Application of pomelo-derived exosomes in preparation of a medicine for preventing and treating vesicular stomatitis virus infection

Abstract

This invention belongs to the field of biomedical technology and discloses the application of grapefruit-derived exosomes in the preparation of drugs for the treatment and / or prevention of rhabdovirus-like virus infections. Through in vitro and in vivo experiments, this invention found that grapefruit-derived exosomes exhibit low cytotoxicity and significant inhibitory effects against vesicular stomatitis virus (VSV). By inhibiting the mRNA expression of the key VSV-P protein and the protein expression of VSV-G in VSV, it inhibits the production of progeny viruses by VSV, thus exerting an antiviral effect. The grapefruit-derived exosomes of this invention can serve as candidate active ingredients for the treatment and / or prevention of VSV infection and have good application value in the development of drugs against VSV infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of grapefruit-derived exosomes in the preparation of drugs for the prevention and treatment of vesicular stomatitis virus infection. Background Technology

[0002] Vesicular stomatitis virus (VSV) belongs to the genus Rhabdovirus of the family Rhabdoviridae and is the causative agent of vesicular stomatitis (VS), prevalent in many countries and regions in the Americas and Asia. In humans, VSV occasionally infects, especially among people in close contact with infected animals. According to the World Health Organization (WHO), VSV causes a large number of livestock infections globally each year, subsequently affecting occupational groups such as farm workers and veterinarians who come into contact with them. VSV is characterized by rapid spread, high infection rate, and transmission via insect vectors, and is considered a zoonotic disease of concern. Human infection with VSV primarily presents with mild, flu-like symptoms, including fever, muscle pain, headache, and fatigue; although serious complications are rare, it can lead to more severe illness in individuals with weakened immune systems. Although vesicular stomatitis virus (VSV) infection is relatively rare in humans, the risk of infection has increased in recent years due to climate change, increased international livestock trade, and closer proximity of wild animals to human habitats. In China, while VSV infection is still rare, the risk of its spread in the human population cannot be ignored given the increasing international trade and population movement.

[0003] Currently, there are no specific antiviral drugs targeting vesicular stomatitis virus (VSV), and treatment for human infection is primarily symptomatic and supportive. Vaccination is an important way to prevent animal diseases, but due to the low infection rate of VSV in humans, there are currently no vaccines specifically for human infection on the market. Furthermore, although some molecules with antiviral activity against VSV have been discovered, due to safety and side effects concerns, there are currently no widely used drugs targeting this virus. Therefore, the discovery of antiviral molecules against VSV has significant biological research and practical implications. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. It provides the application of grapefruit-derived exosomes in the preparation of drugs for the prevention and treatment of vesicular stomatitis virus infection. Through in vitro and in vivo experiments, this invention has found that grapefruit-derived exosomes can significantly inhibit the replication and infection of vesicular stomatitis virus, exhibiting good antiviral activity. Based on the above findings, grapefruit-derived exosomes can serve as candidate active ingredients for the treatment and / or prevention of vesicular stomatitis virus infection, and have good application value in the development of anti-VSV infection drugs.

[0005] The first aspect of this invention aims to provide the use of grapefruit-derived exosomes in the preparation of medicaments for the treatment and / or prevention of rhabdovirus and viroid infections.

[0006] A second aspect of the present invention aims to provide the use of grapefruit-derived exosomes in the preparation of medicaments for the treatment and / or prevention of vesicular stomatitis virus infection.

[0007] A third aspect of the present invention is to provide a pharmaceutical composition.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the invention provides the use of grapefruit-derived exosomes in the preparation of medicaments for the treatment and / or prevention of rhabdovirus viroid infections.

[0009] In some embodiments of the present invention, the rhabdovirus includes at least one of vesicular stomatitis virus, plant rhabdovirus, and fish rhabdovirus.

[0010] A second aspect of the invention provides the use of grapefruit-derived exosomes in the preparation of a medicament for the prevention and treatment of vesicular stomatitis virus infection.

[0011] In some embodiments of the present invention, the grapefruit-derived exosomes inhibit the mRNA expression of the key protein VSV-P and the protein expression of VSV-G in vesicular stomatitis virus.

[0012] In some embodiments of the present invention, the grapefruit-derived exosomes inhibit the production of progeny viruses by vesicular stomatitis virus.

[0013] In some embodiments of the present invention, the effective dose of grapefruit-derived exosomes in the drug is 10. 8 -10 11 particles / mL.

[0014] In some embodiments of the present invention, the effective dose of grapefruit-derived exosomes in the drug is 10. 9 -10 11 particles / mL.

[0015] In some embodiments of the present invention, the effective dose of grapefruit-derived exosomes in the drug is 10. 9 -10 10 particles / mL.

[0016] In some embodiments of the present invention, the method for preparing grapefruit-derived exosomes includes at least one of differential centrifugation, density gradient centrifugation, reagent kit extraction, size exclusion chromatography, filtration, polymer-based precipitation technology, immunoassay, and screening separation.

[0017] In some embodiments of the present invention, the method for preparing the grapefruit-derived exosomes is differential centrifugation.

[0018] In some embodiments of the present invention, the method for preparing grapefruit-derived exosomes includes the following steps: peeling and juicing grapefruit to obtain grapefruit juice, centrifuging to obtain the supernatant, ultracentrifuging to collect the precipitate, and resuspending it with buffer solution to obtain grapefruit-derived exosomes.

