Preparation method and application of plant-derived extruded nano vesicles

Abstract

The invention discloses a preparation method and application of plant-derived extruded nano-vesicles, and relates to the technical field of biological medicines. The preparation method of the plant-derived extruded nano-vesicles comprises the following steps: juicing cleaned plant roots, stems, leaves, flowers or fruits to obtain plant juice, and filtering with gauze to obtain first filtrate; centrifuging the first filtrate at a low speed of 300-6000 * g to obtain a second filtrate; performing 8,000-20,000 * g centrifugation on the second filtrate for 0.5-3 hours at the temperature of 0-5 DEG C, so as to obtain a first precipitate; adding the first precipitate into a buffer solution, carrying out ultrasonic treatment, diluting, and sequentially passing through a filter membrane with the pore diameter of 0.4-10 microns and a filter membrane with the pore diameter of 0.1-0.4 microns, so as to obtain a third filtrate; and centrifuging the third filtrate, taking the precipitate, and resuspending to obtain the extruded nano-vesicles. Experimental detection shows that the extruded vesicles are similar to natural vesicles in characteristics, but the yield is far higher than that of the natural vesicles, and the extruded vesicles can serve as nano-carriers to be applied to a foundation or clinic.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of plant-derived extruded nanovesicles. Background Art

[0002] Extracellular vesicles (EVs) are vesicles with a lipid bilayer structure that are secreted by cells. They range in size from 50 to 1000 nm and contain, in addition to lipids, nucleic acids, proteins, and other small molecules within their lumen. Their primary biological function is to mediate intercellular signaling and the transmission of active molecules. This unique structure and function of EVs also makes them promising nanoparticle drug delivery vehicles. The unique physicochemical properties of EVs offer several advantages as therapeutic nanomaterials: their lipid bilayer structure protects drugs from degradation during circulation; their biological origin offers improved biocompatibility, reduced toxicity, and reduced immunogenicity compared to traditional nanomaterials; and the inclusion of small proteins within their lipid bilayers, combined with their nanoscale size, enhances their ability to evade host immune clearance and cross physiological barriers. Therefore, the development of EVs provides excellent drug delivery vehicles for nanomedicine.

[0003] However, mammalian cell-derived extracellular vesicles (EVs) have several drawbacks that limit their clinical application: (1) extremely low yield; (2) complex production process; and (3) the inability to consistently produce high-quality and uniform VEs. In recent years, plant-derived exosome-like nanoparticles (PELNs) have gradually attracted attention due to their advantages such as wide availability and high yield. They have a morphological structure similar to that of EVs and therefore have the potential to be used as drug delivery systems. In 2013, researchers first obtained PELNs from squeezed grape juice using a method combining differential centrifugation with density gradient centrifugation. They found that PELNs can target intestinal stem cells and mediate intestinal tissue remodeling, thereby resisting dextran sulfate sodium-induced colitis.

[0004] Extraction and preparation methods are key factors influencing extracellular vesicle yield. Currently, the most commonly used methods are differential centrifugation and density gradient centrifugation. Specifically, a liquid sample is first subjected to differential centrifugation to remove most impurities, followed by ultracentrifugation and density gradient centrifugation to obtain relatively pure vesicles. The main advantages of this method are ease of use, large yields, and high purity. However, this method requires high equipment requirements, and vesicle homogeneity cannot be guaranteed.

[0005] In addition to the above methods, a variety of other PELNs extraction methods have been developed, such as ultrafiltration, size exclusion chromatography, co-precipitation, and immunoaffinity. However, these methods also have many limitations, such as low yield and efficiency of size exclusion chromatography and the introduction of new impurities by co-precipitation.

[0006] Therefore, how to increase the yield of small vesicle substances, improve production efficiency, and ensure the uniformity of vesicles has become a difficult problem that needs to be solved urgently in the current production and preparation technology of extracellular vesicle substances. Summary of the Invention

[0007] The present invention provides a method for preparing and applying plant-derived extruded nanovesicles. Using plant roots, stems, leaves, flowers, or fruits, the present invention employs differential centrifugation to extract the precipitate from the juice after centrifugation. This precipitate is then filtered through a filter membrane with gradually decreasing pore size under a specific external pressure to yield a large number of small vesicles, significantly increasing the yield of PELNs to better meet potential clinical needs. This is achieved through the following techniques.

[0008] A method for preparing plant-derived extruded nanovesicles comprises the following steps:

[0009] Taking clean plant roots, stems, leaves, flowers or fruits to squeeze juice to obtain plant juice, and filtering it with gauze to obtain a first filtrate;

[0010] The first filtrate is centrifuged at 0-5°C at 300-1,500 × g and 2,000-6,000 × g, with each centrifugation time being 0.5-3 h, to obtain a second filtrate;

[0011] The second filtrate was centrifuged at 8,000-20,000 × g for 0.5-3 h at 0-5° C. to obtain a first precipitate;

[0012] The first precipitate is added to a buffer solution, sonicated, diluted, and passed through filter membranes with a pore size of 0.4-10 μm and a pore size of 0.1-0.4 μm in sequence under a pressure condition of 0.5-1.5 MPa to obtain a third filtrate;

[0013] The third filtrate is centrifuged at 100,000-200,000 × g for 0.5-3 h at 0-5° C., and the precipitate is resuspended to obtain the extruded nanovesicles.

