Preparation method for industrial production of plant-derived exosomes

Abstract

The invention discloses a preparation method for industrial production of plant-derived exosomes, and relates to the technical field of biological product preparation. Comprising the following steps: S1, pretreatment of raw materials: cleaning and disinfecting plant tissues, mixing the plant tissues with a buffer solution, and homogenizing to obtain a plant crude extract; s2, pre-clarification: sequentially carrying out differential centrifugation and step-by-step filtration on the crude extract obtained in the step S1 to remove large-particle impurities so as to obtain pre-clarified liquid; s3, tangential flow filtration: performing concentration and buffer solution replacement on the pre-clarified solution through a tangential flow filtration system to obtain a primarily purified exosome concentrated solution. According to the method disclosed by the invention, industrial, continuous and scalable production of the plant exosome is realized, the product purity is high, and the batch difference is small; impurities influencing gelation are removed through an optimized purification process, a method for compounding the exosome with polysaccharide hydrogel under a low shear condition is defined, the formed compound preparation improves the structural stability of the exosome by 2-5 times, and local sustained-release administration is realized.

Description

Technical Field

[0001] This invention relates to the field of bioproduct preparation technology, and in particular to a method for preparing plant-derived exosomes for industrial production. Background Technology

[0002] Plant-derived extracellular vesicles (PDEVs) are a class of natural nanocarriers that have attracted widespread attention in recent years, showing promising applications in tissue repair, anti-inflammation, and hair growth. However, the preparation of plant exosomes is currently mostly limited to laboratory scale, and faces the following problems: 1. Large batch-to-batch variation: Plant raw materials are significantly affected by the place of origin, season, and maturity, resulting in unstable exosome yield.

[0003] 2. High impurity content: Plant tissues contain a large amount of polyphenols, polysaccharides, pigments and cell debris, making it difficult to achieve the purity of biological products.

[0004] 3. Lack of scalable technology: Traditional ultracentrifugation cannot achieve production at the scale of hundreds of liters.

[0005] 4. Does not meet GMP requirements: lacks unified process parameters, quality control indicators and documented procedures.

[0006] 5. Incompatibility with formulation systems: Mixing exosomes with hydrogel systems can lead to structural instability and degradation. Therefore, a standardized, GMP-compatible exosome production process suitable for downstream formulations is needed. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for the industrial production of plant-derived exosomes.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing plant-derived exosomes for industrial production includes the following steps: S1: Raw material pretreatment: After cleaning and disinfecting the plant tissue, it is mixed with buffer solution and homogenized to obtain crude plant extract; S2: Pre-clarification: The crude extract obtained in S1 is subjected to differential centrifugation and step-by-step filtration to remove large particulate impurities and obtain a pre-clarified liquid. S3: Tangential flow filtration: The pre-clarified solution is concentrated and buffer replaced by a tangential flow filtration system to obtain a preliminarily purified exosome concentrate; S4: Purification: The exosome concentrate is purified by size exclusion chromatography or density gradient ultracentrifugation to collect high-purity plant exosome components.

[0009] Preferably, in step S1, the homogenization conditions are as follows: homogenize at 8000-10000 rpm for 30 seconds at low temperature, repeat once after an ice bath interval; the mass-to-volume ratio of the plant tissue to the buffer solution is 1:5 g:mL.

[0010] Preferably, in step S2, differential centrifugation includes: Centrifuge at 300×g, 4℃ for 10 minutes to remove unbroken tissue fragments; Centrifuge at 2000×g, 4℃ for 15 minutes to remove cell debris; Centrifuge at 10000×g, 4℃ for 30 minutes to remove large vesicles and fibrous impurities; The step-by-step filtration includes passing the material through filter membranes of 5μm, 0.45μm, and 0.22μm in sequence.

[0011] Preferably, in step S3, the tangential flow filtration system uses a membrane with a molecular weight cutoff of 100-500 kDa, an operating temperature of 4-10°C, a transmembrane pressure controlled at 0.5-1.0 bar, and a shear rate not exceeding 3000 s⁻¹. -1 The concentration factor is 10-20 times, and the solution is replaced with 5-7 system volumes of buffer.

[0012] Preferably, in step S4, the packing material used for the size exclusion chromatography is Sepharose CL-2B, the column bed volume is not less than 10 times the sample loading volume, and the elution flow rate is 0.3-0.5 mL / min; the eluted fraction located in the empty volume region is collected as high-purity exosomes.

[0013] Preferably, after S4, S5 is included: aseptic final treatment and preservation, in which the purified exosome solution is filtered through a 0.22μm aseptic filter and optionally a lyophilization protectant is added for freeze drying; the lyophilization protectant is 5-10% w / v trehalose or 3-5% w / v mannitol.

[0014] Preferably, the plant tissue is selected from mulberry leaves; the mulberry leaves are mature but not lignified functional leaves, and are processed within 4 hours after collection.

