A method for preserving a plant exosome

Abstract

This invention discloses a method for preserving plant exosomes, belonging to the field of biotechnology. This method achieves rapid preservation of plant exosomes through vacuum freeze-drying combined with a special freeze-drying protectant. The method effectively protects the structural integrity and biological activity of plant exosomes, and the resulting dried plant exosome samples exhibit good stability and can be stored at room temperature for a relatively long period.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for preserving exosomes. Background Technology

[0002] Exosomes are membranous vesicles secreted by living cells (including animal and plant cells), with a diameter of approximately 30–150 nm and a density of approximately 1.13–1.19 g / mL. Current methods for preparing and isolating plant cell-secreted exosomes primarily involve directly extracting exosome suspensions by juicing plant cells. However, the various proteins, RNA, and DNA contained in the resulting product are highly sensitive to temperature in their liquid state and are prone to degradation and rupture, thus requiring stable storage. Currently, the main storage method for plant exosomes is cryopreservation of suspensions (storage temperature approximately -20 to -80°C), but this method significantly limits the applications of plant exosomes. Summary of the Invention

[0003] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for the preservation and processing of plant exosomes. This method can achieve rapid preservation of plant exosomes by vacuum freeze-drying in combination with a special freeze-drying protective solution. The method can effectively protect the structural integrity and biological activity of plant exosomes. The resulting dried plant exosome samples have good stability and can be stored at room temperature for a long time.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A method for preserving plant exosomes includes the following steps:

[0006] (1) Gradient pre-freezing treatment:

[0007] Plant exosomes were mixed evenly with the freeze-drying protective solution and filtered to remove bacteria. The resulting mixture A was cooled to 4.5–5.5 °C and held at that temperature for 1.5–2.5 h, then cooled to -18–-22 °C and held at that temperature for 1.5–2.5 h, and finally cooled to -43–-47 °C and held at that temperature for 1.5–2.5 h. The cooling rate was 0.5–0.85 °C / min.

[0008] (2) Single drying process:

[0009] The mixture A after gradient pre-freezing in step (1) was heated to -24 to -26°C under a vacuum of 0.27 to 0.3 mbar and held for 7.5 to 8.5 h, then heated to -14 to -16°C and held for 5.5 to 6.5 h, and finally heated to -4 to -6°C and held for 1.5 to 2.5 h; the heating rate was 0.08 to 0.1°C / min.

[0010] (3) Secondary drying process:

[0011] The sample obtained from the first drying process in step (2) is heated to 9-11℃ and kept at that temperature for 5.5-6.5h, then heated to 24-26℃ and kept at that temperature for 3.5-4.5h, and finally the vacuum degree is reduced to ≤0.01mbar and kept at that temperature for 3.5-4.5h. After the compression treatment, the preservation treatment of plant exosomes is completed; the heating rate is 0.08-0.085℃ / min.

[0012] The freeze-drying protective solution comprises the following components in parts by weight: 0.5-5 parts PEG1500, 1-6 parts glycerol, 2-10 parts mannitol, 1-8 parts trehalose, and 0.01-0.5 parts tetrahydropyrimidine.

[0013] In the preservation and processing method of plant exosomes described in this invention, the plant exosomes are pre-frozen, freeze-dried once, and desorbed twice within the active temperature and humidity range. This process transforms the plant exosomes into freeze-dried powder products with uniform morphology and particle size, resulting in a high appearance qualification rate. The product also has low water content and high biological activity. When the freeze-drying protective solution is mixed with and processed with the plant exosomes, it can effectively act as a permeation regulator and stabilizer for their biofilm and active substances. This ensures that the plant exosomes maintain structural stability after processing and exhibit ideal stability during subsequent storage, resulting in a long service life.

[0014] Meanwhile, the plant exosome preservation and treatment method of the present invention adopts conventional treatment conditions, requires no additional special reagents or treatment equipment, has simple operation steps, and can realize industrial-scale production.

