A plant-derived apoptosis-mimicking extracellular vesicle and its preparation method and application

Abstract

The present invention belongs to the field of nanobiotechnology and discloses a plant-derived apoptosis-mimicking extracellular vesicle, a preparation method thereof, and an application thereof. The preparation method comprises the following steps: extracting plant-derived extracellular vesicles from plant tissues, and fusing the plant-derived extracellular vesicles with a solution containing apoptosis signal phosphatidylserine (PS) to obtain the plant-derived apoptosis-mimicking extracellular vesicles. The plant-derived apoptosis-mimicking extracellular vesicles of the present invention are derived from natural plants and have good biocompatibility compared to apoptotic cell vesicles derived from mammalian cells, and are less likely to cause immune rejection reactions in the body; after modification 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.

Description

Technical Field

[0001] The present 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] Macrophages are activated in different ways Divided into two categories: classic activation (M1 type) and alternatively activated (M2 type). Classically activated (M1 type) is 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 crucial for host resistance to pathogens. (M2 type) is induced by interleukin-4 (IL-4), IL-13 or IL-10 and is generally 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 suppress inflammation, contributing to tissue repair, remodeling, angiogenesis and maintaining homeostasis.

[0003] Plant-derived nanovesicles (PNVs) are lipid bilayer nanovesicles derived from plant cells, with diameters ranging from 30 to 150 nm. They can secrete and deliver a variety of bioactive molecules, such as proteins, nucleic acids, and secondary metabolites, outside the cell, with high biocompatibility and stability. Growing evidence indicates that plant-derived nanovesicles can enter mammalian cells and mediate cross-kingdom gene regulation between plants and animals, suggesting potential medical applications for regulating fundamental biological processes in the human body, such as immune system regulation and drug and nucleic acid delivery. In recent years, increasing research has focused on the potential application of plant-derived nanovesicles in the treatment of inflammatory diseases. Studies have shown that plant-derived nanovesicles can treat inflammatory diseases through various mechanisms, including modulating immune responses, alleviating oxidative stress, and inhibiting inflammatory pathways. Nanovesicles extracted from plants such as ginseng, ginger, grapefruit, and houttuynia cordata have demonstrated anti-inflammatory, antioxidant, and immunomodulatory effects in macrophages, suggesting potential therapeutic applications for a variety of inflammatory diseases, such as lung injury, colitis, and rheumatoid arthritis. Therefore, plant extracellular vesicles (EVs) have great potential as a novel therapeutic strategy and will become a research hotspot in the treatment of inflammatory diseases. However, improving their ability to target and regulate immune cells and achieve satisfactory therapeutic effects remains a challenge that needs 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 receptor binding, regulating Polarization and function promote its transformation to M2 type. This process enhances The phagocytic and anti-inflammatory abilities of ApoVs promote 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 therapy.

[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 needs further research and evaluation. Existing production methods mainly use bioreactors and other equipment for large-scale cell culture. However, when producing on a large scale 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 their therapeutic effects. Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings of the existing technology 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 extracellular vesicles from plant tissues;

[0009] S2. The plant-derived extracellular vesicles described in step S1 are co-fused with a solution containing phosphatidylserine (PS) to obtain plant-derived apoptosis-mimicking extracellular vesicles.

[0010] The present invention's method for preparing plant-derived apoptosis-mimicking extracellular vesicles uses natural plant-derived extracellular vesicles as raw material. These vesicles are modified with phosphatidylserine (PS), enhancing their immunomodulatory capacity. This method provides a scientific basis for biomimetic modification of plant-derived extracellular vesicles. Compared to methods for preparing apoptotic extracellular vesicles from cell cultures, the present method is simpler to operate, and the resulting plant-derived apoptosis-mimicking extracellular vesicles exhibit relatively stable biological activity, demonstrating excellent therapeutic efficacy for 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 30-40° C. for 30-120 min.

