Extraction method, modification method and application of dendrobium nobile outer vesicles

Abstract

The invention relates to an extraction method, a modification method and application of dendrobium nobile outer vesicles. The method comprises the following steps: placing dendrobium nobile leaves in a phosphate buffer solution, crushing and separating to ensure that plant cells are fully broken and outer vesicles are released; then a differential centrifugation method is combined with sucrose gradient centrifugation, finally, the outer vesicles in a sucrose solution with the mass concentration of 30-45% are selected, and the extracted dendrobium nobile outer vesicles are moderate in mass and high in purity. Then cholesterol structural analogues such as sterol compounds and triterpenoid saponin active molecules are embedded into a phospholipid bilayer of the dendrobium nobile outer vesicles to modify the dendrobium nobile outer vesicles, and the modification strategy can effectively improve the stability of the dendrobium nobile outer vesicles in a complex physiological environment; and the treatment effect can be enhanced through the synergism of active molecules and endogenous components of the vesicles.

Description

Technical Field

[0001] The present invention relates to the field of nanobiotechnology, and particularly to a method for extracting extracellular vesicles from Dendrobium nobile, a method for modifying the same, and an application thereof. Background Art

[0002] Plant-derived extracellular vesicles are nanoscale membrane structures with a size of 30 - 150 nm secreted by plant cells, having a lipid bilayer membrane structure, and encapsulating bioactive substances such as proteins, nucleic acids, lipids, and secondary metabolites therein. These vesicles not only mediate intercellular communication and material transport in plants but also exhibit the ability to regulate physiological and pathological processes in animals and humans across species. Due to their natural origin, low immunogenicity, and high biocompatibility, plant-derived extracellular vesicles have become an important research object in the field of novel nanotherapeutic systems.

[0003] As a traditional medicinal plant, the extracts of Dendrobium nobile have been proven to have multiple pharmacological activities such as anti-inflammatory, antioxidant, and immunomodulatory effects. Extracellular vesicles derived from Dendrobium nobile (DONVs) not only completely retain the active ingredients of the parent plant but also have excellent cell affinity. Its lipid bilayer membrane structure can effectively protect the contents, showing unique advantages in the fields of inflammation regulation, tissue repair, etc. Research shows that DONVs can participate in the damage repair process through multi-pathway synergistic effects, which can not only directly promote tissue regeneration but also enhance cell repair ability through immunomodulation. However, the in vivo application of DONVs still faces technical bottlenecks such as insufficient stability and low targeting efficiency. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a method for extracting extracellular vesicles from Dendrobium nobile, a method for modifying the same, and an application thereof, to extract high-purity extracellular vesicles from Dendrobium nobile, modify the extracellular vesicles with cholesterol structural analogs, construct engineered extracellular vesicles with enhanced anti-inflammatory functions, and improve the stability of DONVs in the complex physiological environment in vivo.

[0005] First aspect:

[0006] A method for extracting extracellular vesicles from Dendrobium nobile, comprising the following steps:

[0007] Mix Dendrobium nobile leaves with a phosphate buffer solution to obtain a mixed solution;

[0008] Perform fragmentation treatment on the mixed solution and filter to collect the filtrate of Dendrobium nobile;

[0009] Centrifuge the filtrate of Dendrobium nobile at a centrifugal force of 3000 - 5000 g for 10 - 30 min to collect the first supernatant;

[0010] Centrifuge the first supernatant at a centrifugal force of 130,000 - 150,000 g for 1 - 3 h, and collect the precipitate.

[0011] Using the sucrose gradient centrifugation method, successively set several gradient concentrations of sucrose solutions at a mass concentration of 8 - 60%, then mix with the precipitate, centrifuge at a centrifugal force of 130,000 - 150,000 g for 0.5 - 2 h, intercept the sucrose solution with a mass concentration of 30 - 45%, and remove the sucrose to obtain the exosomes of Dendrobium nobile Lindl.

[0012] In the method for extracting the exosomes of Dendrobium nobile Lindl. of the present invention, first place the leaves of Dendrobium nobile Lindl. in a phosphate buffer solution, and then break and separate them to ensure that plant cells are fully ruptured and exosomes are released; then combine differential centrifugation with sucrose gradient centrifugation, and finally select the exosomes in the sucrose solution with a mass concentration of 30 - 45%. The quality of the extracted exosomes of Dendrobium nobile Lindl. is moderate and the purity is relatively high.