[0015] Preferably, the osmotic pressure of the freeze-drying protective solution is 300–500 mOsm / kg.

[0016] The freeze-drying protective solution under the specified osmotic pressure can effectively protect the structural integrity of plant exosomes.

[0017] Preferably, the solvent of the lyophilization protective solution is at least one of water, physiological saline, and PBS buffer.

[0018] Preferably, the method for preparing the plant exosomes includes: crushing the plant, centrifuging, precipitating, and purifying by chromatography to obtain the plant exosomes.

[0019] More preferably, the plant exosomes are prepared according to the method described in CN114540271A.

[0020] It should be noted that the plant exosomes targeted by the preservation method of the present invention can be self-made products or conventionally available commercial products. The inventors have confirmed through experiments that the expected technical effects can be achieved after processing with the method described in the present invention.

[0021] Preferably, the mass ratio of the plant exosomes to the freeze-drying protective solution is (8.5-9.5):(0.5-1.5).

[0022] Preferably, the method for preserving plant exosomes includes the following steps:

[0023] (1) Gradient pre-freezing treatment:

[0024] Plant exosomes were mixed evenly with freeze-drying protection solution and filtered to remove bacteria. The resulting mixture A was cooled to 5°C and kept at that temperature for 2 hours, then cooled to -20°C and kept at that temperature for 2 hours, and finally cooled to -45°C and kept at that temperature for 2 hours.

[0025] (2) Single drying process:

[0026] The mixture A after the gradient pre-freezing treatment in step (1) was heated to -25°C under a vacuum of 0.27 to 0.3 bar and kept at that temperature for 8 hours, then heated to -15°C and kept at that temperature for 6 hours, and finally heated to -5°C and kept at that temperature for 2 hours.

[0027] (3) Secondary drying process:

[0028] The sample obtained from the first drying process in step (2) is heated to 10°C and kept at that temperature for 6 hours. Then, it is heated to 25°C and kept at that temperature for 4 hours. Finally, the vacuum degree is reduced to 0.01 mbar and kept at that temperature for 4 hours. After the plugging process, the preservation of plant exosomes is completed.

[0029] The beneficial effects of this invention are that it provides a method for preserving plant exosomes. This method uses vacuum freeze-drying in conjunction with a special freeze-drying protective solution to achieve rapid preservation of plant exosomes. The method can effectively protect the structural integrity and biological activity of plant exosomes. The resulting dried plant exosome samples have good stability and can be stored at room temperature for a long time. Detailed Implementation

[0030] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments and comparative examples. The purpose of this description is to provide a detailed understanding of the invention, not to limit its scope. All other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of this invention. Unless otherwise specified, the experimental reagents and instruments involved in the implementation of this invention are commonly used reagents and instruments. In the embodiments and comparative examples, the plant exosomes used are wheat exosomes, which are wheat extract solutions prepared according to the method of Example 1 in CN114540271A.

[0031] Example 1

[0032] An embodiment of the plant exosome preservation method of the present invention includes the following steps:

[0033] Plant exosomes and lyophilization protection solution were mixed at a mass ratio of 9:1 to prepare mixture A. The mixture was then filtered through a 0.22 μm filter membrane for sterilization and transferred to a sterile environment to be placed into lyophilization bottles (2 mL of filtrate per bottle). The processing procedure in Table 1 was carried out, with an initial temperature of 25 °C and a pressure of atmospheric pressure.

[0034] The formulation of the freeze-drying protective solution is shown in Table 2, and the osmotic pressure of the freeze-drying protective solution is approximately 400 mOsm / kg.

[0035] Examples 2-14

[0036] The only difference between Examples 2-14 and Example 1 is the composition of the freeze-drying protective solution, as shown in Table 2.

[0037] Table 1

[0038]

[0039] Table 2

[0040]

[0041]

[0042] Example 15

[0043] The only difference between this embodiment and Embodiment 1 is the processing procedure of the saving method, as shown in Table 3.