[0014] Traditional methods for preparing plant vesicles involve juicing plant materials, followed by low-speed centrifugation, high-speed centrifugation, and ultracentrifugation (sometimes with density gradient centrifugation). During this process, the low-speed precipitate is often discarded as impurities, while the high-speed precipitate is squeezed to obtain ultrahigh-speed nanoparticles. The purpose of using a membrane filter in traditional methods is primarily to sterilize or further remove larger impurities. The membrane filters are small enough to pass through the membrane. These exosome-like nanoparticles, due to their small size, do not undergo deformation or reorganization, but instead pass directly through the membrane pores.

[0015] The present invention is essentially different from the traditional method. The present invention selects the precipitate (first precipitate) after the differential centrifugation of the second filtrate as the subsequent production material. After the first precipitate is passed through filter membranes with pore sizes of 0.4-10 μm and 0.1-0.4 μm in sequence under specific pressure conditions, the precipitate is deformed after being squeezed (rather than simply filtered), and re-self-assembled into small vesicles. The precipitate that has undergone deformation and reorganization is collected. The method of the present invention not only significantly improves the yield of vesicles, but also makes the vesicle structure more uniform and stable; no structural damage occurs after being stored in a buffer solution for 7 days, indicating that the prepared vesicles have good structural stability; no obvious particle size change occurs after being stored in a buffer solution for 14 days, indicating that the prepared vesicles have good particle size stability.

[0016] The present invention can use any plant material to produce uniform, stable, and high-yield extruded nanovesicles. For example, emblica oleracea, Artemisia argyi, and mulberry can be used. Furthermore, fruits or other organs from these plants can be used. These plant materials are primarily composed of thin-walled cells, have widely distributed vacuoles, and are rich in active biofilm components, significantly increasing the yield of extruded nanovesicles.

[0017] Furthermore, the first filtrate is centrifuged at 1,000-1,500 × g and 3,000-5,000 × g at 4° C., with each centrifugation time being 0.5-2 h, to obtain a second filtrate.

[0018] Furthermore, the second filtrate is centrifuged at 12,000-20,000 × g for 0.5-3 h at 4° C. to obtain a first precipitate.

[0019] Furthermore, the ultrasonic treatment method is: 50-650 W treatment for 1-1800 s, stop for 1-1800 s; cycle in sequence, and continue for 10-180 min.

[0020] Furthermore, the ultrasonic treatment method is: 100-200 W treatment for 10-20 s, stop for 30 s; cycle in sequence and continue for 10 min.

[0021] Furthermore, the process of passing through the filter membrane is carried out under a pressure condition of 0.5-1 MPa.

[0022] Furthermore, the third filtrate was centrifuged at 160,000 × g for 1.5 h at 4° C., and the precipitate was resuspended.

[0023] Furthermore, the solution is first passed through a filter membrane with a pore size of 0.4-10 μm 1-10 times, and then passed through a filter membrane with a pore size of 0.1-0.4 μm 1-10 times.

[0024] Furthermore, the solution is first passed through a filter membrane with a pore size of 0.4-10 μm 1-5 times, and then passed through a filter membrane with a pore size of 0.1-0.4 μm 1-2 times to obtain a third filtrate.

[0025] Furthermore, the filter membrane is a polycarbonate membrane. The filter membrane used in the present invention is a specially formulated polycarbonate membrane specifically for extrusion. This membrane has a uniform pore size distribution and essentially cylindrical, straight-through pores, unlike the curved pore structure of conventional pinhole bacteria filters. Therefore, it can be used as a filter membrane material for vesicle extrusion or homogenization.

[0026] Furthermore, a buffer solution is used for the resuspension.

[0027] The present invention also provides the use of extruded nanovesicles prepared by any of the above preparation methods, using the extruded nanovesicles as nano delivery carriers loaded with active ingredients to prepare corresponding preparations.

[0028] The extruded nanovesicles provided by the present invention can be used not only as carriers for pharmaceutical active ingredients to prepare drugs for treating corresponding diseases, but can also serve as nano-delivery carriers for any other active ingredients that require loading, such as inorganic or organic compounds, small peptides, or proteins. In other words, the extruded nanovesicles provided by the present invention can be used in any application requiring a carrier.

[0029] Optionally, the drug is an anti-tumor drug. For example, the active ingredient of the drug is doxorubicin.

[0030] Compared with the existing technology, the benefits of the present invention are: compared with the vesicle structure prepared by the traditional method, the vesicle structure prepared by the present invention is more uniform and stable, with a PDI of 0.157 in the buffer solution, and the structure is not damaged and the particle size does not change significantly after being stored for one week; the yield is increased by 2.07-97.64 times, which is a significant improvement; the prepared vesicle structure has good drug loading capacity and is non-cytotoxic. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the steps of the vesicle preparation method provided by the present invention.

