Plant-derived apoptosis-imitating extracellular vesicle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano biology, and discloses a plant-derived apoptosis-imitating extracellular vesicle as well as a preparation method and application of the plant-derived apoptosis-imitating extracellular vesicle. The preparation method comprises the following steps: extracting a plant source outer vesicle from a plant tissue, and co-fusing the plant source outer vesicle with a phosphatidylserine (PS) solution containing an apoptosis signal to obtain the plant source apoptosis-imitating extracellular vesicle. The plant-derived apoptotic extracellular vesicles disclosed by the invention are derived from natural plants, and compared with apoptotic cell vesicles derived from mammalian cells, the plant-derived apoptotic extracellular vesicles have good biocompatibility and are not easy to cause immunological rejection of organisms; compared with a natural plant-derived extracellular vesicle, the plant-derived apoptotic extracellular vesicle modified by phosphatidylserine (PS) has the advantages that the macrophage targeting property, the macrophage inflammatory reaction inhibition effect and the RA improvement effect of the plant-derived apoptotic extracellular vesicle are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the field of nano-biotechnology, and in particular relates to a plant-derived apoptosis-mimicking extracellular vesicle and a preparation method and application thereof. Background Art

[0002] Depending on the activation mode, macrophages Divided into two categories: classic activation (M1 type) and alternatively activated (M2 type). Classically activated (M1 type) Induced by interferon-γ (IFN-γ) and / or microbial stimulation (such as lipopolysaccharide LPS). M1 macrophages exhibit a pro-inflammatory phenotype and produce cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, which are essential for host resistance to pathogens. (M2 type) is induced by interleukin-4 (IL-4), IL-13 or IL-10 and is usually associated with tissue repair and remodeling. M2 macrophages produce anti-inflammatory cytokines, such as IL-10 and transforming growth factor-β (TGF-β), as well as extracellular matrix proteins that promote tissue repair. When infection or inflammation is severe enough to affect an organ, macrophages first exhibit the M1 phenotype, releasing TNF-α, IL-1β, IL-12 and IL-23 to counteract the stimulus. However, if the M1 phase continues, tissue damage will occur. Therefore, M2 macrophages secrete large amounts of IL-10 and TGF-β to inhibit inflammation, contribute to tissue repair, remodeling, angiogenesis and maintain homeostasis.

[0003] Plant-derived nanovesicles (PNVs) are lipid bilayer membrane nanovesicles from plant cells with a diameter of 30-150 nm. They can secrete and deliver a variety of bioactive molecules, such as proteins, nucleic acids, and secondary metabolites, outside the cells, and have high biocompatibility and stability. Increasing evidence shows that plant-derived nanovesicles can enter mammalian cells and mediate plant-animal cross-kingdom gene regulation, which indicates that plant-derived nanovesicles have potential medical applications in regulating basic biological processes in the human body, such as immune system regulation, drug and nucleic acid delivery. In recent years, more and more studies have focused on the potential application of plant-derived nanovesicles in the treatment of inflammatory diseases. Studies have shown that plant extracellular vesicles can treat inflammatory diseases through multiple mechanisms such as regulating immune responses, reducing oxidative stress, and inhibiting inflammatory pathways. Nanovesicles extracted from plants such as ginseng, ginger, grapefruit, and houttuynia cordata have anti-inflammatory, antioxidant, and immunomodulatory effects in macrophages, and can be used to treat a variety of inflammatory diseases, such as lung injury, colitis, and rheumatoid arthritis. Therefore, plant extracellular vesicles, as a new therapeutic strategy, have great application potential and will become a research hotspot in the field of inflammatory disease treatment. At present, how to improve its ability to target and regulate immune cells and obtain satisfactory therapeutic effects is still a difficult problem to be overcome.

[0004] Apoptotic extracellular vesicles (ApoVs), which are vesicles produced during apoptosis, are important mediators of intercellular communication and play an important role in various biological processes such as cell growth and development, aging, renewal, tumorigenesis, and immune regulation. Surface receptors bind to regulate polarization and function, promoting its transformation to M2 type. This process enhances The phagocytic and anti-inflammatory abilities of ApoVs induce the release of anti-inflammatory factors such as IL-10 and TGF-β, while inhibiting inflammatory factors such as TNF-α and IL-6, thereby effectively inhibiting inflammation and promoting tissue repair. Targeted bioactive materials have broad prospects in regulating inflammatory responses and injury repair treatment.

[0005] However, this also faces some significant problems and challenges. At present, ApoVs used to treat diseases are mainly derived from mammals, and their potential toxicity, including immunogenicity and off-target effects, still need further research and evaluation. Existing production methods mainly use bioreactors and other equipment for large-scale cell culture, but when large-scale production is carried out in bioreactors, it may be more difficult to control factors such as culture medium composition, cell type, and culture time. The separation and purification operations in the bioreactor are also more complicated, resulting in certain fluctuations in the biological activity of the prepared ApoVs, thereby affecting its therapeutic effect. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a preparation method and application of plant-derived apoptosis-mimicking extracellular vesicles.