[0044] Table 3

[0045]

[0046]

[0047] Example 16

[0048] The only difference between this embodiment and Embodiment 1 is the processing procedure of the saving method, as shown in Table 4.

[0049] Table 4

[0050]

[0051] Comparative Examples 1-12

[0052] The only difference between Comparative Examples 1-12 and Example 1 is the composition of the freeze-drying protective solution, as shown in Table 2.

[0053] Comparative Example 13

[0054] The only difference between this comparative example and Example 1 is the processing procedure of the preservation method, as shown in Table 5.

[0055] Table 5

[0056]

[0057] Comparative Example 14

[0058] The only difference between this comparative example and Example 1 is the processing procedure of the preservation method, as shown in Table 6.

[0059] Table 6

[0060]

[0061] Comparative Example 15

[0062] The only difference between this comparative example and Example 1 is the processing procedure of the preservation method, as shown in Table 7.

[0063] Table 7

[0064]

[0065] Comparative Example 16

[0066] The only difference between this comparative example and Example 1 is the processing procedure of the preservation method, as shown in Table 8.

[0067] Table 7

[0068]

[0069]

[0070] Example 1

[0071] To verify the effectiveness of the plant exosome preservation method described in this invention, the freeze-dried plant exosome powder products obtained in each embodiment and comparative example were subjected to the following tests:

[0072] (1) Appearance inspection: The appearance of each product is observed by direct observation. The ideal appearance should be a white, loose block structure with a smooth and delicate surface, without bumps or pits. If there are any problems, the appearance of the product is considered unqualified.

[0073] The appearance pass rate of each product is calculated as follows:

[0074]

[0075] (2) Moisture content test: The moisture content of each product was tested according to the first method of moisture determination method 0832 recorded in the 2020 edition of the Chinese Pharmacopoeia;

[0076] (3) Particle size and concentration analysis: After dissolving each product in water, a portion was taken out and diluted to a concentration of 0.1 mg / mL. The particle size and concentration of the diluted solution were analyzed using a Malvern's NanoSight NS300 instrument. During the analysis, standard samples were used to calibrate the instrument and verify its accuracy. At the same time, the solution was serially diluted to avoid sample blockage.

[0077] The test results are shown in Table 8.

[0078] Table 8

[0079]

[0080]

[0081] As shown in Table 8, the plant exosome freeze-dried powders processed by the methods in each embodiment had a high appearance qualification rate, low moisture content, and relatively uniform and similar particle size and concentration. However, in the comparative examples, the products of Comparative Examples 4-8, 10, and 12-15 had lower appearance qualification rates, while the products of Comparative Examples 4-5, 10, and 12 had higher moisture content, the products of Comparative Examples 9, 13-14, and 16 had larger particle sizes, and the products of Comparative Examples 4-5 and 8 had higher concentrations. Considering the overall appearance qualification rate, moisture content, particle size, and concentration, the products of Examples 1-16, Comparative Examples 1-3, and Comparative Example 11 met the requirements.

[0082] Example 2

[0083] Based on the results of Example 1, the products with better results were further tested for the activity of plant exosomes: the biological activity of plant exosomes in each product was tested by utilizing the principle that plant exosomes have a stimulating effect on the growth of mouse embryonic fibroblasts (BALB / c 3T3 cells) and that the growth status of BALB / c 3T3 cells varies depending on the biological activity of plant exosomes.

[0084] The specific steps are as follows:

[0085] (1) Prepare the required solution:

[0086] Take one packet of RPMI 1640 culture medium powder (1L specification), dissolve and dilute with water to 1000mL, add 105IU of penicillin and 105IU of streptomycin, then add 2.1g of sodium bicarbonate, dissolve, mix well, filter sterilely to obtain RPMI 1640 culture medium, and store at 4℃.

