Preparation method and application of gynura formosana extracellular vesicle targeted drug delivery system with liver injury repairing effect
The drug delivery system constructed by loading glycyrrhizic acid into extracellular vesicles of Scutellaria baicalensis solves the problems of liver damage repair and liver cancer inhibition, achieves better liver targeting and anti-tumor activity, and improves biosafety.
Patent Information
- Application Number
- CN202510829930.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-20
- Publication Date
- 2025-09-19
AI Technical Summary
Existing technologies are difficult to effectively repair liver damage, especially liver cell damage caused by drug-induced liver damage, and traditional drug carriers lack liver targeting and anti-liver cancer activity.
Extracellular vesicles of Bletilla striata were used as carriers to load glycyrrhetinic acid (GA) to construct a GA-GELNs drug delivery system. Extracellular vesicles of Bletilla striata were extracted and purified by ultracentrifugation and differential centrifugation, and then co-incubated with GA to form a targeted drug delivery system.
It achieved effective inhibition of liver cancer cells and repair of liver damage, showed better anti-tumor activity and tumor targeting, and improved biosafety and therapeutic effects.
Smart Images
Figure CN120661688A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to a preparation method and application of a Bletilla striata extracellular vesicle targeted drug delivery system with the function of repairing liver damage. Background Art
[0002] Liver damage is a serious liver injury characterized by metabolic disturbances, internal environmental imbalances, and loss of liver function. It primarily refers to severe liver damage caused by short-term exposure to large doses of exogenous substances such as chemicals, alcohol, drugs, bacteria, and viruses. Long-term liver damage often leads to liver fibrosis and is a key risk factor for cirrhosis and hepatocellular carcinoma.
[0003] Glycyrrhetinic acid (GA), a white powder at room temperature with poor water solubility, is one of the active ingredients extracted from the roots of the traditional Chinese medicinal legume Glycyrrhiza uralensis. It is the aglycone of glycyrrhizic acid (GL) and possesses both hydrophilic and hydrophobic properties. It has two chemical isomers: 18α-GA and 18β-GA. GA exerts multiple pharmacological effects, including anti-acid and anti-inflammatory, antiviral, and anti-tumor activities, by affecting signaling pathways and protein expression. When used as a drug carrier to construct a drug delivery system, GA significantly enhanced its in vitro anti-proliferation activity against human hepatocellular carcinoma cells compared to the effects of either drug alone.
[0004] White Phoenix Gynuraformosana Kitam ) is Asteraceae ( Compositae ) Chrysanthemum Panax notoginseng ( Gynura The whole herb of the perennial plant Baifengcai (Baifengcai), also known as Baizaihuangcao (Baifengcai), is cooling in nature, sweet and mild in flavor, and non-toxic. It enters the liver, lungs, kidneys, and large and small intestine meridians, exhibiting antipyretic and detoxifying properties. Its aerial parts are widely used by the people in Fujian, particularly in southern Fujian, and are a valuable natural Chinese herbal medicine for research and development.
[0005] Plant extracellular vesicles (EVs) are small, membranous vesicles secreted by plant cells. They have a specific phospholipid bilayer structure, are saucer- or cup-shaped, and range in size from 30 to 300 nm. They contain a variety of active ingredients, including lipids, proteins, and nucleic acids. Plant EVs share a similar structure and function to animal exosomes. Exosomes act as messengers for intercellular communication and are involved in the physiological processes of various human diseases. They possess the advantages of good biocompatibility and low immunogenicity, allowing them to cross biological barriers and reflect the physiological and pathological conditions of cells. Summary of the Invention
[0006] Liver injury primarily involves damage to liver cells, leading to abnormalities in liver structure and function. Drug-induced liver injury is the most common form of liver injury, which can lead to the accumulation of toxins in the body, impairing metabolism, detoxification, synthesis, and storage, potentially interfering with the normal function of other organs and even inducing more serious diseases. This study aimed to extract and purify extracellular vesicles (GELNs) from G. elegans (Baifengcai) using a combination of ultracentrifugation and differential centrifugation in C57BL / 6 mice. The morphology and size distribution of the exosomes were observed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and zeta potential. Protein content was also determined based on the structural integrity of the plant exosomes. A drug delivery system loaded with GA was constructed, and its anti-hepatocellular carcinoma activity and liver damage repair effects were investigated using cytological, physiological, and biochemical parameters in a human hepatocellular carcinoma (HepG2) cell model and a mouse model of liver injury.