[0007] The present invention provides a method for preparing plant-derived apoptosis-mimicking extracellular vesicles, comprising the following steps:

[0008] S1. Extracting plant-derived exosomes from plant tissues;

[0009] S2. Co-fusion of the plant-derived extracellular vesicles described in step S1 with a solution containing phosphatidylserine (PS) to obtain plant-derived apoptosis-mimicking extracellular vesicles.

[0010] The preparation method of the plant-derived apoptosis-mimicking extracellular vesicles of the present invention uses natural plant-derived extracellular vesicles as raw materials, and after modification with phosphatidylserine (PS), the immunomodulatory ability of the extracellular vesicles is enhanced, providing a scientific basis for the bionic modification of plant-derived extracellular vesicles. Compared with the preparation method of apoptotic extracellular vesicles cultured by cells, the preparation method of the present invention is simple to operate, and the biological activity of the prepared plant-derived apoptosis-mimicking extracellular vesicles is relatively stable, and has a good therapeutic effect on inflammatory diseases.

[0011] Furthermore, the preparation method further comprises the following steps:

[0012] S3, extruding the plant-derived apoptosis-mimicking extracellular vesicles described in step S2 through a membrane with a pore size of 50 nm to 400 nm to obtain plant-derived apoptosis-mimicking extracellular vesicles of corresponding particle size.

[0013] Furthermore, the PS in step S2 is L-α-phosphatidylserine, dipalmitoylphosphatidylserine (DPPS) or dioleoylphosphatidylserine (DOPS).

[0014] Furthermore, the co-fusion method in step S2 is to mix the plant-derived extracellular vesicles in step S1 with PS-containing liposomes (PSLs), and react at 30 to 40° C. for 30 to 120 minutes.

[0015] Furthermore, the quantitative ratio of the plant-derived extracellular vesicles to PSLs is 3-6:1-3.

[0016] Furthermore, the preparation method of the PSLs comprises the following steps: dissolving and mixing phospholipids and cholesterol, and evaporating to obtain a lipid film; hydrating the lipid film, and ultrasonicating at a power of 100W to 1000W for 1 to 30 minutes to obtain the PSLs.

[0017] Furthermore, the phospholipids include one or more of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PS.

[0018] Furthermore, the molar ratio of PS to PSLs is 0.5-3.0:7.0-9.5.

[0019] The present invention also provides a plant-derived apoptosis-mimicking extracellular vesicle, which is a plant-derived apoptosis-mimicking extracellular vesicle prepared by the preparation method provided by the present invention.

[0020] The plant-derived apoptosis-mimicking extracellular vesicles of the present invention are derived from natural plants, and compared with apoptotic extracellular vesicles derived from mammalian cells, have good biocompatibility and are less likely to cause immune rejection reactions in the body; after being modified with phosphatidylserine (PS), compared with natural plant-derived extracellular vesicles, the plant-derived apoptosis-mimicking extracellular vesicles of the present invention have significantly improved macrophage targeting, inhibition of macrophage inflammatory response and improvement of RA.

[0021] The present invention also provides an application of plant-derived apoptosis-mimicking extracellular vesicles in drugs for treating inflammation.

[0022] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 Transmission electron microscopy images of plant-derived apoptotic extracellular vesicles prepared in Examples 1 and 2;

[0025] Figure 2 Surface characteristics analysis diagram of plant-derived apoptotic extracellular vesicles prepared for Examples 1 and 2;

[0026] Figure 3 The macrophage compatibility evaluation results of the plant-derived apoptotic extracellular vesicles prepared in Examples 1 and 2;

[0027] Figure 4 The results of the evaluation of the macrophage targeting ability of the plant-derived apoptotic extracellular vesicles prepared in Examples 1 and 2;

[0028] Figure 5 The results of the evaluation of the plant-derived apoptosis-mimicking extracellular vesicles prepared in Examples 1 and 2 for inhibiting the inflammatory response of M1 macrophages in vitro, FIG. A shows the effect of the plant-derived apoptosis-mimicking extracellular vesicles on the expression of TNF-α, FIG. B shows the effect of the plant-derived apoptosis-mimicking extracellular vesicles on the expression of IL-1β, FIG. C shows the effect of the plant-derived apoptosis-mimicking extracellular vesicles on the expression of IL-6, and FIG. D shows the effect of the plant-derived apoptosis-mimicking extracellular vesicles on the expression of iNOS;

[0029] Figure 6 The visual image and X-ray image of the left hind foot of a rat after the plant-derived apoptosis-mimicking extracellular vesicles were prepared for the treatment of RA in Example 2. FIG. A is a visual image of the injured part of the left hind foot of the rat, and FIG. B is an X-ray image of the left hind foot of the rat;

