Ginseng-derived vesicle-like nanoparticle suspension as well as preparation method and application thereof

Abstract

The invention relates to a ginseng-derived vesicle-like nanoparticle suspension as well as a preparation method and application thereof, and belongs to the technical field of hydrogel. The preparation method comprises the following steps: firstly, performing cell disruption on clean fresh ginseng, performing primary centrifugation and secondary centrifugation on the disrupted mixture, dropwise adding iodixanol into secondary supernate, and discarding supernate after tertiary centrifugation to obtain crude GDVLNs suspension; the method comprises the following steps: adding GDVLNs into a plurality of layers of iodixanol-containing buffer solutions with different concentrations, carrying out primary centrifugation, adding a yellow suspension in the iodixanol-containing buffer solution with the concentration of 20v / w% into the buffer solution, dropwise adding iodixanol, carrying out secondary centrifugation, and discarding the supernatant to obtain the purified GDVLNs suspension. In the preparation method, the centrifugate has a more obvious GDVLNs layer, the obtained GDVLNs maintain a better form, and the prepared GDVLNs suspension shows a better anti-inflammatory effect and can promote skin wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogels, and specifically relates to a ginseng-derived vesicle-like nanoparticle suspension, a preparation method and application thereof, and in particular relates to the application of the ginseng-derived vesicle-like nanoparticle suspension in the preparation of ginseng exosome hydrogels. Background Art

[0002] Due to the safety, affordability, and good biocompatibility of plant-derived extracellular vesicles, related research has continued to advance in recent years. Vesicle-like nanoparticles have been isolated from many plants, including ginseng, ginger, broccoli, bitter melon, grapes, grapefruit, lemons, and oranges. Among them, multiple studies have shown that ginseng-derived vesicle-like nanoparticles (GDVLNs) have excellent therapeutic effects in various types of diseases.

[0003] Existing methods for obtaining plant-derived vesicle-like nanoparticles primarily include tissue disruption and tissue infiltration centrifugation. The former involves disrupting plant tissue in a blender and subjecting the resulting juice to ultrahigh-speed centrifugation to remove cell debris to extract the nanoparticles. The latter involves using vacuum or pressure to infiltrate vesicles from the isolation buffer (VIB) into the exoplasmic space of plant cells, where they are then centrifuged to obtain the exosomes.

[0004] For GDVLNs, density gradient purification is usually used. The process is as follows: first collect plant tissue, wash it, grind it or homogenize it to make a cell suspension, remove large cell fragments and impurities by low-speed centrifugation, and further precipitate smaller vesicle structures such as exosomes at a higher speed to obtain a crude exosome extract; then use sucrose and heavy water to prepare a density gradient solution to form a continuous or discontinuous density gradient from low to high; then add the crude exosome extract to the top of the density gradient solution and use an ultracentrifuge for centrifugation. Under the action of centrifugal force, the exosomes will move in the gradient solution according to their own density. Exosomes migrate in the solution until they reach a position with the same density as the exosome, thereby separating from impurities with different densities. After centrifugation, different bands or layers can be observed in the density gradient solution. Exosomes are usually located in a specific density area. For example, during sucrose density gradient centrifugation, exosomes are often concentrated in the area with a density of 1.13g / ml-1.21g / ml. Finally, use a syringe or pipette to carefully draw out the solution in this area, collect the components containing exosomes, dilute with an appropriate amount of buffer, and then precipitate the exosomes by high-speed centrifugation to remove the residual gradient medium and impurities to obtain purified exosomes.

[0005] However, density gradient purification usually uses heavy aqueous sucrose solution as the isolation buffer, which has the following disadvantages:

[0006] The osmotic pressure difference between the heavy aqueous sucrose solution and GDVLNs is large, which affects the stability of exosomes and causes structural changes or functional loss;

[0007] Heavy water may be toxic to cells or biological samples in some experiments, affecting the experimental results;

[0008] The cost of heavy water is relatively high and may increase the overall expense of the experiment.