[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 includes 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 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. Compared with apoptotic extracellular vesicles derived from mammalian cells, they have good biocompatibility and are less likely to cause immune rejection reactions in the body. After modification 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 following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Transmission electron micrographs 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 results of macrophage compatibility evaluation of the plant-derived apoptotic extracellular vesicles prepared in Examples 1 and 2;

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

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

[0029] Figure 6 Visual images and X-ray images of the left hind paw of a rat after treatment of RA with plant-derived mimicking apoptosis extracellular vesicles prepared in Example 2. Figure A is a visual image of the injured area of ​​the left hind paw of the rat, and Figure B is an X-ray image of the left hind paw of the rat;

[0030] Figure 7 Evaluation results of the plant-derived mimicking apoptosis extracellular vesicles prepared in Example 2 for the treatment of RA. Figure A is a diagram showing the injury severity score of the left hind paw of a rat, and Figure B is a diagram showing the joint thickness test results of the left hind paw of a rat.

[0031] Figure 8 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 the synovial TNF-α gene, Figure B shows the effect of the plant-derived apoptosis-mimicking extracellular vesicles on the expression of the synovial IL-6 gene, and Figure C shows the effect of the plant-derived apoptosis-mimicking extracellular vesicles on the expression of the synovial iNOS gene.

[0032] Figure 9 Evaluation results of serum inflammatory factors after the treatment of RA with plant-derived apoptosis-mimicking extracellular vesicles prepared in Example 2. 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 following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

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

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

[0036] According to literature reports, plant extracellular vesicles have almost no PS signals. The inventors envisioned that the construction of plant-derived apoptotic-cell-inspired nanovesicles (P-ApoVs) would have the following advantages: plant-derived apoptotic-cell-inspired nanovesicles are derived from natural plants, have good biocompatibility, and are less likely to cause immune rejection reactions in the body; plant-derived apoptotic-cell-inspired nanovesicles carry apoptosis signals on their surface, which can specifically recognize and was Phagocytosis, thereby achieving targeted delivery; plant-derived mimicking apoptosis extracellular vesicles can promote Transformation to M2 type, M2 type They possess phagocytic and anti-inflammatory abilities, releasing anti-inflammatory factors to inhibit inflammatory responses. The constructed plant-derived mimicking apoptotic extracellular vesicles combine the advantages of both plant and apoptotic extracellular vesicles, and are expected to become a novel therapeutic strategy for treating inflammatory diseases.

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

[0038] Wash 5g of Dendrobium officinale stem segments repeatedly with sterile or deionized water to remove surface dust and microorganisms. Allow the washed Dendrobium officinale stem segments to air dry. Mix the dried Dendrobium officinale stem segments with 25mL of phosphate buffer solution (pH 7.0) and incubate for 12h. Add pectinase at a concentration of 1mg / g stem segment and cellulase at a concentration of 2mg / g stem segment for enzymatic hydrolysis for 12h at 24°C. Collect all the juice after enzymatic hydrolysis and centrifuge at 3000g for 30min. Collect the supernatant and centrifuge at 10000g for 1.5h to remove the supernatant. Centrifuge the supernatant in an ultrahigh-speed centrifuge at a speed of 150000g for 2.5h. Take the precipitate after ultrahigh-speed centrifugation. Use a sucrose gradient (8%, 30%, 45%, and 60% (g / v) sucrose solutions) and then centrifuge at 150,000g for 1.5 hours in an ultracentrifuge. Extract and purify Dendrobium officinale exovesicles in the 30-45% gradient. Aspirate 4 mL of the purified extract. Add five volumes of PBS, remove the sucrose using an ultrafiltration tube, and replace with PBS. This procedure yields highly purified Dendrobium officinale-derived nanovesicles (DONVs).

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

[0040] PS liposomes (PSLs) and DONVs suspension were mixed in a 3:1 ratio and incubated at 37°C for 60 minutes with mechanical agitation to promote fusion of the PSLs and DONVs. The resulting mixture was extruded to a uniform particle size of approximately 200 nm, resulting in P-ApoVs1.

