Drug-loaded extracellular vesicle as well as preparation method and application thereof

Abstract

The invention relates to a drug-loaded extracellular vesicle and a preparation method and application thereof, the extracellular vesicle in the drug-loaded extracellular vesicle comes from natural killer cells and is loaded with paclitaxel, and the extracellular vesicle or the natural killer cells are treated by urolithin A. The preparation method of the drug-loaded extracellular vesicles comprises the step of incubating natural killer cells or the extracellular vesicles of the natural killer cells in a culture solution containing paclitaxel and / or urolithin A. By means of the tumor targeting capability of the extracellular vesicles, the medicine can accurately reach tumor tissues, and the concentration of the medicine in the tumor is increased. The extracellular vesicles secreted by the NK cells contain various immune components, can play an immune regulation role through a paracrine way or a circulatory system targeting immune system, enhance the application safety of the paclitaxel at the same time, and can play a drug synergistic role with the paclitaxel.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a drug-loaded extracellular vesicle, a preparation method thereof and an application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Breast carcinoma (BC) is a phenomenon in which breast epithelial cells undergo uncontrolled proliferation under the action of various carcinogenic factors, and can directly threaten the life of patients in the advanced stage. Triple negative breast cancer (TNBC) is a subtype of breast cancer, lacking the expression of estrogen and progesterone receptors as well as human epidermal growth factor receptor, accounting for about 20% of the total number of breast cancer diagnoses globally. TNBC mainly prevails in young women and has characteristics such as high invasiveness, high heterogeneity, and poor prognosis.

[0004] Natural killer cells (NK cells) are cytotoxic lymphocytes of the innate immune system and are becoming a promising direction in cancer clinical research. Although NK cells have shown good effects in cancer treatment, there are also the following problems: (1) The survival time of NK cells injected into patients is short, which limits their therapeutic effect; (2) The microenvironment of solid tumors is prone to inhibiting the function of NK cells, and it is difficult for NK cells to enter the tumor interior and survive; (3) The risk of cytokine storm; (4) The cost of cell amplification / transportation is high, and the storage conditions for live NK cells are demanding, and it is difficult to ensure the homogeneity of different batches of cells.

[0005] Extracellular vesicles (EVs) are small vesicles with a membrane structure actively secreted by cells, generally with a diameter of 30 - 150 nm, and have the dual functions of disease treatment and drug delivery. The extracellular vesicles secreted by NK cells contain a variety of immune components and can play an immunomodulatory role by the paracrine pathway or by targeting the immune system through the circulatory system. Therefore, NK cell-derived extracellular vesicles can be used as an alternative treatment method for NK cells.

[0006] Urolithin A (UA) is a natural small molecule compound produced by the metabolism of dietary ellagitannins (such as components in pomegranates and nuts) by gut microbiota. As a potent mitophagy activator, it can optimize cellular energy metabolism and delay aging-related diseases by clearing dysfunctional mitochondria. Its anti-inflammatory and antioxidant properties (such as inhibiting the NF-κB pathway and activating the Nrf2 pathway) further confer its protective effect against chronic inflammation and oxidative damage.

[0007] Paclitaxel (PTX) is a highly effective, low-toxic, and broad-spectrum natural anti-cancer drug. As a diterpenoid alkaloid compound with anti-cancer activity, it can inhibit the proliferation of cancer cells by inducing cell cycle arrest and mitotic catastrophe. Clinically, PTX has been widely used in the treatment of breast cancer, ovarian cancer, and some head and neck cancers and lung cancers.

[0008] Currently, there are drug-loading schemes for extracellular vesicles and PTX, but how to further improve its therapeutic effect in TNBC remains the research content continuously carried out by scientific researchers. Summary of the Invention

[0009] Aiming at the current technical status, the purpose of the present invention is to provide a drug-loaded extracellular vesicle, its preparation method and application, to prepare an extracellular vesicle derived from natural killer cells pretreated with urolithin A and use it to carry paclitaxel. The aim is to exert the synergistic effect of multiple drugs for the treatment of tumors, especially TNBC, while enhancing the safety of PTX application.

[0010] To achieve the above purpose, the technical solution of the present invention is as follows: In the first aspect, a drug-loaded extracellular vesicle includes an extracellular vesicle, the extracellular vesicle is derived from natural killer cells and is loaded with paclitaxel, and the extracellular vesicle or the natural killer cells are treated with urolithin A.

