collagen with 3-amino-4-methoxybenzoic acid (AMB), followed by crosslinking with 3-aminophenylboronic acid (APBA) in 2-morpholinoethanesulfonic acid (MES) buffer in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) 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 The HC-EVs@BC hydrogel was prepared by adding 10 wt% PVA to a 1:1 volume ratio of the mixture to the PVA solution.

[0012] The present invention's microenvironment-responsive collagen hydrogel loaded with houttuynia cordata vesicles is composed of houttuynia cordata vesicles, modified recombinant human type III collagen, and the polymer PVA. When applied to diabetic wounds, the hydrogel maintains a moist environment. The presence of reactive oxygen species in the environment and the influence of the skin's pH environment promote the cleavage of boronate bonds in the collagen hydrogel, thereby scavenging the reactive oxygen species and releasing the encapsulated houttuynia cordata vesicles into the diabetic wound. This provides a potential and effective treatment for diabetic wounds.

[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 boronate bonds. The boronate 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 the 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 continuously and stably on the wound, reducing the volatility and peak concentration of the drug, avoiding the rapid disappearance of the drug effect, and thus prolonging the duration of the therapeutic effect. 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 enhances the repair and support effect of the hydrogel on cells by reducing inflammation and oxidative stress, 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 B-C. (d) Photos of the transition from solution to hydrogel by mixing PVA solution and B-C solution and the injectability of the hydrogel. (e) The behavior of the hydrogel sample in the diluted state as the shear rate increases. (f) Changes in G' and G" of the hydrogel sample as the strain increases 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 images of HC-EVs. (c) Particle size distribution of HC-EVs. (d) Schematic diagram of the borate ester bond microenvironment response mechanism. (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 performance and biocompatibility of HC-EVs and hydrogels (a) Quantitative analysis of HC-EVs' ABTS radical scavenging activity. (b) Quantitative analysis of HC-EVs' hydroxyl radical scavenging activity. (c) Quantitative comparison of HC-EVs and hydrogels' DPPH radical scavenging activity. (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 Evaluation of the effects of EVs and hydrogels on cell toxicity and cell migration. (a) Illustration of the cell scratch and MTT cytotoxicity assay. (b) Representative MTT experiment images of L929 cell toxicity at 0 and 24 h for different sample groups (scale bar = 200 μm). (c) Representative images of L929 cell scratch healing experiments 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 cytotoxicity experiments for different sample groups. (e) Statistical analysis of L929 wound closure area for different sample groups.

[0022] Figure 6Figure 3. Evaluation of wound healing in vivo using EVs and hydrogels. (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 and three treatment groups at days 0, 3, 7, 10, and 14. (d) Quantitative analysis of wound closure rates across all groups over a 14-day period. (e) Blood glucose measurements in mice at days 0, 7, and 14.

[0023] Figure 7 HC-EVs@B-CH promotes wound healing in vivo. (a) Representative HE-stained images of wound tissue sections in each group on day 14 (scale bars = 200 μm and 50 μm). (b) Quantitative analysis of wound thickness in each group on day 14. (c) Representative Masson trichrome images of wound tissue sections in each group on day 14 (scale bars = 200 μm and 20 μm). (d) Quantification of collagen deposition density in each group on day 14. (e) Sirius red-stained images showing wound tissue in different groups on day 14 (scale bars = 500 μm and 100 μm). (f) Ratio of type III to type I collagen in wound tissue in 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 bars = 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 bars = 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 bars = 50 μm and 20 μm). (f) Quantitative analysis of CD206+ cells.

[0025] Figure 9 Mechanistic analysis of HC-EVs@B-CH in treating 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 upregulated and downregulated genes, respectively. (e) and (f) KEGG pathway enrichment analysis comparing HC-EVs@B-CH and the control group.

