Multi-effect functional exosome aerogel dressing and application thereof

Abstract

The invention discloses a multi-effect functional exosome aerogel dressing and an application thereof. The aerogel dressing is prepared by taking modified plant cellulose as a matrix, loading a ginseng source exosome, compounding with carboxymethyl chitosan, and carrying out directional freezing and freeze-drying processes. Compared with a traditional dressing, the aerogel material disclosed by the invention has good biocompatibility and a light structure; through reasonable design, triple treatment functions of stopping bleeding, resisting bacteria and promoting wound healing can be achieved at the same time. As a biological material with relatively high transformation potential and application prospect, the aerogel disclosed by the invention can be prepared into medical wound dressings, and is particularly suitable for treatment and management of chronic refractory wounds such as diabetic foot, bedsore, ulcer after burn and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of medical dressings, and particularly relates to a multi-functional exosome aerogel dressing and its application in the preparation of wound treatment products, which is particularly suitable for the repair of chronic refractory wounds such as diabetic foot ulcers. Background Art

[0002] With the intensification of global aging, the incidence of chronic wounds (such as diabetic foot ulcers, venous ulcers, pressure sores, etc.) has increased significantly, becoming an important problem seriously threatening public health. Among them, diabetic foot ulcers (DFUs) are one of the most common and destructive complications of diabetic patients, affecting more than 15% of diabetic patients, and having characteristics such as long-term non-healing, high infection rate, high recurrence rate, and high amputation risk, greatly reducing the quality of life of patients and causing a heavy social and medical economic burden. The pathological mechanism of DFUs is complex, involving multiple factors such as hyperglycemia-induced cell dysfunction, chronic inflammatory response, microvascular lesions, tissue hypoxia, and immune imbalance. Although current clinical treatments mainly rely on blood glucose control, infection treatment, surgical debridement, and the use of traditional dressings, the treatment effect is limited, the healing speed is slow and it is easy to relapse, and there is an urgent need to develop new and effective treatment strategies.

[0003] In recent years, extracellular vesicles (EVs) have become an important research hotspot in the field of chronic wound treatment because they naturally carry a variety of bioactive molecules (such as proteins, lipids, nucleic acids, etc.) and have good biocompatibility and the potential to regulate cell behavior. Especially plant-derived extracellular vesicle-like particles (PEVPs), compared with mammalian-derived EVs, have advantages such as wide source, high safety, large-scale production, and low immunogenicity.

[0004] However, plant exosomes still face many challenges when applied to wound treatment, such as the need for ultra-low temperature preservation (usually -80°C), which limits their wide clinical application.

[0005] At the same time, most current chronic wound dressings have a single function and are difficult to simultaneously meet multiple treatment needs such as hemostasis, antibacterial, and promoting healing.

[0006] Therefore, developing a new type of biological dressing that can load and release plant-derived exosomes and simultaneously have multiple functions of hemostasis, antibacterial, and promoting healing has become a key direction that urgently needs to be broken through in the field of diabetic chronic wound treatment. Summary of the Invention

[0007] The present invention aims to solve the current problems in the treatment of chronic wounds (such as diabetic foot). The present invention discloses a multi-functional exosome aerogel dressing and its application, which overcomes the limitations of traditional dressings with single function and slow wound healing, and proposes a new and comprehensive-functional wound dressing with three-in-one functions of hemostasis, antibacterial and wound healing promotion, providing an innovative solution for the treatment of chronic wounds, especially diabetic foot.

[0008] To achieve the above object, the solution of the present invention is: A multi-functional exosome aerogel dressing, which uses carboxylated plant cellulose grafted with cationic polymer as the matrix to load exosomes, and then composites with carboxymethyl chitosan, and is subjected to directional freeze-drying to form an aerogel dressing with a three-dimensional porous structure having hemostasis, antibacterial and wound healing promotion functions; the exosomes are enriched in the cellulose network through electrostatic adsorption and are released during use.

[0009] Furthermore, the released exosomes have a complete structure and a diameter of 100-200 nm; carboxymethyl chitosan not only enhances the adhesion and mechanical strength of the aerogel dressing in a moist wound environment; at the same time, due to its negative charge, it desorbs the negatively charged exosomes from the positively charged cellulose matrix, promotes the release of exosomes; and synergistically provides antibacterial performance.

[0010] Furthermore, the source of the plant cellulose is one or more of ginseng extraction residue, green tea residue, wolfberry residue, grape residue, turmeric residue, citrus peel residue; the cationic polymer is polyethyleneimine PEI with a molecular weight of 10-100 kDa, and the mass ratio of PEI to carboxylated plant cellulose is 1:2-5:1.

[0011] Furthermore, the exosomes are ginseng-derived exosomes; the extraction method of the ginseng exosomes can be selected from any one of the following, including but not limited to: ultracentrifugation method, polyvinyl alcohol precipitation method or commercial exosome extraction kit method.

[0012] Furthermore, the enrichment complex of carboxylated plant cellulose loaded with exosomes and carboxymethyl chitosan are mixed in a mass ratio of 5:1-1:2; the mass concentration of the carboxymethyl chitosan is 1-2%.

[0013] Furthermore, the directional freeze-drying enables the water in the gel to freeze rapidly to form directional ice crystals, and then sublimate and dehydrate under vacuum freeze-drying conditions to form a porous structure arranged along the temperature gradient.

[0014] The application of the aerogel dressing described in any one of the above in the preparation of a product for wound treatment, and the dressing has hemostasis, antibacterial and wound healing promotion functions.

[0015] Further, the wound surface refers to chronic wounds, including but not limited to diabetic foot, bedsore, post-burn ulcer or varicose ulcer.