[0032] Figure 2 (a) and (b) are the TEM image and particle size distribution of the first precipitate in Example 1, respectively.

[0033] Figure 3 (a), (b) and (c) are the TEM images, particle size distribution and zeta potential of the extruded nanovesicles prepared in Example 1 and the natural mulberry vesicles obtained by differential centrifugation in Comparative Example 1, respectively.

[0034] Figure 4 (a) and (b) are TEM images of the extruded nanovesicles prepared in Example 1 and the natural mulberry vesicles obtained by differential centrifugation in Comparative Example 1 after storage for 7 days and 14 days, respectively.

[0035] Figure 5 Toxicity test results of U87MG cells and SH-SY5Y cells treated with buffer (blank control), extruded nanovesicles without doxorubicin (blank vesicles), doxorubicin (DOX), and vesicles formed by high-speed centrifugation precipitation of juice encapsulating doxorubicin (vesicles@DOX) for 24 h.

[0036] Figure 6 This is the standard curve for BCA protein quantification. DETAILED DESCRIPTION

[0037] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] In some embodiments of the present invention, a method for preparing plant-derived extruded nanovesicles is provided, comprising the steps of:

[0039] S1. Taking clean plant roots, stems or fruits, squeezing juice to obtain plant juice, and filtering with gauze to obtain a first filtrate;

[0040] When squeezing juice, it is preferred to use fresh plant roots, stems, leaves, flowers or fruits, wash them and then squeeze the juice. When using dried plant roots, stems, leaves, flowers or fruits, they need to be washed and fully soaked before squeezing the juice.

[0041] S2. Centrifuge the first filtrate at 0-5° C. at 300-1,500 × g and 2,000-6,000 × g, respectively, for 0.5-3 h each time, to obtain a second filtrate;

[0042] Optionally, in step S2, the first filtrate is centrifuged at 1,000-1,500 × g and 3,000-5,000 × g at 4° C., with each centrifugation time being 0.5-2 h, to obtain a second filtrate.

[0043] Specifically, the mixture was centrifuged at 1000 × g and 5000 × g in sequence, with each centrifugation time being 1 h, to obtain a second filtrate.

[0044] S3, centrifuging the second filtrate at 8,000-20,000 × g for 0.5-3 h at 0-5° C. to obtain a first precipitate;

[0045] Optionally, in step S3, the second filtrate is centrifuged at 12,000-20,000 × g for 0.5-3 h at 4° C. to obtain a first precipitate.

[0046] S4, adding the first precipitate to a buffer solution, sonicating, diluting, and sequentially passing through filter membranes with a pore size of 0.4-10 μm and a pore size of 0.1-0.4 μm under a pressure of 0.5-1.5 MPa to obtain a third filtrate;

[0047] Optionally, in step S4, the ultrasonic treatment method is: 50-650 W treatment for 1-1800 s, stop for 1-1800 s; cycle in sequence, and continue for 10-180 min.

[0048] Further optionally, the ultrasonic treatment method is: 100-200 W treatment for 10-20 s, stop for 30 s; cycle in sequence for 10 min.

[0049] The ultrasonic treatment method can be specifically as follows: 50 W treatment for 10 s, stop for 30 s; and cycle in sequence for 10 min.

[0050] Optionally, the solution is first passed through a filter membrane with a pore size of 0.4-10 μm 1-10 times, and then passed through a filter membrane with a pore size of 0.1-0.4 μm 1-10 times.

[0051] Specifically, the third filtrate was obtained by sequentially passing through filter membranes with a pore size of 0.4 μm and a pore size of 0.2 μm.

[0052] Optionally, the filter membrane is a polycarbonate filter membrane.

[0053] S5. Centrifuge the third filtrate at 100,000-200,000 × g for 0.5-3 h at 0-5° C., and resuspend the precipitate to obtain the extruded nanovesicles.

[0054] In some implementation cases of the present invention, the selected plants may be emblica, Artemisia selengensis, mulberry, etc.

[0055] Example 1: Preparation of extruded nanocapsules using fresh black mulberry fruit (berry raw material)

[0056] The preparation method of extruded nanovesicles provided in this embodiment is:

[0057] (1) Take 200 g of fresh black mulberry fruit, wash it with ultrapure water, mix the black mulberry fruit and ultrapure water in a mass ratio of 1:1 (generally, the ratio can be 1:(1-10)), stir and crush to obtain mulberry juice;

[0058] Most of the pulp and insoluble matter were removed by filtering with gauze to obtain the first filtrate.

[0059] (2) Centrifuge the first filtrate at 1,000 × g for 1 h at 4°C and take the supernatant; then centrifuge at 5,000 × g for 1 h and take the supernatant to obtain the second filtrate.

[0060] (3) Centrifuge the second filtrate at 18,500 × g for 3 h at 4°C to obtain the first precipitate.