[0009] In addition, when the density gradient purification method is used to enrich vesicle nanoparticles by ultrahigh-speed centrifugation, the vesicle nanoparticles are easily impacted by centrifugal force, causing changes in the morphology and structure of the vesicles, which may affect subsequent experimental research. After discarding the supernatant, the vesicle particles firmly attached to the bottom of the tube are not conducive to collection. If the vesicles are repeatedly blown with liquid during the collection process, it will not only affect the integrity of the vesicles, but also easily lead to agglomeration and reduced yield. Summary of the Invention

[0010] The present invention aims to solve the technical problems in the prior art, such as the method for obtaining GDVLNs affects the structure, morphology and function of GDVLNs, poor stability, high cost, and difficulty in collecting the product. A ginseng-derived vesicle-like nanoparticle suspension, a preparation method and application thereof are provided.

[0011] In a first aspect, the present invention provides a method for preparing ginseng-derived vesicle-like nanoparticles, comprising the following steps:

[0012] Step 1: Separation of GDVLNs suspension

[0013] First, clean fresh ginseng cells are disrupted in a buffer containing protein inhibitors. The disrupted mixture is centrifuged once to obtain a primary supernatant, which is then centrifuged a second time to obtain a secondary supernatant. Finally, iodixanol is added dropwise to the secondary supernatant. After three centrifugations, the supernatant is discarded to obtain a crude ginseng-derived vesicle-like nanoparticle suspension.

[0014] Step 2: Purification of GDVLNs suspension

[0015] First, a crude ginseng-derived vesicle-like nanoparticle suspension was added to multiple layers of buffer containing iodixanol at different concentrations. After one centrifugation, the yellow suspension in the buffer containing iodixanol at a concentration of 20 v / w% was collected. The yellow suspension was then added to a dilution buffer, iodixanol was added dropwise, and after a second centrifugation, the supernatant was discarded to obtain a purified ginseng-derived vesicle-like nanoparticle suspension.

[0016] The concentration range of the different concentrations is 5v / m%-40v / m%, and the multiple layers of buffer solutions containing iodixanol with different concentrations are distributed from top to bottom in order from low to high concentration.

[0017] Preferably, in step 1, the protein inhibitor is a cocktail (commercial name, a mixed protease inhibitor containing AEBSF, Aprotinin, Bestatin, E-64, Leupeptin and Pepstatin A).

[0018] Preferably, in step 1, the buffer is a phosphate buffer.

[0019] Preferably, in step 1, the ratio of the clean fresh ginseng, protease inhibitor, and buffer is 100-150 g: 2-3 mL: 200-300 mL.

[0020] Preferably, in step 1, the volume ratio of the buffer solution containing protein inhibitors to iodixanol is 200-300:1.

[0021] Preferably, in step 1, the temperature of the centrifugation is 4° C., the speed is 800-1500×g, and the time is 15-20 min.

[0022] Preferably, in step 1, the temperature of the secondary centrifugation is 4° C., the speed is 10,000-20,000×g, and the time is 1-1.5 h.

[0023] Preferably, in step 1, the temperature of the three centrifugations is 4° C., the speed is 100,000-200,000×g, and the time is 1-1.5 h.

[0024] Preferably, in step 2, the ratio of the volume of the crude ginseng-derived vesicle-like nanoparticle suspension to the total volume of multiple layers of buffer solutions containing iodixanol at different concentrations is 1:10-30.

[0025] Preferably, in step 2, the multiple layers of buffer solutions containing iodixanol of different concentrations are, from top to bottom, buffer solutions containing iodixanol of concentrations of 5v / m%, 10v / m%, 20v / m%, and 40v / m%, and the volume of each layer of buffer solution containing iodixanol is equal.

[0026] Preferably, in step 2, the buffer solution in the buffer solution containing iodixanol is a phosphate buffer solution.

[0027] Preferably, in step 2, the volume ratio of the yellow suspension, dilution buffer and iodixanol is 1:10-30:1.

[0028] Preferably, in step 2, the dilution buffer is phosphate buffer.

[0029] Preferably, in step 2, the temperature of the first centrifugation is 4° C., the speed is 100,000-200,000×g, and the time is 1.5-2 h.

[0030] Preferably, in step 2, the temperature of the secondary centrifugation is 4° C., the speed is 100,000-200,000×g, and the time is 1-1.5 h.

[0031] In a second aspect, the present invention also provides a ginseng-derived vesicle-like nanoparticle suspension prepared by the above preparation method.

[0032] In a third aspect, the present invention further provides use of the ginseng-derived vesicle-like nanoparticle suspension in the preparation of a drug for promoting skin wound healing.

[0033] In a fourth aspect, the present invention further provides a ginseng exosome hydrogel comprising 10-15 mL: 3-4.5 g of GDVLNs suspension and PF-127 (Poloxamer F127) powder.