[0016] Further, the aerogel dressing can achieve rapid repair of chronic wounds by promoting angiogenesis.

[0017] By extracting modified cellulose from ginseng residues, adding ginseng exosomes obtained by any exosome extraction method, mixing with carboxymethyl chitosan solution, and finally undergoing directional freezing and freeze-drying treatment, an aerogel dressing with three-in-one functions of hemostasis, antibacterial and wound healing promotion is obtained. Specifically: using ginseng residues as raw materials, extracting cellulose and grafting polyethyleneimine (PEI) molecules to construct a highly reactive cellulose matrix with a cationic surface to achieve stable loading of negatively charged ginseng exosomes to obtain a complex; then mixing the above complex with carboxymethyl chitosan, and treating it through directional freezing and vacuum drying processes to obtain an integrated multi-effect aerogel dressing with a three-dimensional porous structure, excellent adsorption and release properties.

[0018] The aerogel dressing prepared by the present invention has excellent breathability, liquid absorption capacity, exosome release characteristics, and good biocompatibility. It can be used for the treatment of chronic wounds, promote wound healing, and has good storage stability and broad clinical application prospects. Specifically: Compared with the prior art, the present invention has the following beneficial effects: (1) Green circular economy concept: The present invention uses ginseng residues as the source of plant cellulose, which conforms to the green circular economy concept, makes full use of traditional Chinese medicine by-products, reduces resource waste, and at the same time increases the added value of plant residues, with high environmental protection and sustainability.

[0019] (2) Dressing with three-in-one functions: Compared with traditional single-function dressings, the aerogel dressing of the present invention not only has the basic functions of traditional dressings, but also has triple functions of hemostasis, antibacterial and promoting wound healing, and can improve the wound microenvironment in multiple aspects and accelerate the healing of wounds such as diabetes.

[0020] (3) Improvement of storage conditions and applicability: Traditional exosome dressings usually need to be stored at extremely low temperatures, which limits their application scope and popularity. On the contrary, the aerogel dressing prepared by the present invention improves the storage conditions through directional freezing and freeze-drying processes, has high storage stability, and is easy to store and transport at room temperature.

[0021] (4) Excellent exudate absorption and exosome release performance: The aerogel dressing of the present invention has high porosity and good exudate absorption capacity, and can effectively regulate the wound humidity; Moreover, the aerogel dressing of the present invention promotes wound repair through the continuous release of exosomes. Among them, the introduction of chitosan not only enhances the adhesion and mechanical strength of the aerogel dressing in a moist wound environment; at the same time, due to its carboxylation, it is negatively charged. After the aerogel gets wet, the negatively charged chitosan will cause the negatively charged exosomes to desorb from the positively charged modified cellulose, thereby promoting the release of exosomes; and it also provides good antibacterial performance synergistically.

[0022] (5) Excellent biocompatibility and safety: The aerogel dressing of the present invention has excellent biocompatibility and safety (after continuous administration, the levels of serum ALT, AST, γ-GT, BUN, and CREA are all within the normal range, and HE staining of tissues such as the liver and kidney shows no pathological damage).

[0023] (6) Broad clinical application prospects: The aerogel dressing of the present invention is not only applicable to the treatment of chronic wounds such as diabetic foot ulcers, but also has high transformation potential and application value, and can show good treatment effects in other difficult-to-heal wounds (such as pressure ulcers, burn wounds, etc.). Description of the Drawings

[0024] Figure 1 : Nanoparticle tracking analysis image of ginseng exosomes in Test Example 1; Figure 2 : Transmission electron microscope image of ginseng exosomes in Test Example 1 (bar = 100 nm); Figure 3 : Fourier transform infrared spectra of four kinds of celluloses in Test Example 2; Figure 4 : X-ray diffraction patterns of four kinds of celluloses in Test Example 2; Figure 5 : Atomic force microscope image of GEVPs@PEI-GNFs / CMC in Test Example 2; Figure 6 : Scanning electron microscope image of GEVPs@PEI-GNFs / CMC in Test Example 2; Figure 7 : Cck-8 experimental results of GEVPs@PEI-GNFs / CMC on HUVEC in Test Example 3; Figure 8 : Tube formation experimental results of GEVPs@PEI-GNFs / CMC in Test Example 3; Figure 9 : Scanning electron microscope image of the platelet / red blood cell adhesion experiment of the aerogel dressing in Test Example 4; Figure 10 : In vitro coagulation experiment image and statistical results of the aerogel dressing in Test Example 4; Figure 11:Hemostatic effects and statistical results of the aerogel dressing in Test Example 4 in different bleeding models; Figure 12 :In vitro antibacterial effect images and statistical results of the aerogel dressing in Test Example 5 against E. coli and S. aureus; Figure 13 :Biofilm inhibition effect images and statistical results of the aerogel dressing in Test Example 5 against E. coli and S. aureus; Figure 14 :SEM images of the morphological changes of the biofilms of E. coli and S. aureus after treatment with the aerogel dressing in Test Example 5; Figure 15 :Colony number images of the mouse wounds after 1 day of treatment with the aerogel dressing in Test Example 5; Figure 16 :Wound healing conditions and statistical results of different treatment groups in Test Example 6 in mice; Figure 17 :HE staining and Masson staining results of the wound skin in Test Example 6; Figure 18 :Safety analysis results (serum) of the aerogel dressing in Test Example 7.