[0061] (4) Disperse the entire first precipitate in 20 mL of PBS buffer (1 L PBS: 2.9 g sodium hydrogen phosphate dodecahydrate, 296.4 mg sodium dihydrogen phosphate dihydrate, 8.47 g sodium chloride, dilute to 1000 mL with water) and ultrasonicate. The ultrasonication conditions were: 600 W for 10 s, then 30 s, and this cycle lasted for 10 min. The entire ultrasonication process was performed on ice.

[0062] The sonicated sample was diluted with PBS buffer to a final volume of 200 mL.

[0063] (5) Using a liposome extruder (purchased from Zhejiang Microfluidic Nanobiotechnology Co., Ltd., China, trade name: Pneumatic Liposome Extruder-100 mL), the sample after constant volume was passed through a polycarbonate membrane with a pore size of 0.4 μm twice and then through a polycarbonate membrane with a pore size of 0.2 μm once at a pressure of 1.5 MPa to obtain a third filtrate.

[0064] (6) The third filtrate was centrifuged at 160,000 × g for 1.5 h at 4 °C, the supernatant was removed and the precipitate was retained. The precipitate was fully resuspended in 20 mL of PBS buffer to obtain the final extruded nanovesicle product.

[0065] (7) Weigh the final extruded nanovesicle product, add 20 mL of PBS buffer and thoroughly pipette to resuspend, then aspirate 2 μL of the sample for BCA protein quantification. The BCA protein quantification / concentration determination kit used in this example was purchased from Dalian Meilun Biotechnology Co., Ltd., China (Cat. No. MA0082-3).

[0066] In this example, BCA protein quantification can be performed according to the instructions for the BCA protein quantification / concentration assay kit. The specific steps are as follows: Aspirate 2 μL of sample and follow the kit instructions. Specifically, mix Solution A and Solution B in the kit at a 50:1 volume ratio to form a reaction solution. Mix 200 μL of the reaction solution with 20 μL of the sample diluent, incubate at 37°C for 30 minutes, and measure the absorbance at 562 nm. Calculate the protein concentration using the standard curve.

[0067] Example 2: Preparation of extruded nanovesicles using fresh emblica (drupe raw material)

[0068] The preparation method of extruded nanovesicles provided in this embodiment is:

[0069] (1) Take 300 g of fresh emblica fruit, wash it with ultrapure water, remove the core and take out the pulp, mix the emblica pulp with ultrapure water in a mass ratio of 1:1, stir and crush to obtain emblica juice;

[0070] Most of the pulp and insoluble matter were removed by filtering with gauze to obtain the first filtrate.

[0071] (2) Centrifuge the first filtrate at 1,000 × g for 2 h at 4°C and take the supernatant; then centrifuge at 6,000 × g for 1 h and take the supernatant to obtain the second filtrate.

[0072] (3) Centrifuge the second filtrate at 15,000 × g for 1.5 h at 4°C to obtain the first precipitate.

[0073] (4) Disperse the entire first precipitate in 10 mL of PBS buffer and perform ultrasonic treatment. The ultrasonic treatment conditions are: 300 W for 10 s, then 30 s, and repeat this cycle for a total of 60 min. The entire ultrasonic process should be performed on ice.

[0074] The sonicated sample was diluted with PBS buffer to a final volume of 200 mL.

[0075] (5) Using a liposome extruder (purchased from Zhejiang Microfluidic Nanobiotechnology Co., Ltd., China, trade name: Pneumatic Liposome Extruder-100 mL), the sample after constant volume was passed through a polycarbonate membrane with a pore size of 0.4 μm five times and then through a polycarbonate membrane with a pore size of 0.2 μm twice at a pressure of 0.5 MPa to obtain a third filtrate.

[0076] (6) The third filtrate was centrifuged at 100,000 × g for 3 h at 4°C, the supernatant was removed and the precipitate was retained. The precipitate was fully resuspended in 10 mL of PBS buffer to obtain the final extruded nanovesicle product.

[0077] (7) The final extruded nanovesicles were weighed, and 10 mL of PBS buffer was added and thoroughly pipetted to resuspend. 2 μL of sample was aspirated and BCA protein quantification / concentration determination kit was used for BCA protein quantification. The detection method was the same as in Example 1.

[0078] In this example, BCA protein quantification can be performed according to the instructions for the BCA protein quantification / concentration assay kit. The specific steps are as follows: Aspirate 2 μL of sample and follow the kit instructions. Specifically, mix Solution A and Solution B in the kit at a 50:1 volume ratio to form a reaction solution. Mix 200 μL of the reaction solution with 20 μL of the sample diluent, incubate at 37°C for 30 minutes, and measure the absorbance at 562 nm. Calculate the protein concentration using the standard curve.

[0079] Example 3: Preparation of extruded nanocapsules using fresh Artemisia selengensis (herbaceous plant stem and leaf raw material)

[0080] The preparation method of extruded nanovesicles provided in this embodiment is:

[0081] (1) Take 100 g of fresh Artemisia selengensis, wash it with ultrapure water, mix the Artemisia selengensis and ultrapure water in a mass ratio of 1:1, stir and crush to obtain Artemisia selengensis juice;

[0082] Most of the pulp and insoluble matter were removed by filtering with gauze to obtain the first filtrate.