[0034] In a fifth aspect, the present invention also provides a method for preparing the above-mentioned ginseng exosome hydrogel, which comprises mixing the GDVLNs suspension and pre-cooled PF-127 powder according to a ratio, stirring at 4°C until the PF-127 powder is completely dissolved to generate the ginseng exosome hydrogel.

[0035] Preferably, the pre-cooling temperature is 4°C.

[0036] Preferably, the stirring speed is 600-8000xg.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] In the preparation method of the ginseng-derived vesicle-like nanoparticle suspension of the present invention, iodixanol is used to separate and purify GDVLNs, the centrifuged liquid has a more obvious GDVLNs layer, and the obtained GDVLNs maintain a better morphology and a cleaner background, showing a better extraction effect.

[0039] The ginseng-derived vesicle-like nanoparticle suspension of the present invention affects the M1 polarization of macrophages, exhibits a good anti-inflammatory effect, and can promote skin wound healing.

[0040] The ginseng exosome hydrogel of the present invention can promote skin wound healing and has good wound healing efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments and comparative examples of the present invention, the following briefly introduces the drawings required for use in the embodiments and comparative examples. Obviously, the drawings described below are only some embodiments and comparative examples of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 The figures are photos of the centrifuge and the yellow suspension in Example 1 and Comparative Example 1 of the present invention, wherein a and b are photos of the centrifuge obtained by the first centrifugation during the purification of the GDVLNs suspension in step 2 of Comparative Example 1 and Example 1, respectively, and c and d are photos of the yellow suspension obtained in Comparative Example 1 and Example 1, respectively.

[0043] Figure 2 These are transmission electron microscopy (TEM) images of the purified GDVLNs suspensions obtained in Comparative Example 1 and Example 1 of the present invention, wherein a is a transmission electron microscopy image of the purified GDVLNs suspension obtained in Comparative Example 1, and b is a transmission electron microscopy image of the purified GDVLNs suspension obtained in Example 1.

[0044] Figure 3 Graphs showing the concentration and particle size of the purified GDVLNs suspension obtained in Example 1 and Comparative Example 1 of the present invention, wherein a is a graph showing the concentration and particle size of the purified GDVLNs suspension obtained in Example 1, and b is a graph showing the concentration and particle size of the purified GDVLNs suspension obtained in Comparative Example 1.

[0045] Figure 4 Zeta potential (electrokinetic potential) diagrams of the purified GDVLNs suspension obtained in Example 1 of the present invention and Comparative Example 1, wherein a is the Zeta potential diagram of the purified GDVLNs suspension obtained in Example 1, and b is the Zeta potential diagram of the purified GDVLNs suspension obtained in Comparative Example 1.

[0046] Figure 5 These are photographs of the purified GDVLNs suspension prepared in Example 1 of the present invention at 4°C and 25°C, wherein a is a photograph of the purified GDVLNs suspension at 4°C, and b is a photograph of the purified GDVLNs suspension at 25°C.

[0047] Figure 6 These are photos of PF127 hydrogel@ GDVLNs prepared in Example 1 of the present invention at 4°C and 25°C, wherein a is a photo of PF127 hydrogel@ GDVLNs at 4°C, and b is a photo of PF127 hydrogel@ GDVLNs at 25°C.

[0048] Figure 7 The effect of the purified GDVLNs suspension obtained in Example 1 of the present invention on the M1 polarization of macrophages; wherein a is the marker gene interleukin-1a ( IL-1a), b is the effect of the purified GDVLNs suspension prepared in Example 1 on the marker gene interleukin-1b ( IL-1b ), c is the effect of the purified GDVLNs suspension prepared in Example 1 on the marker gene inducible nitric oxide synthase ( NOS2 ), d is the effect of the purified GDVLNs suspension prepared in Example 1 on the marker gene interferon-γ of M1 polarization of macrophages ( IFN-g ); Control represents the untreated group, LPS represents the lipopolysaccharide (LPS)-treated group, and LPS+GDVLNs represents the GDVLNs and LPS-treated group.