[0007] To achieve the above object, the present invention adopts the following technical solutions: A targeted drug delivery system of an extracellular vesicle of Bletilla striata having the effect of repairing liver damage, wherein the targeted drug delivery system of an extracellular vesicle of an Bletilla striata is GA-GELNs, which uses the extracellular vesicles of an Bletilla striata GELNs as a carrier and loads the drug glycyrrhetinic acid GA, thereby constructing the targeted drug delivery system of an extracellular vesicle of an Bletilla striata GA-GELNs.
[0008] Preferably, the preparation method of the extracellular vesicle GELNs of white herb comprises the following steps: adding white herb to phosphate buffer, soaking for 5 hours, first squeezing and crushing the juice with a juicer, filtering the squeezed white herb juice and centrifuging it at 1000×g for 10 min, 3000×g for 20 min, and 10000×g for 30 min at 4°C to obtain the supernatant; using an ultrahigh-speed centrifuge to centrifuge the supernatant at 4°C for 2 h at a speed of 150,000×g to obtain a precipitate; resuspending the precipitate with PBS, and filtering the resulting suspension through a 0.22 μm filter membrane to obtain a purified white herb vesicle GELNs suspension.
[0009] Preferably, the usage ratio of white phoenix and phosphate buffer saline (PBS) is 1:10 g / mL.
[0010] Preferably, the pH of phosphate buffer saline (PBS) is 7.4.
[0011] Preferably, the preparation method of the described extracellular vesicle targeted drug delivery system of white broccoli with the effect of repairing liver damage comprises the following steps: mixing the GELNs suspension and the GA solution in a mass ratio of 1:2, placing them in a shaker at 37°C for 2 h, centrifuging them at 12000 rpm for 10 min, and taking the supernatant to measure the encapsulation efficiency; adding purified water to the precipitate, ultrasonicating for 15 min and stopping for 5 min, repeating three times, and centrifuging again at 12000 rpm for 10 min. After centrifugation, the supernatant is taken and transferred into a sample injection bottle. The supernatant is GA-GELNs. The above sample is taken to measure the drug loading by UV, and the drug loading is 38.76%.
[0012] Preferably, the concentration of the GELNs suspension is 2 mg / mL.
[0013] Preferably, the concentration of the GA solution is 1 mg / mL.
[0014] Preferably, in the GA-GELNs, an extracellular vesicle targeted drug delivery system of Bletilla striata, the drug loading amount of GA is 38.76%.
[0015] Application 1: Application of the described Bletilla striata extracellular vesicle targeted drug delivery system in the preparation of drugs for repairing liver damage.
[0016] Application 2: Application of the described Bletilla striata extracellular vesicle targeted drug delivery system in the preparation of anti-liver cancer drugs.
[0017] The advantages of the present invention are: This study demonstrates for the first time that extracellular vesicles from Bletilla striata can effectively inhibit liver tumor cells and repair liver damage. This system, in which modified Bletilla striata extracellular vesicles are loaded with glycyrrhetinic acid, exhibits superior anti-tumor activity and tumor targeting compared to either Bletilla striata extracellular vesicles or glycyrrhetinic acid alone, achieving a multi-component synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is a photo of the extracellular vesicle suspension of Bletilla striata; Figure 2 This is a transmission electron micrograph of extracellular vesicles of Bletilla striata; Figure 3 This is the particle size distribution diagram of the extracellular vesicles of Baifengcai; Figure 4 Effects of different concentrations of GELNs on the survival rate of HepG2 cells (n=3); Figure 5 Effects of different concentrations of GA on the survival rate of HepG2 cells (n=3); Figure 6 Effects of different concentrations of GA-GELNs on the survival rate of HepG2 cells (n=3); Figure 7This is the appearance of the liver of mice in different groups; Figure 8 is the level of ALT and AST in mouse plasma; Figure 9 The following are the pathological morphological manifestations of liver tissues of mice in each group (HE, ×400). Note: A (blank control group), B (APAP group), C (low-dose group), D (medium-dose group), and E (high-dose group). DETAILED DESCRIPTION
[0019] To make the above features and advantages of the present invention more clearly understood, the following embodiments are given for detailed description. The methods of the present invention are all conventional methods in the art unless otherwise specified.