[0025] Figure 19 :Safety analysis results (liver, kidney and other tissues) of the aerogel dressing in Test Example 7. Detailed implementation manners

[0026] The present invention will be described below in conjunction with the accompanying drawings and embodiments, but not limited thereto. Variations and improvements made by those skilled in the art under the inspiration of the present invention shall fall within the protection scope of the present invention.

[0027] Preparation Example 1: In this embodiment, ginseng cellulose is used as an example, and the specific process is as follows: 1. Preparation of ginseng-modified cellulose Take ginseng residue powder, add sodium hydroxide solution with a mass concentration of 5% (liquid-solid ratio is 20:1), place it at 80 °C for reaction for 2 hours to obtain crude cellulose. Subsequently, bleach it with sodium hypochlorite solution (NaClO, available chlorine content 10%) to obtain purified ginseng cellulose. Disperse the purified ginseng cellulose into the TEMPO catalytic system, adjust the pH to 10, and react at room temperature for 2 hours to obtain carboxylated cellulose (GNFs). Mechanically shear and disperse the GNFs (10000 rpm / 30 minutes). Subsequently, use a high-pressure homogenizer to circulate and process it 6 times at a pressure of 500 bar to obtain a highly pure and dispersed cellulose solution. Then, add EDC and NHS for cross-linking reaction, graft 25 kDa of polyethyleneimine (PEI), and after reacting for 24 hours, remove the unreacted substances by washing to finally obtain a modified cellulose material with PEI grafted on the surface (PEI-GNFs).

[0028] 2. Extraction of Ginseng Exosome GEVPs by Ultracentrifugation Method Take fresh ginseng (Panax ginseng C.A. Meyer) roots and stems, wash them clean, cut them into small pieces after removing impurities, and homogenize them using a homogenizer to obtain a ginseng tissue suspension. Centrifuge the homogenized suspension at a low speed (3000g) for 10 - 15 minutes at 4 °C to remove cells and large particle impurities. Collect the supernatant. Continue to centrifuge the supernatant at a medium speed (10,000 g) for 30 minutes at 4 °C to remove cell debris and larger particles. Transfer the clear supernatant to an ultracentrifuge tube and perform ultracentrifugation (100,000 g) at 4 °C for 1 hour. After centrifugation, collect the precipitate part (i.e., exosomes), and resuspend the exosome precipitate with an appropriate amount of pure water to obtain GEVPs. Use the BCA protein concentration assay method to measure its protein content. The collected exosomes can be aliquoted into cryotubes and stored at -80 °C for later use.

[0029] 3. Preparation of Gel Mixture PEI-GNFs / CMC: Uniformly mix the above-mentioned modified cellulose PEI-GNFs with a 2 w% carboxymethyl chitosan solution at a mass ratio of 1:1.

[0030] GEVPs@PEI-GNFs / CMC: Mix the above-mentioned modified cellulose PEI-GNFs with ginseng exosomes, and then uniformly mix the mixture with a 2 w% carboxymethyl chitosan solution at a mass ratio of 1:1.

[0031] 4. Preparation of Aerogel Dressing The two gels obtained in Step 2 and mixed evenly are respectively placed in a mold, pre-cooled to 4 °C, and then quickly frozen on a liquid nitrogen-cooled copper brick to form a directional ice crystal structure. Subsequently, vacuum freeze-drying technology is used for sublimation dehydration, and finally an aerogel dressing with a directional pore structure is obtained.

[0032] Test Example 1: Characterize the ginseng exosomes obtained in Preparation Example 1.

[0033] Method: (1) Nanoparticle Tracking Analyzer (NTA): Dilute the sample to an appropriate concentration, and use the NTA instrument to scan to measure the particle size distribution and concentration. By tracking the movement of nanoparticles in the liquid, calculate their particle size distribution. NTA data provides information on the average particle size and concentration of exosomes.

[0034] (2) Transmission Electron Microscope (TEM): Place the ginseng exosome sample on a TEM sample grid and observe it using a transmission electron microscope. Obtain the morphological structure of exosomes through high-resolution electron microscope images. Analyze the shape, size, and distribution of exosomes through TEM images.

[0035] Results: (1) The results of NTA analysis show that the particle size distribution of ginseng exosomes is concentrated at about 100 nm, and the average particle size is 116.8 ± 8.2 nm. Figure 1 The particle size distribution curve of ginseng exosomes is shown, and the data indicates that the particle size of the sample is mainly concentrated in the range of 100 - 200 nm, and the sample has good monodispersity.

[0036] (2) Transmission electron microscope images show that ginseng exosomes present a round or oval structure and have a typical bilayer membrane structure. Figure 2 The electron microscope images shown clearly capture the morphology of multiple exosomes, and their size is consistent with the results of NTA analysis.

[0037] Through the characterization of NTA and TEM, the size and morphology of ginseng exosomes are effectively verified. These results provide reliable physical characterization data for subsequent application research.

[0038] Test Example 2: Characterize the four types of cellulose obtained in Preparation Example 1.

[0039] Method: (1)The four cellulose samples in Preparation Example 1 were characterized by Fourier transform infrared spectroscopy (FTIR) to analyze their chemical structures and compositions. By comparing the FTIR spectra, the interactions between different components (such as GEVPs, PEI, CMC, etc.) in the hydrogel and their effects on the properties of the hydrogel can be confirmed, providing a theoretical basis for further applications.

[0040] (2)The structures of the four cellulose samples in Preparation Example 1 were characterized by X-ray diffraction (XRD) technology to analyze their crystallinity and molecular arrangements. By comparing the spectra, the effects of the introduction of different components on the crystal structure and crystallization characteristics of the hydrogel were studied.