[0019] In some embodiments of the present invention, the method for preparing grapefruit-derived exosomes further includes a pre-peeling washing step.

[0020] In some embodiments of the present invention, the buffer solution includes PBS buffer.

[0021] In some embodiments of the present invention, the centrifugation conditions are: 300-600×g for 5-10 min, 1500-3000×g for 15-25 min, 4000-6000×g for 25-40 min, and 30000-50000×g for 50-70 min.

[0022] In some embodiments of the present invention, the centrifugation conditions are: 500×g centrifugation for 10 min, 2000×g centrifugation for 20 min, 5000×g centrifugation for 30 min, and 40000×g centrifugation for 1 h.

[0023] In some embodiments of the present invention, the ultracentrifugation conditions are: 120,000-200,000 × g for 1.5-3 h.

[0024] In some embodiments of the present invention, the ultracentrifugation conditions are: centrifugation at 150000×g for 2 hours.

[0025] In some embodiments of the present invention, the method for preparing grapefruit-derived exosomes includes the following steps: washing grapefruit with water, peeling, and juicing; centrifuging the obtained juice sequentially at 500×g for 10 min, 2000×g for 20 min, 5000×g for 30 min, and 40000×g for 1 h to remove residual pulp and fibrous material, and collecting the supernatant; ultracentrifuging the supernatant at 150000×g for 2 h to obtain a precipitate containing exosomes, and resuspending it in PBS solution to obtain grapefruit-derived exosomes.

[0026] In some embodiments of the present invention, the particle size of the grapefruit-derived exosomes is 30-300 nm.

[0027] In some embodiments of the present invention, the particle size of the grapefruit-derived exosomes is 50-150 nm.

[0028] In some embodiments of the present invention, the grapefruit-derived exosomes have a cup-like vesicle structure.

[0029] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0030] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of diluents, binders, wetting agents, disintegrants, lubricants, solubilizers, pH adjusters, isotonic adjusters, stabilizers, and preservatives.

[0031] In some embodiments of the present invention, the diluent includes at least one selected from starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, and calcium carbonate.

[0032] In some embodiments of the present invention, the adhesive comprises at least one selected from starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, and polyethylene glycol.

[0033] In some embodiments of the present invention, the wetting agent includes at least one of water, ethanol and isopropanol.

[0034] In some embodiments of the present invention, the disintegrant includes at least one of starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium, sodium carboxymethyl starch and polyoxyethylene.

[0035] In some embodiments of the present invention, the lubricant includes at least one of talc, silica, stearate, liquid paraffin, and polyethylene glycol.

[0036] In some embodiments of the present invention, the solubilizer includes at least one selected from ethanol, isopropanol, propylene glycol, polyethylene glycol, poloxamer, lecithin, and hydroxypropyl-β-cyclodextrin.

[0037] In some embodiments of the present invention, the pH adjuster further includes an acid-base adjuster and a buffer.

[0038] In some embodiments of the present invention, the acid-base regulator includes at least one of hydrochloric acid, sodium hydroxide, potassium hydroxide, citric acid, and lactic acid.

[0039] In some embodiments of the present invention, the buffer includes at least one selected from disodium hydrogen phosphate, sodium dihydrogen phosphate, citrate, sodium bicarbonate, acetate, citrate, lactate, histidine, glutamic acid, and glycine.

[0040] In some embodiments of the present invention, the isotonic regulator includes at least one of sodium chloride, mannitol, glycerol, sorbitol, xylitol, propylene glycol, glucose, phosphate, citrate, and acetate.

[0041] In some embodiments of the present invention, the stabilizer includes at least one of xanthan gum, sodium alginate, trehalose, and mannitol.

[0042] In some embodiments of the present invention, the preservative includes at least one of phenoxyethanol, sodium dehydroacetate, sodium benzoate, potassium sorbate, methylparaben, ethylparaben, propylparaben, butylparaben, and methylchloroisothiazolinone.

[0043] In some embodiments of the present invention, the dosage form of the drug includes at least one of injection, lyophilized powder, tablet, gel, and microsphere.

[0044] In some embodiments of the present invention, the route of administration of the drug includes at least one of oral, nasal, skin, intravenous, intramuscular, subcutaneous, and intraperitoneal perfusion and injection.

[0045] A third aspect of the invention provides a pharmaceutical composition comprising grapefruit-derived exosomes and pharmaceutically acceptable excipients.

[0046] In some embodiments of the invention, the pharmaceutical composition further includes other drugs for treating vesicular stomatitis virus infection.

[0047] In some embodiments of the present invention, the drug for treating vesicular stomatitis virus infection includes an antiviral drug.

[0048] In some embodiments of the present invention, the antiviral drug includes at least one of ribavirin, acyclovir, and ganciclovir.

[0049] In some embodiments of the present invention, the grapefruit-derived exosomes achieve antiviral effects by inhibiting the replication or release of vesicular stomatitis virus.