[0083] (2) Centrifuge the first filtrate at 900 × g for 1 h at 4°C and take the supernatant; then centrifuge at 4,000 × g for 1 h and take the supernatant to obtain the second filtrate.

[0084] (3) Centrifuge the second filtrate at 12,000 × g for 3 h at 4°C to obtain the first precipitate.

[0085] (4) Disperse the entire first precipitate in 10 mL of PBS buffer and perform ultrasonic treatment. The ultrasonic treatment conditions are: 130W for 10 s, then 30 s, and repeat this cycle for a total of 180 min. The entire ultrasonic process should be performed on ice.

[0086] The sonicated sample was diluted with PBS buffer to a final volume of 200 mL.

[0087] (5) Using a liposome extruder (purchased from Zhejiang Microfluidic Nanobiotechnology Co., Ltd., China, trade name: Pneumatic Liposome Extruder-100 mL), the sample after constant volume was passed through a polycarbonate membrane with a pore size of 0.4 μm five times and then through a polycarbonate membrane with a pore size of 0.2 μm five times at a pressure of 1 MPa to obtain a third filtrate.

[0088] (6) The third filtrate was centrifuged at 200,000 × g for 1 h at 4°C, the supernatant was removed and the precipitate was retained. The precipitate was fully resuspended in 10 mL of PBS buffer to obtain the final extruded nanovesicle product.

[0089] (7) Add 20 mL of PBS buffer to the final extruded nanovesicles and thoroughly vortex to resuspend them. Take 2 μL of sample and use the BCA protein quantification / concentration determination kit for BCA protein quantification. The detection method is the same as in Example 1.

[0090] In this experiment, BCA protein quantification was performed according to the instructions for the BCA protein quantification / concentration assay kit. The specific steps were as follows: Aspirate 2 μL of sample and follow the kit instructions. Specifically, mix Solution A and Solution B in the kit at a 50:1 volume ratio to form a reaction solution. Mix 200 μL of the reaction solution with 20 μL of the sample diluent, incubate at 37°C for 30 minutes, and measure the absorbance at 562 nm. Calculate the protein concentration using the standard curve.

[0091] Example 4: Preparation of extruded nanovesicles using rose petals (flower raw materials)

[0092] The preparation method of extruded nanovesicles provided in this embodiment is:

[0093] (1) Take 200 g of rose petals, wash them with ultrapure water, mix the rose petals and ultrapure water in a mass ratio of 1:1 (generally, 1:(1-10) is acceptable), squeeze, stir and crush to obtain rose petal puree;

[0094] Most of the pulp and insoluble matter were removed by filtering with gauze to obtain the first filtrate.

[0095] (2) Centrifuge the first filtrate at 1,000 × g for 1 h at 4°C and take the supernatant; then centrifuge at 5,000 × g for 1 h and take the supernatant to obtain the second filtrate.

[0096] (3) Centrifuge the second filtrate at 20,000 × g for 0.5 h at 4°C to obtain the first precipitate.

[0097] (4) Disperse the entire first precipitate in 20 mL of PBS buffer and perform ultrasonic treatment. The ultrasonic treatment conditions are: 600 W for 10 s, then 30 s, and repeat this cycle for a total of 30 min. The entire ultrasonic process should be performed on ice.

[0098] The sonicated sample was diluted with PBS buffer to a final volume of 200 mL.

[0099] (5) Using a liposome extruder (purchased from Zhejiang Microfluidic Nanobiotechnology Co., Ltd., China, trade name: Pneumatic Liposome Extruder-100 mL), the sample after constant volume was passed through a polycarbonate membrane with a pore size of 0.4 μm five times and then through a polycarbonate membrane with a pore size of 0.2 μm once at a pressure of 1.5 MPa to obtain a third filtrate.

[0100] (6) The third filtrate was centrifuged at 160,000 × g for 1 h at 4°C, the supernatant was removed and the precipitate was retained. The precipitate was fully resuspended in 20 mL of PBS buffer to obtain the final extruded nanovesicle product.

[0101] (7) The final extruded nanovesicles were weighed, and 20 mL of PBS buffer was added and thoroughly pipetted for resuspending. 2 μL of the sample was aspirated for BCA protein quantification. The BCA protein quantification / concentration determination kit used in this example was purchased from Dalian Meilun Biotechnology Co., Ltd., China (Cat. No. MA0082-3).

[0102] In this example, BCA protein quantification can be performed according to the instructions for the BCA protein quantification / concentration assay kit. The specific steps are as follows: Aspirate 2 μL of sample and follow the kit instructions. Specifically, mix Solution A and Solution B in the kit at a 50:1 volume ratio to form a reaction solution. Mix 200 μL of the reaction solution with 20 μL of the sample diluent, incubate at 37°C for 30 minutes, and measure the absorbance at 562 nm. Calculate the protein concentration using the standard curve.