[0049] Figure 8 This is the effect of the purified GDVLNs suspension obtained in Example 1 of the present invention on the migration ability of mouse fibroblasts; wherein, a, b, c, and d respectively represent the cell migration effects of fibroblasts and untreated macrophages after co-culture for 0 h, 12 h, 24 h, and 48 h (Control), e, f, g, and h respectively represent the cell migration effects of fibroblasts and LPS-treated macrophages after co-culture for 0 h, 12 h, 24 h, and 48 h (LPS), and i, j, k, and l respectively represent the cell migration effects of fibroblasts and the GDVLNs obtained in Example 1 and LPS-treated macrophages after co-culture for 0 h, 12 h, 24 h, and 48 h (LPS+GDVLNs).

[0050] Figure 9 The effect of PF127 hydrogel@GDVLNs of Example 1 of the present invention on wound healing in mice; wherein, a, b, c, and d respectively represent the effects of normal saline on wound healing in mice on 1 day (Day), 5 days, 10 days, and 14 days (Control), e, f, g, and h respectively represent the treatment effects of PF127 hydrogel on wound healing in mice on 1 day, 5 days, 10 days, and 14 days (PF127hydrogel), i, j, k, and l respectively represent the treatment effects of the purified GDVLNs suspension of Example 1 on wound healing in mice on 1 day, 5 days, 10 days, and 14 days (GDVLNs), and m, n, o, and p respectively represent the treatment effects of PF127 hydrogel@GDVLNs of Example 1 on wound healing in mice on 1 day, 5 days, 10 days, and 14 days (PF127 hydrogel@GDVLNs). DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the following embodiments and comparative examples.

[0052] Unless otherwise specified, the reagents and materials described in the following examples are commercially available; the test methods are conventional methods unless otherwise specified. Among them, the experimental mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., male, 6 - 8 weeks old, model number SCXK(Beijing)2021 - 0006; mouse fibroblasts (L929) were obtained from the American Type Culture Collection. The transmission electron microscope (TEM) model is Tecnai Spirit Biotwin 120K; the ZETA potential analyzer is the ZetaView nanoparticle tracking analyzer, model Particle Metrixq; the PCR instrument used for quantitative PCR (qPCR) detection is from Bio - Rad, USA.

[0053] Example 1

[0054] Step 1: Isolation of GDVLNs

[0055] After cutting 100 g - 150 g of clean fresh ginseng roots into small pieces, place them in a blender and add 200 - 300 mL of phosphate - buffered saline containing 2 - 3 mL of cocktail protease inhibitor for cell disruption. Centrifuge the disrupted mixture at 4°C at 800 - 1500 xg for 15 - 20 min, discard the precipitate, transfer the supernatant to a new centrifuge tube, then centrifuge at 4°C at 10000 - 20000 xg for 1 - 1.5 h, discard the precipitate, and transfer the supernatant to a new centrifuge tube again. Next, slowly add 1 ml of iodixanol along the centrifuge tube wall and then centrifuge at 4°C at 100000 - 200000 xg for 1 - 1.5 h. After discarding the supernatant, the yellow suspension at the bottom of the tube is the crude GDVLNs suspension.

[0056] Step 2: Purification of GDVLNs

[0057] Phosphate buffer was used as a solvent to prepare buffer solutions containing iodixanol at concentrations of 5%, 10%, 20%, and 40% v / m. Equal volumes of buffer solutions containing iodixanol at concentrations of 5%, 10%, 20%, and 40% v / m were slowly added along the side of a centrifuge tube. Subsequently, the crude GDVLNs suspension was slowly added to the iodixanol buffer solution along the side of the centrifuge tube. The ratio of the crude GDVLNs suspension volume to the total volume of the iodixanol buffer solution was 1:20. The mixture was centrifuged at 4°C at 100,000-200,000 x g for 1.5-2 hours to obtain a centrifuge solution. The yellow suspension in the buffer containing iodixanol at a concentration of 20 v / m% was collected and transferred to a centrifuge tube containing phosphate buffer. Iodixanol was then slowly added along the wall of the tube. The volume ratio of the yellow suspension, phosphate buffer, and iodixanol was 1:20:1. The tube was centrifuged at 4°C at 100,000-200,000 x g for 1-1.5 hours. The supernatant was discarded. The yellow suspension at the bottom of the tube was the purified GDVLNs suspension. The photos of the purified GDVLNs suspension at 4°C and 25°C are shown in Figure 2. Figure 5 As shown in a and b, the concentration of the extracted and purified GDVLNs suspension was determined by BCA method and recorded.