[0050] The beneficial effects of this invention are: This invention is the first to demonstrate that grapefruit-derived exosomes have a significant inhibitory effect on vesicular stomatitis virus (VSV). The antiviral effect is achieved by inhibiting VSV replication, thus providing a new research foundation and candidate direction for developing naturally derived, safe drugs against VSV infection. This invention also provides the application of grapefruit-derived exosomes in the preparation of drugs for the prevention and treatment of VSV infection. Grapefruit-derived exosomes exhibit low cytotoxicity and good inhibitory effect on VSV, showing a clear dose-dependent trend. This application not only broadens the biomedical applications of grapefruit-derived exosomes and provides crucial theoretical and data support for their clinical translation, but also offers new technical approaches for the prevention and treatment of VSV-related diseases, possessing significant scientific and clinical value and broad application prospects. Attached Figure Description

[0051] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The figures shown are characterization results of grapefruit-derived exosomes from Example 1. In the figures, A is a transmission electron microscope image of grapefruit-derived exosomes, B is a Zeta potential detection result of grapefruit-derived exosomes, C is a nanoparticle tracking analysis result of grapefruit-derived exosomes, and D is a dynamic light scattering detection result of grapefruit-derived exosomes.

[0052] Figure 2 This image shows the results of detecting the antiviral activity of grapefruit-derived exosomes using a fluorescence inverted microscope (A) and a multifunctional microporous detection system (B) in Example 4. Scale bar: 100 μm.

[0053] Figure 3 The figure shows the results of real-time quantitative PCR detection of the mRNA inhibition level of the key VSV protein VSV-L by grapefruit-derived exosomes in Example 5.

[0054] Figure 4 This is a graph showing the results of Western blotting analysis in Example 6 to detect the protein expression level of the key VSV virus protein VSV-G in grapefruit-derived exosomes.

[0055] Figure 5 Example 7: Grapefruit-derived exosomes (10 10The figure shows the inhibitory effect of the particle / mL assay on progeny virus production in the serum of mice infected with VSV. Each group was replicated three times, and whole-well plaque imaging results were obtained using a plaque assay, mainly to show the formation of plaques by infectious virus particles in the serum of different treatment groups. Detailed Implementation

[0056] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0057] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0058] HeLa cells, a type of human cervical cancer cell, are susceptible to vesicular stomatitis virus (VSV) infection and are one of the most commonly used cell types in VSV research. The HeLa cells (TCHu187), 293T cells (GNHu17), and Vero cells (SCSP-520) used in the experiment were purchased from the Cell Bank of the Chinese Academy of Sciences; the MTT assay kit for cell proliferation and cytotoxicity was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (C0009S); fetal bovine serum was purchased from Suzhou Ecosun Biotechnology Co., Ltd. (FCS500); cell culture plates were purchased from Corning Incorporated, Inc.; and DMEM medium was purchased from Thermo Fisher Scientific, Inc. (11965092).

[0059] This invention first examines the cytotoxicity of grapefruit-derived exosomes on HeLa cells. Different concentrations of grapefruit-derived exosomes were applied to HeLa cells to understand cell survival rates at different concentrations, providing reference data for subsequent experiments on the inhibitory effect of grapefruit-derived exosomes on VSV. Different concentrations (10... 8 10 9 10 10Grapefruit-derived exosomes (particles / mL) were applied to HeLa cells infected with VSV. The inhibitory efficiency of grapefruit-derived exosomes against the virus was obtained from four aspects: cell viability, viral protein level, mRNA level, and viral progeny yield. In this invention, the concentration of grapefruit-derived exosomes used was within the range that ensured a HeLa cell viability of over 80%, thus eliminating the possibility that excessively high concentrations of grapefruit-derived exosomes would affect the analysis of experimental data.

[0060] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0061] Example 1: Extraction and characterization of grapefruit-derived exosomes 1. Extraction of exosomes from grapefruit The specific steps are as follows: Grapefruit (genus: Citrus; order: Citrusales; subclass: Archaeocitrus; class: Dicotyledons; species: Pomelo) was purchased from a farmers' market. The grapefruit was washed three times with distilled water, peeled, and juiced. The juice was centrifuged sequentially at 500×g for 10 min, 2000×g for 20 min, 5000×g for 30 min, and 40000×g for 1 h to remove residual pulp and fiber. The supernatant was collected. The supernatant was then ultracentrifuged at 150000×g for 2 h to obtain a precipitate containing exosomes. This precipitate was resuspended in PBS solution to obtain grapefruit-derived exosomes (EVs). Pomelo ), stored at -80℃.

[0062] 2. Characterization of grapefruit-derived exosomes The specific steps are as follows: (1) Observation of exosome morphology using transmission electron microscopy (TEM): Take EV Pomelo The suspension was dropped onto a 300-mesh copper grid and allowed to settle at room temperature for 15 minutes to allow the sample to fully adhere to the grid surface. Excess liquid was then gently blotted away from the grid edges with filter paper, followed by negative staining with 5% phosphotungstic acid solution for 1 minute. After staining, excess staining solution was blotted away, and the grid was gently rinsed once with ultrapure water to remove any residual staining. The treated copper grid was then placed in a well-ventilated and dry place overnight. The following day, the grid was placed in the TEM sample chamber, the accelerating voltage was adjusted, and different fields of view were selected for observation. Clear images were captured to record the morphology of the exosomes.