[0087] Take 4 mL of newborn calf serum and add RPMI 1640 culture medium to a final volume of 1000 mL to prepare maintenance culture medium;

[0088] Take 100 mL of newborn calf serum and add RPMI 1640 culture medium to a final volume of 1000 mL to prepare a complete culture medium.

[0089] Weigh out 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of potassium dihydrogen phosphate, dissolve in water and dilute to 1000mL, sterilize at 121℃ for 15min to obtain PBS solution;

[0090] Weigh 0.10 g of thiazolyl blue (MTT) powder, dissolve it in 20 mL of PBS solution, filter it through a 0.22 μm filter membrane to sterilize it, and store it at 4 °C in the dark.

[0091] (2) Preparation of standards and test samples:

[0092] Under aseptic conditions, the commercial human epidermal growth factor standard was reconstituted according to the instructions and diluted with maintenance culture medium to a concentration of 50 IU per mL. In a 96-well cell culture plate, a 4-fold serial dilution was performed, resulting in a total of 8 dilutions, with 2 wells for each concentration.

[0093] Under aseptic conditions, the test samples from each example and comparative example were reconstituted to the labeled amount and then diluted with maintenance culture medium to approximately 50 IU per mL. Serial dilutions of 4-fold were performed in 96-well cell culture plates, resulting in 8 dilutions, with 2 wells for each concentration.

[0094] (3) Test:

[0095] Under aseptic conditions, the commercial BALB / c 3T3 cell line was cultured in complete culture medium at 37°C and 5% carbon dioxide, with the cell concentration controlled at 1.0 × 10⁻⁶ cells per mL. 5 ~5.0×10 5 Cells were passaged and used for biological activity assays 24–36 hours later. The culture medium in the culture flask was discarded, the cells were digested and collected, and reconstituted with complete culture medium to a concentration of 5.0 × 10⁶ cells per mL. 4 ~8.0×10 4Cell suspensions of 100 μL were seeded into 96-well cell culture plates and cultured at 37°C and 5% CO2 for 24 h. Afterward, the medium was replaced with maintenance medium and cultured at 37°C and 5% CO2 for another 24 h. The maintenance medium was discarded from the prepared cell culture plates, and 100 μL of standard solution and test solution were added to each well. The plates were then cultured at 37°C and 5% CO2 for 64–72 h. 20 μL of thiazolyl blue solution was added to each well, and the plates were cultured at 37°C and 5% CO2 for 5 h. After discarding the liquid in the culture plates, 100 μL of dimethyl sulfoxide was added to each well, mixed well, and the absorbance was measured at 570 nm using a microplate reader with 630 nm as the reference wavelength. The results were recorded.

[0096] Experimental data were processed using a computer program or a four-parameter regression method, and the results were calculated using the following formula:

[0097]

[0098] In the formula, Pr represents the biological activity of the standard, in IU / mL;

[0099] Ds is the pre-dilution factor of the test sample;

[0100] Dr represents the pre-dilution factor of the standard;

[0101] Es is the dilution factor of the test sample relative to half the effective amount of the standard;

[0102] Er is the dilution factor of the half-effective dose of the standard.

[0103] The test results are shown in Table 9.

[0104] Table 9

[0105]

[0106]

[0107] As shown in Table 9, the products obtained from each example treatment exhibit good biological activity and can effectively stimulate and influence cell lines. However, the products from Comparative Examples 1-3 show relatively low biological activity.

[0108] Example 3

[0109] Based on the results of Example 2, the products with better performance will be further tested for the stability of plant exosomes: plant exosomes are highly sensitive to temperature and are generally stored at 2-8℃. If accelerated stability tests are performed on them at higher room temperatures, it can be determined whether they have stability.

[0110] The specific steps are as follows: Three batches of products were prepared according to the methods of each embodiment and comparative example. Five groups of products were taken from each batch, for a total of 15 parallel products. They were placed in an incubator with set light and constant temperature conditions (temperature 25℃±2℃, relative humidity 60%±5%) for storage for 6 months. Samples were taken at the end of the 3rd and 6th months of the experiment to examine the appearance qualification rate, moisture content, particle size and concentration, and biological activity of the plant exosome freeze-dried powder products. The test methods were the same as those in Example 1 and 2.