[0020] Example 1 1 Extraction and identification of vesicles from Baifengcai 1.1 Extraction experiment of vesicles from Baifengcai 1.5 kg of B. chinensis was added to phosphate buffered saline (PBS, pH 7.4) precooled to 4°C at a ratio of 1:10. After soaking for 5 hours, the juice was squeezed and crushed using a juicer. The juice was filtered and centrifuged at 4°C at 1000 × g for 10 minutes, 3000 × g for 20 minutes, and 10,000 × g for 30 minutes. The supernatant was collected and centrifuged at 150,000 × g for 2 hours at 4°C. The pellet was resuspended in 10 mL of PBS and filtered through a 0.22 μm filter to obtain the purified B. chinensis vesicle suspension.
[0021] 1.2 Identification of extracellular vesicles of B. The vesicles of the white broccoli obtained by centrifugation were identified by transmission electron microscopy. The specific experimental process is as follows: Use a pipette to transfer 10 μL of the vesicle suspension onto a wax plate. Place a copper mesh so that the surface of the support membrane is in contact with the surface of the suspension. Let it sit for 5 minutes. Remove the mesh, remove any excess liquid with filter paper, and allow it to dry briefly. Place a small drop of 3% phosphotungstic acid solution onto the wax plate. Place the copper mesh with the sample on the surface of the dye solution (contacting the sample and the dye solution) and let it sit for 3-5 minutes. Remove the mesh, remove any excess liquid with filter paper, and allow it to dry briefly. Take an electron microscope image.
[0022] Nanoparticle analysis of extracellular vesicles of B. truncatula was performed. The specific operation process is as follows: The size of GELNs was analyzed using nanoparticle tracking analysis technology. After the extracted GELNs suspension was diluted to an appropriate concentration with PBS buffer, the particle number, particle size and potential information could be obtained.
[0023] 1.3 Preparation of GA-GELNs The GELNs suspension with a concentration of 2 mg / mL was mixed evenly with the GA solution of 1 mg / mL in a volume ratio of 2:1, placed in a shaker at 37 °C for incubation for 2 h, centrifuged at 12000 rpm for 10 min, and the supernatant was taken to measure the encapsulation efficiency; 0.5 g of the precipitate was added with 1 mL of purified water, ultrasonicated for 15 min and stopped for 5 min, repeated three times, and centrifuged again at 12000 rpm for 10 min. After centrifugation, the supernatant was taken and transferred to the injection bottle. The supernatant was GA-GELNs. The above sample was taken to measure the drug loading by UV, and the drug loading was 38.76%.
[0024] 2 Evaluation of the inhibitory effect of GA-GELNs on hepatocellular carcinoma 2.1 Cell assay 2.1.1 HepG2 cell culture HepG2 cells were grown in a medium containing DMEM (high-glucose medium) with serum and penicillin-streptomycin (100:10:1) at 37°C and 5% CO2. They typically become confluent within 48–72 hours and can be passaged or used for subsequent experiments.
[0025] 2.1.2 Cytotoxicity assay Experimental group: cells were treated with three groups of drugs, namely GELNs, GA, and GA-CELNs, and culture medium containing drugs of different concentrations was prepared.
[0026] Control group: cells were added with complete culture medium.
[0027] Blank group: no cells, only complete culture medium was added.
[0028] HepG2 cells in the logarithmic growth phase were seeded in 96-well plates at a number of 1000 per well (n=3). After the cells adhered to the wall, three groups of drugs, namely GELNs, GA, and GA-CELNs, were added and cultured for 24 h. Then, CCK-8 solution was added and incubated for 1 h. The absorbance (OD) was measured at a wavelength of 450 nm to analyze the cell survival rate under different conditions.
[0029] 3 Effects of GA-GELNs on repairing liver damage 3.1 Animal grouping and treatment After 40 C57BL / 6J mice were adaptively raised for 2 weeks, 8 mice were randomly selected as the blank group, and the remaining 32 C57BL / 6 mice were intraperitoneally injected with APAP (400 mg·kg -1 After the drug-induced liver injury model was established, the patients were randomly divided into APAP model group and GA-GELNs low, medium and high doses (10 μg·kg -1 , 30 μg·kg -1、 80 μg·kg -1 The mice were administered via the tail vein (or tail vein) for 7 consecutive days, with 8 mice per group. Dosing was performed once daily, and the mice's survival was observed. Twenty-four hours after the last administration, the mice were anesthetized, their eyes were enucleated, and blood was collected in 1.5 mL tubes. After 2 hours of rest, the tubes were centrifuged at 3500 × g for 20 minutes at 4°C. The supernatant was collected and stored at -80°C. Liver tissue was collected and HE staining was used to examine the pathological morphology of the liver tissue. This study adhered to the "3R" principle.