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

[0042] Example 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 nanonovesicles (CFNVs) and P-ApoVs2. The TEM morphology of the CFNVs and P-ApoVs2 was as follows: Figure 1 As shown, the morphology of P-ApoVs2H and CFNVs is spherical. The particle size was measured by particle size analyzer to be 202.26±3.55nm and 183.51±1.09nm, respectively, and the surface potential was -28.72±0.54mV and -8.49±0.21mV, respectively. Figure 1 ).

[0044] Example 3: Experimental evaluation of plant-derived apoptotic extracellular vesicles of Examples 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, and IL-6 play a key role. They are produced by activated immune cells (such as macrophages and T lymphocytes) and mediate inflammatory responses, leading to pathological changes such as congestion, edema, and exudation in the synovial membrane of the joints. These inflammatory factors can also promote the formation of pannus and cartilage destruction, further exacerbating 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 RA patients and the treatment effect can be assessed. 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 targeting M1 macrophages has become one of the important strategies for the treatment of RA.

[0046] Evaluation Experiment 1: Surface Property 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 cell membrane lipid bilayer. 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, exposing it to the extracellular environment. Annexin V, as a phospholipid-binding protein, can specifically bind to the everted PS. Therefore, the early stages of cell apoptosis can be detected by Annexin V. Annexin V is usually labeled with a fluorescein such as FITC (Annexin V-FITC) and used as a fluorescent probe in combination with flow cytometry or fluorescence microscopy to detect cell apoptosis.

[0048] Procedure: Prepare 100 μL of samples from experimental groups 1-2, control groups 1-2, and a blank group from Experiment 1 (the grouping scheme for Experiment 1 is shown in Table 1). Place the samples in EP tubes, add an equal volume of Annexin binding buffer from the apoptosis kit, and mix thoroughly. Add 5 μL of Annexin V-FITC, mix thoroughly, and incubate at room temperature in the dark for 30 min. After incubation, wash with 500 μL of PBS and concentrate using a 100 kDa cutoff ultrafiltration centrifuge tube (MilliPore) to remove excess Annexin V-FITC solution. Collect 100 μL of the sample, add 200 μL of PBS solution, mix thoroughly, and analyze on the flow cytometer. Data were collected using a flow cytometer (BD FACSCanto) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Data were analyzed using 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 cells / mL) Control group 2 (CFNVs) CFNVs solution (108 cells / mL) Blank group (PS absent-NPs) Liposomes without PS (equal volume)

[0051] Results: As 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) was very small, and the values ​​of the three groups were all around 1%, indicating that there was 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), the proportion of surface-bound Annexin V nanoparticles in the experimental group 1 (P-ApoVs1) and the experimental group 2 (P-ApoVs2) was 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 had apoptosis signal phosphatidylserine (PS) on the surface.

[0052] Evaluation experiment 2: Cytocompatibility evaluation

[0053] CCK-8, or Cell Counting Kit-8, is a cell proliferation and cytotoxicity assay based on a water-soluble tetrazolium salt (WST-8). The WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonatophenyl)-2H-tetrazolium monosodium salt) in the CCK-8 kit is reduced by mitochondrial dehydrogenases to a highly water-soluble orange-yellow formazan product under the action of an electron carrier (such as 1-methoxy-5-methylphenazinium dimethyl sulfate, 1-Methoxy PMS). The amount of formazan generated is proportional to the number of viable cells, so its absorbance (at a wavelength of 450 nm) can be used to indirectly reflect the number of viable cells. The calculation formula is: Cell viability (%) = [(A1 - A0) / A0] × 100%. Among them, A0: absorbance value of blank control, A1: absorbance value of sample treatment group.