[0030] Figure 7 The evaluation results of preparing plant-derived apoptosis-mimicking extracellular vesicles for treating RA in Example 2, FIG. A is a scoring diagram of the injury degree of the left hind foot of a rat, and FIG. B is a test result of the joint thickness of the left hind foot of a rat;

[0031] Figure 8 The evaluation results of the plant-derived apoptosis-mimicking extracellular vesicles prepared in Example 2 for inhibiting the inflammatory response of RA synovial tissue, Figure A shows the expression of synovial TNF-α gene, Figure B shows the effect of plant-derived apoptosis-mimicking extracellular vesicles on synovial IL-6 gene expression, and Figure C shows the effect of plant-derived apoptosis-mimicking extracellular vesicles on synovial iNOS gene expression;

[0032] Fig. 9 The evaluation results of serum inflammatory factors after the plant-derived apoptosis-mimicking extracellular vesicles were prepared in Example 2 to treat RA. Figure A shows the effect of plant-derived apoptosis-mimicking extracellular vesicles on serum TNF-α gene expression, and Figure B shows the effect of plant-derived apoptosis-mimicking extracellular vesicles on serum IL-6 gene expression. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] For purposes of the present invention, the following terms are defined below.

[0035] Throughout the specification and claims, unless otherwise required, the following words "comprise" and variations "comprising" and "including" will be understood to imply the inclusion of a stated integer or step, or a group of integers or steps, but not the exclusion of any other integer or step, or other group of integers or steps.

[0036] According to literature reports, plant extracellular vesicles have almost no PS signals. The inventors envision that constructing plant-derived apoptotic-cell-inspired nanovesicles (P-ApoVs) will have the following advantages: plant-derived apoptotic-cell-inspired nanovesicles are derived from natural plants, have good biocompatibility, and are not likely to cause immune rejection reactions in the body; plant-derived apoptotic-cell-inspired nanovesicles carry apoptosis signals on their surfaces, which can specifically recognize and Phagocytosis, thereby achieving targeted delivery; plant-derived apoptotic extracellular vesicles can promote Transformation to M2 type, M2 type It has phagocytic and anti-inflammatory abilities, can release anti-inflammatory factors and inhibit inflammatory responses. The constructed plant-derived mimicking apoptotic extracellular vesicles will combine the advantages of plant extracellular vesicles and apoptotic extracellular vesicles, and is expected to become a new therapeutic strategy for the treatment of inflammatory diseases.

[0037] Embodiment 1: Preparation of plant-derived apoptotic extracellular vesicles (P-ApoVs1)

[0038] Wash 5g of Dendrobium officinale stem segments repeatedly with sterile water or deionized water to remove surface dust and microorganisms. Let the washed Dendrobium officinale stem segments dry naturally. Mix the dried Dendrobium officinale stem segments with 25mL phosphate buffer solution (pH 7.0) and incubate for 12h. Add pectinase at a concentration of 1mg / g stem segments and cellulase at a concentration of 2mg / g stem segments for enzymatic hydrolysis for 12h at a temperature of 24°C. Collect all the juice after enzymatic hydrolysis and centrifuge at 3000g for 30min. Collect the supernatant and centrifuge it at 10000g for 1.5h to take the supernatant. Use an ultrahigh-speed centrifuge to centrifuge the supernatant for 2.5h at a speed of 150000g. Take the precipitate after ultrahigh-speed centrifugation. Sucrose gradient (sucrose solution is 8%, 30%, 45% and 60% (g / v)) centrifugation method and ultra-high speed centrifuge at 150000g for 1.5h. Select the 30-45% layer to extract the purified Dendrobium officinale exovesicles, and draw 4mL. Add 5 times the volume of PBS, remove the sucrose with an ultrafiltration tube and replace it with PBS. Through the above steps, high-purity Dendrobium officinale-derived nanovesicles (DONVs) can be obtained.

[0039] 1 μmol of dioleoylphosphatidylserine (DOPS), 7 μmol of phosphatidylcholine and 2 μmol of cholesterol were mixed and dissolved in 10 mL of organic solvent; the organic solvent was chloroform and ethanol, the volume ratio was chloroform: ethanol = 9:1, and the mixture was shaken and mixed evenly. The organic solvent was evaporated by rotary evaporator (40°C, 20 min) to obtain a lipid film. The lipid film was hydrated with 1 mL of PBS solution to obtain a turbid PS liposome solution. Use a probe sonicator to sonicate for 1 min, 150 W, until the solution is clear and transparent. The particle size of the PS liposome was homogenized by an extruder and maintained at about 200 nm.

[0040] PS liposomes (PSLs) and DONVs suspension were mixed in a quantitative ratio of 3:1 and incubated at 37°C for 60 minutes. Mechanical stirring was performed during the incubation process to promote the fusion of PSLs and DONVs. The co-fused mixture was passed through an extruder to homogenize the particle size and maintain it at about 200 nm, which is P-ApoVs1.