[0063] (2) The concentration and particle size of exosomes were detected using a nano-tracking analyzer: Nanoparticle tracking analysis (NTA) was used to detect EVs. PomeloThe particle size distribution and particle concentration were determined. Before testing, the EVs were... Pomelo The sample was thoroughly mixed with sterile PBS buffer and diluted 1:1000 to a suitable detection concentration to reduce the impact of particle overlap and aggregation on the detection results. The nanoparticle tracking analyzer was then turned on and calibrated. The diluted sample was slowly injected into the sample cell, avoiding the formation of air bubbles. Detection parameters were set according to the particle motion state, and each sample was tested three times under the same conditions, with each acquisition time being 60 seconds. After detection, the instrument software automatically analyzed the particle size distribution and particle concentration based on the Brownian motion trajectory of the particles, and calculated the original EV based on the dilution factor. Pomelo Particle concentration of the sample.

[0064] (3) The size distribution and zeta potential were measured using dynamic light scattering (DLS) combined with Zetasizer Nano ZS: EV detection using dynamic light scattering method Pomelo The hydrated particle size distribution was analyzed, and EV was evaluated using Zeta potential analysis. Pomelo The surface charge characteristics of exosomes from 293T cells. Before detection, EVs... Pomelo The sample was diluted with deionized water at a ratio of 1:10 to a suitable detection concentration, and thoroughly mixed to reduce the impact of particle agglomeration on the detection results. The treated sample was then slowly added to a Malvern cuvette, taking care to avoid air bubbles and contamination. The cuvette was placed in a Zetasizer Nano ZS instrument, and appropriate detection temperature and scattering angle were set. The particle size distribution and zeta potential of the sample were measured sequentially. Each sample was measured three times under the same conditions, and the final result was the average of the three measurements for analysis.

[0065] The extraction method for exosomes from 293T cells was as follows: 293T cells were cultured in DMEM medium containing 10% fetal bovine serum to 80%-90% confluence; the old medium was discarded, the cells were washed twice with PBS, and the medium was replaced with serum-free and exosome-free medium for another 48 hours. The culture supernatant was collected, centrifuged at 3000 rpm for 15 minutes at 4°C to remove cell debris, then centrifuged at 12000 rpm for 30 minutes, and finally ultracentrifuged at 100000 rpm for 70 minutes. The precipitate was resuspended in PBS to obtain 293T cell exosomes, which were stored at 4°C for a short period.

[0066] The results of characterization of the extracted grapefruit-derived exosomes are as follows: Figure 1 As shown. Transmission electron microscopy results indicate that the exosomes derived from grapefruit exhibit a cup-like vesicle structure with a relatively intact membrane structure. Figure 1(A). Zeta potential detection results showed that the surface potential of exosomes derived from grapefruit was approximately 20 mV ( Figure 1 (B) Nanoparticle tracking analysis results showed that the average particle size of exosomes derived from grapefruit was 118.5 nm, and the particle size distribution was relatively concentrated. Figure 1 (C). Dynamic light scattering analysis showed that the dispersion index (PDI) of exosomes derived from grapefruit was 0.215, indicating good dispersibility and relatively uniform particle size distribution. Figure 1 (D).

[0067] Example 2: Toxicity of grapefruit-derived exosomes on HeLa cells HeLa cells were treated with different concentrations of grapefruit-derived exosomes, and the cell survival rate at these concentrations was detected. This provides reference data for subsequent experiments investigating the inhibitory effect of grapefruit-derived exosomes on VSV. The specific experimental steps are as follows: (1) Inoculation of HeLa cells: Prepare a single cell suspension in DMEM medium containing 10% (V / V) fetal bovine serum, and inoculate 10,000 cells per well into a 96-well cell culture plate with an inoculation volume of 100 μL per well; (2) Culture HeLa cells: Culture at 37℃ and 5% CO2 for 24 h; (3) Add grapefruit-derived exosomes: Discard the culture medium from each well and add 100 μL of DMEM medium diluted with 10% (V / V) fetal bovine serum to each well to the corresponding concentration (10 μL / V). 6 10 7 10 8 10 9 10 10 10 11 Grapefruit-derived exosomes (particles / mL) were added to the control wells, and 100 μL of DMEM medium containing 10% (V / V) fetal bovine serum without exosomes was added. (4) Color development: After culturing for 24 hours, add 10 μL of 5 mg / mL MTT solution to each well and continue culturing for 4 hours at 37°C and 5% CO2. Then add DMSO and observe under a regular optical microscope to find that Formazan (formazan, the product generated after MTT is reduced by living cells) has completely dissolved. (5) Measurement and calculation: The absorbance value was measured at 570 nm. Cell viability of grapefruit-derived exosomes at different concentrations = absorbance value at that concentration / absorbance value of the control well × 100%.

[0068] The results are shown in Table 1. The concentration of exosomes from grapefruit was 10. 10 At a particle / mL concentration, the survival rate of HeLa cells was 92.5%; the concentration of grapefruit-derived exosomes was 10.11 At a concentration of particles / mL, the survival rate of HeLa cells was 89.9%. No cytotoxicity was observed in HeLa cells after treatment with different concentrations of grapefruit-derived exosomes, indicating that grapefruit-derived exosomes have low cytotoxicity.

[0069] Table 1. Toxicity of different concentrations of grapefruit-derived exosomes on HeLa cells.