[0058] Step 3. Preparation of ginseng exosome hydrogel

[0059] Add 13 ml of purified GDVLNs suspension to a clean beaker, then add 4 g of PF-127 powder pre-cooled to 4°C. Then, stir at 600-800 x g using a magnetic stirrer at 4°C until the PF-127 powder is completely dissolved, forming a complex of purified GDVLNs suspension and PF-127 hydrogel, namely ginseng exosome hydrogel, recorded as PF127 hydrogel@GDVLNs, and store at 4°C in the dark. Photos of PF127 hydrogel@GDVLNs at 4°C and 25°C are shown in the figure below. Figure 6 As shown in a and b.

[0060] Comparative Example 1

[0061] Step 1: Isolation of GDVLNs

[0062] Cut 100-150g of clean, fresh ginseng root into small pieces and place them in a cell disruptor. Add 200-300mL of phosphate buffered saline (PBS) containing 2-3mL of a protease inhibitor cocktail to disrupt the cells. Centrifuge the disrupted mixture at 800-1500xg for 15-20min at 4°C. Discard the pellet and transfer the supernatant to a fresh centrifuge tube. Subsequently, centrifuge at 10,000-20,000xg for 1-1.5h at 4°C. Discard the pellet and transfer the supernatant to a fresh centrifuge tube. Centrifuge again at 100,000-200,000xg for 1-1.5h at 4°C. Discard the supernatant and add 1mL of phosphate buffered saline to the bottom of the tube. Resuspend the mixture by pipetting to obtain a yellow suspension, which is the crude GDVLN suspension.

[0063] Step 2: Purification of GDVLNs

[0064] Using deuterated water as the solvent, deuterated sucrose solutions at concentrations of 5%, 10%, 20%, and 40% were prepared. Equal volumes of the deuterated sucrose solutions at concentrations of 5%, 10%, 20%, and 40% were slowly added along the side of the centrifuge tube. The crude GDVLNs suspension was then added to the top layer of the solution. The ratio of the crude GDVLNs suspension to the total volume of the deuterated sucrose solution was 1:20. The mixture was centrifuged at 4°C at 100,000-200,000 x g for 1.5-2 hours to obtain a centrifuge solution. Collect the yellow suspension in the 20 v / m% layer and transfer it to a centrifuge tube filled with phosphate buffer. Centrifuge at 4°C at 100,000-200,000 x g for 1-1.5 hours. Discard the supernatant and add phosphate buffer to the bottom of the tube. The volume ratio of the yellow suspension, the buffer in the centrifuge tube, and the added phosphate buffer is 1:20:1. Resuspend the yellow suspension by pipetting to obtain the purified GDVLNs suspension. Determine the concentration of the extracted and purified GDVLNs suspension using the BCA assay and record the concentration.

[0065] The purified GDVLNs suspensions prepared in Example 1 and Comparative Example 1 were tested.

[0066] 1. During the purification of the GDVLNs suspension in step 2 of Comparative Example 1 and Example 1, the centrifuge obtained by the first centrifugation was as follows: Figure 1 As shown in a and b, the yellow suspension obtained is as Figure 1 As shown in Figures c and d, it can be seen that the method of Example 1 has a more obvious GDVLNs layer when separating and purifying GDVLNs.

[0067] 2. The morphology of the purified GDVLNs obtained in Comparative Example 1 and Example 1 was observed using a transmission electron microscope (TEM). Figure 2 As shown in a and b, it can be seen that the GDVLNs obtained in Example 1 have a cleaner background and a more complete morphology.

[0068] Nanoparticle tracking analysis (NTA) was used to analyze the diameter and concentration of the purified GDVLNs obtained in Example 1 and Comparative Example 1. The results are as follows: Figure 3 As shown in a and b, it can be seen that the GDVLNs obtained in Example 1 have more uniform sizes and the number of particles obtained is significantly improved.

[0069] The potential of the purified GDVLNs obtained in Example 1 and Comparative Example 1 was analyzed using a ZETA potentiometer. Figure 4 As shown in a and b, it can be seen that the potentials of the purified GDVLNs obtained in Comparative Example 1 and Example 1 are relatively close, and both have good dispersion characteristics.