[0026] Figure 10 是In vitro analysis of the regulatory effect of HC-EVs@B-CH on the NF-κB / YAP signaling pathway. (a) Western blot analysis of the protein expression levels of p65, p-p65, IκBα, p-IκBα, YAP, and p-YAP in the control, HC-EVs, B-CH, and HC-EVs@B-CH groups. (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] Figure 11 is a representative picture of hydrogel adhesion.

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

[0029] Figure 13 Schematic diagram of a microenvironmentally responsive smart recombinant human type III collagen hydrogel loaded with fishtail vesicles, designed for diabetic wound healing. (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, alleviating oxidative stress, stimulating cell proliferation, reducing inflammation, and modulating the NF-κB / YAP signaling pathway. DETAILED DESCRIPTION

[0030] The technical scheme of the present invention will be further explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the invention. Where specific conditions are not specified in the examples, the procedures are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, 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 against deionized water for 3 days and then lyophilized to obtain AC.

[0033] Next, 2 g of A-C was dissolved in 20 mL of 2-morpholinoethanesulfonic acid buffer (MES) (pH = 5.0). Meanwhile, 0.52 g of 3-aminophenylboronic acid (APBA), 0.32 g of l-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, B-C was obtained by lyophilization.

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

[0035] Before preparing the hydrogel, B-C 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 rapidly mixed at a volume ratio of 1:1 to prepare B-C hydrogel (B-CH), and the mixture was then placed in a refrigerator at -20°C for three freeze-thaw cycles.

[0036] Preparation of Houttuynia cordata vesicles of Example 3

[0037] Houttuynia cordata slices were purchased from Fengxie (Foshan) Herbal Slice Co., Ltd. of China National Pharmaceutical Group. 200 g of Houttuynia cordata slices were weighed and added to 500 ml of high-pressure sterile PBS buffer (1X, pH 7.2-7.4, purchased from Adamas life). The juice was obtained by using a juicer and centrifuged at 500 x g for 20 minutes, 2000 x g for 30 minutes, and 10000 x g for 60 minutes in a centrifuge (Baiyang, R18, Beijing, China) to remove large plant tissues and cell debris to obtain Houttuynia cordata vesicles. Subsequently, the vesicles were collected by ultracentrifugation at 100,000 x g for 70 minutes and resuspended in 50 mL of PBS. The resuspended vesicles were filtered through a 0.22 μm sterile filter to obtain Houttuynia cordata vesicles (HC-EVs), and the resulting liquid was finally stored at -80°C for standby use.

[0038] Encapsulation of Houttuynia cordata vesicles of Example 4

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

[0040] Characterization of HC-EVs of Example 5

[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-coated 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 wire and their morphology was observed using a transmission electron microscope (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 nuclear magnetic resonance ( 1 H NMR) spectroscopy ( Figure 1 (b)).

[0047] Example 8 In vitro drug release

[0048] To investigate the microenvironment responsiveness of hydrogels, protein nanoparticles with a similar size to HC-EVs (100 nm ovalbumin nanoparticles) were used to simulate the release of HC-EVs in different environments. These protein nanoparticles were labeled with RhB to detect their release. 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 soaked 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), and group 6 (pH 7.4 + 4 g / L glucose) Figure 2 (e.f), Figure 3 ).

[0049] Example 9 In vitro anti-hemolytic activity

[0050] 100 μL HC-EVs samples (1 x 10 10 particles mL -1 ), 100 μL 100 mg B-CH extract, and 100 μL 100 mg HC-EVs@B-CH extract (extract preparation method: after the hydrogels were soaked in pH 7.4 PBS for 72 h, the supernatant was taken) were incubated with 2% fresh mouse red blood cells (rbc) for 2 hours. 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 effect of hydrogels on L929 cells was detected by the MTT method; 1 g of hydrogel was soaked in 5 mL of complete cell culture medium (pH 5.0, containing 0.2 mM H2O2) for 72 h Figure 5 (b.d)).