[0070] Example 3: Detection of antiviral activity of grapefruit-derived exosomes using the MTT assay (1) 50,000 HeLa cells were inoculated into each well of a 96-well cell culture plate. After 24 hours, the cells grew to a monolayer, covering approximately 80%-90% of the bottom of the well. The culture medium was aspirated, and the cells were washed once with PBS. 200 μL of serum-free DMEM diluted VSV-GFP virus sample (MOI=0.001) was then inoculated and incubated at 37°C for 1 hour. After incubation, the virus solution in each well was aspirated, and the cells were washed once with PBS. The VSV-GFP virus sample was prepared as follows: 293T cells were cultured in DMEM medium containing 10% fetal bovine serum to 80%-90% confluence. The cells were transfected with the VSV-GFP-related helper plasmid (Addgene, 11912), incubated at 37°C for 5-6 hours, and then replaced with fresh complete culture medium, and cultured for another 48 hours. The supernatant of transfected cells was transferred to another batch of cultured 293T cells and incubated at 37°C for 48-72 h. GFP expression was observed. After 40%-100% of cells showed pathological effects, the supernatant was collected and centrifuged at 450×g for 10 min or filtered through a 0.45μm filter membrane to remove cell debris, thus obtaining the amplified VSV-GFP virus.

[0071] (2) Add 100 μL of DMEM medium containing 10% (V / V) fetal bovine serum to each well to the specified concentration (10 μL / V). 8 10 9 10 10 Grapefruit-derived exosomes (particles / mL) were added to the virus control group, and 100 μL of DMEM medium containing 10% (V / V) fetal bovine serum was added and cultured at 37℃ under 5% CO2 conditions.

[0072] (3) After 24 hours, when obvious cytopathic effects appeared in the cells, 10 μL of 5 mg / mL MTT solution was added to each well, and the cells were cultured at 37°C and 5% CO2 for another 4 hours. Then, DMSO was added, and under a regular optical microscope, it was observed that Formazan (formazan, a product generated after MTT is reduced by living cells) was completely dissolved. The absorbance value was measured at 570 nm. Inhibition rate = (1 - absorbance value at grapefruit-derived exosome concentration / absorbance value of virus control group) × 100%.

[0073] The results are shown in Table 2. The inhibition rate of grapefruit-derived exosomes against VSV was within 10%. 10 At particle / mL, it has a good inhibitory effect on VSV infection of cells.

[0074] Table 2. Effects of different concentrations of grapefruit-derived exosomes on the survival rate of VSV-infected HeLa cells.

[0075] Example 4: Detection of antiviral activity of grapefruit-derived exosomes using a fluorescence inverted microscope and a multifunctional microporous detection system. HeLa cells were used at 10 5 Seeds were inoculated per well in 24-well plates and cultured overnight. The DMEM medium was discarded, and the plates were washed three times with PBS by gentle shaking. 200 μL of serum-free DMEM diluted VSV-GFP (MOI = 0.001) was added, and the plates were incubated at 37°C for 1 h. The plates were then washed three times with PBS. The exosomes to be tested were serially diluted with DMEM medium containing 2% (v / v) fetal bovine serum to a concentration of 10⁻⁶. 8 10 9 and 10 10 Particles / mL, 200 μL per well. A blank group (no VSV-GFP infection), a PBS group (VSV-GFP infection), and a ribavirin (Shanghai Taoshu Biotechnology Co., Ltd., T0684) positive drug group were set up. After incubation for 24 h, cell fluorescence was observed and photographed under a fluorescence inverted microscope (Nikon Corporation, Japan). The green fluorescence intensity was then quantified using a multifunctional microwell detection system (Excitation / Emission = 488 / 509 nm).

[0076] The results are as follows Figure 2 As shown in Figure A, cells in the PBS group exhibited a significant green fluorescent signal, indicating active viral replication. With the development of grapefruit-derived exosomes (EVs)... Pomelo Concentration from 10 8 The particles / mL gradually increased to 10 10As the number of particles / mL decreased, the GFP fluorescence intensity gradually decreased, reaching 10. 10 The fluorescence signal was significantly reduced in the particles / mL group, approaching the level of the blank group. The ribavirin group, as a positive control, also showed a significant inhibitory effect. Quantitative analysis of the fluorescence signal was performed ( Figure 2 (B) indicates that, compared with the PBS group (VSV-GFP infection), EV Pomelo The fluorescence intensity decreased significantly in all dose groups. P <0.0001), and showed a clear dose-dependent trend, 10 10 The particles / mL group showed the most significant inhibitory effect.

[0077] Example 5: Real-time quantitative PCR detection of the mRNA inhibitory level of grapefruit-derived exosomes on the key VSV protein VSV-L. VSV virus mainly contains five structural proteins: G, L, P, N, and M. The structural protein L is crucial, not only catalyzing RNA synthesis but also collaborating with the P protein to add and modify the RNA cap structure, directly determining the virus's reproductive capacity. This invention further confirms the inhibitory effect of grapefruit-derived exosomes on VSV by detecting the mRNA inhibition level of the L protein by different concentrations of grapefruit-derived exosomes. The specific experimental steps are as follows: (1) HeLa cells were inoculated into 6-well cell culture plates at a density of 100,000 cells / well. After 24 hours, the cells grew into a monolayer, covering approximately 80%-90% of the bottom of the well. The DMEM medium was aspirated, and the cells were washed once with PBS. 200 μL of serum-free DMEM medium was added to dilute the VSV-GFP virus sample (MOI=0.001), and the sample was incubated at 37°C for 1 hour. After incubation, the virus solution in each well was aspirated, and the cells were washed once with PBS.