[0011] Optionally, each 1×10 10 drug-loaded extracellular vesicle contains 10 - 20 ng of paclitaxel; to achieve a good therapeutic effect.

[0012] In the second aspect, a preparation method of the above drug-loaded extracellular vesicle includes incubating natural killer cells or extracellular vesicles of natural killer cells in a culture solution containing paclitaxel and / or urolithin A.

[0013] Optionally, the preparation method includes a natural killer cell incubation method, including the steps: S11. Take natural killer cells and co-incubate them in a culture solution containing urolithin A and paclitaxel for a set time; S12. Take the cell supernatant, filter it through a filter membrane and concentrate it, and then resuspend it after treatment by high-speed differential centrifugation and ultra-high-speed centrifugation to obtain the drug-loaded extracellular vesicles.

[0014] Optionally, in S11, the ratio of natural killer cells, paclitaxel, and urolithin A in each milliliter of the culture medium is (10 7 ~10 10 ) cells: (0.01 - 10) μg: (2 - 20) μg, and the incubation time is 1 - 48 h to obtain a set drug loading.

[0015] Optionally, in S12, filter through a 0.20 - 0.30 μm filter membrane to prepare extracellular vesicles.

[0016] Optionally, in S12, the high-speed differential centrifugation method includes: centrifuging the concentrated cell supernatant at 300 - 500 g for 10 - 20 min, collecting the first supernatant; centrifuging the first supernatant at 2000 - 3000 g for 10 - 20 min, collecting the second supernatant; centrifuging the second supernatant at 12000 - 13000 g for 30 - 60 min, collecting the third supernatant; centrifuging the third supernatant at 120000 - 140000 g for 70 - 90 min, resuspending the precipitate with PBS buffer; transferring the resuspended precipitate to a new ultracentrifugation tube, centrifuging at 120000 - 140000 g for 70 - 90 min, and resuspending the precipitate with PBS buffer to collect and obtain the drug-loaded extracellular vesicles.

[0017] Optionally, the preparation method includes an extrusion method, which includes the steps: S21. Take natural killer cells, co-incubate them in a culture medium containing urolithin A and paclitaxel for a set time, centrifuge and resuspend them, and collect the cells; S22. Extrude the cells sequentially using filter membranes from thick to thin; S23. Ultracentrifuge and resuspend the extruded liquid to obtain the drug-loaded extracellular vesicles.

[0018] Optionally, in S21, the ratio of natural killer cells, paclitaxel, and urolithin A in each milliliter of the culture medium is (10 7 ~10 10 ) cells: (0.01 - 10) μg: (2 - 20) μg, and the incubation time is 1 - 48 h.

[0019] Optionally, in S21, after centrifugation, resuspend the cells to (5 - 6) × 10 6 cells / mL for the extrusion step in S22.

[0020] Optionally, in S22, a filter membrane with a specification of 0.2 - 1 μm is selected, and an extruder is used to extrude through the filter membrane in order from thick to thin, and each specification of the filter membrane is extruded 3 - 20 times, thereby preparing extracellular vesicles of a set size.

[0021] Optionally, in S23, the method of ultra-high-speed centrifugation includes: centrifuging at 120,000 - 140,000 g for 70 - 90 min, discarding the supernatant, and resuspending the precipitate with PBS buffer, thereby collecting and obtaining drug-loaded extracellular vesicles.

[0022] Optionally, the preparation method includes an extracellular vesicle incubation method, which includes the steps: S31. Treat natural killer cells with urolithin A, take the supernatant, filter it through a filter membrane, and concentrate it; S32. Treat the concentrated cell supernatant by high-speed differential centrifugation and ultra-high-speed centrifugation methods, and resuspend it to obtain extracellular vesicles derived from natural killer cells pretreated with urolithin A; S33. Mix and incubate the extracellular vesicles obtained in S32 with paclitaxel, and after ultra-high-speed differential centrifugation, resuspend it to obtain the drug-loaded extracellular vesicles.

[0023] Optionally, in S31, the concentration of urolithin A is 2 - 20 μg / mL (molar concentration is about 10 - 80 μM); filter through a 0.20 - 0.30 μm filter membrane.