[0103] Example 5: Preparation of extruded nanovesicles using Mirabilis jalapa root (root raw material)

[0104] The preparation method of extruded nanovesicles provided in this embodiment is:

[0105] (1) Take 300 g of fresh Mirabilis jalapa roots, wash them with ultrapure water, cut the roots and mix them evenly with ultrapure water at a mass ratio of 1:1 (generally, 1:(1-10) is acceptable), squeeze and stir to obtain Mirabilis jalapa root puree;

[0106] Most of the pulp and insoluble matter were removed by filtering with gauze to obtain the first filtrate.

[0107] (2) Centrifuge the first filtrate at 1,000 × g for 1 h at 4°C and take the supernatant; then centrifuge at 5,000 × g for 1 h and take the supernatant to obtain the second filtrate.

[0108] (3) Centrifuge the second filtrate at 12,000 × g for 3 h at 4°C to obtain the first precipitate.

[0109] (4) Disperse the entire first precipitate in 20 mL of PBS buffer and perform ultrasonic treatment. The ultrasonic treatment conditions are: 600 W for 10 s, then 30 s, and repeat this cycle for a total of 30 min. The entire ultrasonic process should be performed on ice.

[0110] The sonicated sample was diluted with PBS buffer to a final volume of 200 mL.

[0111] (5) Using a liposome extruder (purchased from Zhejiang Microfluidic Nanobiotechnology Co., Ltd., China, trade name: Pneumatic Liposome Extruder-100 mL), the sample after the fixed volume was passed through a polycarbonate membrane with a pore size of 0.4 μm once and then through a polycarbonate membrane with a pore size of 0.2 μm once at a pressure of 1.0 MPa to obtain a third filtrate.

[0112] (6) The third filtrate was centrifuged at 120,000 × g for 1.5 h at 4°C, the supernatant was removed and the precipitate was retained. The precipitate was fully resuspended in 30 mL of PBS buffer to obtain the final extruded nanovesicle product.

[0113] (7) Weigh the final extruded nanovesicle product, add 20 mL of PBS buffer and thoroughly pipette to resuspend, then aspirate 2 μL of the sample for BCA protein quantification. The BCA protein quantification / concentration determination kit used in this example was purchased from Dalian Meilun Biotechnology Co., Ltd., China (Cat. No. MA0082-3).

[0114] In this example, BCA protein quantification can be performed according to the instructions for the BCA protein quantification / concentration assay kit. The specific steps are as follows: Aspirate 2 μL of sample and follow the kit instructions. Specifically, mix Solution A and Solution B in the kit at a 50:1 volume ratio to form a reaction solution. Mix 200 μL of the reaction solution with 20 μL of the sample diluent, incubate at 37°C for 30 minutes, and measure the absorbance at 562 nm. Calculate the protein concentration using the standard curve.

[0115] Comparative Example 1: Preparation of extruded nanovesicles from mulberry, emblica, Artemisia selengensis, rose petals and Mirabilis jalapa roots by differential centrifugation

[0116] (1) Take 200 g of fresh mulberries, rinse them with ultrapure water, mix the mulberries with ultrapure water at a mass-to-volume ratio of 1:1, and squeeze and crush them to obtain mulberry juice;

[0117] 300 g of fresh emblica fruit were rinsed with ultrapure water, the pulp was removed and the pulp was mixed with ultrapure water in a mass-to-volume ratio of 1:5, and the emblica juice was obtained after squeezing and crushing.

[0118] Take 100 g of fresh Artemisia selengensis, rinse it with ultrapure water, mix the Artemisia selengensis with ultrapure water in a mass-to-volume ratio of 1:10, and squeeze and crush it to obtain Artemisia selengensis juice;

[0119] (2) Use gauze to filter to remove most of the pulp and insoluble matter;

[0120] (3) Centrifuge at 1,000 × g for 1 h at 4°C and collect the supernatant. Then centrifuge at 5,000 × g for 1 h and collect the supernatant.

[0121] (4) Centrifuge at 10,000 × g for 1 h at 4°C and collect the supernatant.

[0122] (5) Centrifuge at 80,000 × g for 1 h at 4°C and collect the supernatant.

[0123] (6) Filter with a 0.45 μm pinhole filter;

[0124] (7) Centrifuge at 165,000 × g for 1 h at 4°C, remove the precipitate, and wash 2-3 times with PBS.

[0125] (8) Finally, the sample was resuspended in PBS to obtain exosome-like nanoparticles derived from mulberry, emblica, and Artemisia selengensis. 2 μL of the sample was aspirated for BCA protein quantification. The BCA protein quantification kit used in this example was purchased from Dalian Meilun Biotechnology Co., Ltd., China, and the trade name is BCA protein quantification / concentration determination kit. BCA protein quantification was performed according to the instructions of the BCA protein quantification kit, and the specific steps were the same as those in Examples 1-3.

[0126] Test example: Performance testing of extruded nanovesicles

[0127] In this test example, the extruded nanovesicles prepared in Example 1 were used as test samples, and the micromorphology, particle size, Zeta potential, yield, stability, drug encapsulation concentration, and cytotoxicity after drug encapsulation were tested.