[0041] (3)Atomic force microscopy (AFM) was used to characterize GEVPs@PEI-GNFs / CMC obtained in Preparation Example 1. By scanning the surface of the sample, height and phase images were obtained to observe the roughness, particle size and surface structure characteristics of the sample. The scanning range of AFM measurement was 2 μm × 2 μm, and the scanning resolution was set to be relatively high to obtain a finer image.

[0042] (4)Scanning electron microscopy (SEM) was used to observe the surface morphology of the GEVPs@PEI-GNFs / CMC composite material. The sample was uniformly coated on a conductive substrate by a standard sample preparation method and then observed in the SEM. The voltage was set to 3.0 kV and the magnification was 80,000 times. The position of exosomes was marked by an arrow in the image.

[0043] Results: (1) As Figure 3 shown, the spectrum of GNFs exhibited typical absorption peaks of cellulose. At approximately 3420 cm - ⁻¹, a broad peak of -OH (hydroxyl) stretching vibration appeared, reflecting the presence of hydroxyl groups in cellulose. At approximately 1100 cm - ⁻¹, the absorption peak of C-O showed the characteristics of ether bonds in cellulose molecules, while there was a relatively weak C=O stretching vibration peak near 1600 cm - ⁻¹, indicating that there was no significant chemical change in the cellulose groups in this sample.

[0044] In the spectrum of PEI-GNFs, the -OH stretching vibration peak at 3420 cm - ⁻¹ was similar to that of GNFs, indicating that the characteristics of cellulose were still retained in the sample. The C=O stretching vibration peak at 1600 cm - ⁻¹ was relatively obvious, showing that amide groups appeared in the material after PEI modification. The absorption peak at 1100 cm -The C-O absorption peak at ¹ is also relatively significant, indicating that the grafting of PEI did not significantly affect the ether bond structure of the cellulose backbone. However, due to the introduction of PEI, the intensity of the spectral peak increased, indicating the successful grafting of PEI.

[0045] Next, the spectrum of PEI-GNFs / CMC showed obvious changes. The C=O stretching vibration peak at 1600 cm - ¹ was significantly enhanced, indicating that the chemical interaction between CMC and PEI-GNFs strengthened the presence of amide groups. In addition, the C-O absorption peak near 1100 cm - ¹ remained clear, proving that the ether bond characteristics in the cellulose structure were still retained. However, due to the introduction of CMC, some new absorption characteristics appeared in the spectrum, indicating the successful addition of CMC.

[0046] Finally, the spectrum of GEVPs@PEI-GNFs / CMC showed the most complex absorption characteristics. The C=O stretching vibration peak at 1600 cm -1 was also clearly present and had a high intensity, suggesting the interaction between exosomes and the PEI-GNFs / CMC complex. In particular, unique absorption peaks appeared in the spectrum in the range of about 1000 cm - ¹, which may be related to the phospholipid membrane structure of exosomes, indicating the successful introduction of exosomes into the hydrogel.

[0047] The results of FTIR analysis indicated the successful addition and interaction of each component in the four samples. GNFs exhibited the typical structural characteristics of cellulose, PEI-GNFs showed the characteristics of amide groups introduced after PEI grafting, while the spectrum of the PEI-GNFs / CMC sample further demonstrated the influence of CMC on the structure. The spectrum of GEVPs@PEI-GNFs / CMC confirmed the successful introduction of exosomes. In summary, this experiment successfully demonstrated the addition of each component through FTIR spectroscopy and revealed their chemical interactions and structural changes in the hydrogel.

[0048] (2) Different diffraction peaks of the four samples could be observed from Figure 4 The XRD pattern of GNFs showed obvious diffraction peaks at approximately 22° and 34°, indicating its high crystallinity. With the introduction of PEI, the intensity of the diffraction peaks decreased, showing a decrease in crystallinity, indicating that the crystal structure of cellulose changed to some extent after PEI modification. In the PEI-GNFs / CMC sample, the diffraction peaks continued to weaken, indicating that the addition of CMC further affected the crystallinity of cellulose. Finally, the pattern of GEVPs@PEI-GNFs / CMC showed that the diffraction peaks almost disappeared, indicating that the addition of exosomes significantly affected the crystalline structure of the hydrogel, resulting in a significant decrease in its crystallinity.

[0049] (3) If Figure 5 , the height image of GEVPs@PEI-GNFs / CMC shows the presence of particles of different sizes on the sample surface, with a maximum height difference of 189 nm, indicating that ginseng exosomes are evenly distributed on the sample surface. The change in height indicates that these exosomes maintain a relatively complete structure and there is no obvious structural damage or uneven distribution. The phase difference of the phase image of GEVPs@PEI-GNFs / CMC shows that there are changes in the physical properties of the sample surface, which may be related to the membrane structure of the exosomes. The presence of phase changes indicates that the sample surface may contain multiple components or structures, indicating that the exosomes still retain their original structural characteristics in the composite material.

[0050] (4) If Figure 6 , the surface of the GEVPs@PEI-GNFs / CMC composite material showed multiple clear granular structures, and the particles indicated by the arrows in the figure were exosomes. Exosomes showed typical spherical or quasi-spherical structures with a diameter of about 100-200nm, which is consistent with the typical size range of exosomes. In addition, the surface of the exosomes was smooth and uniform, without obvious aggregation or structural damage. These surface features indicate that the exosomes maintained their intact structure.

[0051] Test Example 3: Cell experiments were performed on the GEVPs@PEI-GNFs / CMC obtained in Preparation Example 1.