[0013] As a preferred solution, it further includes the following steps: wash the leaves of Dendrobium nobile Lindl. with sterile water or deionized water, then dry them in an environment at 20 - 30°C with a relative humidity of 50 - 70%, and then mix with the phosphate buffer solution; after the breaking treatment, filter with a 20 - 50 mesh filter to obtain the filtrate of Dendrobium nobile Lindl.

[0014] Wash the picked leaves of Dendrobium nobile Lindl. with sterile water or deionized water multiple times to remove the dust, pollutants and potential microorganisms attached to the surface, and ensure that no exogenous pollutants are introduced during the subsequent treatment process. Control the appropriate temperature and humidity when drying the leaves of Dendrobium nobile Lindl. to ensure that the water in the plant tissue tends to be balanced and avoid damage to the exosome structure.

[0015] As a preferred solution, after intercepting the sucrose solution with a mass concentration of 30 - 45%, place it in a filter tube with a molecular weight cut-off of 30 kDa, add the phosphate buffer solution, centrifuge at a centrifugal force of 3,000 - 5,000 g for 0.5 - 1 h, and collect the second supernatant, which includes the exosomes of Dendrobium nobile Lindl. By adding the phosphate buffer solution and centrifuging to remove sucrose molecules, the purity of the exosomes of Dendrobium nobile Lindl. is improved, and at the same time, the sucrose solution is replaced with the phosphate buffer solution.

[0016] As a preferred solution, the mass ratio of the leaves of Dendrobium nobile Lindl. to the phosphate buffer solution is 1:2 - 5, and after mixing, place it in an environment at 0 - 4°C for 12 - 16 h; the pH of the phosphate buffer solution is 6.0 - 7.5. Place the leaves of Dendrobium nobile Lindl. in the phosphate buffer solution in a low-temperature environment to ensure the stability of exosomes and make the exosomes slowly released. Further, slow stirring can be carried out during this process to improve the release rate of exosomes.

[0017] Second aspect:

[0018] A method for modifying the exosomes of Dendrobium nobile Lindl. described in the first aspect, comprising the following steps:

[0019] Dissolve the sterol compound or triterpenoid saponin active molecule in a mixed solvent, and under the protection of an inert gas, perform ultrasonic treatment to obtain a lipid solution with a concentration of 1-2 mg / mL. The mixed solvent is chloroform:ethanol with a volume ratio of 3:1;

[0020] Place the lipid solution in a 37°C constant temperature water bath and evaporate it under vacuum to form a lipid film;

[0021] Mix the lipid film with the exosomes of Dendrobium nobile Lindl., and place it in a constant temperature environment of 50-60°C for heat preservation for 4-10 h to obtain a hybrid membrane composite system;

[0022] Extrude the hybrid membrane composite system with a membrane extruder to obtain functionalized modified exosomes of Dendrobium nobile Lindl.

[0023] In the present invention, cholesterol structural analogues such as sterol compounds and triterpenoid saponin active molecules are embedded in the phospholipid bilayer of the exosomes of Dendrobium nobile Lindl. to modify the exosomes of Dendrobium nobile Lindl. This modification strategy can not only effectively improve the stability of the exosomes of Dendrobium nobile Lindl. in a complex physiological environment, but also enhance the therapeutic effect through the synergy of the active molecules and the endogenous components of the vesicles.

[0024] When modifying the exosomes of Dendrobium nobile Lindl. in the present invention, first dissolve the sterol compound or triterpenoid saponin active molecule in a mixed solvent to make it completely dissolve and fully disperse; then assemble by the film-hydration method, evaporate the lipid solution under vacuum to form a lipid film, and reconstruct the structure of the lipid film and the exosomes of Dendrobium nobile Lindl. During this process, the exogenous lipid film spontaneously inserts into the phospholipid bilayer of the exosomes through hydrophobic interaction to form a stable hybrid membrane composite system; finally, extrude the hybrid membrane composite system with a membrane extruder.

[0025] As a preferred embodiment, it further comprises the following steps: first place the hybrid membrane composite system in an environment of 0-4°C for pulsed ultrasonic treatment, and then extrude it with a membrane extruder. Pulsed ultrasonic treatment of the hybrid membrane composite system can eliminate the aggregates of the hybrid membrane composite system, facilitating the extrusion of functionalized modified exosomes of Dendrobium nobile Lindl. with uniform particle size.