[0078] (2) Add to DMEM medium containing 10% (V / V) fetal bovine serum diluted to the specified concentration (10 8 10 9 10 10 Grapefruit-derived exosomes (particles / mL) were cultured at 37°C under 5% CO2 conditions. A blank control group (not infected with VSV virus) and a virus control group (infected with VSV virus) were set up.

[0079] (3) After 24 hours, when obvious cytopathic effects appeared in the cells, total RNA was collected: 1 mL of Trizol reagent (15596-026, Ambion, Inc.) was added to each well of a 6-well cell culture plate, and after incubation at room temperature for 5 min, the supernatant was transferred to a 1.5 mL Eppendorf tube; 0.2 mL of chloroform was added to each tube, the plate was shaken, incubated at room temperature for 15 min, centrifuged at 12000 rpm at 4℃ for 15 min, and the upper phase was transferred to another 1.5 mL Eppendorf tube. Eppendorf tubes; add 0.5 mL isopropanol, vortex, incubate at room temperature for 10 min, centrifuge at 12000 rpm for 10 min at 4℃; discard the supernatant, add 1 mL of 75% (V / V) ethanol, wash the precipitate, centrifuge at 12000 rpm for 10 min at 4℃, discard the supernatant; air dry at room temperature until transparent; add 20 μL of DEPC-treated triple-distilled water to dissolve the RNA, store at -80℃ for later use; measure the 260 / 280 absorbance ratio with a UV spectrophotometer and calculate the RNA concentration.

[0080] (4) Reverse transcription of mRNA into cDNA: The reaction system consisted of 1 mg of RNA obtained in step (3), 4 μL of 5×PrimeScriptRT Master Mix (A6001, TAKARA), and DEPC-treated triple-distilled water to a total volume of 20 μL. After careful mixing, the mixture was 37℃ for 15 min and then 85℃ for 5 s.

[0081] (5) Real-time quantitative PCR detection of viral structural protein L RNA levels in each sample: Reaction system (10 v): 1 μL cDNA template from step (4), 5 μL GoTaq® qPCR Master Mix (Promega), 0.4 μL forward and reverse primers (10 μM) (Table 3), and 3.2 μL DEPC-treated triple-distilled water. After mixing, pre-denaturation was performed at 95℃ for 10 min, followed by 95℃ for 15 s and 60℃ for 1 min (40 cycles).

[0082] Table 3. Primers for target RNA and internal control

[0083] (6) Calculation: Based on the Ct value of each sample, use 2 -ΔΔCt The RNA levels of each sample relative to the virus control group were calculated using this method.

[0084] The results are as follows Figure 3 As shown, with increasing concentrations of grapefruit-derived exosomes, the mRNA level of viral structural protein L gradually decreased. 10Grapefruit-derived exosomes (particles / mL) completely inhibited the mRNA level of vesicular stomatitis virus (VSDV) structural protein L. This indicates that grapefruit-derived exosomes can suppress the mRNA level of the key protein L in VSDV.

[0085] Example 6: Western blot analysis of protein expression levels of VSV-G protein, a key VSV virus protein, in grapefruit-derived exosomes. VSV virus mainly contains five structural proteins: G, L, P, N, and M. VSV-G plays an important role in VSV virus replication and amplification. Therefore, this study investigated the inhibitory effect of different concentrations of grapefruit-derived exosomes on the VSV-G protein to further confirm the inhibitory effect of grapefruit-derived exosomes. This can be cross-validated with mRNA data to verify the inhibitory effect of grapefruit-derived exosomes on VSV virus. The specific experimental steps are as follows: (1) HeLa cells were inoculated into 6-well cell culture plates. After 24 hours, the cells grew to a monolayer, covering approximately 80%-90% of the bottom of the wells. The culture medium was aspirated, and the cells were washed once with PBS. 200 μL of serum-free DMEM medium was added to dilute the VSV-GFP virus sample (MOI=0.001), and the sample was incubated at 37°C for 1 hour. After incubation, the virus solution in each well was aspirated, and the cells were washed once with PBS.

[0086] (2) Add to DMEM medium containing 10% (V / V) fetal bovine serum diluted to the specified concentration (10 8 10 9 10 10 Grapefruit-derived exosomes (particles / mL) were cultured at 37°C under 5% CO2 conditions. A blank control group (not infected with VSV virus) and a virus control group (infected with VSV virus) were set up.

[0087] (3) After 24 hours, when obvious cytopathic effects appeared in the cells, total cell protein was collected: After HeLa cells were treated with drugs and vesicular stomatitis virus (VSV), protein was extracted 24 hours later: The cells were washed with cold PBS, and 100 μL of RIPA lysis buffer (Jiangsu Kaiji Biotechnology Co., Ltd., KGP702), phosphatase inhibitor (Jiangsu Kaiji Biotechnology Co., Ltd., KGB5101-2) and protease inhibitor (Jiangsu Kaiji Biotechnology Co., Ltd., KGB5101-100) were added to each well of a 6-well plate. The cells were scraped off and collected in 1.5 mL Eppendorf tubes, centrifuged at 12000×g for 15 min at 4℃, and the supernatant was transferred to a new Eppendorf tube.

