Exosome based on modified cellulose material and enrichment method thereof

Abstract

The invention provides an exosome enrichment method based on a modified cellulose material, which comprises the following steps: carrying out TEMPO oxidation on plant residue cellulose to prepare a cellulose material with a carboxyl structure; the cellulose structure is further refined by adopting mechanical shearing and high-pressure homogenizing means, so that the specific surface area and the dispersion stability are remarkably improved; through polyethyleneimine PEI grafting, a large number of amino functional groups are introduced, the surface electropositivity of the material is enhanced, and efficient adsorption and enrichment of exosomes with negative charges are achieved. The invention also provides the exosome prepared by the method. The modified cellulose material disclosed by the invention is wide in source, simple and convenient to prepare and mild in enrichment process, the separation purity and the enrichment efficiency of the exosome can be remarkably improved, and the modified cellulose material has good biocompatibility and environmental friendliness; compared with existing ultracentrifugation, density gradient centrifugation and immunoaffinity separation methods, the method has the advantages that the operation process is simplified, the equipment dependence and reagent cost are reduced, and the potential of large-scale production and clinical application is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome separation and purification, and particularly relates to a method for efficiently enriching exosomes by using a modified cellulose material. Background Art

[0002] Exosomes are a class of nanoscale vesicles released into the extracellular environment by exocytosis after the fusion of multivesicular bodies (MVBs) with the cell membrane, with a diameter usually ranging from 30 to 150 nm. Exosomes are widely present in body fluids such as blood, urine, saliva, milk, and cerebrospinal fluid derived from mammals, and also in media such as fruit juice, extracts, and plant tissue culture media derived from plants. Exosomes contain various active molecules such as proteins, lipids, DNA, mRNA, and miRNA, and can transmit signals between cells, regulate immune responses, and participate in physiological and pathological processes such as cell migration, proliferation, and differentiation. With the in-depth application research of exosomes in the fields of tumor diagnosis, immunotherapy, tissue repair, and drug delivery, their potential as liquid biopsy markers and drug carriers is being widely concerned.

[0003] In recent years, exosomes derived from plants have become a research hotspot. Plant exosomes have a wide source, are easy to obtain, and the extraction process is relatively mild and low-cost. Plant exosomes not only have good biocompatibility and biodegradability, but also are rich in plant-derived functional active components such as polyphenols, flavonoids, and specific small RNAs, showing excellent anti-inflammatory, antioxidant, and tissue repair-promoting functions. Plant exosomes have shown unique application values in the fields of tumor targeted therapy, inflammation regulation, and intestinal microecology regulation. However, compared with mammalian exosomes, plant exosomes have a more complex particle size distribution, high compositional heterogeneity, and complex source matrix, thus posing higher requirements for separation and enrichment methods.

[0004] Currently, there are still significant deficiencies in the mainstream separation methods for exosomes derived from mammals and plants: As a standard separation technique, ultracentrifugation is complex in operation and prone to causing exosome rupture. Especially when dealing with plant tissue homogenate samples, the impurity content is high and it is difficult to guarantee the purity of exosomes; the polymer precipitation method is prone to introducing polymer residue contamination, affecting subsequent exosome function verification and downstream applications; although density gradient centrifugation and immunoaffinity separation can improve purity, they have problems such as low separation efficiency, strong antibody dependence, and difficulty in large-scale application. For plant exosomes, due to the obvious differences in their surface structure and composition, traditional separation methods are insufficient in terms of separation selectivity and purity control.

[0005] In summary, there is currently a lack of a general and efficient separation and enrichment technology that can adapt to the diverse exosomes from mammalian and plant sources and simultaneously meet the application requirements of high separation efficiency, high purity, low cost, and large-scale production. Therefore, there is an urgent need to develop a new separation and enrichment method that can not only adapt to the treatment of complex matrix samples but also maintain the structural and functional integrity of exosomes, especially taking into account the separation and enrichment requirements of exosomes from mammalian and plant sources. Summary of the Invention

[0006] The present invention aims to solve the problems existing in the existing exosome separation methods, such as complex operation, high cost, strong equipment dependence, low enrichment efficiency, and easy damage to the exosome structure. It provides an exosome enrichment method with simple process, high enrichment efficiency, good purity, and scalable production, that is, an exosome enrichment method based on a modified cellulose material.

[0007] The present invention also provides the exosomes obtained by the above method.

[0008] To achieve the above object, the solution of the present invention is as follows: An exosome enrichment method based on a modified cellulose material, comprising the following steps: Extract cellulose from plant sources; Subject the cellulose from plant sources to alkali extraction and bleaching treatment; Obtain carboxylated cellulose by TEMPO-catalyzed oxidation; Graft polyethyleneimine (PEI) onto the carboxylated cellulose by the EDC / NHS crosslinking method to obtain a modified cellulose material with PEI grafted on its surface, and the obtained cellulose material has a positive charge on its surface; Mix and incubate the modified cellulose material with the original exosome solution to be treated to selectively enrich exosomes, and obtain an enrichment complex of the modified cellulose material and exosomes; Collect and elute the enrichment complex to release the exosomes from the modified cellulose material; then further purify by ultrafiltration concentration and impurity removal filtration to obtain high-purity exosomes.