[0041] Its TEM morphology is as follows Figure 1 As shown, the morphology of P-ApoVs1 and DONVs is spherical, and the particle size measured by the particle size analyzer is 191.12±2.43nm and 223.62±2.90nm, respectively, and the surface potential is -19.81±0.68mV and -6.69±0.46mV, respectively. Figure 1 ).

[0042] Embodiment 2: Preparation of plant-derived apoptotic extracellular vesicles (P-ApoVs2)

[0043] 5 g of Clematis filamentosa leaves were repeatedly washed with sterile water or deionized water to remove surface dust and microorganisms. Other operating conditions were exactly the same as in Example 1 to prepare Clematis filamentosa-derived nanovesicles (CFNVs) and P-ApoVs2. The TEM morphology of the CFNVs and P-ApoVs2 was as follows: Figure 1 As shown in the figure, the morphology of P-ApoVs2H and CFNVs are both spherical. The particle sizes measured by the particle size analyzer were 202.26±3.55nm and 183.51±1.09nm, respectively, and the surface potentials were -28.72±0.54mV and -8.49±0.21mV, respectively. Figure 1 ).

[0044] Embodiment 3: Experimental evaluation of plant-derived apoptotic extracellular vesicles of Example 1-2

[0045] RA (rheumatoid arthritis) is a chronic systemic disease characterized by inflammatory synovitis. In the pathogenesis of RA, a variety of serum inflammatory factors such as TNF, IL-1, IL-6, etc. play a key role. They are produced by activated immune cells (such as macrophages, T lymphocytes, etc.) and mediate inflammatory responses, leading to pathological changes such as synovial congestion, edema, and exudation. These inflammatory factors can also promote the formation of pannus and cartilage destruction, further aggravating joint swelling, osteoporosis, and cartilage damage. The level of inflammatory factors in serum is often closely related to the disease activity and severity of RA. By detecting the level of inflammatory factors in serum, the progression of the disease and the treatment effect of RA patients can be evaluated. For example, elevated levels of inflammatory factors such as TNF-α and IL-6 usually indicate that RA is in the active stage, and after effective treatment, the levels of these factors will gradually decrease. Targeted therapy for M1 macrophages has become one of the important strategies for the treatment of RA.

[0046] Evaluation experiment 1: Surface characteristics evaluation

[0047] Annexin V is a reagent for detecting cell apoptosis. In normal cells, phosphatidylserine (PS) is only distributed on the inner side of the lipid bilayer of the cell membrane. However, in the early stages of apoptosis, PS flips from the inner side of the cell membrane to the outer side of the cell membrane and is exposed to the extracellular environment. Annexin V, as a phospholipid-binding protein, can specifically bind to the everted PS. Therefore, the early stages of apoptosis can be detected by Annexin V. Annexin V is usually labeled with a fluorescent dye such as FITC (Annexin V-FITC) and used as a fluorescent probe to detect cell apoptosis in combination with flow cytometry or fluorescence microscopy.

[0048] Operation steps: Prepare 100 μL samples of experimental group 1-2, control group 1-2 and blank group of evaluation experiment 1 (the grouping scheme of evaluation experiment 1 is shown in Table 1) and place them in EP tubes, add a medium volume of Annexin binding buffer of the apoptosis kit, and mix well. Add 5 μL Annexin V-FITC and mix well, and incubate at room temperature in the dark for 30 minutes. After incubation, add 500 μL PBS to wash, and use a 100 kDa molecular cutoff ultrafiltration centrifuge tube (MilliPore) for concentration to remove excess Annexin V-FITC solution. Collect 100 μL of sample, then add 200 μL PBS solution and mix well, and detect on the machine. Detect by flow cytometry (BD FACSCanto), excitation wavelength Ex = 488 nm, emission wavelength Em = 525 nm, collect data. Data are analyzed by FlowJo_V10 software.

[0049] Table 1: Grouping scheme for evaluation experiment 1

[0050] Group sample Experimental group 1 (P-ApoVs1) P-ApoVs1 solution (108 cells / mL) Experimental group 2 (P-ApoVs2) P-ApoVs2 solution (108 cells / mL) Control group 1 (DONVs) DONVs solution (108 / mL) Control group 2 (CFNVs) CFNVs solution (108 cells / mL) Blank group (PS absent-NPs) Liposomes without PS (equal volume)

[0051] Results: Figure 2 As shown, compared with the blank group (PS absent-NPs), the difference in the proportion of surface-bound Annexin V nanoparticles between the control group 1 (DONVs) and the control group 2 (CFNVs) is very small, and the values ​​of the three groups are all around 1%, indicating that there is almost no apoptosis signal phosphatidylserine (PS) on the surface of the plant-derived extracellular vesicles; compared with the control group 1 (DONVs) and the control group 2 (CFNVs), respectively, the proportion of surface-bound Annexin V nanoparticles in the experimental group 1 (P-ApoVs1) and the experimental group 2 (P-ApoVs2) is more than 95%, indicating that the plant-derived apoptosis-mimicking extracellular vesicles obtained by modifying the plant-derived extracellular vesicles by the preparation method of the present invention have apoptosis signal phosphatidylserine (PS) on the surface.