[0024] Optionally, in S32, the high-speed differential centrifugation method includes: centrifuging the concentrated cell supernatant at 300 - 500 g for 10 - 20 min, collecting the first supernatant; centrifuging the first supernatant at 2000 - 3000 g for 10 - 20 min, collecting the second supernatant; centrifuging the second supernatant at 12,000 - 13,000 g for 30 - 60 min, collecting the third supernatant; centrifuging the third supernatant at 120,000 - 140,000 g for 70 - 90 min, resuspending the precipitate with PBS buffer; after resuspending the precipitate, transfer it to a new ultracentrifuge tube, and centrifuge at 120,000 - 140,000 g for 70 - 90 min, resuspending the precipitate with PBS buffer to generate extracellular vesicles pretreated with urolithin A.

[0025] Optionally, in S33, in each milliliter of the culture solution, the ratio of natural killer cells to paclitaxel is (10 9 ~10 15 ) cells : (0.01 - 20) mg, incubate at room temperature for 1 - 48 h to complete the drug loading process of paclitaxel by the extracellular vesicles pretreated with urolithin A.

[0026] Optionally, in S33, the ultra-high speed differential centrifugation method includes: centrifuging the incubated liquid at 120,000 - 140,000 g for 70 - 90 min, discarding the supernatant, and resuspending the precipitate with PBS buffer; then centrifuging at 12,000 - 13,000 g for 30 - 60 min to collect the first supernatant; centrifuging the first supernatant at 12,000 - 13,000 g for 30 - 60 min to collect the second supernatant; the second supernatant contains the drug-loaded extracellular vesicles at a set concentration; thus, the drug-loaded extracellular vesicles are collected and obtained.

[0027] In a third aspect, the above-mentioned drug-loaded extracellular vesicles are used in at least one of the following (a1) - (a3); (a1) Preparing a product for inhibiting the proliferation of breast cancer cells; (a2) Preparing a product for inhibiting the growth and / or migration of breast cancer; (a3) Preparing a product for treating breast cancer.

[0028] The beneficial effects of the present invention are as follows: 1. The present invention provides an extracellular vesicle derived from natural killer cells and loaded with urolithin A and paclitaxel in view of the disadvantages such as short survival time of cells in the treatment of breast cancer with natural killer cells, difficulty for cells to enter and survive inside tumors, risk of cytokine storm, and high cost of cell amplification / transportation. With the tumor targeting ability of extracellular vesicles, the drug can accurately reach the tumor tissue and increase the drug concentration inside the tumor. The extracellular vesicles secreted by NK cells contain various immune components, which can play an immune regulatory role through the paracrine pathway or target the immune system through the circulatory system, while enhancing the safety of paclitaxel application and exerting a drug synergistic effect with paclitaxel.

[0029] 2. The extracellular vesicles derived from natural killer cells treated with urolithin A contain more perforin 1 and granzyme B, and the obtained extracellular vesicles have an obvious inhibitory effect on triple-negative breast cancer cells and can inhibit tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0031] Figure 1 It is a transmission electron micrograph in Example 4. (A) represents the transmission electron micrograph of extracellular vesicles derived from natural killer cells pretreated with urolithin A, and (B) represents the transmission electron micrograph of extracellular vesicles derived from natural killer cells pretreated with urolithin A carrying PTX.

[0032] Figure 2It is the particle size distribution diagram in Example 4. (A) represents the particle size distribution diagram of urothelial A-pretreated natural killer cell-derived extracellular vesicles, and (B) represents the particle size distribution diagram of urothelial A-pretreated natural killer cell-derived extracellular vesicles carrying PTX.

[0033] Figure 3 It is the diagram of biomarker detection results in Example 4.

[0034] Figure 4 It is the diagram of the detection results of granzyme B content in Example 4.

[0035] Figure 5 It is the diagram of the detection results of perforin 1 content in Example 4.

[0036] Figure 6 It is the diagram of the detection results of PTX content in Example 4.

[0037] Figure 7 It is the diagram of the inhibition results of PTX on breast cancer cells in Example 5.

[0038] Figure 8 It is the diagram of the inhibition results of urothelial A-pretreated natural killer cell-derived extracellular vesicles on breast cancer cells in Example 5.

[0039] Figure 9 It is the diagram of the inhibition results of natural killer cell-derived extracellular vesicles carrying PTX on breast cancer cells in Example 5.

[0040] Figure 10 It is the diagram of the inhibition results of urothelial A-pretreated natural killer cell-derived extracellular vesicles carrying PTX on breast cancer cells in Example 5.