[0030] 3.2 Repair test of liver damage in mice using drug delivery system Plasma levels of alanine aminotransferase (GPT) and aspartate aminotransferase (GOT) in each group of mice were measured using a GPT / ALT activity assay kit and a GOT / AST activity assay kit, respectively, according to the manufacturer's instructions. The specific steps were as follows: Reagent 1 was prepared by dissolving the lyophilized powder in 2 mL of distilled water. A 20 μmol / mL sodium pyruvate solution was diluted with distilled water to 1, 0.8, 0.6, 0.4, 0.2, 0.1, and 0.05 μmol / mL, and 30 μL of each solution was pipetted into seven EP tubes, labeled standard tubes 1 to 7. One EP tube, labeled blank, was added with 30 μL of distilled water for plotting the standard curve. The standard and blank tubes were incubated at 37°C for 30 min, followed by 25 μL of reagent 2, which was then incubated at 37°C for 20 min. For each plasma sample, two EP tubes were prepared, labeled as the assay tube and the control tube. Add 5 μL of plasma and 25 μL of Reagent 1 to the assay tube, and 25 μL of Reagent 1 to the control tube. Mix the two tubes thoroughly and incubate at 37°C for 30 min. Add 25 μL of Reagent 2 to each tube, and add 5 μL of plasma to the control tube. Mix the two tubes thoroughly and incubate again at 37°C for 20 min. Add 240 μL of Reagent 3 to each tube, mix thoroughly, and incubate at room temperature for 10 min. Pipette 200 μL of each reaction solution into a 96-well plate and measure the absorbance at 505 nm using a microplate reader. These values are designated as A assay tube, A control tube, A standard tube, and A blank tube. Calculate ΔA standard = A standard tube - A blank tube, and ΔA = A assay tube - A control tube. Plot a standard curve with the concentration of each standard on the x-axis and ΔA standard on the y-axis, yielding the equation y = kx + b. Substitute ΔA into the equation to determine the value of x (μmoL / mL). GPT / GOT activity (U / mL) = 12x.
[0031] Liver tissue was obtained and fixed in 4% paraformaldehyde solution; the tissue was removed from the paraformaldehyde, the formaldehyde solution was absorbed with absorbent paper, and the tissue surface was smoothed with a blade; the trimmed tissue was immersed in 70% (4-12h), 80% (4-12h), 90% (1-2h), and 100% (1-2h) ethanol solutions in sequence to replace the formaldehyde solution in the tissue; the tissue was immersed in xylene solution to replace the ethanol in it. When the edge of the tissue became translucent, the immersion was stopped; an oven containing paraffin was opened in advance to melt the paraffin, and the tissue was immersed in paraffin for 3h, and then placed in another oven containing paraffin for 1h to replace the xylene in the tissue. Pour melted paraffin into an embedding frame. Quickly remove the tissue from the paraffin and place it in the embedding frame, cut surface facing down. Label the frame with label paper. Keep the paraffin melted during the process. To obtain continuous and smooth sections during sectioning, pre-cool the wax block containing the tissue sample in a -20°C refrigerator before slicing. Secure the wax block to the microtome and perform continuous sectioning. Carefully transfer the sections to 40°C warm water for spreading. Use a slide to pick up the sections and place them in an oven to bake. Baking prevents the sections from falling off the slide during staining. Once baked, staining can be performed. If you do not stain the sections immediately after baking, wait for the sections to cool to room temperature, then place them in a sectioning box and store them in a -20°C refrigerator. They will need to be baked again before staining.
[0032] Tissue sections were stained with a hematoxylin and eosin (HE) staining kit. Hematoxylin staining causes cell nuclei to appear blue and cytoplasm to appear pink or red, allowing for visualization of tissue or lesions. Sample processing: Paraffin sections were dewaxed in xylene for 5–10 minutes. Fresh xylene was then used, followed by another dewax treatment for 5–10 minutes, followed by anhydrous ethanol for 5 minutes, 90% ethanol for 2 minutes, 80% ethanol for 2 minutes, 70% ethanol for 2 minutes, and distilled water for 2 minutes. Hematoxylin and eosin staining: Samples treated as described above were stained with hematoxylin solution for 5–10 minutes. Excess stain was then rinsed off in tap water for approximately 10 minutes, followed by another wash with distilled water, followed by staining with eosin solution for 30 seconds to 2 minutes. At this point, if direct observation is desired, sections can be washed twice with 70% ethanol. If not, dehydration, clearing, and mounting are still necessary after the 70% ethanol wash. After staining, images of each tissue group were acquired using a microscope.