[0052] Evaluation experiment 2: Cytocompatibility evaluation

[0053] CCK-8, the full name of Cell Counting Kit-8, is a cell proliferation and cytotoxicity detection kit based on water-soluble tetrazolium salt (WST-8). WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolyl monosodium salt) in the CCK-8 kit is reduced to a highly water-soluble orange-yellow formazan product (Formazan) by dehydrogenase in the cell mitochondria under the action of electron carriers (such as 1-methoxy-5-methylphenazinium dimethyl sulfate, 1-Methoxy PMS). The amount of formazan generated is proportional to the number of living cells, so the number of living cells can be indirectly reflected by measuring its light absorption value (at a wavelength of 450nm). The calculation formula is: Cell survival rate (%) = [(A1-A0) / A0]×100%. Among them, A0: absorbance value of blank control, A1: absorbance value of sample treatment group.

[0054] Procedure: 10,000 RAW264.7 cells were plated in a 96-well plate and placed in a CO 2Culture in an incubator overnight. Take samples from the experimental group 1-2 and the control group 1-2 of the evaluation experiment 2 (the grouping scheme of the evaluation experiment 2 is shown in Table 2), and dilute them into a series of sample dilutions of different concentrations (0.5×108 cells / mL, 1×108 cells / mL, 2×108 cells / mL, 4×108 cells / mL, 8×108 cells / mL) with DMEM culture medium. Treat the cultured RAW264.7 cells with the sample dilution for 48 hours. Take 10μL CCK-8 reagent and add it to the culture medium of each well, and culture it in an incubator at 37°C and 5% CO2 for 2 hours. Detect the absorbance value of each well at a wavelength of 450nm.

[0055] Table 2: Grouping scheme for evaluation experiment 2

[0056] Group sample Experimental group 1 (P-ApoVs1) P-ApoVs1 suspension Experimental group 2 (P-ApoVs2) P-ApoVs2 suspension Control group 1 (DONVs) DONVs suspension Control group 2 (CFNVs) CFNVs suspension

[0057] Results: Figure 3 As shown, at 0.5×10 8 / mL to 8×10 8 Within the concentration range of P-ApoVs1 / mL, after incubation with macrophages for 48 hours, the cell survival rates of experimental group 1 (P-ApoVs1), experimental group 2 (P-ApoVs2), control group 1 (DONVs) and control group 2 (CFNVs) were all maintained above 85%, indicating that the plant-derived extracellular vesicles and the plant-derived apoptosis-mimicking extracellular vesicles modified by the preparation method of the present invention showed good cytocompatibility with macrophages.

[0058] Evaluation experiment 3: Evaluation of macrophage targeting ability

[0059] DAPI, the full name of which is 4',6-diamidino-2-phenylindole, is a nuclear staining reagent that can stain DNA. It releases blue fluorescence after being embedded in double-stranded DNA.

[0060] Fluorescein-DHPE, the full name of which is N-(fluorescein-5-thiocarbamide)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, triethylammonium acetate, is a phospholipid fluorescent dye used to mark cell membranes or specific structures within cells and release green fluorescence.

[0061] Lyso-Tracker Red is a lysosome red fluorescent probe that can selectively remain in acidic lysosomes, thereby achieving specific red fluorescent labeling of lysosomes.

[0062] Lysosomes are important degradation systems in cells that can degrade various substances that enter the cell, including nano-exosomes. Due to their small particle size and specific surface properties, exosomes are often more easily taken up by cells. Once entering the cell, the co-localization degree of exosomes and lysosomes is low, reflecting that they can escape capture and degradation by lysosomes, thus stably existing in the cell and having a certain concentration in the cell. This stability increases the interaction time and opportunity between exosomes and targets, improving the targeting and efficacy of exosomes.

[0063] Procedure: Plate 10 5 RAW264.7 cells / well were placed in CO 2 Incubate overnight in an incubator. The cultured RAW264.7 cells were treated with samples of experimental group 1-2 and control group 1-2 of evaluation experiment 2 labeled with Fluorescein-DHPE (concentration of 108 cells / mL) (the grouping scheme of evaluation experiment 3 is shown in Table 3) for 24 hours. After 24 hours, all the culture medium was aspirated and washed three times with PBS, each time for 5 minutes, and attention was paid to avoid light. Add 4% paraformaldehyde to fix for 15 minutes and wash three times with PBS. Add 10uM Lyso-Tracker Red and act at room temperature for 20 minutes, and wash three times with PBS. Stain with 10μg / mL LDAPI solution for 10 minutes. Add anti-fluorescence quencher and take pictures with a confocal microscope (LSM 880 with AiryScan, Carl Zeiss) in a short time. Set the wavelength, green light Ex488, Em535; red light Ex543, Em611. Image analysis was performed using ZEN2.3 (Blue Edition, Carl Zeiss) software.