[0009] Further, the collection and elution of the exosomes include any one or a combination of the following methods: syringe method, chromatography column method, elution method, enzymatic digestion method, ultrasonic method, temperature regulation method, solvent dissolution method, centrifugation method, pH regulation method.

[0010] After the exosomes are collected and eluted, they can be further purified using a filter membrane with a pore size of 0.45 μm or less.

[0011] Further, the cellulose from plant sources includes, but is not limited to: ginseng cellulose, bamboo pulp cellulose, corn straw cellulose, lignin-treated residue.

[0012] Furthermore, for the alkali extraction and bleaching treatment: Add cellulose from plant sources into a sodium hydroxide solution with a mass fraction of 2% - 10%, with a liquid-solid ratio of 10:1 - 30:1, react at 60°C - 90°C for 1 - 4 hours to remove lignin, pectin, and hemicellulose, obtaining a crude cellulose product after alkali extraction; place the crude cellulose product after alkali extraction into an aqueous solution of sodium hypochlorite with a mass percentage of 1% - 10%, with a liquid-solid ratio of 10:1 - 30:1, react at 60°C - 90°C for 15 - 30 minutes to obtain bleached cellulose.

[0013] Furthermore, for obtaining carboxylated cellulose by TEMPO-catalyzed oxidation: Disperse the bleached cellulose in a TEMPO-catalyzed reaction system, with a liquid-solid ratio of 10:1 - 30:1; the TEMPO-catalyzed reaction system includes 0.5 mM - 1.5 mM of TEMPO, 1 mM - 10 mM of NaBr, and 5 mM - 50 mM of NaClO; adjust the pH value to 10 - 11, and react at 20°C - 30°C for 2 - 6 hours.

[0014] Furthermore, subject the carboxylated cellulose suspension to shear treatment, with a shear rotation speed of 8000 - 15000 rpm and a shear time of 10 - 60 minutes; after shear, perform high-pressure homogenization treatment with a homogenization pressure of 600 bar - 1200 bar to obtain a high-purity cellulose material, and then conduct PEI grafting.

[0015] Furthermore, for the PEI grafting: Add PEI into the suspension of the high-purity cellulose material, with a mass ratio of PEI to cellulose of 1:2 - 5:1, the molecular weight range of PEI is 10 - 100 kDa, the reaction time is 4 - 24 hours, and the reaction temperature is 20°C - 30°C to obtain a modified cellulose material with PEI grafted on the surface. Use an EDC / NHS cross-linking reaction, with a mass ratio of EDC to NHS of 1:0.5 - 1:2.

[0016] Furthermore, the exosome stock solution to be treated includes, but is not limited to, exosomes from plant sources, exosomes from animal sources, or exosomes from microbial sources.

[0017] The present invention is applicable to enriching exosome samples with diverse sources, including, but not limited to, exosomes from plant sources (such as ginseng, wolfberry, grape, tea, etc.), exosomes from animal sources (serum, plasma, milk, urine, etc.), exosomes from microbial sources (vesicle-like particles secreted by bacteria, yeast, fungi, etc.), and is applicable to various complex samples such as plant juices, plant tissue culture media, animal body fluids, and cell culture supernatants.

[0018] Furthermore, mix the modified cellulose material with surface grafted with PEI and the supernatant of the to-be-treated exosome stock solution without ultracentrifugation treatment, and incubate at 4°C to 37°C for 0.5 to 6 hours to achieve the enrichment of exosomes. The enriched exosomes are negatively charged.

[0019] The enrichment complex of the modified cellulose material and exosomes can be used for the enrichment, concentration, storage or transportation of exosomes.

[0020] An exosome enriched by the method based on any one of the above, the exosome is spherical or nearly spherical, with clear edges, a particle size of 30 to 150 nm, and a complete structure.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention significantly improves the surface functionality of the cellulose material through TEMPO oxidation and polyethyleneimine graft modification, realizing the efficient adsorption of exosomes, and is particularly suitable for the enrichment of exosomes in complex plant stock solutions without ultracentrifugation treatment.

[0022] Specifically: By performing TEMPO oxidation treatment on plant residue cellulose (such as ginseng residue), a cellulose material with a carboxyl structure is prepared, and then the cellulose structure is further refined by mechanical shearing and high-pressure homogenization means, significantly improving its specific surface area and dispersion stability. On this basis, through polyethyleneimine (PEI) grafting reaction, a large number of amino functional groups are introduced to enhance the positive charge on the surface of the material, thereby realizing the efficient adsorption and enrichment of negatively charged exosomes.