[0070] 3. Obtaining and culturing mouse bone marrow-derived macrophages (BMDMs)

[0071] M-CSF (macrophage colony-stimulating factor) was added to RPMI 1640 medium, and the medium was prewarmed to 37°C to facilitate the subsequent isolation of mouse BMDMs. For this procedure, autoclaved scissors, forceps, and disposable syringes were prepared to ensure a sterile environment. After sacrificing the mouse, it was immediately disinfected with 75% ethanol to ensure all manipulations were performed under sterile conditions. After disinfection, the mouse's abdomen was carefully incised along the groin, and the hind limbs were removed along the spine. The excised sections were further disinfected in 75% ethanol to prevent contamination. Next, the skin, muscle, femur, and tibia were isolated from the mouse. The isolated bones were transferred to a clean bench, where scissors were used to cut the femur and tibia at both ends. Prewarmed RPMI 1640 medium was then drawn into a sterile disposable syringe and flushed into the bone cavity until the bone became translucent, indicating that the bone marrow cells had been effectively released into the culture medium.

[0072] Extracted mouse BMDMs were cultured in RPMI 1640 medium supplemented with 10 wt% fetal bovine serum (FBS), supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin (1 wt%) to provide necessary antimicrobial protection. The culture flasks were placed in a cell culture incubator at 5% CO2 and a constant temperature of 37°C to ensure a suitable environment for cell growth. On the third day of culture, cell growth was monitored and the medium was changed to remove metabolic waste and dead cells. After one week, once the cells had grown well and reached an appropriate density, drug treatment and subsequent experiments could be initiated.

[0073] 4. Verification of the anti-inflammatory effect of GDVLNs

[0074] To investigate whether GDVLNs affect the M1 polarization of macrophages and exert anti-inflammatory effects, a blank group (Control), a lipopolysaccharide (LPS) group, and an LPS+GDVLNs group were constructed. The blank group received no treatment. In the LPS group, BMDMs were treated with LPS (1 mg / ml) for 24 hours. In the LPS+GDVLNs group, BMDMs were treated with LPS for 24 hours to establish an M1 macrophage polarization model. The cells were then treated with a purified GDVLNs suspension (20 μg / mL) for 24 hours. M1 polarization marker genes were detected by qPCR. IL-1a, IFN-g, IL-1b, NOS2 The changes in transcription levels of Figure 7 As shown in a, b, c, and d (in the figure, * indicates P < 0.05 compared with WT; ** indicates P < 0.01 compared with WT; *** indicates P < 0.001 compared with WT; **** indicates P < 0.0001 compared with WT). Figure 7 It can be seen that compared with the LPS group, the M1 BMDMs IL-1a, IFN-g, IL-1b, NOS2 The transcription levels of α-glucan and β-glucan were significantly reduced, showing a good anti-inflammatory effect.

[0075] 5. Do GDVLNs promote wound healing by affecting macrophages?

[0076] To investigate whether GDVLNs promote wound healing by influencing macrophages, this study co-incubated BMDMs treated with a suspension of purified GDVLNs with mouse fibroblasts L929. The effects on fibroblast migration were then assessed using a wound wound healing assay. Three groups were established: a blank control, a lipopolysaccharide (LPS) group, and an LPS+GDVLNs group. The blank group received no treatment. In the LPS group, BMDMs were treated with LPS (1 mg / ml) for 24 hours. In the LPS+GDVLNs group, BMDMs were treated with LPS for 24 hours to induce M1 macrophage polarization, and then treated with a purified GDVLNs suspension (20 μg / mL) for 24 hours. Simultaneously, after confluent 6-well plates containing mouse fibroblasts L929 were vertically wounded with a pipette tip. BMDMs and L929 cells were then co-incubated in chambers for 24 hours, and L929 cell migration was assessed.

[0077] The results are as follows Figure 8 As shown, from Figure 8 It can be seen that GDVLNs significantly accelerated the migration ability of co-cultured fibroblasts, resulting in a faster healing speed.

[0078] 6. PF127 hydrogel@GDVLNs improves wound healing

[0079] To confirm the improvement effect of PF127 hydrogel@ GDVLNs on wound healing in vivo, a mouse skin laser thermal injury model was established in this study. Physiological saline, PF127 hydrogel, the purified GDVLNs suspension of Example 1, and PF127 hydrogel@ GDVLNs of Example 1 were applied to the wounds of the mice once in the morning and evening respectively. The wound healing of the mice was observed on the 1st, 5th, 10th, and 14th days. The results are shown in Figure 2. Figure 9 As shown, from Figure 9 It can be seen that PF127hydrogel@GDVLNs has faster wound healing efficiency compared with other treatment groups.