[0064] Table 3: Grouping scheme for evaluation experiment 3

[0065] Group sample Experimental group 1 (P-ApoVs1) P-ApoVs1 Experimental group 2 (P-ApoVs2) P-ApoVs2 Control group 1 (DONVs) DONVs Control group 2 (CFNVs) CFNVs

[0066] Results: Figure 4As shown, green fluorescence can be detected in experimental group 1 (P-ApoVs1), experimental group 2 (P-ApoVs2), control group 1 (DONVs) and control group 2 (CFNVs), and the overlap between green fluorescence and red fluorescence is low, indicating that plant-derived extracellular vesicles and plant-derived apoptotic-mimicking extracellular vesicles modified by the preparation method of the present invention can be phagocytosed by macrophages, and after entering the cells, the co-localization degree between the extracellular vesicles and lysosomes is low, and they have a certain targeting property; it is worth noting that, compared with control group 1 (DONVs) and control group 2 (CFNVs), the green fluorescence intensity of experimental group 1 (P-ApoVs1) and experimental group 2 (P-ApoVs2) is significantly improved, indicating that the plant-derived apoptotic-mimicking extracellular vesicles modified by the preparation method of the present invention have higher targeting property.

[0067] Evaluation experiment 4: Inhibition of inflammatory response of M1 macrophages in vitro

[0068] Lipopolysaccharide (LPS) is a common endotoxin that can activate monocytes, macrophages, endothelial cells, epithelial cells, etc. through the cell signal transduction system in the body, synthesize and release a variety of cytokines and inflammatory mediators, and then cause a series of reactions in the body.

[0069] Interferon-γ (INF-γ) induces the expression of multiple inflammatory genes by activating the JAK / STAT signaling pathway, which play an important role in immune response and inflammatory reaction.

[0070] Steps:

[0071] Experimental group 1 (P-ApoVs1): 106 RAW264.7 cells were plated in a 6-well plate and placed in CO 2 Culture in an incubator overnight. Replace the culture medium, add 100 ng / mL LPS and 20 ng / mL INF-γ and continue culturing for 12 hours to induce M1 macrophages. 8 The cells were treated with 5% paraformaldehyde (P < 0.05) / mL for 24 hours, and the expressions of TNF-α, IL-6, IL-1β and iNOS mRNA were detected by qPCR.

[0072] Control group 1 (DONVs): After induction with LPS+INF-γ, the cells were diluted with DONVs sample solution (concentration of 10 8 / mL) for 24 hours, and other operation steps were the same as those of experimental group 1.

[0073] Experimental group 2 (P-ApoVs2): After induction with LPS+INF-γ, the cells were diluted with P-ApoVs2 sample solution (concentration of 108 / mL) for 24 hours, and other operation steps were the same as those of experimental group 1.

[0074] Control group 2 (CFNVs): After induction with LPS+INF-γ, the cells were treated with CFNVs sample dilution (concentration of 108 cells / mL) for 24 hours. Other operation steps were the same as those of experimental group 1.

[0075] Model group (PBS): After induction with LPS+INF-γ, the cells were treated with an equal volume of PBS solution for 24 hours. Other steps were the same as those in experimental group 1.

[0076] Blank group (Control): plate 10 6 RAW264.7 cells / well were placed in CO 2 Cultured overnight in an incubator. The culture medium was replaced without LPS+INF-γ treatment and cultured for 12 hours. The cells were treated with an equal volume of PBS solution for 24 hours, and the expression of TNF-α, IL-6, IL-1β and iNOS mRNA was detected by qPCR.

[0077] Results: Figure 5 As shown, compared with the blank group (Control), the expression levels of TNF-α, IL-6, IL-1β and iNOS in the model group (PBS) were significantly increased, indicating that LPS+INF-γ can significantly promote the expression of inflammatory genes after acting on macrophages, and the model was successfully established; compared with the model group (PBS), the expression levels of TNF-α, IL-6, IL-1β and iNOS in the experimental groups 1-2 and the control groups 1-2 were significantly reduced, indicating that the plant-derived extracellular vesicles and the plant-derived apoptotic extracellular vesicles modified by the preparation method of the present invention can inhibit the expression of inflammatory genes to a certain extent; it is worth noting that compared with the control group 1, the experimental group 1 and the experimental group 2 were compared with the control group 2, and the expression levels of TNF-α, IL-6, IL-1β and iNOS were significantly reduced, indicating that the plant-derived apoptotic extracellular vesicles modified by the preparation method of the present invention have an enhanced inhibitory effect on the expression of inflammatory genes.