[0015] A plant-derived exosome prepared by the above method, wherein the plant-derived exosome has an average particle size distribution of 80-150 nm, and has a nanovesicle structure and a clear lipid bilayer membrane.

[0016] An exosome-hydrogel composite formulation comprising the above-mentioned plant-derived exosomes and a polymeric hydrogel matrix.

[0017] The beneficial effects of this invention are as follows: 1. This invention enables the industrial, continuous, and scale-up (≥100L) production of plant exosomes, with high product purity and small batch-to-batch variation (CV≤15%).

[0018] 2. The process parameters of this invention are controllable and meet GMP documentation requirements; the optimized purification process removes impurities that affect gelation, and clarifies the method of compounding with polysaccharide hydrogel under low shear conditions. The resulting composite formulation improves the structural stability of exosomes by 2-5 times, realizes local sustained-release drug delivery, and broadens the application scenarios.

[0019] 3. This invention can be used in the fields of androgenetic alopecia, water-light products, and tissue repair. Attached Figure Description

[0020] Figure 1 This is an electron microscope image of exosomes derived from mulberry leaves in this invention; Figure 2 This is a particle size distribution diagram of exosomes derived from mulberry leaves in this invention; Figure 3 This is a diagram of an in vitro uptake experiment of mulberry leaf-derived exosomes co-cultured with HUVECs according to the present invention. Figure 4 This is a comparison image of SDS-PAGE of mulberry leaf-derived exosomes from this invention with other exosomes. Detailed Implementation

[0021] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0022] Example 1: A method for preparing plant-derived exosomes for industrial production, using mulberry leaves (Morus alba L.) as the specific example, provides a detailed description of the preparation method. The method, while ensuring GMP compatibility and industrial scalability, yields high-purity and highly stable mulberry leaf-derived exosomes; the specific steps include: (1) Raw material pretreatment: Fresh mulberry leaves were selected as raw materials, preferably mature but non-lignified functional leaves. The mulberry leaves were processed within 4 hours after collection. First, they were rinsed with running deionized water to remove surface mud and impurities, then rapidly surface-sterilized with 75% (v / v) ethanol for 30 seconds, and then rinsed 3 times with sterile PBS (pH 7.4). The processed mulberry leaves were added to pre-cooled sterile PBS buffer at a mass-to-volume ratio of 1:5 (g:mL), and the tissue was homogenized using a high-speed homogenizer under low temperature conditions. The homogenization parameters were 8000-10000 rpm, and the homogenization was carried out continuously for 30 seconds. After standing in an ice bath for 30 seconds, the process was repeated once to obtain crude mulberry leaf extract.

[0023] (2) Low-speed differential centrifugation and pre-clarification: The crude mulberry leaf extract was subjected to the following centrifugation steps in sequence: 300×g, 4℃, 10min, to remove insufficiently broken tissue blocks; 2000×g, 4℃, 15min, to remove cell debris; 10000×g, 4℃, 30min, to remove large vesicles and fibrous impurities. The supernatant was then filtered sequentially through 5μm, 0.45μm and 0.22μm filter membranes to further remove plant cell wall residues, polysaccharide aggregates and suspended impurities, to obtain a pre-clarified solution.

[0024] (3) Tangential Flow Filtration (TFF) Concentration and Desalination: The pre-clarified liquid is introduced into a tangential flow filtration system for continuous treatment. The TFF membrane material used is polyethersulfone (PES), with a molecular weight cutoff of 300 kDa (selectable within the range of 100-500 kDa). The operating temperature is controlled at 4-10℃, the transmembrane pressure (TMP) is controlled at 0.5-1.0 bar, and the shear rate is not higher than 3000 s. -1 The sample was concentrated 10-20 times by TFF and then replaced with sterile PBS at a volume of 5-7 system volumes to effectively remove small molecule salts, soluble proteins, polyphenols and metabolites, thus obtaining a preliminarily purified mulberry leaf exosome concentrate.

[0025] (4) Purification Steps: To further improve the purity of exosomes, size exclusion chromatography (SEC) is preferred for purification. The packing material used is Sepharose CL-2B or an equivalent material, the column bed volume is not less than 10 times the sample loading volume, the eluent is PBS, and the flow rate is controlled at 0.3-0.5 mL / min. The eluted fraction located in the void volume region of the elution curve is collected as the high-purity mulberry leaf-derived exosome fraction.

[0026] (5) Aseptic final processing and preservation: The exosome solution purified by SEC is aseptically filtered through a 0.22 μm filter to obtain mulberry leaf-derived exosome stock solution that can be directly used for formulation or subsequent processing. If necessary, 5-10% (w / v) trehalose or 3-5% (w / v) mannitol can be added to the exosome stock solution as a freeze-drying protectant, and vacuum freeze-drying is performed after pre-freezing at −80℃ to improve its storage stability.