[0026] As a preferred embodiment, the following steps are further included. The membrane extruder is equipped with a polycarbonate membrane with a pore size of 100 nm, and under a nitrogen environment, the functionalized modified Dendrobium nobile Lindl. exocyst is extruded cyclically. By equipping the membrane extruder with a polycarbonate membrane with a pore size of 100 nm, the particle size distribution of the functionalized modified Dendrobium nobile Lindl. exocyst is controlled to converge to 80 - 150 nm, improving the consistency. When the particle size distribution of the functionalized modified Dendrobium nobile Lindl. exocyst is within 80 - 150 nm, it is beneficial for entering cells and enhancing the drug efficacy.

[0027] As a preferred embodiment, the following steps are further included. The functionalized modified Dendrobium nobile Lindl. exocyst is extruded through the membrane extruder, first filtered through a 100 - 300 kDa ultrafiltration membrane for the first time, then filtered through a 100 - 300 kDa filter membrane for the second time, and finally filtered through a sterile filter membrane with a pore size not greater than 0.22 μm for the third time. The first filtration removes unbound lipid membranes and other free molecules, the second filtration replaces the buffer solution with physiological saline, and the third filtration sterilizes.

[0028] As a preferred embodiment, the sterol compound includes ruscoside, and the triterpenoid saponin active molecule includes asiatic acid.

[0029] Third aspect:

[0030] Use of a functionalized modified Dendrobium nobile Lindl. exocyst prepared as in the second aspect in anti - inflammatory treatment. Brief Description of the Drawings

[0031] Figure 1 Scanning electron microscope images of the morphology of Dendrobium nobile Lindl. exocyst and functionalized Dendrobium nobile Lindl. exocyst;

[0032] Figure 2 Pie chart of the lipid composition of Dendrobium nobile Lindl. exocyst;

[0033] Figure 3 Stability evaluation chart of Dendrobium nobile Lindl. exocyst and functionalized modified Dendrobium nobile Lindl. exocyst;

[0034] Figure 4 Cell compatibility evaluation chart of Dendrobium nobile Lindl. exocyst and functionalized modified Dendrobium nobile Lindl. exocyst;

[0035] Figure 5 Macrophage internalization efficiency chart of Dendrobium nobile Lindl. exocyst and functionalized modified Dendrobium nobile Lindl. exocyst;

[0036] Figure 6 In vitro inhibition efficiency chart of Dendrobium nobile Lindl. exocyst functionalized modified Dendrobium nobile Lindl. exocyst on macrophage inflammatory response;

[0037] Figure 7Functional modification of Dendrobium nobile Lindl. extracellular vesicles and the efficiency diagram of Dendrobium nobile Lindl. extracellular vesicles regulating macrophage to promote the proliferation of lung epithelial cells;

[0038] Figure 8 Biological safety evaluation diagram of functional modification of Dendrobium nobile Lindl. extracellular vesicles;

[0039] Figure 9 Therapeutic effect diagram of functional modification of Dendrobium nobile Lindl. extracellular vesicles on acute lung injury;

[0040] Among them, the "*" in the bar chart represents significance in statistics. p < 0.05 is one star, p < 0.01 is two stars, and p < 0.001 is three stars. Detailed implementation method

[0041] Example 1

[0042] This example provides a method for extracting Dendrobium nobile Lindl. extracellular vesicles, which includes the following steps:

[0043] Pretreatment of Dendrobium nobile Lindl. leaves

[0044] Take 5 g of Dendrobium nobile Lindl. leaves, and wash them repeatedly with sterile water or deionized water. After washing, place the Dendrobium nobile Lindl. leaves in an environment at 25 °C and a relative humidity of 60% to air dry naturally to ensure that there is no residual moisture on the leaf surface.

[0045] Initial extraction

[0046] Fully mix the air-dried Dendrobium nobile Lindl. leaves with 10 mL of phosphate buffer solution (PBS) with pH = 7.0, and slowly stir in an ice-water bath at 4 °C for 12 h. Then place the mixture in a juicer for crushing. The operation process is to crush for 1 min and stop for 1 min, and repeat the operation 5 times to ensure complete extraction. Filter the crushed mixture through a 30-mesh sieve to remove large residues, collect the Dendrobium nobile Lindl. filtrate and dispense it into centrifuge tubes, and place it in an ice-water bath at 4 °C for standby.

[0047] Centrifugation treatment

[0048] Centrifuge the collected Dendrobium nobile Lindl. filtrate at 3000 g for 30 min, and collect the supernatant; centrifuge the supernatant at 10000 g again for 1.5 h, and collect the new supernatant; use an ultra-high-speed centrifuge to centrifuge the obtained supernatant at 150000 g for 2.5 h. After centrifugation, collect the precipitate part to obtain the preliminarily extracted Dendrobium nobile Lindl. extracellular vesicle precipitate.