[0088] (4) Protein concentration detection: The protein concentration assay kit (P0010, Beyotime Biotechnology Co., Ltd.) was used for detection according to its instructions. First, BSA protein standards were prepared and diluted to a 0.5 mg / mL standard solution. BCA working solution was prepared at a ratio of A:B = 50:1 and thoroughly mixed. Different volumes of BSA standards were added to 96-well plates and diluted to 20 μL to prepare standard gradients of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL. An appropriate amount of protein sample was added to each well and diluted to 20 μL. Then, 200 μL of BCA working solution was added to each well and incubated at 37°C for 20-30 min. After the reaction, the absorbance at 562 nm was measured using a microplate reader. The protein concentration of the samples was calculated based on the BSA standard curve. Data processing was performed by adding the appropriate volume of RIPA lysis buffer and loading buffer to each sample to ensure a consistent protein concentration. Denature at 100℃ for 5 minutes, then store at -20℃ for later use.

[0089] (5) Western blot analysis of protein expression differences in vesicular stomatitis virus proteins after treatment with grapefruit-derived exosomes at gradient concentrations: a) Preparation of 10% SDS-polyacrylamide gel: Prepare separating and stacking gels according to experimental requirements. The total volume of the separating gel is 48 mL. During preparation, add 16 mL of 30% Acrylamide-bis, 12 mL of 1.5 M Tris-HCl (pH 8.8), 19.2 mL of ddH2O, 480 μL of 10% SDS, and 720 μL of 10% APS sequentially. Finally, add 24 μL of TEMED, mix quickly, and pour into a gel casting plate to allow it to polymerize naturally.

[0090] The total volume of the stacking gel is 18 mL. To prepare it, add 2.25 mL of 30% Acrylamide-bis, 4.53 mL of 0.5 M Tris-HCl (pH 6.8), 10.8 mL of ddH2O, 190 μL of 10% SDS, and 270 μL of 10% APS in sequence, and finally add 18 μL of LTEMED. Mix quickly and pour the mixture onto the polymerized separating gel, insert a comb, and allow it to solidify completely before using it for electrophoresis experiments.

[0091] b) Electrophoresis: Turn on the power and start electrophoresis with a constant voltage of 80V. After the dye front enters the separating gel, change to 120V and run for 75 minutes.

[0092] c) Transfer: Remove the gel from the glass plate, cut a PVDF membrane to the size of the gel, and soak it in methanol for 1 min, then soak it in transfer buffer (1×Tris / Glycine Buffer, G800880-5kg, BT350-5kg) for 5 min. Immerse the gel, filter paper, and PVDF membrane in the transfer buffer. Assemble the transfer apparatus in the following order: negative electrode - cotton pad - filter paper - gel - PVDF membrane - filter paper - cotton pad - positive electrode, stacking each item carefully. Place the transfer apparatus in the transfer chamber, confirm the electrodes are correct, add transfer buffer until it covers the entire apparatus, and cover the transfer chamber and top with ice. Turn on the power and transfer at a constant voltage of 100V for approximately 70 min. After completion, remove the PVDF membrane.

[0093] d) Protein blocking: The PVDF membrane was placed in 5% skim milk blocking solution (PS112L, Shanghai Yamei Biomedical Technology Co., Ltd.) and soaked on a shaker at room temperature for 1 hour to block non-specific antigens. After blocking, the membrane was washed three times with TBST on a shaker at room temperature for 5 minutes each time, followed by incubation with primary antibody.

[0094] e) Primary antibody hybridization: Place the PVDF membrane protein side up in the small box, add 5% (w / v) skim milk buffer prepared with TBST containing the primary antibody for the vesicular stomatitis virus structural protein VSV-G (Anti-VSV-G antibody, 8G5F11, Kerafast), and incubate overnight at 4°C with shaking. Wash the membrane six times with PBST at room temperature with shaking, 5 min each time.

[0095] f) Secondary antibody hybridization: Place the PVDF membrane protein side up in a small box, add 5% (w / v) skim milk buffer containing secondary antibody (mouse antibody, FDM007, Hangzhou Fude Biotechnology Co., Ltd.), and shake at room temperature for 1 hour. Wash the membrane six times with TBST at room temperature for 5 minutes each time.

[0096] g) Chemiluminescence development: Add ECL developer A / B solution (P10100, Suzhou Xinsaimei Biotechnology Co., Ltd.) evenly according to the ratio, immerse the PVDF membrane in the luminescent solution, and react for 2 minutes. Remove the PVDF membrane, place it in a dark box, expose it in a dark room, place an X-ray film, expose it again, remove and develop the film, dry the film, record the data, and store it.

[0097] The results are as follows Figure 4 As shown, with increasing concentrations of grapefruit-derived exosomes, the protein level of the viral structural protein VSV-G gradually decreased. 10 The particles / mL assay can inhibit the expression of the viral structural protein VSV-G. This indicates that grapefruit-derived exosomes can suppress the expression level of the key protein VSV-G in vesicular stomatitis virus.

[0098] Example 7 Plaque inhibition experiment of grapefruit-derived exosomes on VSV progeny virus production Plaque inhibition assay is an important indicator for detecting viral titer and infectivity. After viral infection of cells, due to the limitation of the solid medium, the released virus can only spread from the initially infected cells to the surrounding areas. After several proliferation cycles, a localized lesion cell area is formed, which is the viral plaque. After staining and washing, the viral plaque will not be stained, thus appearing as a blank, while the remaining cells remain intact and appear blue. Whole blood was collected from mice, and the supernatant was centrifuged for viral plaque assay to further confirm the levels of viral content, titer, and infectivity in mouse serum after treatment with grapefruit-derived exosomes, and to determine the anti-VSV progeny virus effect of grapefruit-derived exosomes. The specific experimental steps are as follows: (1) C57BL / 6 mice were fixed with their abdomens facing upwards. The needle was inserted into the lower lateral abdomen, avoiding the internal organs. A solution of 10... 10 Inject 200 μL of grapefruit-derived exosomes (prepared with PBS solution) with particles / mL. Gently massage the abdomen after needle removal to prevent leakage. Inject once daily. Collect samples 7 days after injection.