[0041] Figure 11 It is the diagram of the treatment results of urothelial A-pretreated natural killer cell-derived extracellular vesicles carrying PTX on breast cancer model mice. (A) represents the in vivo bioimaging results of urothelial A-pretreated natural killer cell-derived extracellular vesicles carrying PTX inhibiting mouse tumors, and (B) represents the in vivo fluorescence intensity of urothelial A-pretreated natural killer cell-derived extracellular vesicles carrying PTX inhibiting mouse tumors.

[0042] Figure 12 It is the diagram of the results of enhancing the uptake of PTX by breast cancer cells in Example 5. (A) represents the observation of the uptake of PTX by confocal microscopy, (B) represents the detection of the fluorescence intensity of PTX after Cy5.5 labeling in cells by flow cytometry, and (C) represents the statistical chart of the fluorescence intensity of PTX after Cy5.5 labeling. Detailed implementation manners

[0043] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0044] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0045] The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions. All raw materials and reagents used in the following examples, unless otherwise specified, can be obtained commercially.

[0046] Example 1 A drug-loaded extracellular vesicle, the preparation method being the natural killer cell incubation method, comprising the following steps: S11. Take natural killer cells and co-incubate them in a culture medium containing urolithin A and paclitaxel for 48 h. In each milliliter of the culture medium, the ratio of natural killer cells, paclitaxel, and urolithin A is 10 10 cells: 0.5 μg: 10 μg.

[0047] S12. Take the cell supernatant, filter it through a 0.22-μm filter membrane, and then concentrate it using a tangential flow ultrafiltration system. Then, perform high-speed differential centrifugation and ultra-high-speed centrifugation at 4°C, including: centrifuging the concentrated cell supernatant at 300 g for 10 min, collecting the first supernatant, and discarding the precipitate; centrifuging the first supernatant at 2000 g for 10 min, collecting the second supernatant, and discarding the precipitate; centrifuging the second supernatant at 12000 g for 30 min, collecting the third supernatant, and discarding the precipitate; centrifuging the third supernatant at 120000 g for 70 min, discarding the supernatant, and resuspending the precipitate with PBS buffer; after resuspending the precipitate, transfer it to a new ultracentrifuge tube, centrifuge it at 120000 g for 70 min, discard the supernatant, and resuspend the precipitate with PBS buffer to obtain the drug-loaded extracellular vesicle.

[0048] Among them, the tangential flow ultraconcentration system is an existing technology in the art, and the present invention has no technical improvement on it.

[0049] Example 2 A drug-loaded extracellular vesicle, the preparation method being the extrusion method, comprising the following steps: S21. Take natural killer cells and co-incubate them in a culture medium containing urolithin A and paclitaxel for 48 h. In each milliliter of the culture medium, the ratio of natural killer cells, paclitaxel, and urolithin A is 10 10 cells: 0.5 μg: 10 μg. After centrifuging the incubation liquid, resuspend it with PBS buffer until the cell number reaches 5×10 6 cells / mL.

[0050] S22. Use an extruder to extrude the resuspended cells successively through filter membranes with specifications of 1 μm, 0.4 μm, and 0.2 μm, and perform 11 extrusions for each specification of the filter membrane.

[0051] S23. Perform ultra-high-speed centrifugation on the extruded liquid at 4°C at 120000 g for 70 min, then discard the supernatant and resuspend the precipitate with PBS buffer to obtain the drug-loaded extracellular vesicles.

[0052] Example 3 A kind of drug-loaded extracellular vesicle, the preparation method is the extracellular vesicle incubation method, including the following steps: S31. Add urolithin A during the culture of natural killer cells to make the final concentration 9.12 μg / mL, continue to culture for 48 h, take the cell supernatant, filter it through a 0.22-μm filter membrane, and concentrate it using a tangential flow ultrafiltration system.

[0053] S32. Process the concentrated cell supernatant by high-speed differential centrifugation and ultra-high-speed centrifugation at 4°C, including: centrifuge at 300 g for 10 min, collect the first supernatant, and discard the precipitate; centrifuge the first supernatant at 2000 g for 10 min, collect the second supernatant, and discard the precipitate; centrifuge the second supernatant at 12000 g for 30 min, discard the precipitate, and collect the third supernatant; centrifuge the third supernatant at 120000 g for 70 min, discard the supernatant, and resuspend the precipitate with PBS buffer; transfer the resuspended precipitate to a new ultracentrifuge tube, centrifuge at 120000 g for 70 min, and resuspend the precipitate with PBS buffer to obtain extracellular vesicles derived from natural killer cells pretreated with urolithin A.