[0054] Procedure: Plate 10,000 RAW264.7 cells per well in a 96-well plate and culture overnight in a CO2 incubator. Samples from Experimental Groups 1-2 and Control Groups 1-2 of Evaluation Experiment 2 (the grouping scheme for Evaluation Experiment 2 is shown in Table 2) were taken and diluted with DMEM medium to form a series of sample dilutions with different concentrations (0.5×108 cells / mL, 1×108 cells / mL, 2×108 cells / mL, 4×108 cells / mL, and 8×108 cells / mL). The cultured RAW264.7 cells were treated with the sample dilutions for 48 hours. 10 μL of CCK-8 reagent was added to the culture medium in each well and incubated in a 37°C, 5% CO2 incubator for 2 hours. The absorbance of each well was measured at a wavelength of 450 nm.

[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: As Figure 3 As shown, at 0.5×10 8 cells / mL to 8×10 8 Within the concentration range of 1:100 cells / 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 exhibited 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 intercalated into 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 label 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 intracellular degradation systems, capable of degrading a variety of intracellular substances, including nano-exosomes. Due to their small size and specific surface properties, exosomes are often more readily absorbed by cells. Once inside cells, the colocalization between exosomes and lysosomes is low, reflecting their ability to evade capture and degradation by lysosomes, allowing them to remain stable within the cell and maintain a certain concentration. This stability increases the interaction time and opportunities between exosomes and targets, improving their targeted nature and therapeutic efficacy.

[0063] Procedure: Plate 10 5 RAW264.7 cells were plated at 10 cells / well and cultured in a CO2 incubator overnight. The cultured RAW264.7 cells were treated with Fluorescein-DHPE-labeled samples from Experimental Groups 1-2 and Control Groups 1-2 of Evaluation Experiment 2 (at a concentration of 108 cells / mL) (the grouping scheme for Evaluation Experiment 3 is shown in Table 3) for 24 hours. After 24 hours, all culture medium was aspirated and the cells were washed three times with PBS for 5 minutes each, protecting from light. Fixation was performed with 4% paraformaldehyde for 15 minutes and three washes with PBS. Lyso-Tracker Red (10 μM) was added for 20 minutes at room temperature and then washed three times with PBS. Staining was performed with a 10 μg / mL LDAPI solution for 10 minutes. Anti-quenching agents were added and images were captured using a confocal microscope (LSM 880 with AiryScan, Carl Zeiss) within a short time. The wavelengths were set to Ex 488 and Em 535 for green light and Ex 543 and Em 611 for red light. 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: As 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 the plant-derived extracellular vesicles and the plant-derived apoptosis-mimicking extracellular vesicles modified by the preparation method of the present invention can be phagocytosed by macrophages, and after entering the cells, the degree of co-localization between the extracellular vesicles and lysosomes is low, and they have a certain degree of targeting; 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 apoptosis-mimicking extracellular vesicles modified by the preparation method of the present invention have higher targeting.

[0067] Evaluation Experiment 4: Inhibition of the 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) activates the JAK / STAT signaling pathway to induce the expression of multiple inflammatory genes, 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 cultured overnight in a CO2 incubator. The culture medium was replaced and 100 ng / mL LPS and 20 ng / mL INF-γ were added and cultured for 12 hours to induce M1 macrophages. 8 The cells were treated with 4% paraformaldehyde (P<0.05) for 24 h, 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 The cells / mL were treated 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 10 8The cells / mL were treated 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 procedures were the same as those in experimental group 1.

[0076] Blank group (Control): 10 6 RAW264.7 cells were cultured overnight in a CO2 incubator. The culture medium was replaced without LPS and INF-γ treatment and cultured for 12 hours. The cells were treated with an equal volume of PBS for 24 hours, and TNF-α, IL-6, IL-1β, and iNOS mRNA expression was measured by qPCR.