[0023] (2) The modified cellulose material provided by the present invention has a wide source, is easy to prepare, and the enrichment process is mild, which can significantly improve the separation purity and enrichment efficiency of exosomes, and has good biocompatibility and environmental friendliness.

[0024] (3) The exosome enrichment method provided by the present invention has a simple process, avoids complex operations such as ultracentrifugation and density gradient centrifugation, reduces equipment dependence and production costs; has high enrichment efficiency, good exosome purity, and can release exosomes in different ways according to needs while maintaining the structural and functional integrity of exosomes, and has good application prospects and industrialization value.

[0025] (4) The enrichment method of the present invention can be applied to exosomes derived from plants, animals or microorganisms, including but not limited to. Description of the Drawings

[0026] Figure 1 : FTIR spectra of cellulose grafted with PEI of different molecular weights and ungrafted cellulose (GNFs); Figure 2: XRD patterns of cellulose grafted with PEI of different molecular weights and ungrafted cellulose (GNFs); Figure 3 : AFM image of 25 kDa PEI-GNFs; Figure 4 : SEM images of cellulose grafted with PEI of different molecular weights (bar = 1 μm); Figure 5 : AFM image of the mixture of ginseng exosomes enriched by 25 kDa PEI-GNFs; Figure 6 : SEM image of the mixture of ginseng exosomes enriched by 25 kDa PEI-GNFs; Figure 7 : Comparative graph of Zeta potential of cellulose grafted with PEI of different molecular weights and their complexes enriched with ginseng exosomes; Figure 8 : NTA images of exosomes obtained by cellulose enrichment method (left) and ultracentrifugation method (right); Figure 9 : TEM images of exosomes obtained by cellulose enrichment method (left) and ultracentrifugation method (right) (bar = 100nm); Figure 10 : Schematic diagram of the assembly of the syringe filtration device for enriching exosomes with modified cellulose; Figure 11 : Schematic diagram of the use of the syringe filtration device for enriching exosomes with modified cellulose; Figure 12 : TEM image of the exosomes of bacterial origin obtained by the cellulose enrichment method. Detailed implementation mode

[0027] The present invention will be described below in conjunction with the accompanying drawings and embodiments, but not limited thereto. Modifications and improvements made by those skilled in the art under the inspiration of the present invention shall fall within the protection scope of the present invention.

[0028] Preparation Example 1: In this example, ginseng cellulose is used as an example, and the specific process is as follows: 1. Preparation of ginseng cellulose (1) Select the residue after squeezing juice from ginseng (Panax ginseng C.A. Meyer), wash, air-dry and crush it to a particle size not greater than 1 mm.

[0029] (2) Weigh 20 g of ginseng residue powder, add it to a sodium hydroxide (NaOH) solution with a mass fraction of 5%, with a liquid-solid ratio of 20:1 (mL / g), and react at 80 °C for 2 hours to remove impurities such as lignin, pectin and hemicellulose.

[0030] After the reaction is completed, the residue is repeatedly washed with deionized water until the washing liquid is neutral, and the crude cellulose after alkali extraction is obtained.

[0031] (4) Weigh the crude cellulose after alkali extraction, add deionized water according to a liquid-solid ratio of 10:1 (mL / g), add sodium hypochlorite (NaClO) with a mass fraction of 5% (effective chlorine content 10%), and react at 80 °C for 15 - 20 minutes. After the reaction is completed, repeatedly rinse with deionized water until neutral to obtain bleached and purified ginseng cellulose.

[0032] 2. Preparation of carboxylated cellulose Weigh 20 g of bleached and purified ginseng cellulose, disperse it in a TEMPO catalytic reaction system (1 mM TEMPO, 1 mM NaBr, and 10 mM NaClO), and dissolve it in 200 mL of deionized water. Adjust the pH value of the reaction system to 10 and react at room temperature for 2 hours. After the reaction is completed, add ten times the volume of deionized water to terminate the reaction, and repeatedly wash with deionized water until the washing liquid is neutral to obtain carboxylated ginseng cellulose GNFs.

[0033] 3. Preparation of modified cellulose materials The carboxylated ginseng cellulose suspension is mechanically sheared and dispersed at a shearing speed of 12000 rpm for 30 minutes. Subsequently, it is continuously circulated and processed 5 times under a pressure of 600 bar using a high-pressure homogenizer to obtain highly pure ginseng cellulose with good dispersibility.

[0034] Weigh 2 g of dry cellulose, add 1 g of EDC and 1 g of NHS, and stir and activate at room temperature (25 °C) for 1 hour. Subsequently, add 4 g of a 50% polyethyleneimine (PEI) solution with a PEI molecular weight of 25 kDa. Using an EDC / NHS cross-linking reaction with a mass ratio of EDC to NHS of 1:0.5 - 1:2, the reaction system continues to stir at room temperature for 24 hours to complete the grafting reaction of PEI.