[0128] 1. Transmission electron microscopy

[0129] The polished surface of a copper mesh was placed upside down on the exosome nanoparticle sample and allowed to adsorb for 5 minutes. After the mesh was dried, it was negatively stained with 2% uranyl acetate and examined under a transmission electron microscope.

[0130] like Figure 2 As shown in (a), in the preparation method of Example 1 of the present invention, the first precipitate appears as a large vesicle with a double lipid membrane structure under an electron microscope. Figure 3 As shown in (a), the extruded nanovesicles after being squeezed through the filter membrane showed a well-defined cup-shaped structure under transmission electron microscopy, which had a similar morphological structure to the natural vesicles obtained by differential centrifugation.

[0131] 2. Particle size and Zeta potential measurement

[0132] 100 μg of sample was dissolved in 1 mL of PBS buffer, and the vesicle size and zeta potential data were measured using a nanoparticle size analyzer.

[0133] like Figure 2 As shown in (b), in the preparation method of Example 1, the average particle size of the first precipitate is 350.0 nm, its peak value peak1 is 501.7 nm, and its polydispersity index (PDI) is 0.312. This result further indicates that the first precipitate contains a large number of large vesicles.

[0134] like Figure 3 As shown in (b), the extruded nanovesicles prepared using the preparation method of Example 1 have an average particle size of 250.0 nm and an aggregation index (PDI) of 0.157 in PBS. This result indicates that the extruded nanovesicles described in the present invention have good uniformity.

[0135] like Figure 3 As shown in (c), the extruded nanovesicles prepared by the preparation method of Example 1 have an average zeta potential of -10.19 mV in PBS, which is similar to the zeta potential of natural vesicles obtained by differential centrifugation of -11.7 mV.

[0136] 3. Yield detection of extruded nanovesicles

[0137] The total protein content was used as the basis for calculating the yield of extruded nanovesicles. The yield data of extruded nanovesicles from three different plant sources (Examples 1-3) and natural vesicles (Comparative Example 1) were statistically analyzed, and the improvement factor was calculated.

[0138] The specific test method is: aspirate 2 μL of sample for BCA protein quantification. The BCA protein quantification kit used in this example was purchased from Dalian Meilun Biotechnology Co., Ltd. in China, and the trade name is BCA Protein Quantification / Concentration Determination Kit.

[0139] Preparation of standard curve: Add 0, 4, 8, 12, 16, 18, and 20 μL of protein standard (5 mg / mL, reagent included in the kit) to different wells of a 96-well plate, and then add PBS to each well to make up to 20 μL.

[0140] In this embodiment, BCA protein quantification can be performed according to the instructions of the BCA protein quantification kit. Specifically, solution A and solution B in the kit are mixed in a ratio of 50:1 to prepare a reaction solution. 200 μL of the reaction solution is mixed with 20 μL of sample and the standard bottle diluent of each concentration, incubated at 37°C for 30 min, and the absorbance at 562 nm is measured.

[0141] The protein concentration was calculated based on the standard curve. Figure 6 The yield of extruded nanovesicles / natural vesicles was calculated according to the following formula:

[0142] The yield of extruded nanovesicles / natural vesicles (mg / kg) = protein amount (mg) / weight of raw materials (g) × 1000. The calculated results are shown in Table 1.

[0143] Table 1

[0144]

[0145] From the results in Table 1 above, it can be seen that the yields of extruded nanovesicles prepared from mulberry, emblica fruit, Artemisia selengensis rose petals and Mirabilis jalapa roots using the preparation method of the present invention are 97.6, 2.0, 37.4, 5.02 and 30.0 times that of the natural vesicles extracted by the corresponding traditional method (Comparative Example 1), respectively, with higher yields.

[0146] 4. Stability Assessment of Extruded Nanovesicles

[0147] The extruded nanovesicle samples prepared in Example 1 were resuspended in PBS buffer, and the particle size distribution was monitored on days 0, 4, 7, 10, and 14, and transmission electron microscopy was performed on day 7.

[0148] like Figure 4 As shown in (a), the morphological structure of the extruded nanovesicles prepared by the preparation method of Example 1 did not change significantly on the 7th day. This shows that the extruded nanovesicles prepared by the preparation method of the present invention can maintain the particle size and structural stability of nanoparticles.

[0149] like Figure 4 As shown in (b), the average particle size of the extruded nanovesicles prepared using the method of Example 1 remained unchanged after 14 days of storage in PBS. This demonstrates that the nanoparticles formed by high-speed centrifugal precipitation and extrusion of juice prepared using the method of the present invention can maintain their size and structural stability.