[0054] S33. Mix the extracellular vesicles obtained in S32 with PBS buffer containing PTX, incubate at room temperature for 24 h, and the ratio of extracellular vesicles to PTX in each milliliter of the culture medium is 10 10 cells: 0.5 μg; after incubation, centrifuge the mixed liquid at 4°C at 120000 g for 70 min, discard the supernatant, resuspend the precipitate with PBS buffer, transfer the resuspended precipitate to a new ultracentrifuge tube, centrifuge at 13000 g for 30 min, collect the first supernatant, discard the precipitate, and centrifuge the first supernatant at 13000 g for 30 min, collect the second supernatant, and discard the precipitate to obtain the drug-loaded extracellular vesicles.

[0055] In the above Examples 1 to 3, the culture supernatant was derived from the culture supernatant obtained by the amplification culture of human umbilical cord blood NK cells, and the culture amplification method of the Chinese patent with the application number 2023111277604 (Preparation and Application of Extracellular Vesicles of NK Cells) was adopted, and there was no improvement in this technical solution compared with it.

[0056] In Examples 1 to 3, drug-loaded extracellular vesicles carrying urolithin A and paclitaxel can be prepared, and the size range and drug loading amount of the drug-loaded extracellular vesicles obtained in each example are the same. The following examples are carried out using the drug-loaded extracellular vesicles prepared in Example 3.

[0057] Example 4 Identification of the obtained drug-loaded extracellular vesicles includes: Observation of the morphology of extracellular vesicles derived from natural killer cells pretreated with urolithin A (obtained from step S32 in Example 3, abbreviated as extracellular vesicles treated with urolithin A) and extracellular vesicles derived from natural killer cells pretreated with urolithin A carrying PTX (drug-loaded extracellular vesicles) finally obtained in Example 3 under TEM (transmission electron microscopy): Take 20 μL of each of the two types of extracellular vesicles and drop them onto a copper mesh. After 1 minute, blot them dry with filter paper; then add a drop of 1% (m / m, mass percentage) uranyl acetate solution and blot it dry with filter paper after 1 minute; observe and take pictures under TEM after drying. The observation results are as Figure 1 shown, Figure 1 in (A) represents the extracellular vesicles treated with urolithin A, Figure 1 in (B) represents the finally obtained drug-loaded extracellular vesicles, and both of their morphologies are in the typical saucer shape with a middle depression, meeting the morphological standard of extracellular vesicles.

[0058] Determination of the particle size of extracellular vesicles treated with urolithin A and the finally obtained drug-loaded extracellular vesicles by a nanoparticle tracking analyzer (NTA): Dilute the extracellular vesicles treated with urolithin A with ultrapure water to about 1×10 7 per mL, and use a nanoparticle tracking analyzer to detect the particle size. The results are as Figure 2 shown, Figure 2 in (A) represents the particle size of the extracellular vesicles treated with urolithin A, Figure 2 in (B) represents the particle size of the drug-loaded extracellular vesicles, both of which are about 100 nm.

[0059] Western blotting analysis of specific proteins in urolithin A-treated extracellular vesicles and the finally obtained drug-loaded extracellular vesicles: Isolate and purify the two types of extracellular vesicles, add 5-fold concentrated loading buffer, boil at 100 °C for 5 min, and cool to room temperature. After separation by 12% (mass concentration) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transfer the proteins in the gel to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA), seal with 5% skim milk powder and react at room temperature for 1 h. Incubate overnight at 4 °C with rabbit anti-human CD63 monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:1000), rabbit anti-human CD81 monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:1000), rabbit anti-human TSG101 monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:1000), and rabbit anti-human Calnexin monoclonal antibody (diluted with antibody diluent at a volume ratio of 1:1000). After washing the membrane with Tris-buffered saline with Tween-20 (TBST) containing Tween 20 (5 min × 3 times), incubate with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody at room temperature for 2 h, and wash the membrane with TBST (5 min × 5 times). Finally, develop using a super-sensitive chemiluminescence (ECL) kit. The results are as Figure 3 shown. Both urolithin A-treated extracellular vesicles and the finally obtained drug-loaded extracellular vesicles expressed CD63, CD81, and TSG101, and lacked the expression of Calnexin, meeting the identification criteria for extracellular vesicles.