[0027] (6) Combination with hydrogel formulations: Under low shear conditions, the prepared mulberry leaf-derived exosomes are added to a polysaccharide hydrogel system mainly composed of sodium carboxymethyl cellulose, sodium alginate or hyaluronic acid to form an exosome-hydrogel composite formulation, so as to avoid the destruction of exosome structure and achieve local sustained-release drug delivery.

[0028] Example 2: Preparation and characterization of exosomes derived from mulberry leaves like Figure 1-4 As shown, mulberry leaf-derived exosomes were prepared according to the above method, and the obtained products were characterized. Nanoparticle tracking analysis (NTA) was used to detect their particle size and concentration. The results showed that the average particle size of the exosomes was distributed in the range of 80–150 nm, exhibiting a unimodal distribution, with a particle concentration of 5.5 × 10¹⁰ particles / mL. Transmission electron microscopy revealed that they possessed typical spherical or cup-shaped nanovesicle structures with a clearly defined lipid bilayer membrane. Cellular uptake experiments demonstrated that the exosomes extracted by this method exhibited specific biological activity and could be taken up by target cells and absorbed into the cytoplasm to exert their biological functions.

[0029] SDS-PAGE analysis of the protein composition revealed the presence of proteins with particle sizes of 50 kDa and <20 kF, with no obvious contamination bands from plant-soluble proteins. The coefficients of variation for particle size and protein content were all less than 20% for each batch, indicating that this method has good stability and reproducibility. The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing plant-derived exosomes for industrial production, characterized in that, Includes the following steps: S1: Raw material pretreatment: After cleaning and disinfecting the plant tissue, it is mixed with buffer solution and homogenized to obtain crude plant extract; S2: Pre-clarification: The crude extract obtained in S1 is subjected to differential centrifugation and step-by-step filtration to remove large particulate impurities and obtain a pre-clarified liquid. S3: Tangential flow filtration: The pre-clarified solution is concentrated and replaced with buffer solution through a tangential flow filtration system to obtain a preliminarily purified exosome concentrate; S4: Purification: The exosome concentrate is purified by size exclusion chromatography or density gradient ultracentrifugation to collect high-purity plant exosome components.

2. The method for preparing plant-derived exosomes for industrial production according to claim 1, characterized in that, In S1, the homogenization conditions are as follows: homogenize at 8000-10000 rpm for 30 seconds at low temperature, repeat once after an ice bath interval; the mass-to-volume ratio of the plant tissue to the buffer solution is 1:5 g:mL.

3. The method for preparing plant-derived exosomes for industrial production according to claim 1, characterized in that, In S2, differential centrifugation includes: Centrifuge at 300×g, 4℃ for 10 minutes to remove unbroken tissue fragments; Centrifuge at 2000×g, 4℃ for 15 minutes to remove cell debris; Centrifuge at 10000×g, 4℃ for 30 minutes to remove large vesicles and fibrous impurities; The step-by-step filtration includes passing the material through filter membranes of 5μm, 0.45μm, and 0.22μm in sequence.

4. The method for preparing plant-derived exosomes for industrial production according to claim 1, characterized in that, In step S3, the tangential flow filtration system uses a membrane with a molecular weight cutoff of 100-500 kDa, an operating temperature of 4-10°C, a transmembrane pressure controlled at 0.5-1.0 bar, and a shear rate not exceeding 3000 s⁻¹. -1 The concentration factor is 10-20 times, and the solution is replaced with 5-7 system volumes of buffer.

5. The method for preparing plant-derived exosomes for industrial production according to claim 1, characterized in that, In step S4, the size exclusion chromatography uses Sepharose CL-2B packing material, the column bed volume is not less than 10 times the sample loading volume, and the elution flow rate is 0.3-0.5 mL / min; the eluted fraction located in the empty volume region is collected as high-purity exosomes.

6. The method for preparing plant-derived exosomes for industrial production according to claim 5, characterized in that, The process after S4 includes S5: aseptic final treatment and preservation, in which the purified exosome solution is filtered through a 0.22 μm aseptic filter and optionally a lyophilization protectant is added for freeze drying; the lyophilization protectant is 5-10% w / v trehalose or 3-5% w / v mannitol.

7. The method for preparing plant-derived exosomes for industrial production according to claim 1, characterized in that, The plant tissue was selected from mulberry leaves; the mulberry leaves were mature but not lignified functional leaves, and were processed within 4 hours after collection.

8. A plant-derived exosome prepared by the method of any one of claims 1 to 7.

9. The plant-derived exosomes according to claim 8, characterized in that, The plant-derived exosomes have an average particle size distribution of 80-150 nm and possess a nanovesicle structure and a clear lipid bilayer membrane.

10. An exosome-hydrogel composite formulation, characterized in that, It comprises plant-derived exosomes as described in claim 8 or 9 and a polymeric hydrogel matrix.

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