[0035] After the reaction is completed, add deionized water for dilution and wash 5 times to remove unreacted substances, and obtain a modified ginseng cellulose material (25 kDa PEI - GNFs) with 25 kDa PEI grafted on the surface.

[0036] Preparation Example 2: Other operations are the same as in Example 1, except that the molecular weight of the PEI is 10 kDa, and the remaining conditions remain unchanged.

[0037] Preparation Example 3: Other operations are the same as in Example 1, except that the molecular weight of the PEI is 70 kDa, and the remaining conditions remain unchanged.

[0038] Example 1: In this example, the prepared example 1 and ginseng juice are used as examples, and the specific process is as follows: 1. Enrichment of ginseng exosomes Ginseng stock solution was prepared by chopping and homogenizing ginseng roots. The supernatant was taken after centrifugation at 10000 g for 30 minutes, and the process was repeated 3 times to remove large particle impurities, obtaining ginseng stock solution without ultracentrifugation treatment.

[0039] The modified ginseng cellulose material obtained in Preparation Example 1 was added to the ginseng stock solution without ultracentrifugation treatment.

[0040] The modified ginseng cellulose was mixed with the ginseng stock solution without ultracentrifugation treatment, and slowly shaken and incubated at 4 °C for 30 minutes to enrich ginseng exosomes, obtaining an enrichment complex of 25 kDa PEI-GNFs and exosomes (GEVPs@25 kDa PEI-GNFs).

[0041] 2. Elution and collection of ginseng exosomes After enrichment, the enrichment complex was added to a citric acid buffer solution with a pH of 5.5, and slowly shaken and incubated for 30 minutes to disrupt the electrostatic adsorption between the cellulose material and ginseng exosomes, releasing ginseng exosomes.

[0042] The eluate was filtered through a 0.45 μm filter to obtain ginseng exosomes with a particle size of 80 - 120 nm.

[0043] Test Example 1: Testing and characterization of ginseng cellulose obtained in Preparation Examples 1 - 3 In this test example, the structural characterization of cellulose materials grafted and modified with polyethyleneimine (PEI) of different molecular weights was carried out. The selected PEI molecular weights were 10 kDa, 25 kDa, and 70 kDa, namely 10 kDa PEI-GNFs, 25 kDa PEI-GNFs, and 75 kDa PEI-GNFs. The ungrafted sample GNFs (carboxylated ginseng cellulose) was used as a control group.

[0044] (1) Fourier transform infrared spectroscopy (FTIR) After the freeze-dried cellulose samples (GNFs and samples grafted with PEI of different molecular weights) were ground evenly, they were mixed with dry KBr at a mass ratio of 1:100 and pressed into tablets to make infrared transparent tablets. A Fourier transform infrared spectrometer (FTIR) was used to scan in the wavenumber range of 4000 - 500 cm⁻¹ to analyze the changes in surface functional groups of the samples.

[0045] As Figure 1As shown, the GNFs sample shows an obvious absorption peak of –OH stretching vibration near 3330 cm⁻¹, the absorption peak of carboxyl (C=O) stretching vibration at 1720 cm⁻¹, and the characteristic absorption peak of C–O–C near 1060 cm⁻¹. This spectrum confirms the existence of carboxyl groups on the surface of GNFs. In contrast, the PEI-grafted samples all show an absorption peak of N–H bending vibration near 1640 cm⁻¹, and at the same time, characteristic peaks of –NH2 and –CH2 appear near 1560 cm⁻¹ and 1450 cm⁻¹, indicating that polyethyleneimine has been successfully grafted onto the cellulose surface. As the molecular weight of PEI increases, the intensity of its characteristic absorption peak gradually increases, especially in the 70 kDa sample, which indicates that high molecular weight PEI has a higher grafting density. In addition, the –OH / NH stretching vibration peaks in the range of 3300~3500 cm⁻¹ of the PEI-grafted samples show a broadening of the peak shape, further confirming the enhanced hydrogen bond interaction and the introduction of PEI.

[0046] The test results show that PEI has been successfully grafted onto the surface of carboxylated cellulose, and the higher the molecular weight, the more significant the grafting degree.

[0047] (2)X-ray Diffraction (XRD) To further verify the effect of polyethyleneimine (PEI) grafting on the crystal structure of cellulose, the crystallization properties of GNFs and three PEI-grafted cellulose samples with different molecular weights were analyzed by XRD. After the cellulose samples were fully dried, they were pressed into tablets or spread on the sample tray, and diffraction analysis was carried out using an X-ray diffractometer (XRD) with a scanning range of 2θ = 5°~70° and a scanning rate of 5° / min.