[0052] method: (1) CCK-8 experiment Human umbilical vein endothelial cells (HUVEC) (Cybecon Biotech Co., Ltd.) were cultured in a HUVEC cell-specific medium (Cybecon Biotech Co., Ltd.) containing 10% fetal bovine serum (FBS). The cell growth conditions were 37°C and 5% CO2, and cultured to the logarithmic growth phase. Subsequently, the cells were seeded in a 96-well plate. After the cells grew to about 80% confluence, different concentrations of GEVPs@PEI-GNFs / CMC complexes were added, with concentrations of 0, 31.25, 62.5, 125, 250, and 500 µg / mL (cellulose concentration) plus 10 µg / mL of ginseng exosomes, and the treatment was carried out for 48 hours. After 48 hours, the cells in each treatment group were added with CCK-8 reagent and cultured for another hour. The absorbance (OD value) was measured at a wavelength of 450 nm using an enzyme reader. The cell survival rate was calculated by comparing the OD values ​​of each group. Cell survival rate = OD value of the treatment group / OD value of the control group × 100%.

[0053] (2) Tube forming experiment Coat the bottom of a 96-well plate with Matrigel (356230, Corning, USA), and allow it to solidify at 37 °C for 30 minutes to form a basal layer. Add HUVEC cells to the well plate containing the basal layer at a concentration of 2×10 4 cells / mL, add media containing different ratios (complex: media) of GEVPs@PEI-GNFs / CMC complexes (containing 15 mg / mL PEI-GNFs / CMC and 1 mg / mL GEVPs), and culture in an incubator. Regularly observe the cell morphology and the formation of tubular structures in each group using a microscope. The formation of tubular structures is evaluated by the number, length, and connectivity of the tubes. The effect of GEVPs@PEI-GNFs / CMC complexes on the tube formation ability of HUVEC cells is evaluated by analyzing the tubular structures of cells on the Matrigel substrate.

[0054] Results: (1) Detect the changes in cell viability after treatment with different concentrations of aerogel extracts (31.25, 62.5, 125, 250, and 500 μg / mL) using a CCK-8 reagent. The results are as Figure 7 shown. After treatment with the low-concentration groups (31.25, 62.5, 125 μg / mL), the cell viability was close to or slightly higher than that of the control group (Control), with no obvious inhibitory effect (P>0.05), and the cell viability in the 62.5 μg / mL group increased slightly. As the concentration of the extract further increased, the cell viability in the 250 μg / mL and 500 μg / mL groups showed a certain degree of decline, and the cell viability in the 500 μg / mL group decreased significantly compared with the control group (P<0.001), but the cell viability still remained above 80%, indicating that the material as a whole has good cell compatibility and no obvious cytotoxicity even at higher concentrations.

[0055] (2) As Figure 8 , at different concentrations, GEVPs@PEI-GNFs / CMC showed a concentration-dependent effect on the tube formation ability of HUVEC cells. In the control group, fewer tubular structures were formed by HUVEC cells, the cell distribution was relatively loose, and there were no obvious tubular structures. As the concentration of GEVPs@PEI-GNFs / CMC increased (from 1:320 to 1:80), the ability of cell aggregation and tube formation gradually increased. The cells formed more tubular structures, the connections between cells were tight, and the tubular network appeared, showing a better tube formation effect. As the concentration was further increased to 1:40, the tubular structures between cells were slightly reduced compared with the 1:80 concentration, and the tubular network was not as tight as that in the 1:80 concentration group. Finally, in the highest concentration group of 1:20, the tubular structures of the cells were the loosest, showing the worst tube formation effect.

[0056] Test Example 4: The hemostatic effects of the aerogel dressing obtained in Preparation Example 1 were evaluated in vitro and in vivo.

[0057] Methods: (1)Blood cell / platelet adhesion experiment Healthy ICR mice aged 6 - 8 weeks (GemPhar-matech, Nanjing, China) were selected, and whole blood samples were collected. Platelet-rich plasma and red blood cells were obtained by centrifugation respectively, and 100 μL of each was dropped onto the dressing samples in 24-well plates (groups: medical gauze group, PEI-GNFs group, PEI-GNFs / CMC group, and GEVPs@PEI-GNFs / CMC group). After incubation at 37 °C for 30 min, they were washed three times with PBS and then subjected to SEM testing.

[0058] (2)In vitro coagulation experiment Healthy ICR mice aged 6 - 8 weeks (GemPhar-matech, Nanjing, China) were selected, and whole blood samples were collected. The whole blood was mixed with 0.1% CaCl2 at a ratio of 9:1 to prepare purified blood, and 100 μL of the purified blood was dropped onto the dressing samples in 24-well plates (groups: blank control group, medical gauze group, PEI-GNFs group, PEI-GNFs / CMC group, and GEVPs@PEI-GNFs / CMC group). After incubation at 37 °C, 1 mL of deionized water was added at different time points, and then photos were taken and the supernatant was transferred to a 96-well plate. The absorbance was measured at 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the blood coagulation index was calculated.

[0059] (3)In vivo hemostasis experiment Healthy ICR mice aged 6 - 8 weeks (GemPhar-matech, Nanjing, China) were selected to establish liver laceration, tail amputation, and femoral artery bleeding models. According to different dressings, the experiments were divided into five groups: blank control group, gauze group, PEI-GNFs group, PEI-GNFs / CMC group, and GEVPs@PEI-GNFs / CMC group. Under the exposed liver, a clean gauze and a quantitative filter paper were laid. The liver was cut with a scalpel to create a laceration with a depth of 3 mm and a length of 1 cm. After making the laceration, the dressing was immediately covered, and the hemostasis time and blood loss were recorded (the weight difference of the filter paper before and after hemostasis was measured by the weighing method); the tail was cut at the proximal 1 / 3, and massaged from the tail root to the tail tip, and blood flowed out from the cut end. The dressing was immediately covered, and the hemostasis time and blood loss were recorded; the skin at the groin was incised, and the subcutaneous deep and superficial fascia tissues at the groin were bluntly separated up and down. The femoral artery was cut short, and the dressing was immediately covered, and the hemostasis time and blood loss were recorded. The in vivo hemostatic effects were verified through three animal bleeding models.