[0150] 5. Drug encapsulation concentration of blank vesicles and cytotoxicity evaluation of drug-loaded vesicles

[0151] (1) Cell culture:

[0152] U87MG cells (human astroglioblastoma cells) and SH-SY5Y cells (human neuroblastoma cells) were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0153] (2) Drug encapsulation concentration detection and cytotoxicity evaluation of drug-loaded vesicles

[0154] 200 μg of the extruded nanovesicle test sample prepared in Example 1 was mixed with 100 μg of doxorubicin (DOX). Ultrasonic cell disruptor was used for sonication at 130 W for 10 s on, 30 s off, for a total of 20 min. The sonicated sample was then incubated at 37°C for 30 min to restore the vesicle membrane structure. The sample was then centrifuged at 120,000 × g for 0.5 h at 4°C, the pellet washed twice with PBS, and finally resuspended in 100 μL of PBS. 10 μL of the sample was mixed with 90 μL of RIPA lysis buffer and incubated on ice for 30 min. The absorbance at 490 nm was measured, and the amount of DOX contained in the vesicles was calculated.

[0155] The DOX concentration within the vesicles was calculated using the formula: DOX content (μg DOX / μg extruded nanovesicles) = total DOX content / total vesicle protein content. The calculated result was 0.41 μg DOX / μg extruded nanovesicles.

[0156] 5×10 3 U87MG cells and SH-SY5Y cells were plated into 96-well plates and cultured in a 37°C, 5% CO2 incubator for 24 hours. PBS (blank control), vesicles without doxorubicin (blank vesicles), doxorubicin (DOX), and vesicles encapsulated with DOX (DOX concentration of 10 μg / mL) were added and treated for 24 hours. The volume of U87MG cells and SH-SY5Y cells in each group was calculated to assess the cell-killing effect. CCK8 reagent was added to each well at a concentration of 10%, and absorbance at 450 nm was measured after incubation at 37°C for 45 minutes. Statistical analysis was performed using data analysis software.

[0157] The results are as follows Figure 5 As shown, the extruded nanovesicles themselves are non-toxic to cells, but after being encapsulated with DOX, they have strong cytotoxicity. This shows that the extruded nanovesicles described in the present invention have the ability to effectively load drugs.

[0158] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A method for preparing plant-derived extruded nanovesicles, characterized in that: The following steps are involved: Taking clean plant roots, stems, leaves, flowers or fruits to squeeze juice to obtain plant juice, and filtering it with gauze to obtain a first filtrate; The first filtrate is centrifuged at 0-5°C at 300-1,500 × g and 2,000-6,000 × g, with each centrifugation time being 0.5-3 h, to obtain a second filtrate; The second filtrate was centrifuged at 8,000-20,000 × g for 0.5-3 h at 0-5° C. to obtain a first precipitate; The first precipitate is added to a buffer solution, sonicated, diluted, and passed through filter membranes with a pore size of 0.4-10 μm and a pore size of 0.1-0.4 μm in sequence under a pressure condition of 0.5-1.5 MPa to obtain a third filtrate; The third filtrate is centrifuged at 100,000-200,000 × g for 0.5-3 h at 0-5° C., and the precipitate is resuspended to obtain the extruded nanovesicles.

2. The method for preparing plant-derived extruded nanovesicles according to claim 1, characterized in that: The first filtrate was centrifuged at 1,000-1,500 × g and 3,000-5,000 × g at 4° C., with each centrifugation time being 0.5-2 h, to obtain a second filtrate.

3. The method for preparing plant-derived extruded nanovesicles according to claim 1, characterized in that: The second filtrate was centrifuged at 12,000-20,000 × g for 0.5-3 h at 4° C. to obtain a first precipitate.

4. The method for preparing plant-derived extruded nanovesicles according to claim 1, characterized in that: The ultrasonic treatment method is: 50-650 W treatment for 1-1800 s, stop for 1-1800 s; cycle in sequence, and continue for 10-180 min; Furthermore, the ultrasonic treatment method is: 100-200 W treatment for 10-20 s, stop for 30 s; cycle in sequence and continue for 10 min.

5. The method for preparing plant-derived extruded nanovesicles according to claim 1, characterized in that: The process of passing through the filter membrane is carried out under a pressure condition of 0.5-1 MPa.

6. The method for preparing plant-derived extruded nanovesicles according to claim 1, characterized in that: First pass through a filter membrane with a pore size of 0.4-10 μm 1-10 times, and then pass through a filter membrane with a pore size of 0.1-0.4 μm 1-10 times.

7. The method for preparing plant-derived extruded nanovesicles according to claim 1, characterized in that: First, pass through a filter membrane with a pore size of 0.4-10 μm 1-5 times, and then pass through a filter membrane with a pore size of 0.1-0.4 μm 1-2 times to obtain a third filtrate; Furthermore, the filter membrane is a polycarbonate filter membrane.

8. The method for preparing plant-derived extruded nanovesicles according to claim 1, characterized in that: The third filtrate was centrifuged at 120,000-180,000 × g for 1.5-3 h at 4° C., and the precipitate was resuspended.

9. The method for preparing plant-derived extruded nanovesicles according to claim 1, characterized in that: The resuspension is performed using a buffer.

10. Use of the extruded nanovesicles prepared by the preparation method according to any one of claims 1 to 9, characterized in that: The extruded nanovesicles are used as nano delivery carriers loaded with active ingredients to prepare corresponding preparations.

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