[0033] Results and Discussion 1 Extraction and separation of extracellular vesicles from Bletilla striata The extracellular vesicle suspension of B. chinensis extracted by differential centrifugation combined with ultracentrifugation was yellow-green (e.g. Figure 1 As shown), there are no large particles or fibers visible to the naked eye after filtering through the membrane.
[0034] 1.2 Identification of extracellular vesicles of B. (1) Transmission electron microscopy (TEM) Figure 2 The morphological characterization results of TEM showed that the extracellular vesicles of white broccoli have the typical characteristics of plant extracellular vesicles, with obvious lipid bilayers, a saucer-shaped structure, a diameter of about 150nm and uniform distribution.
[0035] (2) Nanoparticle Tracking Analysis (NTA) Figure 3 The particle size analysis results of extracellular vesicles of Bletilla striata showed that the particle size distribution of extracellular vesicles of Bletilla striata was relatively uniform, with an average particle size of about 153.4 nm, and most of them were distributed between 100 and 200 nm.
[0036] 2 Evaluation of the inhibitory effect of GELNs on hepatocellular carcinoma 2.1 Cell assay 21.1 Evaluation of the Inhibitory Effect of GA-GELNs on HepG2 Cell Proliferation The experimental results showed that the GELNs group, GA group, and GA-GELNs group showed different degrees of proliferation inhibition on HepG2 cells compared with the blank control group, and the inhibitory effect was concentration-dependent. By fitting, the IC values of GELNs, GA, and GA-GELNs can be obtained. 50 The values were 2.045 mg / mL, 87.93 μg / mL, and 60.18 μg / mL, respectively, and the IC 50 The IC value is used to measure the concentration of the drug at which half of the inhibitory effect is produced. 50 The smaller the value, the better the inhibitory effect on HepG2 cells; therefore, the inhibitory effect of GA-GELNs on HepG2 cells is better than that of GELNs and GA (e.g. Figure 4-Figure 6 shown).
[0037] 3.2 Effect of GA-GELNs on repairing liver damage In the control group, the liver was light red, the shape was intact, the surface was smooth and delicate, and the texture was soft; in the model group, the liver became darker in color, the surface became rough and uneven, with obvious dark red bleeding spots, and the texture became obviously hard; in the low-dose group, the liver was light red in color, the surface was relatively smooth, the bleeding spots were significantly reduced, and the texture was slightly hardened; in the medium and high-dose groups, the appearance of the liver almost returned to normal (such as Figure 7 shown).
[0038] The degree of liver cell damage can be determined by measuring ALT and AST. Under normal circumstances, the levels of ALT and AST in the blood are relatively stable. When liver cells are damaged, enzymes are released and transferred into the blood, and the levels of ALT and AST increase significantly. Figure 8 It can be seen that compared with the APAP group, the ALT and AST levels of each drug-treated group were significantly reduced, and the ALT and AST levels of the medium and high-dose groups were significantly lower than those of the model group. This shows that there are significant differences in ALT and AST levels between different groups, and each drug-treated group has a repair effect on liver cell damage caused by APAP. The differences are statistically significant (ALT: F = 318.4, P <0.001; AST:F=124.0, P <0.001).
[0039] HE staining results showed that the hepatocytes of mice in the blank control group were closely arranged, and the hepatic sinusoids did not show obvious expansion or compression, and no obvious inflammatory changes were observed. Figure 9 Middle A: In the APAP model group, mice had extensive liver necrosis, with a large number of hepatocyte nuclei condensed, darkly stained, fragmented or dissolved, accompanied by inflammation. Figure 9 Middle B: The liver tissue of mice in the low-dose group showed necrosis, with hepatocyte nuclei shrinking, dark staining, fragmentation or dissolution, accompanied by inflammation. Figure 9 Middle C: The liver cells in the medium-dose group showed pyknosis and dark staining, accompanied by a small amount of inflammatory cell infiltration. Figure 9 In the high-dose group, the liver tissue and hepatic sinusoids of mice showed no obvious expansion or compression, but slight inflammatory changes were observed. Figure 9 Middle E.