[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, approximately 20 g) were acclimated for 1 week and then fasted for 24 hours. A 10 mg / mL solution of STZ was prepared in 0.1 mol / L sodium citrate buffer (pH 4.0). Hyperglycemia was induced by intraperitoneal injection of 100 mg / kg STZ. Blood glucose levels were measured using an Accu-Chek Active blood glucose meter. Mice with blood glucose levels exceeding 15 mmol / L were selected for study, and their blood glucose levels were monitored throughout the treatment period. Dorsal tissue was harvested from the selected mice, and isoflurane anesthesia was induced. A 1 cm long elliptical full-thickness skin wound was created on the upper back using a disposable biopsy punch to establish a chronic diabetic wound model. The diabetic mice were divided into four groups according to treatment: (I) control group (saline), (II) HC-EVs group, (III) B-CH group, and (IV) HC-EVs@B-CH group. Wound closure was recorded using a digital camera, and wound area was analyzed using ImageJ software (NIH, ImageJ 2.1.1, USA). The animals were sacrificed 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 sliced ​​into 6 μm thick slices. The wound surface was histologically analyzed using hematoxylin-eosin (HE) staining and Masson trichrome staining. After staining, images were captured using a microscope (ECLIPSE Ti2, Nikon, Japan) and analyzed using 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. They were then stained with DAPI for 10 minutes. Finally, images were captured using a microscope (ECLIPSE Ti2, Nikon, Japan) and processed using 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). Libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, USA), and sequencing was performed using the Illumina HiSeq X Ten platform (Shanghai Aoyi Biotechnology Co., Ltd., China). Raw reads were processed using Trimmomatic to remove low-quality reads, and 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 thresholds of P < 0.05 and |log2 fold-change| ≥ 1.0. Functional annotation and pathway analysis were performed using the DAVID (Database for annotation, Visualization, and Integrated Discovery) bioinformatics resource (version 6.8, https: / / david.ncifcrf.gov / summary.jsp) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations. Three animals were included in each experimental group ( Figure 9 ).

[0061] Example 15 Western Blot

[0062] Animal tissues were lysed using RIPA lysis buffer, and protein concentrations were determined using a BCA assay. 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 signals were detected by chemiluminescence (Millipore, Bedford, MA, USA) and quantified using ImageJ software ( Figure 10 ).

[0063] With the above-described preferred embodiments of the present invention as a guide, and with reference to the above description, relevant personnel are fully capable of making various changes and modifications without departing from the technical scope of this invention. The technical scope of this invention is not limited to the contents of the specification and 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 is composed of houttuynia cordata vesicles HC-EVs, modified recombinant human type III collagen BC and polyvinyl alcohol; wherein the houttuynia cordata vesicles are extracted from houttuynia cordata; The modified recombinant human type III collagen BC is 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, i.e., BC.

2. The modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 1, characterized in that: The preparation method of the Houttuynia cordata vesicles comprises the following steps: (1) Weigh Houttuynia cordata slices, add high-pressure sterile PBS, and squeeze the juice with a juicer. The resulting juice is centrifuged at 4°C to remove large plant tissues and cell debris to obtain Houttuynia cordata vesicles HC-EVs; (2) Collect the Houttuynia cordata vesicles, ultracentrifuge them, resuspend them in PBS, filter them through 0.22 μm sterilization filter, and finally store them in aliquots at -80°C for later 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 preparation method of modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles is as follows: BC is dissolved in HC-EVs solution, the mixture is allowed to stand for 30 minutes, and then mixed with PVA, and freeze-thawed to prepare HC-EVs@BC hydrogel.

6. The modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 5, characterized in that: The mass volume ratio of BC to HC-EVs solution was 250 mg:1 mL; the volume ratio of the mixture to PVA solution was 1:

1.

7. The modified recombinant type III collagen hydrogel loaded with Houttuynia cordata vesicles according to claim 6, characterized in that The concentration of HC-EVs solution was 10 10 Particles / mL, the mass concentration of PVA solution is 10%.

8. A use 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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