[0049] Sucrose gradient purification

[0050] Prepare sucrose solutions with mass concentrations of 8%, 30%, 45%, and 60%, layer them into centrifuge tubes in sequence, add the exosome precipitate, then ultracentrifuge at 150,000 g for 1.5 h, and collect the liquid between the 30% and 45% sucrose layers to obtain purified Dendrobium officinale exosome liquid.

[0051] Remove sucrose

[0052] Add 5 volumes of PBS (pH = 7.0) to the purified Dendrobium officinale exosome liquid, place it in an ultrafiltration tube with a molecular weight cut-off of 30 kDa, centrifuge at a centrifugal force of 3000 - 5000 g for 0.5 - 1 h, and collect the supernatant; then add 5 volumes of PBS (pH = 7.0) to the supernatant, repeat centrifugation 3 times to remove sucrose, and finally replace the solution with PBS; then filter through a 100 nm polycarbonate membrane 3 times to control the particle size of Dendrobium officinale exosomes and improve dispersibility, and then filter through a 0.22 μm microporous membrane to sterilize, finally obtaining highly pure Dendrobium officinale-derived exosomes (DONVs).

[0053] Characterization of Dendrobium officinale exosomes

[0054] Characterize the performance of the extracted DONVs, such as yield, particle size, and surface potential:

[0055] The yield of DONVs measured by the BCA method was 0.85 mg / g.

[0056] As shown in Table 1, the detection results of the particle size analyzer showed that the surface potential of DONVs was -24.11 ± 1.07 mV, which was consistent with that of most plant exosomes, indicating that the extracted Dendrobium officinale exosomes were intact; the average particle size was about 121.97 ± 7.09 nm, and the polydispersity index (PDI) value was 0.126, indicating good control of particle size and good surface dispersibility.

[0057] Table 1 Particle size and surface potential analysis of Dendrobium officinale exosomes and functionalized Dendrobium officinale exosomes

[0058]

[0059] As Figure 1 shown, (the scale bar is 200 nm) the transmission electron microscope observation results showed that the extracted vesicles were round, and their size was consistent with the detection results of the particle size analyzer. Through the above steps, highly pure Dendrobium officinale-derived exosomes can be extracted and their physicochemical properties can be effectively characterized.

[0060] Example 2

[0061] The lipids (2 mg) in the DONVs of Example 1 were sent to Beijing Bio-Tech Pack Technology Co., Ltd. (Beijing, China) for lipid component analysis. Specifically, a triple quadrupole mass spectrometer (Q-TRAP, Applied Biosystems, California, USA) was used to study the lipid composition of DONVs. After normalizing the lipidomics data to the internal standard of the same class of lipids, it was expressed as the percentage of the total signal of molecular species.

[0062] The analysis results are as Figure 2 shown. The lipid components of the outer vesicles of DONVs mainly include: by mass fraction, diacylglycerol (DG) accounts for about 23%, triglyceride (TG) and phosphatidylcholine (PC) each account for about 10%; monohexosyldiacylglycerol ether (EtherMGDG) and free fatty acid (FA) each account for 6 - 7%, phosphatidylethanolamine (PE) accounts for about 5%, monoglyceride (MGDG) and ether diglyceride (Ether DG) each account for about 4%, phosphatidylinositol (PI) accounts for 3 - 4%, and the other components account for a relatively small proportion. It is worth noting that among them, PI, PC, and PE, as key phospholipids participating in the antioxidant and anti-inflammatory processes, mainly play biological functions through mechanisms such as regulating cell signal transduction pathways and maintaining membrane structure integrity.

[0063] Example 3

[0064] This example provides a method for modifying Dendrobium nobile Lindl. outer vesicles, including the following steps:

[0065] Construction of lipid membrane composite system

[0066] Take high-purity cholesterol (purity ≥ 98%), asiatic acid, and ruscogenin and dissolve them in a mixed solvent respectively. The mixed solvent is chloroform:ethanol with a volume ratio of 3:1. Under the protection of inert gas, ultrasonic dispersion treatment (40 kHz, 200 W, 10 min) is carried out to form a homogeneous lipid solution. Adjust the concentration of the cholesterol lipid solution to 5 mM, the concentration of the asiatic acid lipid solution to 1 mg / mL, and the concentration of the ruscogenin lipid solution to 2 mg / mL, and perform ultrasonic treatment again (40 kHz, 150 W, 15 min) to ensure complete dissolution.