[0078] Evaluation experiment 5: Evaluation of treatment in rat RA model

[0079] The rat RA (rheumatoid arthritis) model is an animal model commonly used in medical research, among which the more common ones are adjuvant-induced models, such as the Freund's adjuvant-induced arthritis model (AIA).

[0080] The arthritis model induced by Complete Freund's Adjuvant (CFA) can show persistent inflammation accompanied by systemic changes, as well as synovial hyperplasia, the appearance of "arthritis" nodules in the corneal wings, ears and tail, etc. It has many similarities with human arthritis and is suitable for the study of pathogenesis and the evaluation of the efficacy of new drugs.

[0081] Steps:

[0082] Experimental group (P-ApoVs2): SD rats were injected with 50 μL of 10 mg / mL complete Freund's adjuvant into the left hind foot pad. The first signs of inflammation were observed on the 10th day, and 200 μL of 4×10 8 P-ApoVs2 / mL. Treatment was performed 5 times, once every other day. During the experiment, health status and body weight were monitored, and the degree of ankle swelling was scored (scoring range 0-4 points, corresponding to normal to severe). On the 10th and 20th days of the experiment, the right foot of the rats was photographed and X-rayed to evaluate the degree of redness, swelling and cartilage damage.

[0083] Control group (CFNVs): After treatment with complete Freund's adjuvant, 200 μL of CFNVs with a concentration of 4×108 / mL were locally injected into the joint cavity of the lacquer joint. Other operation steps were the same as those of the experimental group.

[0084] Model group (Saline): After treatment with complete Freund's adjuvant, 200 μL of normal saline was injected locally into the lacuna joint cavity. Other operation steps were the same as those of the experimental group.

[0085] Blank group (Control): SD rats were not treated with complete Freund's adjuvant. On the 10th day, the initial signs of inflammation were observed and 200 μL of normal saline was injected locally into the joint cavity of the lacquer joint. The injection was performed 5 times, once every other day, and the subsequent operation steps were the same as those of the experimental group.

[0086] Results: Figure 6 As shown, Figure A is a visual image of the injured part of the left hind foot of the rat, and Figure B is an X-ray image of the left hind foot of the rat. Compared with the blank group (Control), the left hind foot pad of the SD rats in the model group (Saline) was obviously swollen and thickened ten days after the injection of complete Freund's adjuvant. Figure 6 A), X-rays show joint swelling, osteoporosis, and cartilage damage ( Figure 6 B), indicating that the rat RA model was successfully established. It is worth noting that compared with the model group (Saline) and the control group (CFNVs), the experimental group (P-ApoVs2) significantly inhibited joint swelling, improved joint osteoporosis and promoted cartilage damage repair.

[0087] like Figure 7 As shown, Figure A is a diagram of the injury degree score of the left hind foot of the rat, and Figure B is a test result of the joint thickness of the left hind foot of the rat. Compared with the model group (Saline), the injury degree score and joint thickness of the control group (CFNVs) and the experimental group (P-ApoVs2) decreased significantly with the treatment time, indicating that the plant-derived extracellular vesicles and the plant-derived apoptotic extracellular vesicles modified by the preparation method of the present invention have the effect of improving joint redness and swelling and inhibiting swelling. Moreover, compared with the control group (CFNVs), the injury degree score and joint thickness of the experimental group (P-ApoVs2) decreased more significantly with the treatment time, indicating that the plant-derived apoptotic extracellular vesicles modified by the preparation method of the present invention have an enhanced effect on improving joint redness and swelling and inhibiting swelling.

[0088] Evaluation experiment 6: Inhibition of synovial inflammatory response in rat RA model

[0089] DNase / RNase refers to DNA enzyme (deoxyribonuclease) and RNA enzyme (ribonuclease). Since the sample contains DNA or RNA components, the absence of DNase / RNase can avoid contamination of the experimental samples.

[0090] Procedure: Based on the procedure of evaluation experiment 5, rats were euthanized on the 20th day of the experiment. The knee joints of rats in each group were collected and stored in RNase / DNase-free solution. Then, the synovial tissue of the knee joint was collected using a sterile surgical blade, and total RNA was extracted. The expression of TNF-α, IL-6, and iNOS genes was detected by qRT-PCR.

[0091] Results: Figure 8 As shown, compared with the model group (Saline), the expression levels of TNF-α, IL-6, and iNOS genes in the control group (CFNVs) and the experimental group (P-ApoVs2) were significantly decreased, indicating that the plant-derived extracellular vesicles and the plant-derived apoptotic extracellular vesicles modified by the preparation method of the present invention can inhibit the synovial inflammatory response to a certain extent; it is worth noting that compared with the control group (CFNVs), the expression levels of TNF-α, IL-6, and iNOS in the experimental group (P-ApoVs2) decreased more significantly, indicating that the plant-derived apoptotic extracellular vesicles modified by the preparation method of the present invention have an enhanced inhibitory effect on the synovial inflammatory response.