[0048] As Figure 2 shown, obvious diffraction peaks appear near 2θ ≈ 16.5°, 22.7° and 34.5° for GNFs, corresponding to the (110), (200) and (004) crystal planes of cellulose type I crystals respectively, which are typical diffraction characteristics of natural cellulose crystals. After grafting PEI, the main crystal plane characteristic peaks are still retained in each grafted sample, but as the molecular weight of PEI increases, the intensity of the diffraction peaks gradually weakens, especially in the 70 kDa grafted sample, indicating that high molecular weight PEI grafting will partially destroy the cellulose crystal structure and effectively reduce the crystallinity. In addition, the width of the diffraction peaks increases, indicating that the crystal grain size of cellulose decreases and the amorphous region increases after grafting modification. This change trend shows that the grafting of PEI disturbs the microscopic arrangement structure of cellulose and affects its crystallization behavior, and the higher the molecular weight, the more significant the effect.

[0049] The test results show that by regulating the molecular weight of PEI, the regulation of the crystallinity and structural order of cellulose can be achieved, providing a basis for the structural regulation of its functional applications.

[0050] (3)Atomic Force Microscopy Images (AFM) To further analyze the surface structure changes of PEI-grafted cellulose, Atomic Force Microscopy (AFM) was used to perform imaging analysis of the height and phase diagrams of the samples. An appropriate amount of 25 kDa PEI-GNFs sample suspension was dropped on a dry and flat mica sheet, allowed to stand at room temperature and dry, and then placed on the AFM test platform. The tapping mode was used for scanning, the scanning range was set to 1μm × 1μm, and the surface topological height and phase changes were recorded as Figure 3 shown. The sample showed a typical fiber entanglement structure in the Height diagram, with moderate surface roughness and clear fiber contours. The Phase diagram showed a uniform phase distribution, indicating uniform surface modification and consistent interfacial energy distribution of the material.

[0051] The results show that PEI-grafted cellulose exhibits good surface structure consistency and distribution uniformity at the micro-nano scale, which is beneficial to the interfacial interaction with exosomes.

[0052] (4)Scanning Electron Microscopy Images (SEM) To study the effect of graft modification of different molecular weight polyethyleneimine (PEI) on the microstructure of cellulose materials, the samples were freeze-dried and sputter-coated with gold, and their surface morphology and structure were observed under an electron microscope (Hitachi SU8100 field emission scanning electron microscope).

[0053] As Figure 4 shown, GNFs (ungrafted cellulose) presented as a loose filamentous network with a flat surface, rich pores but weak intermolecular forces. After grafting 10 kDa PEI, the inter-fiber gaps of 10 kDa PEI-GNFs shrank, and the surface was uniformly coated with polymers, but still maintained a relatively large network structure. After grafting 25 kDa PEI, the fiber network of 25 kDa PEI-GNFs was more stable and the pore size was moderate, and a uniform positive charge layer was formed on the surface, which could achieve good adsorption while ensuring the effective entry of exosomes into the network interior. Theoretically, it had the best enrichment effect. When the molecular weight of grafted PEI was increased to 75 kDa, a large amount of aggregation and adhesive layer coating appeared on the fiber surface of 70 kDa PEI-GNFs, resulting in the closure of some pores and too high network density. The steric effect made it difficult for exosomes to enter and adsorb efficiently, and the enrichment ability might be weaker theoretically.

[0054] In summary, the graft modification of PEI with different molecular weights significantly regulates the surface morphology and structural compactness of cellulose, providing a structural basis for the subsequent selective enrichment of exosomes.

[0055] Test Example 2: Characterization of the enrichment complex of ginseng exosomes enriched by the ginseng cellulose obtained in Example 1 (1) AFM images To further observe the composite morphology and structure of modified cellulose with exosomes, atomic force microscopy (AFM) was used to analyze the morphology of the complex formed by 25 kDa PEI-GNFs and exosomes obtained in Example 1. The sample was prepared as an AFM test sample by drop-coating and drying, and scanned in tapping mode to record the height map (Height) and phase map (Phase).

[0056] As Figure 5 shown, the 25 kDa PEI-GNFs enriched exosome complex showed spherical-like particles distributed on the surface of the cellulose network in the Height map, with clear morphology. Obvious phase difference distributions were visible in the Phase map, indicating the existence of a physical adsorption or electrostatic composite interface between the grafted cellulose and exosomes, and a relatively high degree of structural integration.

[0057] The experimental results show that a stable composite system can be formed between the modified cellulose and exosomes.

[0058] (2) Scanning electron microscope (SEM) images To verify the enrichment effect of modified cellulose on exosomes, SEM was used to observe the morphology and particle size distribution characteristics of exosomes after enrichment. The sample of the mixture of 25 kDa PEI-GNFs enriched ginseng exosomes obtained in Example 1 was dropped onto the SEM sample holder adhered with conductive adhesive, dried at room temperature, and then the sample was put into an ion sputtering coater for gold or platinum sputtering treatment, and imaged on a Hitachi SU8100 scanning electron microscope.