[0067] Assembly by thin film-hydration method

[0068] Transfer 2 mL of the above lipid solution to a rotary evaporator and evaporate it under vacuum (pressure ≤ 5 mbar) in a 37 °C constant temperature water bath to form a uniform lipid film. Take 15 mg of the DONVs extracted in Example 1 (calculated by protein concentration) and place it in 30 mL of PBS (pH = 7.4). Add 10 mL of PBS containing DONVs to the above lipid film and hydrate it gradiently in a 55 °C constant temperature shaker (120 rpm) for 4 h to complete membrane reconstruction. During this process, exogenous lipophilic molecules spontaneously insert into the outer vesicle phospholipid bilayer through hydrophobic interaction to form a stable hybrid membrane composite system.

[0069] Nanostructure optimization

[0070] The hybrid membrane composite system was placed in a 4 °C ice bath and subjected to ultrasonic pulse treatment (amplitude 30%, working for 2 s, intermittent for 3 s), and 10 cycles were continued to eliminate aggregation. Subsequently, a membrane extruder (Avanti Polar Lipids) was assembled with a 100 nm pore size polycarbonate membrane, and extrusion was carried out 10 times under the drive of nitrogen pressure to converge the particle size distribution of the functionalized modified Dendrobium officinale outer vesicles to the range of 80 - 150 nm.

[0071] Gradient purification

[0072] Successively use a 300 kDa ultrafiltration membrane to remove free molecules, a 100 kDa ultrafiltration module to replace the buffer with physiological saline, and finally sterilize it through a 0.22 μm sterile filter membrane. Obtain DONVs modified with cholesterol, asiatic acid, and ruscoside, and name them CHOL@DONVs, AA@DONVs, and Rus@DONVs respectively.

[0073] Characterization and analysis of functionalized outer vesicles

[0074] As shown in Table 1, it was detected and confirmed by dynamic light scattering (Malvern Zetasizer Nano ZS90) that the Zeta potential of the product was stably maintained at -15 to -25 mV, and the polydispersity index (PDI) ≤ 0.2. Particle size analysis showed that the average particle sizes of CHOL@DONVs, AA@DONVs, and Rus@DONVs were 118.97 ± 8.03 nm, 115.97 ± 5.97 nm, and 120.97 ± 6.88 nm respectively, indicating that the system had good dispersibility and met the stability requirements of nano-drug carriers.

[0075] As Figure 1 shown, (the scale is 200 nm) the transmission electron microscope observation results showed that the prepared modified vesicles were round and their sizes were consistent with the detection results of the particle size analyzer.

[0076] Stability evaluation

[0077] The particle size change is asFigure 3 As shown in A, after 4 weeks of storage at 4°C, the particle size of the functionalized group was maintained within 200 nm, significantly better than the 300 nm of the unmodified group; the surface potential changes were as shown in Figure 3 As shown in B, after storage at 4°C for 4 weeks, the potentials of AA@DONVs and Rus@DONVs remained within -30 mV, and that of CHOL@DONVs was approximately -35 mV, both higher than -40 mV of the unmodified group.

[0078] Example 4

[0079] Cytocompatibility experiment of extracellular vesicles of Dendrobium nobile before and after functional modification

[0080] RAW264.7 cells were cultured at 1×10 4 The cells were inoculated in a 96-well plate at a density of 100 / well and cultured in a 5% CO2 incubator for 24 hours. The DONVs extracted in Example 1 and the functionalized extracellular vesicles (CHOL@DONVs, AA@DONVs, Rus@DONVs) prepared in Example 3 were diluted with DMEM culture medium to prepare 2.5-200 μg / mL concentration gradient solutions (2.5, 5, 10, 20, 50, 100, 200 μg / mL). After removing the original culture medium, different concentrations of sample solutions were added to treat the cells for 24 hours. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for 2 hours at 37°C and 5% CO2. The absorbance value (OD value) at 450 nm was measured using an enzyme reader.

[0081] The cell survival rate was calculated by the following formula: survival rate (%) = [(A1-A0) / A0] × 100, where A0 is the OD value of the blank control group and A1 is the OD value of the experimental group.

[0082] The results of cytotoxicity experiments were as follows Figure 4 As shown in the figure, when the concentration of DONVs and their functionalized products was ≤200 μg / mL, the survival rate of RAW264.7 cells after 24 hours of treatment was higher than 80%. This result shows that the exosomes derived from Dendrobium nobile maintained excellent cell compatibility before and after functionalization.