[0077] Results: As 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 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 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 significantly reduced compared with the control group 2, 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 Complete Freund's Adjuvant (CFA)-induced arthritis model can exhibit persistent inflammation accompanied by systemic changes, as well as synovial hyperplasia, and the appearance of "arthritis" nodules in the corneal wings, ears, and tail. 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 footpad. On the 10th day, the first signs of inflammation were observed. 200 μL of 4×10 8 P-ApoVs2 was expressed in 1000 cells / mL. Treatments were administered five times every other day. During the experiment, health status and body weight were monitored, and ankle swelling was scored on a scale of 0-4, ranging from normal to severe. On the 10th and 20th days of the experiment, the rats' right feet were photographed and X-rayed to assess 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 cells / mL were locally injected into the lacquer joint cavity. 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 articular cavity of the lacrimal joint. Other procedures were the same as those of the experimental group.

[0085] Control group: SD rats were not treated with complete Freund's adjuvant. On day 10, when initial signs of inflammation were observed, 200 μL of normal saline was injected locally into the lacrimal joint cavity. This was repeated five times, every other day. All other procedures were the same as those in the experimental group.

[0086] Results: As 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 red, swollen and thickened ten days after the injection of complete Freund's adjuvant. Figure 6 A), X-ray images show joint swelling, osteoporosis, and cartilage damage ( Figure 6 B), indicating that the rat RA model was successfully established. Notably, 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 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 sample.

[0090] Procedure: Following the same procedures as in Experiment 5, rats were euthanized on day 20. Knee joints from each group were harvested and stored in an RNase- / DNase-free solution. Synovial tissue was then collected using a sterile scalpel blade, and total RNA was extracted. TNF-α, IL-6, and iNOS gene expression was assessed by qRT-PCR.

[0091] Results: As Figure 8 As shown in the data, 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 mimetic 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 mimetic 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] An ELISA kit, or Enzyme-Linked Immunosorbent Assay (ELISA), is a biological detection technique based on antigen-antibody binding reactions. ELISA kits are biological reagents used to detect antigens or antibodies. The basic principle is to bind the antigen or antibody to an enzyme to form an enzyme-labeled antigen or antibody. 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 catalyzes a substrate reaction, producing a color change. The degree of this color change can be used to semi-quantitatively or quantitatively detect the antigen or antibody in the sample.

[0094] Procedure: Following the same protocol as in Experiment 5, rats were euthanized on day 20. Blood samples were collected by cardiac puncture, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3500 rpm to collect serum. The samples were then diluted according to the ELISA kit instructions to determine the levels of the cytokines TNF-α and IL-6.

[0095] Results: As Figure 9 As shown in the data, 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 mimetic apoptosis 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 mimetic apoptosis 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 this application description belong to common knowledge of those skilled in the art.

[0097] Throughout the specification and claims, the term "including" is an open-ended term and should be interpreted as "including but not limited to." "Substantially" means that within an acceptable error range, a person skilled in the art can solve the technical problem and substantially achieve the technical effect.

[0098] It should also be noted that the terms "include," "comprises," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a product or system comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such product or system. In the absence of further limitations, an element defined by the phrase "comprises a..." does not exclude the presence of other identical elements in the product or system comprising the element.

[0099] The above description shows and describes several preferred embodiments of the present application. However, as previously mentioned, it should be understood that the present application is not limited to the form disclosed herein and should not be construed as excluding other embodiments. Instead, the present application can be used in various other combinations, modifications, and environments and can be modified within the scope of the inventive concept described herein through the above teachings or through techniques or knowledge in the relevant field. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present application are intended to be protected by the claims appended hereto.