[0111] The test results are shown in Table 10.

[0112] Table 10

[0113]

[0114]

[0115]

[0116]

[0117] It can be seen that the plant exosome freeze-dried powder obtained by the method described in this invention has good stability. Even after being stored at room temperature for up to 6 months, it still maintains the standards of all technical indicators, with minimal changes in these indicators before and after storage. In contrast, the product prepared in Comparative Example 11 has poor stability.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preserving and processing plant exosomes, characterized in that, Includes the following steps: (1) Gradient pre-freezing treatment: Plant exosomes were mixed evenly with the freeze-drying protective solution and filtered to remove bacteria. The resulting mixture A was cooled to 4.5~5.5℃ and kept at this temperature for 1.5~2.5h, then cooled to -18~-22℃ and kept at this temperature for 1.5~2.5h, and finally cooled to -43~-47℃ and kept at this temperature for 1.5~2.5h. The cooling rate was 0.5~0.85℃ / min. (2) Primary drying process: The mixture A after gradient pre-freezing in step (1) is heated to -24 to -26°C under a vacuum of 0.27 to 0.3 mbar and held for 7.5 to 8.5 h, then heated to -14 to -16°C and held for 5.5 to 6.5 h, and finally heated to -4 to -6°C and held for 1.5 to 2.5 h; the heating rate is 0.08 to 0.1°C / min. (3) Secondary drying treatment: The sample obtained from the first drying process in step (2) is heated to 9~11℃ and kept at that temperature for 5.5~6.5h, then heated to 24~26℃ and kept at that temperature for 3.5~4.5h, and finally the vacuum degree is reduced to ≤0.01mbar and kept at that temperature for 3.5~4.5h. After the plugging process, the preservation of plant exosomes is completed; the heating rate is 0.08~0.085℃ / min. The freeze-drying protective solution comprises the following components in parts by weight: 0.5-2.5 parts PEG1500, 3-6 parts glycerol, 3-6 parts mannitol, 5-8 parts trehalose, and 0.01-0.5 parts tetrahydropyrimidine.

2. The method for preserving and processing plant exosomes as described in claim 1, characterized in that, The osmotic pressure of the freeze-drying protective solution is 300~500 mOsm / kg.

3. The method for preserving and processing plant exosomes as described in claim 1, characterized in that, The solvent of the lyophilization protection solution is at least one of water, physiological saline, and PBS buffer.

4. The method for preserving and processing plant exosomes as described in claim 1, characterized in that, The mass ratio of plant exosomes to freeze-drying protectant is (8.5~9.5):(0.5~1.5).

5. The method for preserving and processing plant exosomes as described in claim 1, characterized in that, Includes the following steps: (1) Gradient pre-freezing treatment: Plant exosomes were mixed evenly with freeze-drying protection solution and filtered to remove bacteria. The resulting mixture A was cooled to 5°C and kept at that temperature for 2 hours, then cooled to -20°C and kept at that temperature for 2 hours, and finally cooled to -45°C and kept at that temperature for 2 hours. (2) Primary drying process: The mixture A after the gradient pre-freezing treatment in step (1) was heated to -25°C under a vacuum of 0.27~0.3 bar and kept at that temperature for 8 hours, then heated to -15°C and kept at that temperature for 6 hours, and finally heated to -5°C and kept at that temperature for 2 hours. (3) Secondary drying treatment: The sample obtained from the first drying process in step (2) is heated to 10°C and kept at that temperature for 6 hours. Then, the temperature is increased to 25°C and kept at that temperature for 4 hours. Finally, the vacuum degree is reduced to 0.01 mbar and kept at that temperature for 4 hours. After the plugging process, the preservation of plant exosomes is completed.

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