[0083] Example 5

[0084] Experiment on the cellular efficiency of functionalized extracellular vesicles of Dendrobium nobile

[0085] RAW264.7 cells were cultured at 1×10 5Cells were seeded at a density of

[0086] per well in confocal dishes and incubated in a 5% Figure 5 CO2 incubator for 24 h. RAW264.7 cells were treated with CHOL@DONVs, AA@DONVs, and Rus@DONVs labeled with Fluorescein-DHPE (prepared in Example 3, at a concentration of 20 μg / mL) for 4 h. After discarding the medium, the cells were gently washed three times with pre-cooled PBS (5 min each time), and the operation was carried out in the dark throughout the process. The cells were fixed with 4% paraformaldehyde solution for 15 min (at room temperature), and then washed three times with PBS again. After dropping the anti-fluorescence quenching mounting medium, confocal microscopy (LSM 880 with AiryScan, Carl Zeiss) was immediately used for observation and image acquisition. The DAPI channel parameters were set as follows: excitation wavelength Ex = 358 nm, emission wavelength Em = 461 nm; FITC channel parameters: excitation wavelength Ex = 488 nm, emission wavelength Em = 535 nm. After acquisition, multi-channel image fusion processing was performed using ZEN software. Image analysis was completed using ZEN 2.3 (Blue Edition, Carl Zeiss) software, and the quantitative analysis of fluorescence intensity was performed by statistically analyzing the average fluorescence intensity of individual cells using ImageJ software. Figure 5 As shown in

[0087] Example 6

[0088] In vitro anti-inflammatory experiment of functionalized Dendrobium nobile exosomes

[0089] RAW264.7 cells were seeded at a density of 1×10 5Inoculate at a density of [number] / well in a 6-well plate and culture in a 5% CO₂ incubator for 24 h. After replacing the fresh medium, add 100 ng / mL lipopolysaccharide (LPS) to stimulate the cells for 12 hours to induce M1 macrophage polarization. Using PBS as the control group, treat the cells with the DONVs extracted in Example 1 and the functionalized extracellular vesicles prepared in Example 3 (CHOL@DONVs, AA@DONVs, Rus@DONVs) for 24 hours (at a concentration of 20 μg / mL). Collect the cells to extract total RNA, and use real-time quantitative PCR (qPCR) to detect the mRNA expression levels of the inflammatory factor TNF-α, the M1 macrophage marker iNOS, and the M2 macrophage marker Arg-1.

[0090] The experimental results are as Figure 6 shown: In the LPS-stimulated group, the mRNA expressions of TNF-α and iNOS in RAW264.7 cells were significantly increased (p < 0.05), and the expression of Arg-1 was significantly decreased (p < 0.05). After treatment with DONVs and their functionalized products, the mRNA levels of TNF-α and iNOS were significantly downregulated compared with the model group (p < 0.05), and the expression of Arg-1 was significantly restored (p < 0.05). Quantitative analysis showed that: compared with the DONVs group, the inhibition rates of CHOL@DONVs on TNF-α and iNOS were increased by 12.4% and 18.5% respectively, and those of AA@DONVs and Rus@DONVs were increased by 27.0% - 35.2% respectively. This result confirmed that functionalization modification could significantly enhance the inhibition of LPS-induced excessive release of inflammatory factors by DONVs and effectively regulate the polarization state of macrophages.

[0091] Example 7

[0092] Experiment on the effect of functionalized Dendrobium nobile Lindl. extracellular vesicles on promoting macrophage repair

[0093] Establish a Transwell co-culture system to study the macrophage-mediated repair effect of functionalized extracellular vesicles. The steps include: inoculate RAW264.7 cells ([2×10 4 cells / well) in the upper chamber of Transwell, and MLE-12 cells ([5×10 4(cells / well) were inoculated into the lower chamber and cultured for 24 h. The RAW264.7 cells in the upper chamber and the MLE-12 cells in the lower chamber were stimulated with 100 ng / mL LPS for 12 h, and then the cells in the upper chamber were intervened. In the experimental group, the DONVs extracted in Example 1 and the functionalized exosomes prepared in Example 3 (CHOL@DONVs, AA@DONVs, Rus@DONVs, concentration 20 μg / mL) were added and continuously treated for 48 h; the unstimulated and untreated cells were used as the normal control group. The MLE-12 cells in the lower chamber were collected, CCK-8 reagent was added, and after incubation at 37 °C and 5% CO2 for 2 h, the OD value was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0094] The calculation formula for cell viability: viability (%) = [(A1 - A0) / A0] × 100, where A0 is the OD value of the blank control group and A1 is the OD value of the experimental group.