[0060] Detection of granzyme and perforin content: After ultrasonic disruption of extracellular vesicles derived from natural killer cells, urolithin A-treated extracellular vesicles, and drug-loaded extracellular vesicles, use an ELISA kit for detection. The results are as Figure 4 and Figure 5 shown. Figure 4 represents the content of granzyme B. The urolithin A-treated extracellular vesicles and the drug-loaded extracellular vesicles increased by 52.01% and 15.12% respectively compared to the extracellular vesicles derived from natural killer cells. Figure 5 represents the content of perforin 1. The urolithin A-treated extracellular vesicles and the drug-loaded extracellular vesicles increased by 116.07% and 90.57% respectively compared to the extracellular vesicles derived from natural killer cells.

[0061] Detection of PTX content in drug-loaded extracellular vesicles: Take 50 μL of the concentrated storage solution of drug-loaded extracellular vesicles, add 50 μL of acetonitrile solution and ultrasonicate for 30 min, then add docetaxel solution as the internal standard substance. After filtration through a 0.22 μm filter membrane, the PTX concentration is detected using a liquid chromatography-mass spectrometry instrument. The results are as Figure 6 shown. The peaks representing the internal standard and the PTX in the drug-loaded extracellular vesicles obtained by the liquid chromatography-mass spectrometry instrument. The peak value of the drug-loaded extracellular vesicles is 3.52×10 4 . By calculation, the absolute concentration of PTX can be obtained as 13.11 ng of PTX per 1×10 10 drug-loaded extracellular vesicles.

[0062] Example 5 Detection of the inhibitory effect of the prepared drug-loaded extracellular vesicles on breast cancer cells, including the following content.

[0063] Detection of the inhibitory effect of drug-loaded extracellular vesicles on breast cancer cells. The CCK-8 detection kit (Dojindo) was used to measure the inhibition rates of PTX, urolithin A-treated extracellular vesicles, natural killer cell-derived extracellular vesicles carrying only PTX (with the same PTX loading as the drug-loaded extracellular vesicles prepared in Example 3), and the preparation of drug-loaded extracellular vesicles on 4T1 cells: 4T1 cells (1×10 4 cells / well) were seeded into a 96-well culture plate and incubated for 24 hours; then the medium was replaced with 100 μL of serum-free DMEM containing different concentrations of PTX, urolithin A-treated extracellular vesicles, or drug-loaded extracellular vesicles, and cells treated with DMEM were used as the control group; after incubation for 48 hours, the cells were washed with PBS buffer and incubated with 100 μL of DMEM containing 10 μL of CCK-8 reagent at 37 °C for 4 hours; then the absorbance was measured using a spectrophotometric microplate reader at a wavelength of 450 nm. All experiments were repeated three times, and the inhibition rate was expressed as a percentage relative to the control group. The results are as Figure 7 , Figure 8 and Figure 9 shown. Figure 7 It shows that the IC50 of PTX on 4T1 cells is 8.02 μg / mL. Figure 8 It shows that the IC50 of urolithin A-treated extracellular vesicles on 4T1 cells is 1.37×10 9 particles / mL. Figure 9 It shows that the IC50 of natural killer cell-derived extracellular vesicles carrying only PTX on 4T1 cells is 8.65×10 8 particles / mL. Figure 10The IC50 of the drug-loaded extracellular vesicles carrying both urolithin A and PTX against 4T1 cells was 1.93×10 8 particles / mL. After calculation, the synergy index of the extracellular vesicles treated with urolithin A and the extracellular vesicles derived from natural killer cells carrying only PTX was 0.36, indicating that the extracellular vesicles treated with urolithin A and the extracellular vesicles derived from natural killer cells carrying only PTX exerted a strong synergistic effect.