[0092] Evaluation experiment 7: Inhibition of serum inflammatory factor expression in rat RA model

[0093] ELISA kit, the full name of which is Enzyme-Linked Immunosorbent Assay kit, is a biological detection technology based on antigen-antibody binding reaction. ELISA kit is a biological reagent used to detect antigens or antibodies. Its basic principle is to combine antigens or antibodies with enzymes to form enzyme-labeled antigens or antibodies. When these enzyme-labeled antigens or antibodies react with the corresponding antibodies or antigens in the sample, an antigen-antibody-enzyme complex is formed. This complex can catalyze the reaction of the substrate and produce a color change. The degree of color change can be used to semi-quantitatively or quantitatively detect the antigen or antibody in the sample.

[0094] Procedure: Based on the procedure of evaluation experiment 5, rats were euthanized on the 20th day of the experiment. Blood samples were collected by cardiac puncture, left to stand at room temperature for 30 minutes, and centrifuged at 3500 rpm to collect serum. The samples were then diluted according to the instructions of the ELISA kit to determine the levels of cytokines TNF-α and IL-6.

[0095] Results: Fig. 9 As shown, compared with the model group (Saline), the expression levels of TNF-α and IL-6 genes in the control group (CFNVs) and the experimental group (P-ApoVs2) were significantly decreased, indicating that the plant-derived extracellular vesicles and the plant-derived apoptotic extracellular vesicles modified by the preparation method of the present invention can inhibit the expression of serum inflammatory factors to a certain extent; it is worth noting that compared with the control group (CFNVs), the expression levels of TNF-α and IL-6 genes in the experimental group (P-ApoVs2) decreased more significantly, indicating that the plant-derived apoptotic extracellular vesicles modified by the preparation method of the present invention have a more significant inhibitory effect on the expression of serum inflammatory factors.

[0096] The contents not described in detail in the present application description belong to the common knowledge of those skilled in the art.

[0097] As mentioned in the entire specification and claims, "including" is an open term and should be interpreted as "including but not limited to". "Substantially" means that within an acceptable error range, those skilled in the art can solve the technical problem within a certain error range and basically achieve the technical effect.

[0098] It should also be noted that the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a product or system including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such a product or system. In the absence of more restrictions, the elements defined by the sentence "comprises a ..." do not exclude the existence of other identical elements in the product or system including the elements.

[0099] The above description shows and describes several preferred embodiments of the present application, but as mentioned above, it should be understood that the present application is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the invention concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present application, and should be protected by the claims attached to the present application.

Claims

1. A method for preparing plant-derived apoptosis-mimicking extracellular vesicles, characterized in that: The steps include: S1. Extracting plant-derived exosomes from plant tissues; S2. Co-fusion of the plant-derived extracellular vesicles described in step S1 with a solution containing phosphatidylserine (PS) to obtain the plant-derived apoptosis-mimicking extracellular vesicles.

2. The preparation method according to claim 1, characterized in that: The following steps are also included: S3, extruding the plant-derived apoptosis-mimicking extracellular vesicles described in step S2 through a membrane with a pore size of 50 nm to 400 nm to obtain plant-derived apoptosis-mimicking extracellular vesicles of corresponding particle size.

3. The preparation method according to claim 1, characterized in that: The PS in step S2 is L-α-phosphatidylserine, dipalmitoylphosphatidylserine (DPPS) or dioleoylphosphatidylserine (DOPS).

4. The preparation method according to claim 1, characterized in that: The co-fusion method in step S2 is to mix the plant-derived extracellular vesicles in step S1 with PS-containing liposomes (PSLs), and react at 30 to 40° C. for 30 to 120 minutes.

5. The preparation method according to claim 4, characterized in that: The quantitative ratio of the plant-derived extracellular vesicles to the PSLs is 3-6:1-3.

6. The preparation method according to claim 4, characterized in that: The preparation method of PSLs comprises the following steps: dissolving and mixing phospholipids and cholesterol, and evaporating to obtain a lipid film; hydrating the lipid film, and ultrasonicating at a power of 100W to 1000W for 1 to 30 minutes to obtain the PSLs.

7. The preparation method according to claim 6, characterized in that: The phospholipids include one or more of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PS.

8. The preparation method according to claim 7, characterized in that: The molar ratio of PS to PSLs is 0.5-3.0:7.0-9.

5.

9. A plant-derived apoptosis-mimicking extracellular vesicle prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the plant-derived apoptosis-mimicking extracellular vesicles as claimed in claim 9 in drugs for treating inflammation.

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