[0095] The experimental results are as Figure 7 shown: The viability of MLE-12 cells in the LPS-stimulated group was significantly decreased compared with that in the normal control group (p < 0.05). After treatment with DONVs and their functionalized derivatives, the cell viability was significantly restored compared with that in the LPS model group (p < 0.05).

[0096] Quantitative analysis showed that: compared with the DONVs group, CHOL@DONVs increased the cell survival recovery rate by 8.67% (p < 0.05), and AA@DONVs and Rus@DONVs increased it by 22.09% and 18.01% respectively (p < 0.01). This result confirmed that the modification with asiatic acid and ruscoside could significantly enhance the repair-promoting effect of DONVs mediated by macrophages.

[0097] Example 8

[0098] Bio-safety experiment of functionalized Dendrobium nobile Lindl. exosomes

[0099] The in vivo safety of exosomes was evaluated by aerosol inhalation. The DONVs extracted in Example 1 and the functionalized exosomes prepared in Example 3 (AA@DONVs, CHOL@DONVs, Rus@DONVs) were dissolved in PBS, and aerosol droplets were formed by a medical air compression nebulizer (Yuwell, 403D). The dosage for each mouse was 4 mL (concentration 100 μg / mL), and the control group was the PBS group. After 24 h of administration, the mice were sacrificed, whole blood samples were obtained by orbital blood collection, serum was taken after centrifugation at 3000 rpm for 15 min, and the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected using an ELISA kit.

[0100] The experimental results are as Figure 8Shown as follows: There were no significant differences in serum AST and ALT levels between the DONVs group and each functionalized modification group (CHOL@DONVs, AA@DONVs, Rus@DONVs) compared with the healthy control group (p > 0.05). This data confirmed that the functionalized modification did not change the in vivo biocompatibility of Dendrobium officinale exosomes, nor did it change the biosafety of Dendrobium officinale exosomes.

[0101] Example 9

[0102] Experiment on the repair effect of functionalized Dendrobium officinale exosomes on acute lung injury (ALI)

[0103] A mouse ALI model was constructed by intratracheal instillation. After male BALB / c mice (7 - 8 weeks old, 17 - 22 g, n = 8) were anesthetized, the experimental group was perfused with 50 μL of sterile normal saline containing LPS (10 mg / kg) through the airway, and the control group was perfused with an equal volume of PBS. 2 h after modeling, the DONVs extracted in Example 1 and the functionalized exosomes (AA@DONVs) prepared in Example 3 were used for aerosol intervention. A medical air compression nebulizer (Yuwell 403D) was used to prepare aerosol droplets (concentration of 100 μg / mL, and the administration volume for each mouse was 4 mL). The mice were sacrificed 24 h after the intervention. The lung tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and then 5-μm sections were prepared for H&E staining to observe the histological changes. The expression levels of M1 / M2 macrophage markers CD86 and CD206 were detected by immunohistochemistry, and the mRNA expression levels of inflammatory factor TNF-α and M1 marker iNOS and M2 marker ARG-1 were quantitatively analyzed by qPCR.

[0104] As Figure 9 shown in Figure A, the results of H&E staining showed that: the alveolar structure in the control group was intact and evenly distributed, the alveolar structure was disordered with widened interlobular septa in the PBS group, the clarity of the alveolar wall structure in the AA@DONVs intervention group was significantly improved compared with the DONVs group, and the thickness of the alveolar septum decreased.

[0105] Immunohistochemical analysis showed that: compared with the control group, the proportion of CD86-positive cells in the PBS group was significantly increased, while the proportion of CD206-positive cells was significantly decreased. After AA@DONVs intervention, the CD86 positive rate was significantly lower than that in the DONVs group, and at the same time, the CD206 positive rate was significantly higher than that in the DONVs group.

[0106] As Figure 9As shown in B, the qPCR results showed that the expression levels of TNF-α and iNOS mRNA in the AA@DONVs group were decreased by 71% and 64% respectively compared with the PBS group, and the inhibition efficiency was 1.5 times and 1.8 times that of the DONVs group respectively. At the same time, the expression level of ARG-1 mRNA was increased by 1.6 times compared with the DONVs group (p<0.01), which confirmed that it significantly promoted the polarization of M2 macrophages.

[0107] The above results proved that the functional modification endows DONVs with the ability to enhance the regulation of macrophage polarization, and effectively improves the pathological process of ALI by synergistically inhibiting the M1 pro-inflammatory phenotype (CD86+ / iNOS+) and activating the M2 anti-inflammatory phenotype (CD206+ / ARG-1+).