[0059] As Figure 6 shown, the enriched exosomes were spherical or nearly spherical in shape, with clear edges, and the diameter was mainly distributed in the range of 80 - 120 nm, and they were evenly distributed in the cellulose network background, without obvious aggregation or structural damage, indicating that the modified cellulose material used has good affinity and slow release ability for exosomes.

[0060] This result verifies that the material system based on PEI-modified cellulose constructed in the present invention can achieve efficient and gentle enrichment of exosomes and maintain their structural integrity.

[0061] (3) Zeta potential To evaluate the surface charge characteristics of polyethyleneimine (PEI) grafted cellulose materials with different molecular weights and their effects on the exosome adsorption ability, the Zeta potential (ζ-potential) of each sample was measured. The Zeta potential of a total of five materials in the deionized water dispersion state in Preparation Example 1 and Example 1 was tested respectively. The sample concentration was uniformly adjusted to 0.1 mg / mL. After ultrasonic dispersion, the Zeta potential was measured using a laser particle size analyzer (Malvern Zetasizer Nano ZS), and the average value was taken after measuring three times for each group.

[0062] As Figure 7 shown, the surface potential of GNFs was negative (about -25 mV). As the molecular weight of PEI increased, the Zeta potential of the grafted material surface gradually increased. Among them, 70 kDa PEI-GNFs reached the highest positive potential (about +45 mV), indicating that the grafting density of high molecular weight PEI was greater, the cation load was enhanced, which was beneficial to enrich negatively charged exosomes. The Zeta potential of the complex of 25 kDa PEI-GNFs and exosomes (GEVPs@25 kDa PEI-GNFs) decreased significantly to near neutral, about +5 mV, indicating that exosomes were successfully adsorbed on the material surface, resulting in surface charge neutralization.

[0063] This result shows that the change in Zeta potential can be used as an important indicator to judge the grafting efficiency and exosome complex efficiency, and at the same time reflects the colloidal stability of the complex system.

[0064] Test Example 3: Test and characterization of ginseng exosomes obtained in Preparation Example 1 (1) Detection of particle size distribution by Malvern nanoparticle size analyzer (NTA) To evaluate the particle size and distribution characteristics of exosomes obtained by the modified cellulose enrichment strategy and compare them with exosomes obtained by the traditional ultracentrifugation method.

[0065] In the experiment, the ginseng exosome samples prepared by the modified cellulose enrichment method in Example 1 and the ginseng exosome samples purified by the traditional ultracentrifugation (100,000 g, 90 min) method were used respectively, and diluted to 1×10 8 particles / mL and then subjected to NTA detection. The samples were measured three times at room temperature using a NanoSight NS300 system and the average value was taken, and the particle size distribution and average particle size were recorded.

[0066] As Figure 8As shown, the average particle size of exosomes obtained by the cellulose enrichment method (left figure) is 113.4 ± 2.8 nm. The particle size distribution is slightly wider, with a certain number of large and small particles, but overall it shows a spherical-like distribution, indicating that this method can completely retain the particle size heterogeneity of the original vesicles. In contrast, the average particle size of exosomes obtained by ultracentrifugation (right figure) is 114.3 ± 3.0 nm, the particle size distribution is concentrated, and the main peak is sharper, showing a certain degree of particle screening and size uniformity.

[0067] The results show that: the modified cellulose material described in the present invention can enrich exosomes under mild conditions, retain their natural size and heterogeneity characteristics, and is a new separation method that can replace ultracentrifugation.

[0068] (2)Observation of morphology by transmission electron microscopy (TEM) To compare the structural morphology differences of exosomes obtained by the modified cellulose enrichment method and the traditional ultracentrifugation method described in the present invention, transmission electron microscopy (TEM) was used to image and analyze the ginseng exosomes obtained by the two methods.

[0069] Samples of ginseng exosome solutions obtained in Example 1 and ginseng exosome samples purified by ultracentrifugation (100,000 g, 90 min) were taken respectively. After appropriate dilution with pure water, they were dropped on copper grids, allowed to dry naturally, negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscopy.

[0070] As Figure 9 shown, exosomes in both the cellulose enrichment group and the ultracentrifugation group showed a typical spherical-like structure. The particle size was mainly concentrated in the range of 80 - 120 nm, the surface was smooth, the boundary was clear, and there was no obvious damage or collapse phenomenon, indicating that the structural morphology of exosomes obtained by the two methods was highly consistent.

[0071] The experimental results verified that: the method of enriching exosomes with modified cellulose can obtain exosomes with a quality comparable to that of the ultracentrifugation method, and has the advantages of simple operation, little influence on the exosome structure, and is suitable for subsequent functional research and application development.