Claims

1. A method for preparing plant-derived apoptosis-mimicking extracellular vesicles, characterized in that: The steps include: 5 g of Dendrobium officinale stem segments were repeatedly washed with sterile water or deionized water to remove surface dust and microorganisms; the washed Dendrobium officinale stem segments were naturally dried; the dried Dendrobium officinale stem segments were mixed with 25 mL of phosphate buffer solution, pH 7.0, and incubated for 12 h; pectinase at a concentration of 1 mg / g stem segment and cellulase at a concentration of 2 mg / g stem segment were added for enzymatic hydrolysis for 12 h at a temperature of 24°C; the juice after enzymatic hydrolysis was collected and centrifuged at 3000 g for 30 min; the supernatant was collected and then centrifuged at 10000 g for 1.5 h. The supernatant was centrifuged in an ultracentrifuge at 150,000 g for 2.5 h; the precipitate was collected after ultracentrifugation; sucrose gradient centrifugation was performed and then ultracentrifuged at 150,000 g for 1.5 h, with the sucrose solutions being 8% g / v, 30% g / v, 45% g / v, and 60% g / v; 4 mL of purified Dendrobium officinale exovesicles were extracted from the 30-45% layer; 5 volumes of PBS were added, and the sucrose was removed using an ultrafiltration tube and replaced with PBS; through the above steps, high-purity Dendrobium officinale exovesicles (DONVs) were obtained; 1 μmol of dioleoylphosphatidylserine (DOPS), 7 μmol of phosphatidylcholine, and 2 μmol of cholesterol were dissolved in 10 mL of an organic solvent consisting of chloroform and ethanol in a volume ratio of chloroform to ethanol = 9:1, and the mixture was shaken and mixed thoroughly. The organic solvent was evaporated to dryness on a rotary evaporator at 40°C for 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. The solution was sonicated using a probe sonicator for 1 min at 150 W until the solution became clear and transparent. The particle size of the PS liposomes was homogenized by extrusion to maintain a uniform size of approximately 200 nm. PS liposomes (PSLs) and DONVs suspension were mixed in a 3:1 ratio and incubated at 37°C for 60 minutes. Mechanical stirring was used during the incubation process to promote the fusion of PSLs and DONVs. The fused mixture was extruded to homogenize the particle size to about 200 nm, which is P-ApoVs1. or, 5 g of Glechoma longituba leaves were repeatedly washed with sterile or deionized water to remove surface dust and microorganisms; the washed Glechoma longituba leaves were naturally dried; the dried Glechoma longituba leaves were mixed with 25 mL of phosphate buffer solution, pH 7.0, and incubated for 12 h; pectinase at a concentration of 1 mg / g Glechoma longituba leaves and cellulase at a concentration of 2 mg / g Glechoma longituba leaves were added for enzymatic hydrolysis for 12 h at 24°C; the juice after enzymatic hydrolysis was collected and centrifuged at 3000 g for 30 min; the supernatant was collected and then centrifuged at 10000 g for 1.5 h. The supernatant was centrifuged in an ultracentrifuge at 150,000 g for 2.5 h; the precipitate was collected after ultracentrifugation; sucrose gradient centrifugation was performed and then ultracentrifuged at 150,000 g for 1.5 h, with sucrose solutions of 8% g / v, 30% g / v, 45% g / v, and 60% g / v; the 30-45% layer was selected to extract and purify the extracellular vesicles of Acanthocereus, and 4 mL was aspirated; 5 volumes of PBS were added, and the sucrose was removed using an ultrafiltration tube and replaced with PBS; through the above steps, high-purity extracellular vesicles of Acanthocereus CFNVs were obtained; 1 μmol of dioleoylphosphatidylserine (DOPS), 7 μmol of phosphatidylcholine, and 2 μmol of cholesterol were dissolved in 10 mL of an organic solvent consisting of chloroform and ethanol in a volume ratio of chloroform to ethanol = 9:1, and the mixture was shaken and mixed thoroughly. The organic solvent was evaporated to dryness on a rotary evaporator at 40°C for 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. The solution was sonicated using a probe sonicator for 1 min at 150 W until the solution became clear and transparent. The particle size of the PS liposomes was homogenized by extrusion to maintain a uniform size of approximately 200 nm. 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-fusion mixture was passed through an extruder to homogenize the particle size and maintain it at around 200 nm, which is P-ApoVs2.

2. A plant-derived apoptosis-mimicking extracellular vesicle prepared by the preparation method according to claim 1.

3. Use of the plant-derived apoptosis-mimicking extracellular vesicles according to claim 2 in the preparation of anti-inflammatory drugs.

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