[0099] (2) Obtaining serum samples: Carefully trim the mouse whiskers with scissors to reduce the risk of hemolytic reaction. Gently press the skin around the mouse's eyes with tweezers to gradually protrude the eyeballs. Then, quickly grasp the eyeballs with curved tweezers and guide the blood into the pre-prepared EP tubes. After blood collection, wait for the blood to stop flowing, and immediately euthanize the mouse using the spinal dislocation method.

[0100] (3) Serum sample processing: The collected blood samples were allowed to stand at room temperature for 30 minutes to promote natural coagulation. Then, the blood samples were centrifuged at 3000 rpm for 15 minutes. After centrifugation, the supernatant (i.e., serum) was carefully aspirated and aliquoted into appropriate containers. The aliquoted serum samples were immediately stored in a -80°C ultra-low temperature freezer for subsequent experimental use or other processing.

[0101] (4) Plaque assay for viral titer in serum: One day before serum sampling, Vero cells were seeded at an appropriate density in 24-well plates to ensure the cells were in the logarithmic growth phase at the start of the experiment. The serum samples were diluted 10-fold to reduce the potential impact of impurities in the serum on the experimental results. The diluted serum samples were filtered through a 0.2 μm pore size filter membrane to remove any cell debris and other macromolecules.

[0102] The filtered serum samples were serially diluted 10-fold to obtain different concentrations. Each dilution was then added to a 24-well plate pre-coated with Vero cells, with at least three replicates per dilution. The 24-well plates were incubated at 37°C for 1 hour to allow for sufficient virus contact and adsorption to the cells.

[0103] Culture and Plaque Formation: After 1 hour of culture, add an appropriate amount of semi-solid culture medium (containing 0.8% methylcellulose) to maintain normal cell growth and limit viral spread. Continue culturing the 24-well plate in a 37°C incubator for 48-72 hours to promote viral replication and plaque formation within the cells.

[0104] After plaque formation, the methylcellulose covering medium was removed and the cells were fixed with 4% paraformaldehyde (G1101-500ML, Wuhan Saiweier Biotechnology Co., Ltd.). The cells were then stained with 1% (w / v) crystal violet. After 2 hours, the crystal violet was rinsed off with running water, and the cells were dried and scanned in 12-well plates. The inhibitory effect of grapefruit-derived exosomes on the production of progeny viruses in Vero cells infected with VSV was determined based on the number of plaques.

[0105] like Figure 5 As shown, the viral progeny replication level gradually decreased with increasing grapefruit-derived exosome concentration, with the grapefruit-derived exosome concentration at 10... 10 Viral progeny could not be released when the concentration of particles / mL was low. This indicates that grapefruit-derived exosomes can inhibit the replication level of vesicular stomatitis virus progeny.

[0106] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of grapefruit-derived exosomes in the preparation of drugs for the treatment and / or prevention of rhabdovirus and viroid infections.

2. Application of grapefruit-derived exosomes in the preparation of drugs for the treatment and / or prevention of vesicular stomatitis virus infection.

3. Use according to claim 2, characterized in that, The grapefruit-derived exosomes inhibited the mRNA expression of VSV-P and the protein expression of VSV-G, key proteins in vesicular stomatitis virus. Preferably, the grapefruit-derived exosomes inhibit the production of progeny viruses by vesicular stomatitis virus.

4. Use according to claim 1 or 2, characterized in that, The effective dose of the grapefruit-derived exosomes in the drug is 10 8 -10 11 particles / mL.

5. The application according to any one of claims 1-3, characterized in that, The preparation method of grapefruit-derived exosomes includes at least one of differential centrifugation, density gradient centrifugation, reagent kit extraction, size exclusion chromatography, filtration, polymer-based precipitation technology, immunoassay, and screening separation.

6. The application according to claim 5, characterized in that, The method for preparing grapefruit-derived exosomes includes the following steps: peeling grapefruit and juicing it to obtain grapefruit juice, centrifuging to collect the supernatant, ultracentrifuging to collect the precipitate, and resuspending it with buffer solution to obtain grapefruit-derived exosomes.

7. The application according to claim 6, characterized in that, The centrifugation conditions are as follows: 300-600×g for 5-10 min, 1500-3000×g for 15-25 min, 4000-6000×g for 25-40 min, and 30000-50000×g for 50-70 min. Preferably, the ultracentrifugation conditions are: 120,000-200,000 × g for 1.5-3 hours.

8. The application according to claim 1 or 2, characterized in that, The exosomes derived from grapefruit have a particle size of 30-300 nm.

9. The application according to claim 1 or 2, characterized in that, The drug also includes pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include at least one of diluents, binders, wetting agents, disintegrants, lubricants, solubilizers, pH adjusters, isotonic adjusters, stabilizers, and preservatives.

10. A pharmaceutical composition comprising grapefruit-derived exosomes and pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition further includes other drugs for treating vesicular stomatitis virus infection.

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