[0060] Results: (1)Blood cell / platelet adhesion experiment As Figure 9It can be seen that in the platelet and red blood cell adhesion experiments, scanning electron microscope (SEM) images showed that different materials had different adhesion to red blood cells (RBCs) and platelets. The control group (gauze) showed only a small amount of adhesion of red blood cells and platelets, indicating a weak adsorption ability for blood cells. The PEI-GNFs group showed obvious adhesion of red blood cells and platelets, especially platelets, indicating a strong adsorption ability. In the PEI-GNFs / CMC group with the further addition of CMC, the adhesion of platelets and red blood cells increased significantly, indicating that CMC enhanced the adsorption of platelets. In the GEVPs@PEI-GNFs / CMC group, the adhesion effect of red blood cells and platelets was the most significant, especially platelets, indicating that this composite material had the strongest blood cell adsorption ability. Generally speaking, the GEVPs@PEI-GNFs / CMC aerogel dressing showed significant blood cell adhesion, which helped to promote the coagulation process, had strong blood compatibility and potential hemostatic effects.

[0061] (2) In vitro coagulation experiment As Figure 10 shown, the GEVPs@PEI-GNFs / CMC group showed the best coagulation effect at all time points (1, 2, 3, 4 minutes), and the liquid state of the blood quickly changed into a clot, showing a significant hemostatic effect. The PEI-GNFs / CMC group and the PEI-GNFs group also showed good coagulation effects, but were inferior to the GEVPs@PEI-GNFs / CMC group. In contrast, the gauze group and the control group failed to effectively promote blood coagulation for a long time, and the coagulation efficiency was low, indicating a weak hemostatic effect. Through the statistical chart, the coagulation index (BCI%) of the GEVPs@PEI-GNFs / CMC group was significantly higher than that of other groups, further verifying the superior performance of this composite material in promoting coagulation. In summary, the GEVPs@PEI-GNFs / CMC composite material had a significant in vitro coagulation effect, could effectively promote blood coagulation, and had strong hemostatic potential.

[0062] (2) In vivo hemostasis experiment As Figure 11, the gauze treatment group showed certain hemostatic effects in all three models, but the hemostasis time was relatively long (about 130 s in all cases), and the local bleeding area was large. In contrast, PEI-GNFs and PEI-GNFs / CMC significantly shortened the hemostasis time, demonstrating the good auxiliary effect of the nanofiber structure on blood adsorption and aggregation. Notably, GEVPs@PEI-GNFs / CMC achieved rapid hemostasis within 60 s in all three models. Its hemostasis time was significantly better than that of all other control groups. Especially in the arterial hypertension bleeding model, it could still quickly form a clot and achieve stable hemostasis, showing extremely excellent emergency hemostatic ability. A dense clot rapidly formed on the wound surface, and there was basically no visible blood overflow, indicating that it could rapidly exert multiple synergistic effects of adsorption, compression, and coagulation promotion on the wound surface.

[0063] Test Example 5: The antibacterial effects of the aerogel dressing obtained in Preparation Example 1 were evaluated in vitro and in vivo.

[0064] Method: (1) In vitro antibacterial experiment Plate counting: The bacterial strains used for antibacterial performance testing were Staphylococcus aureus and Escherichia coli (both purchased from Ningbo Mingzhou Biotechnology Co., Ltd.). PEI-GNFs, PEI-GNFs / CMC, and GEVPs@PEI-GNFs / CMC were respectively mixed with the bacterial suspensions of Escherichia coli and Staphylococcus aureus at 104 CFU / mL, and cultured in a constant temperature incubator for 12 h. Then, 100 μL of the co-culture solution was spread on a solid nutrient agar medium, and the bacterial solution was evenly spread on the medium. The petri dish was covered and placed in the incubator for inverted culture at 37 °C for 24 h. Bacterial suspensions of Staphylococcus aureus and Escherichia coli at 104 CFU / mL were inoculated with the same method as a control group. Finally, the cultured bacterial solution was diluted (1:10000) and transferred to an LB plate, and cultured at 37 °C for 24 h. The colonies on the plate surface were photographed and counted to calculate the antibacterial rate of each group.

[0065] Crystal violet staining: To observe the anti-biofilm ability, crystal violet staining was used to semi-quantitatively analyze the biofilm disruption ability of the hydrogel. Escherichia coli and Staphylococcus aureus suspensions with a concentration of 106 CFU / mL (OD = 0.1) were added to a 24-well plate, 1 mL per well, and cultured at 37 °C for 24 h to form biofilms. After biofilm formation, the culture medium was removed, and the sample (diluted with PBS) was added and cultured for another 24 h. The biofilms were rinsed 3 times with PBS to remove dead planktonic bacteria. Next, the bacteria were fixed with 99% methanol for 20 min, then stained with 0.1% crystal violet at room temperature for 20 min, and photographed. Finally, the excess crystal violet was rinsed off with PBS, decolorized with dimethyl sulfoxide, and the absorbance value at 590 nm was measured with a microplate reader. The biofilm inhibition rate was calculated by the following formula: Biofilm inhibition rate = (1 - ODsample / ODblank) × 100%, where ODsample is the absorbance value of the sample and ODblank is the absorbance value without any treatment.