[0040] Plant-derived extracellular vesicles (EVs) not only exert pharmacological effects as active ingredients but also offer unique advantages as drug carriers. This study found that EVs from B. elegans have a therapeutic effect on liver injury. To further investigate their potential as anti-hepatocellular carcinoma agents, this study used differential ultracentrifugation to extract, purify, and characterize EVs from B. elegans. A co-incubation method was used to construct EVs loaded with glycyrrhetinic acid (APAP). A mouse model of liver injury was established with intraperitoneal injection of APAP, followed by tail vein injection to investigate their in vitro antitumor activity and in vivo biosafety. Structural characterization of GELNs demonstrated that the GELNs prepared in this study met the evaluation criteria for extracellular vesicles. The modified B. elegans EV-loaded with glycyrrhetinic acid demonstrated superior antitumor activity and tumor targeting compared to other systems. HE staining and serum analysis demonstrated their therapeutic effect on liver injury in mice, achieving both enhanced therapeutic efficacy and improved biosafety.
[0041] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A targeted drug delivery system of extracellular vesicles of Bletilla striata with the effect of repairing liver damage, characterized in that: The Bleu truncatum extracellular vesicle targeted drug delivery system is GA-GELNs, which uses Bleu truncatum extracellular vesicles GELNs as a carrier and loads the drug glycyrrhetinic acid GA, thereby constructing the Bleu truncatum extracellular vesicle targeted drug delivery system GA-GELNs.
2. The extracellular vesicle targeted drug delivery system of Bletilla striata with the function of repairing liver damage according to claim 1, characterized in that: The preparation method of extracellular vesicle GELNs of white herb includes the following steps: adding white herb to phosphate buffer, soaking for 5 hours, first squeezing and crushing the juice with a juicer, filtering the squeezed white herb juice, and centrifuging at 4°C at 1000×g for 10 minutes, 3000×g for 20 minutes, and 10,000×g for 30 minutes in sequence to obtain the supernatant; using an ultrahigh-speed centrifuge to centrifuge the supernatant at 4°C for 2 hours at a speed of 150,000×g to obtain a precipitate; resuspending the precipitate with PBS, and filtering the obtained suspension through a 0.22 μm filter membrane to obtain a purified white herb vesicle GELNs suspension.
3. The extracellular vesicle targeted drug delivery system of Bletilla striata with the function of repairing liver damage according to claim 2, characterized in that: The usage ratio of white mustard and phosphate buffer saline (PBS) is 1:10 g / mL.
4. The extracellular vesicle targeted drug delivery system of Bletilla striata with the effect of repairing liver damage according to claim 2, characterized in that: Phosphate buffered saline (PBS) has a pH of 7.
4.
5. The method for preparing the extracellular vesicle targeted drug delivery system of Bletilla striata with the function of repairing liver damage according to claim 1, characterized in that: The GELNs suspension was mixed with the GA solution in a mass ratio of 1:2, incubated in a shaker at 37°C for 2 h, centrifuged at 12,000 rpm for 10 min, purified water was added to the precipitate, sonicated for 15 min and then stopped for 5 min, repeated three times, and centrifuged again at 12,000 rpm for 10 min. After centrifugation, the supernatant was transferred to a sample vial. The supernatant was GA-GELNs.
6. The method for preparing the extracellular vesicle targeted drug delivery system of Bletilla striata with the function of repairing liver damage according to claim 5, characterized in that: The concentration of the GELNs suspension was 2 mg / mL.
7. The method for preparing the extracellular vesicle targeted drug delivery system of Bletilla striata with the function of repairing liver damage according to claim 5, characterized in that: The concentration of GA solution was 1 mg / mL.
8. The method for preparing the extracellular vesicle targeted drug delivery system of Bletilla striata with the function of repairing liver damage according to claim 5, characterized in that: The drug loading capacity of GA in GA-GELNs was 38.76%.
9. Use of the extracellular vesicle targeted drug delivery system of Bletilla striata according to claim 1 in the preparation of a drug for repairing liver damage.
10. Use of the extracellular vesicle targeted drug delivery system of Bletilla striata according to claim 1 in the preparation of anti-liver cancer drugs.