[0108] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for extracting exosomes of Dendrobium nobile Lindl., characterized in that, It includes the following steps: Mix the leaves of Dendrobium nobile Lindl. with a phosphate buffer solution to obtain a mixed solution; Crush the mixed solution and filter it to collect the filtrate of Dendrobium nobile Lindl.; Centrifuge the filtrate of Dendrobium nobile Lindl. at a centrifugal force of 3000 - 5000g for 10 - 30 min to collect the first supernatant; Centrifuge the first supernatant at a centrifugal force of 130000 - 150000g for 1 - 3 h to collect the precipitate; Using the sucrose density gradient centrifugation method, sequentially set several gradient concentrations of sucrose solutions at a mass concentration of 8 - 60%, then mix with the precipitate, centrifuge at a centrifugal force of 130000 - 150000g for 0.5 - 2 h, intercept the sucrose solution with a mass concentration of 30 - 45%, and remove sucrose to obtain the extracellular vesicles of Dendrobium nobile Lindl.; 2. The extraction method of Dendrobium nobile Lindl. exovesicles according to claim 1, characterized in that It also includes the following steps: wash the leaves of Dendrobium nobile Lindl. with sterile water or deionized water, then dry them in an environment of 20 - 30°C and a relative humidity of 50 - 70%, and then mix with the phosphate buffer solution; after crushing, filter with a 20 - 50 mesh filter to obtain the filtrate of Dendrobium nobile Lindl.; 3. The extraction method of Dendrobium officinale outer vesicles according to claim 1, characterized in that, After intercepting the sucrose solution with a mass concentration of 30 - 45%, place it in a filter tube with a molecular weight cut-off of 30 kDa, add the phosphate buffer solution, centrifuge at a centrifugal force of 3000 - 5000g for 0.5 - 1 h to collect the second supernatant, and the second supernatant contains the extracellular vesicles of Dendrobium nobile Lindl.; 4. The extraction method of Dendrobium nobile Lindl. outer vesicles according to claim 1, characterized in that, The mass ratio of the leaves of Dendrobium nobile Lindl. to the phosphate buffer solution is 1:2 - 5, and after mixing, place it in an environment of 0 - 4°C for 12 - 16 h; the pH of the phosphate buffer solution is 6.0 - 7.5; 5. A modification method of the exosomes of Dendrobium nobile Lindl. according to any one of claims 1 to 4, characterized in that, It includes the following steps: Dissolve the sterol compound or triterpenoid saponin active molecule in a mixed solvent, and under the protection of an inert gas, perform ultrasonic treatment to obtain a lipid solution with a concentration of 1 - 2 mg / mL. The mixed solvent is chloroform:ethanol with a volume ratio of 3:1; Place the lipid solution in a 37°C constant temperature water bath and evaporate it under vacuum to form a lipid film; Mix the lipid film with the extracellular vesicles of Dendrobium nobile Lindl., and keep it warm in a constant temperature environment of 50 - 60°C for 4 - 10 h to obtain a hybrid membrane composite system; Extrude the hybrid membrane composite system with a membrane extruder to obtain functionalized modified extracellular vesicles of Dendrobium nobile Lindl.; 6. A modification method of Dendrobium nobile Lindl. exocytosis vesicles according to claim 5, characterized in that It also includes the following steps: first place the hybrid membrane composite system in an environment of 0 - 4°C for pulsed ultrasonic treatment, and then extrude it with a membrane extruder; 7. A method for modifying exosomes of Dendrobium nobile Lindl., according to claim 6, characterized in that, It also includes the following steps: the membrane extruder is equipped with a polycarbonate membrane with a pore size of 100 nm, and under a nitrogen environment, cyclically extrude the functionalized modified extracellular vesicles of Dendrobium nobile Lindl.; 8. A method for modifying exosomes of Dendrobium nobile Lindl., according to claim 7, characterized in that, It also includes the following steps: extrude the functionalized modified extracellular vesicles of Dendrobium nobile Lindl. through the membrane extruder, first filter it through a 100 - 300 kDa ultrafiltration membrane for the first time, then filter it through a 100 - 300 kDa filter membrane for the second time, and finally filter it through a sterile filter membrane with a pore size not greater than 0.22 μm for the third time; 9. A modification method of Dendrobium nobile Lindl exotoxin vesicles according to claim 5, characterized in that, The sterol compound includes ruscoside aglycone, and the triterpenoid saponin active molecule includes asiatic acid.

10. Use of the functionalized modified Dendrobium nobile Lindl. exocyst described in any one of claims 4 to 9 in anti-inflammatory treatment.

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