[0066] SEM morphological observation: To further evaluate the antibacterial performance, the SEM method was used to observe the morphological changes of Escherichia coli and Staphylococcus aureus after treatment. Specifically, Escherichia coli and Staphylococcus aureus were cultured in LB medium at 37 °C for 12 - 24 h to achieve bacterial proliferation. The cultured bacterial solution was centrifuged (3000 g, 10 min), the supernatant was discarded, and the bacteria were washed 2 times with PBS buffer (pH 7.4) to remove the culture medium components and sample residues. At room temperature, the bacteria were fixed with an electron microscope fixative for 1 - 2 h. After fixation, the bacterial suspension was transferred to a new tube and washed 2 more times with PBS. Finally, the morphological structures of Escherichia coli and Staphylococcus aureus were observed by SEM.

[0067] (2) In vivo antibacterial experiment Healthy 6 - 8-week-old Balb / c mice (GemPhar-matech, Nanjing, China) were selected to establish a subcutaneous Staphylococcus aureus infection model. The experiment was divided into four groups: blank control group (treated with gauze), PEI-GNFs group, PEI-GNFs / CMC group, and GEVPs@PEI-GNFs / CMC group. After the mice were anesthetized, the skin with a diameter of 1 cm was cut off on the back, and 20 μL of Staphylococcus aureus suspension (concentration 10^8 CFU / mL) was dropped subcutaneously. After 24 hours post-operation, the aerogel material was applied locally. The next day, a cotton swab soaked with PBS was used to smear the infected wound for sampling. After gradient dilution, it was spread on an agar plate to calculate the number of colony-forming units per gram of tissue (CFU / g) to evaluate its antibacterial effect.

[0068] Results: (1) In vitro antibacterial experiment As Figure 12, compared with the blank control group (Control), each treatment group showed antibacterial effects to varying degrees. For Escherichia coli (E. coli), the antibacterial rate of the PEI-GNFs group was approximately 30%. After introducing carboxymethyl chitosan, the antibacterial rate increased significantly to above approximately 90%, and it remained at a high level after loading GEVPs. For Staphylococcus aureus (S. aureus), a similar trend was also observed. The antibacterial rate of the PEI-GNFs group was approximately 65%, while the antibacterial rates of the PEI-GNFs@CMC group and the GEVPs@PEI-GNFs@CMC group were both close to or exceeded 95%, showing excellent broad-spectrum antibacterial performance.

[0069] To further explore its intervention effect on bacterial adhesion and biofilm formation, crystal violet staining was used for semi-quantitative analysis of the biofilm on the material surface. As Figure 13 shown, compared with the control group, the crystal violet absorbance of E. coli and S. aureus after treatment with GEVPs@PEI-GNFs / CMC decreased significantly, indicating its good inhibitory effect on bacterial adhesion and biofilm formation.

[0070] As Figure 14 , in the SEM images, the effects of different treated materials on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were analyzed. In the control group (Model), the bacteria maintained their typical rod-shaped (E. coli) or spherical (S. aureus) morphology, and no obvious structural changes were observed. In the PEI-GNFs group, the cell membranes of E. coli and S. aureus showed certain ruptures and morphological changes, and dissolution was observed on the surface of some cells. The bacterial membranes in the PEI-GNFs / CMC group were more damaged, and obvious dissolution and deformation on the bacterial surface were more prominent. The GEVPs@PEI-GNFs / CMC group showed the strongest bacterial destructive effect, especially for E. coli, whose cell membrane was almost completely dissolved, and the morphology of the bacteria was severely distorted or disintegrated, indicating that the composite material had a significant destructive effect on the bacterial membrane.

[0071] (2) In vivo antibacterial experiment As Figure 15 shown, from the culture results, the GEVPs@PEI-GNFs / CMC group showed the strongest antibacterial effect, with almost no bacterial growth, and there was almost no bacterial growth area on the culture medium. In contrast, the PEI-GNFs / CMC group and the PEI-GNFs group also showed certain antibacterial effects, but were not as good as the GEVPs@PEI-GNFs / CMC group, and there was still a certain amount of bacterial growth. The antibacterial effects of the control group (gauze) and the alginate dressing group were poor, and obvious bacterial growth was observed on the culture medium.

[0072] This indicates that the GEVPs@PEI-GNFs / CMC composite has significant antibacterial effects and can effectively inhibit the growth of S. aureus, further demonstrating the potential of this composite in wound treatment.

[0073] Test Example 6: The promoting healing effect of the aerogel dressing obtained in Preparation Example 1 on chronic infectious wounds was evaluated.

[0074] Method: Healthy Balb / c mice aged 6 - 8 weeks (GemPhar-matech, Nanjing, China) were selected. The experimental mice were fed in an environment of about 25 °C, with natural light every day, free access to water and food, and adaptively fed for three days. To establish a diabetic mouse model, the mice were weighed and marked according to the random number table method. Streptozotocin was dissolved in sodium citrate buffer and used immediately after preparation, paying attention to avoiding light. The mice were intraperitoneally injected with streptozotocin at a dose of 50 mg / Kg on an empty stomach for 5 consecutive days. The mice showed polyphagia, polydipsia, polyuria, and accompanied by weight loss. And the random blood glucose measured by tail cutting was greater than 16.7 mmol / L after the 3rd day, which were defined as diabetic mice.