[0072] Test Example 4: Analysis of the enrichment efficiency of ginseng exosomes in Example 1 To evaluate the enrichment ability and reusability of the grafted 25 kDa PEI-GNFs material for ginseng exosomes, a filtration enrichment experiment was designed. Different concentrations of crude ginseng juice were used as treatment solutions, and the change in exosome protein concentration after each round of filtration was measured to calculate the enrichment efficiency.

[0073] The experimental procedure is as follows: 1) Using the crude ginseng extract without ultracentrifugation (centrifuged at 10,000 g to remove large particle impurities, and the supernatant was taken) as the enrichment target, and the concentrations of the original solution were set at 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL (BCA protein concentration); 2) Connect a syringe and a cellulose enrichment component with a 0.8 μm pore size filter membrane ( Figure 10 ), and evenly spread 3 mL of grafted 25 kDa PEI-GNFs on the filter membrane to form a hydrogel-like filtration layer; 3) Take 5 mL of crude extracts with different concentrations and perform cyclic filtration in three groups ( Figure 11 ), record the protein concentration of the filtrate after each round of filtration, and detect it by the BCA method; 4) Each round of filtration continues until the syringe cannot be pushed, indicating that the adsorption has reached saturation.

[0074] The results show (Table 1): At the crude extract concentrations of 0.5 mg / mL and 1.0 mg / mL, as the number of cyclic filtration increases, the protein concentration in the filtrate gradually decreases, and the enrichment efficiency correspondingly increases, indicating that exosomes are gradually adsorbed onto the cellulose surface, and cycling helps to improve the overall enrichment efficiency. Among them, at the concentration of 1.0 mg / mL, the enrichment efficiency of the third cycle reaches 52.57%, which is the highest among the three groups, indicating that multiple rounds of filtration at a moderate concentration are most helpful for improving the exosome capture efficiency. At the concentration of 1.5 mg / mL, the enrichment efficiency of the first round is only 41.89%, and more cycles cannot be successfully completed, suggesting that at a higher protein background, the material surface quickly saturates, which is not conducive to further enrichment. This shows that within an appropriate concentration range, by setting continuous multiple rounds of cyclic filtration, the overall enrichment efficiency of cellulose materials for exosomes can be improved, which helps to achieve deep recovery of low-concentration samples. In contrast, at the high concentration of 1.5 mg / mL, the enrichment efficiency decreases after the first round, suggesting that this concentration is close to the upper limit of the adsorption capacity.

[0075] Table 1: Exosome Enrichment Efficiency of Crude Ginseng Extract after Cyclic Filtration through Modified Cellulose Material (Unit: %)

[0076] The results indicate that the modified cellulose can be used for efficient enrichment of plant exosomes and is a natural source adsorption material with both efficiency and economy.

[0077] Example 2: In this example, the ginseng cellulose prepared in Preparation Example 1 was used to enrich bacterial exosomes, and the specific process is as follows: Lachnoclostridium bacteria (DSMZ, 107473) were cultured under anaerobic conditions until the optical density (600 nm) reached 1.5, and exosomes were isolated from the bacterial culture.

[0078] First, bacteria were removed by centrifugation at 300 xg for 10 minutes at 4°C, then at 3,000 xg for 20 minutes, and finally at 12,000 xg for 30 minutes at 4°C. Subsequently, the supernatant was mixed with the modified ginseng cellulose obtained in Preparation Example 1 and slowly shaken and incubated at 4°C for 30 minutes. After enrichment, the enriched complex was added to a citric acid buffer solution with a pH of 5.5 and slowly shaken and incubated for 30 minutes to release extracellular vesicles of bacteria. Subsequently, high-purity extracellular vesicles of bacteria were obtained by filtration through a 0.45 μm filter membrane.

[0079] Test Example 5: TEM characterization of the morphology of the extracellular vesicles of bacteria obtained in Example 2 A sample of the extracellular vesicle solution obtained in Example 2 was taken, diluted with an appropriate amount of pure water, dropped onto a copper grid, air-dried naturally, negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscopy.

[0080] As Figure 12 shown, the obtained particles were round or quasi-round, with a clear edge membrane structure, a light gray and translucent interior, and the particle size distribution was mainly concentrated in the range of 80 - 120 nm. The morphology was consistent with that of typical extracellular vesicles, and no obvious aggregation, collapse or membrane rupture was observed, indicating that this method can stably obtain extracellular vesicles of bacteria with a complete structure.

[0081] In summary, the present invention provides a method for enriching extracellular vesicles based on a modified cellulose material, which has the advantages of natural structural origin, adjustable surface function, high enrichment efficiency, mild operation process, and high consistency with traditional methods. The modified cellulose material can achieve rapid and efficient enrichment of extracellular vesicles from plants. The enriched product has a complete morphology and a stable protein component, and has good application prospects and expansion space. The present invention is not only applicable to the preparation of plant extracellular vesicles, but can also be extended to the separation and enrichment of other source nanovesicles, proteins or nucleic acid particles, and has broad industrialization and functionalization application potential.