[0080] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the embodiments. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all embodiments here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing a ginseng-derived vesicle-like nanoparticle suspension, characterized in that: Here are the steps: Step 1: Separation of ginseng-derived vesicle-like nanoparticle suspension: First, clean fresh ginseng is disrupted in a buffer containing a protein inhibitor, the disrupted mixture is centrifuged once to obtain a primary supernatant, and then centrifuged twice to obtain a secondary supernatant. Finally, iodixanol is added dropwise to the secondary supernatant, and after three centrifugations, the supernatant is discarded to obtain a crude ginseng-derived vesicle-like nanoparticle suspension; Step 2: Purification of ginseng-derived vesicle-like nanoparticle suspension: First, the crude ginseng-derived vesicle-like nanoparticle suspension is added to multiple layers of buffer solutions containing iodixanol at different concentrations. After one centrifugation, the yellow suspension in the buffer solution containing iodixanol at a concentration of 20 v / w% is collected. The yellow suspension is then added to a dilution buffer solution, iodixanol is added dropwise, and after a second centrifugation, the supernatant is discarded to obtain a purified ginseng-derived vesicle-like nanoparticle suspension. The concentration range of the different concentrations is 5v / m%-40v / m%, and the multiple layers of buffer solutions containing iodixanol at different concentrations are distributed from top to bottom in order from low to high concentrations.

2. The method for preparing the ginseng-derived vesicle-like nanoparticle suspension according to claim 1, characterized in that: In step 1, the protein inhibitor is a cocktail; The buffer is a phosphate buffer; The ratio of the clean fresh ginseng, protease inhibitor and buffer is 100-150 g: 2-3 mL: 200-300 mL; The volume ratio of the buffer solution containing protein inhibitors to iodixanol is 200-300:

1.

3. The method for preparing the ginseng-derived vesicle-like nanoparticle suspension according to claim 1, characterized in that: In step one, The centrifugation temperature is 4°C, the speed is 800-1500xg, and the time is 15-20min; The temperature of the secondary centrifugation is 4°C, the speed is 10000-20000xg, and the time is 1-1.5h; The temperature of the three centrifugations is 4°C, the speed is 100000-200000xg, and the time is 1-1.5h.

4. The method for preparing the ginseng-derived vesicle-like nanoparticle suspension according to claim 1, characterized in that: In step 2, The centrifugation temperature is 4°C, the speed is 100000-200000xg, and the time is 1.5-2h; The temperature of the secondary centrifugation is 4°C, the speed is 100000-200000xg, and the time is 1-1.5h.

5. The method for preparing the ginseng-derived vesicle-like nanoparticle suspension according to claim 1, characterized in that: In step 2, the ratio of the volume of the crude ginseng-derived vesicle-like nanoparticle suspension to the total volume of multiple layers of buffer solutions containing iodixanol at different concentrations is 1:10-30; The multiple layers of buffer solutions containing iodixanol of different concentrations are buffer solutions containing iodixanol of concentrations of 5v / m%, 10v / m%, 20v / m%, and 40v / m% from top to bottom, and the volume of each layer of buffer solution containing iodixanol is equal; The buffer in the buffer containing iodixanol is a phosphate buffer; The volume ratio of the yellow suspension, the dilution buffer and iodixanol is 1:10-30:1; The dilution buffer is phosphate buffer.

6. A ginseng-derived vesicle-like nanoparticle suspension prepared according to the preparation method according to any one of claims 1 to 5.

7. Use of the ginseng-derived vesicle-like nanoparticle suspension according to claim 6 in the preparation of a medicament for promoting skin wound healing.

8. Ginseng exosome hydrogel, characterized in that: Includes 10-15mL: 3-4.5g of ginseng-derived vesicle-like nanoparticle suspension and PF-127 powder; The ginseng-derived vesicle-like nanoparticle suspension is the ginseng-derived vesicle-like nanoparticle suspension according to claim 6.

9. The method for preparing ginseng exosome hydrogel according to claim 8, characterized in that: The ginseng-derived vesicle-like nanoparticle suspension and pre-cooled PF-127 powder were mixed according to the ratio and stirred at 4°C until the PF-127 powder was completely dissolved to form ginseng exosome hydrogel.

10. The method for preparing ginseng exosome hydrogel according to claim 9, characterized in that: The precooling temperature is 4°C; The stirring speed is 600-8000xg.

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