[0075] To establish an infectious chronic wound model (wound diameter 1 cm, deep to the fascia layer), 60 diabetic mice were randomly divided into five groups: blank control group (treated with gauze), positive control group (treated with alginate dressing), PEI-GNFs group, PEI-GNFs / CMC group, and GEVPs@PEI-GNFs / CMC group, with 12 mice in each group. The hair on the back was completely removed, and a 1 cm full-thickness skin excision wound was cut out. The wound was fixed with a rubber ring, and 20 μL of Staphylococcus aureus bacterial solution was added dropwise. After one day, the dressings of each group were covered on the wounds. After the operation, the animals were housed separately in cages, paying attention to keeping the wound area clean, changing the dressings every other day, taking pictures to observe the wound healing situation and recording the body weight. On the 12th day, the animals were sacrificed, and the wound tissues were taken for histological analysis. The epidermal regeneration and granulation tissue thickness were observed by HE staining, and the arrangement and deposition density of collagen fibers were evaluated by Masson staining. The wound area of each group was calculated using Image J software, and the wound closure percentage of each group was calculated as (A0-An / A0)×100% (A0: the wound area on the 0th day; An: the wound area on the nth day), to systematically evaluate the mechanism of the aerogel dressing accelerating wound repair.

[0076] Results: As Figure 16 , in terms of wound closure, the wound closure rate of the GEVPs@PEI-GNFs / CMC group on the 3rd day was comparable to that of the commercially available alginate dressing group, but significantly better than that of the PEI-GNFs / CMC group, PEI-GNFs group, and gauze treatment group; From the 3rd day, the GEVPs@PEI-GNFs / CMC group began to show a trend of being superior to the commercially available alginate dressing group, and was significantly superior to the PEI-GNFs / CMC group, the PEI-GNFs group, and the gauze treatment group; By the 12th day, the wound closure rate of the GEVPs@PEI-GNFs / CMC group was significantly higher than that of the commercially available alginate dressing group, and the cortical continuity at the wound edge was restored more completely, being significantly superior to the PEI-GNFs / CMC group, the PEI-GNFs group, and the gauze treatment group.

[0077] Such as Figure 17 , HE staining showed that the granulation tissue of the wounds of mice treated with GEVPs@PEI-GNFs / CMC thickened significantly, and the collagen fibers were arranged orderly, indicating that it could better promote the ability of wound extracellular matrix remodeling and enhance the healing effect of diabetic wounds. In addition, Masson staining showed that the infiltration of inflammatory cells in the aerogel group was significantly reduced, indicating its obvious multi-dimensional curative effect in promoting healing.

[0078] Test Example 7: Verification of the in vivo safety of the aerogel dressing obtained in Preparation Example 1.

[0079] Method: Healthy Balb / c mice aged 6 - 8 weeks (GemPhar-matech, Nanjing, China) were selected and continuously administered for 7 days. Serum ALT, AST, γ-GT, BUN, and CREA were detected, and HE staining pathological analysis was performed on the main organs.

[0080] Result: Such as Figure 18 And Figure 19 , after continuous administration for 7 days, the levels of serum ALT, AST, γ-GT, BUN, and CREA were all within the normal range, and no pathological damage was seen in the HE staining of tissues such as the liver and kidney.

Claims

1. A multi-functional exosome aerogel dressing, characterized in that: The dressing: uses carboxylated plant cellulose grafted with cationic polymer as a matrix to load exosomes, which is then compounded with carboxymethyl chitosan, and subjected to directional freeze-drying to form an aerogel dressing with a three-dimensional porous structure having hemostatic, antibacterial and healing-promoting functions; The exosomes are accumulated in the cellulose network by electrostatic adsorption and released during use.

2. The aerogel dressing according to claim 1, characterized in that The released exosomes are structurally intact and have a diameter of 100-200 nm; Carboxymethyl chitosan not only enhances the adhesion and mechanical strength of aerogel dressings in moist wound environments, but also desorbs negatively charged exosomes from the positively charged cellulose matrix due to its negative charge, promoting the release of exosomes, and synergistically provides antibacterial properties.

3. The aerogel dressing according to claim 1 or 2, characterized in that: The source of the plant cellulose is one or more of ginseng extraction residue, green tea residue, wolfberry residue, grape residue, turmeric residue, and citrus peel residue; the cationic polymer is polyethyleneimine PEI with a molecular weight of 10-100 kDa, and the mass ratio of PEI to carboxylated plant cellulose is 1:2-5:

1.

4. The aerogel dressing according to claim 1 or 2, characterized in that: The exosomes are ginseng-derived exosomes; the extraction method of the ginseng exosomes can be selected from any one of the following, including but not limited to: ultracentrifugation, polyvinyl alcohol precipitation or a commercial exosome extraction kit.

5. The aerogel dressing according to claim 1 or 2, characterized in that: The enriched complex after the exosomes are loaded on the carboxylated plant cellulose as a matrix is ​​mixed with carboxymethyl chitosan at a mass ratio of 5:1-1:2; the mass concentration of the carboxymethyl chitosan is 1-2%.

6. The aerogel dressing according to claim 1 or 2, characterized in that: The directional freeze drying allows the water in the gel to freeze rapidly to form directional ice crystals, which then sublimate and dehydrate under vacuum freeze drying conditions to form a porous structure arranged along a temperature gradient.

7. Use of the aerogel dressing according to any one of claims 1 to 6 in preparing a product for wound treatment, characterized in that: The dressing is hemostatic, antibacterial and promotes wound healing.

8. The use according to claim 7, characterized in that The wound surface refers to a chronic wound, including but not limited to diabetic foot, bedsore, post-burn ulcer or varicose ulcer.

9. The use according to claim 7 or 8, characterized in that The aerogel dressing can achieve rapid repair of chronic wounds by promoting angiogenesis.

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