[0082] It should be understood that the above embodiments are only used to illustrate the principles and methods of the present invention, and are intended to help understand the core technical ideas of the present invention. For those skilled in the art, without departing from the spirit and essence of the present invention, various forms of modification, substitution or equivalent transformation can be made to it, and these should all be regarded as belonging to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims, rather than being limited to the specific embodiments in the specification.

Claims

1. An exosome enrichment method based on a modified cellulose material, characterized in that, The method includes the following: Extract cellulose from plants; Subject the cellulose from plants to alkali extraction and bleaching treatment; Obtain carboxylated cellulose through TEMPO-catalyzed oxidation; Use the EDC / NHS crosslinking method to graft polyethyleneimine (PEI) onto the carboxylated cellulose to obtain a modified cellulose material with PEI grafted on its surface, and the resulting cellulose material has a positive charge on its surface; Mix and incubate the modified cellulose material with the exosome stock solution to be treated to selectively enrich exosomes, obtaining an enrichment complex of the modified cellulose material and exosomes; Collect and elute the enrichment complex to release the exosomes from the modified cellulose material; then further purify by ultrafiltration concentration and impurity removal filtration to obtain high-purity exosomes.

2. The exosome enrichment method based on a modified cellulose material according to claim 1, wherein, The collection and elution of the exosomes include any one or a combination of multiple methods such as the syringe method, chromatography column method, elution method, enzymatic hydrolysis method, ultrasonic method, temperature regulation method, solvent dissolution method, centrifugation method, and pH regulation method.

3. A method for exosome enrichment based on a modified cellulose material according to any one of claims 1-3, characterized in that, The cellulose from plants includes but is not limited to: ginseng cellulose, bamboo pulp cellulose, corn straw cellulose, and lignin-treated residue.

4. A method for exosome enrichment based on a modified cellulose material according to any one of claims 1-3, characterized in that, The alkali extraction and bleaching treatment: Add the cellulose from plants to a sodium hydroxide solution with a mass fraction of 2% - 10%, with a liquid-solid ratio of 10:1 - 30:1, and react at 60°C - 90°C for 1 - 4 hours to remove lignin, pectin, and hemicellulose, obtaining a crude cellulose product after alkali extraction; Place the crude cellulose product after alkali extraction in an aqueous solution of sodium hypochlorite with a mass percentage of 1% - 10%, with a liquid-solid ratio of 10:1 - 30:1, and react at 60°C - 90°C for 15 - 30 minutes to obtain bleached cellulose.

5. A method for exosome enrichment based on a modified cellulose material according to any one of claims 1-3, characterized in that, The obtaining of carboxylated cellulose through TEMPO-catalyzed oxidation: Disperse the bleached cellulose in a TEMPO-catalyzed reaction system, with a liquid-solid ratio of 10:1 - 30:1; the TEMPO-catalyzed reaction system includes 0.5 mM - 1.5 mM of TEMPO, 1 mM - 10 mM of NaBr, and 5 mM - 50 mM of NaClO; adjust the pH value to 10 - 11 and react at 20°C - 30°C for 2 - 6 hours.

6. A method for exosome enrichment based on a modified cellulose material according to any one of claims 1-3, characterized in that, Subject the carboxylated cellulose suspension to shearing treatment, with a shearing speed of 8000 - 15000 rpm and a shearing time of 10 - 60 minutes; after shearing, perform high-pressure homogenization treatment with a homogenization pressure of 600 bar - 1200 bar to obtain a high-purity cellulose material, and then carry out PEI grafting.

7. A method for exosome enrichment based on a modified cellulose material according to any one of claims 1-3, characterized in that, The PEI grafting: Add PEI to the suspension of the high-purity cellulose material, with a mass ratio of PEI to cellulose of 1:2 - 5:1, a molecular weight range of PEI of 10 - 100 kDa, a reaction time of 4 - 24 hours, and a reaction temperature of 20°C - 30°C to obtain a modified cellulose material with PEI grafted on its surface.

8. A method for enriching exosomes based on a modified cellulose material according to any one of claims 1-3, characterized in that, The exosome stock solution to be treated includes but is not limited to exosomes from plants, exosomes from animals, or exosomes from microorganisms.

9. The exosome enrichment method based on a modified cellulose material according to claim 8, wherein, Mix the modified cellulose material with PEI grafted on its surface with the supernatant of the untreated exosome stock solution without ultracentrifugation treatment, and incubate at 4°C to 37°C for 0.5 to 6 hours to achieve exosome enrichment. The enriched exosomes are negatively charged.

10. An exosome obtained by enrichment based on the method according to any one of claims 1 to 9, characterized in that, The exosomes are spherical or nearly spherical, with clear edges, a particle size of 30 to 150 nm, and a complete structure.

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