[0064] Therapeutic effect of extracellular vesicles treated with urolithin A on breast cancer tumor model mice: A breast cancer tumor model was constructed by subcutaneously injecting 4T1-Luc cells (1×10 6 cells / mouse) into female Balb / C mice; and the mice were divided into 4 groups and injected respectively with: PBS buffer (control group), extracellular vesicles treated with urolithin A (1×10 10 particles / mL), extracellular vesicles derived from natural killer cells carrying only PTX (1×10 10 particles / mL), drug-loaded extracellular vesicles carrying both urolithin A and PTX (1×10 10 particles / mL), once every 3 days, for a total of 4 injections; during the treatment period, the tumor fluorescence emission imaging was performed using the IVIS® imaging system at different time points (day 1, day 4, day 7, day 10, day 13) to observe the development of the tumor, and the results were as Figure 11 shown, Figure 11 in (A) indicates that the extracellular vesicles derived from natural killer cells pretreated with urolithin A carrying PTX can significantly inhibit the growth of breast cancer tumors in vivo, Figure 11 in (B) indicates that the drug-loaded extracellular vesicles carrying both PTX and urolithin A can significantly reduce the fluorescence intensity of breast cancer tumors in vivo.

[0065] Effect of extracellular vesicles treated with urolithin A on the uptake of PTX by breast cancer cells: 1×10 5 4T1 cells were seeded in 20 mm confocal dishes. After culturing for 24 h, the medium was replaced with serum-free DMEM containing cy5.5-labeled PTX (100 ng / mL) and drug-loaded extracellular vesicles (the concentration of cy5.5-labeled PTX was 100 ng / mL) and incubated for 0.5 h. The cells treated with DMEM were used as the control group. After incubation, the cells were fixed with 500 μL of paraformaldehyde for 25 min, and then stained with 400 μL of DAPI for 10 min. After staining, confocal microscopy imaging was used for detection. The results were as Figure 12 shown in (A). Compared with PTX alone, the accumulation of PTX in breast cancer cells increased significantly after PTX was encapsulated with extracellular vesicles treated with urolithin A.

[0066] Inoculate 2×10 5 4T1 cells into a six-well plate. After culturing for 24 h, replace the medium with serum-free DMEM containing PTX labeled with cy5.5 (100 ng / mL) and drug-loaded extracellular vesicles (where the concentration of PTX labeled with cy5.5 is 100 ng / mL) and incubate for 0.5 h. Use the cells treated with DMEM as the control group. After the incubation, collect the cells for flow cytometry detection. Then, the fluorescence intensity of cy5.5 represents the PTX concentration in the cells. The results are as Figure 12 (B) in shows that compared with PTX alone, after PTX is encapsulated in extracellular vesicles derived from natural killer cells pretreated with urolithin A, the fluorescence intensity of PTX-cy5.5 in breast cancer cells increases significantly. Figure 12 (C) in shows that compared with PTX alone, after PTX is encapsulated in extracellular vesicles pretreated with urolithin A, the mean fluorescence intensity of PTX-cy5.5 in breast cancer cells increases significantly.

[0067] According to the above content, the present invention encapsulates the broad-spectrum anti-cancer drug PTX into extracellular vesicles derived from natural killer cells pretreated with urolithin A to prepare a novel extracellular vesicle loaded with PTX and pretreated with urolithin A, which can play a role in the synergistic treatment of breast cancer. Compared with free PTX and extracellular vesicles derived from natural killer cells pretreated with urolithin A without PTX, the extracellular vesicles derived from natural killer cells pretreated with urolithin A and loaded with PTX of the present invention have a better inhibitory effect on breast cancer cells and can be applied to the preparation of drugs for treating breast cancer.

[0068] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A drug-loaded extracellular vesicle, characterized in that: The invention comprises extracellular vesicles, wherein the extracellular vesicles are derived from natural killer cells and loaded with paclitaxel, and the extracellular vesicles or the natural killer cells are treated with urolithin A.

2. The drug-loaded extracellular vesicles according to claim 1, characterized in that Each 1×10 10 Each drug-loaded extracellular vesicle contains 10~20ng of paclitaxel.

3. A method for preparing drug-loaded extracellular vesicles according to any one of claims 1-2, characterized in that: The method comprises incubating natural killer cells or the extracellular vesicles of natural killer cells in a culture medium containing paclitaxel and / or urolithin A.

4. The method for preparing drug-loaded extracellular vesicles according to claim 3, characterized in that: The method comprises a natural killer cell incubation method, comprising the steps of: S11, taking natural killer cells and incubating them in a culture medium containing urolithin A and paclitaxel for a set time; S12, taking the cell supernatant, filtering it through a filter membrane and concentrating it, and then treating it by high-speed differential centrifugation and ultra-high-speed centrifugation and resuspending it to obtain the drug-loaded extracellular vesicles.

5. The method for preparing drug-loaded extracellular vesicles according to claim 4, characterized in that: In S11, the concentration of urolithin A was 10-80 μM, and the ratio of natural killer cells, paclitaxel, and urolithin A per ml of culture medium was (10 7 ~10 10 ) pieces: (0.01-10) μg: (2~20) μg, incubation time is 1~48h; or, in S12, filtered through a 0.20-0.30 μm filter membrane; Or, in S12, the high-speed differential centrifugation method includes: centrifuging the concentrated cell supernatant at 300-500 g for 10-20 min to collect a first supernatant; The first supernatant was centrifuged at 2000-3000 g for 10-20 min to obtain the second supernatant; the second supernatant was centrifuged at 12000-13000 g for 30-60 min to obtain the third supernatant; the third supernatant was centrifuged at 120000-140000 g for 70-90 min and the precipitate was resuspended with PBS buffer; after the precipitate was resuspended, it was transferred to a new ultracentrifuge tube, centrifuged at 120000-140000 g for 70-90 min and the precipitate was resuspended with PBS buffer.

6. The method for preparing drug-loaded extracellular vesicles according to claim 3, characterized in that: The preparation method includes an extrusion method, comprising the steps of: S21, taking natural killer cells, incubating them in a culture medium containing urolithin A and paclitaxel for a set time, centrifuging and resuspending them, and collecting the cells; S22, using filter membranes in order from coarse to fine to squeeze the cells; S23, ultra-centrifuging and resuspending the squeezed liquid to obtain the drug-loaded extracellular vesicles.

7. The method for preparing drug-loaded extracellular vesicles according to claim 6, characterized in that: In S21, the concentration of urolithin A was 10-80 μM, and the ratio of natural killer cells, paclitaxel, and urolithin A per ml of culture medium was (10 7 ~10 10 ) pieces: (0.01-10) μg: (2~20) μg, incubation time is 1~48h; Or, in S21, resuspend the cells to (5-6) × 10 6 Pieces / mL; Or, in S22, the extruder is used to sequentially extrude through filter membranes of 1 to 0.2 μm, and each specification of the filter membrane is extruded 3 to 20 times; Or, in S23, the ultrahigh-speed centrifugation method includes: centrifuging at 120,000-140,000 g for 70-90 min, discarding the supernatant, and resuspending the precipitate with PBS buffer.

8. The method for preparing drug-loaded extracellular vesicles according to claim 3, characterized in that: The preparation method comprises a natural killer cell-derived extracellular vesicle incubation method, comprising the steps of: S31, treating natural killer cells with urolithin A, taking the supernatant and filtering it through a filter membrane and concentrating it; S32, treating the concentrated cell supernatant by high-speed differential centrifugation, resuspending the cell supernatant and treating the cell supernatant by ultra-high-speed centrifugation, and obtaining extracellular vesicles derived from natural killer cells pretreated with urolithin A after resuspending the cell supernatant; S33, mixing the extracellular vesicles obtained in S32 with paclitaxel, and resuspending after ultra-high-speed differential centrifugation to obtain the drug-loaded extracellular vesicles.

9. The method for preparing drug-loaded extracellular vesicles according to claim 8, characterized in that: In S31, the concentration of urolithin A is 2-20 μg / mL; filtered through a 0.20-0.30 μm filter membrane; Or, in S32, the high-speed differential centrifugation method includes: centrifuging the concentrated cell supernatant at 300-500 g for 10-20 min to collect a first supernatant; centrifuging the first supernatant at 2000-3000 g for 10-20 min to collect a second supernatant; centrifuging the second supernatant at 12000-13000 g for 30-60 min to collect a third supernatant; centrifuging the third supernatant at 120000-140000 g for 70-90 min, and resuspending the precipitate with PBS buffer; after resuspending the precipitate, transferring it to a new ultracentrifuge tube, centrifuging it at 120000-140000 g for 70-90 min, and resuspending the precipitate with PBS buffer; Or, in S33, the ratio of natural killer cells to paclitaxel per ml of culture medium is (10 9 ~10 15 ) pieces: (0.01-20) mg, incubate at room temperature for 1~48h; Or, in S33, the ultra-high-speed differential centrifugation method includes: centrifuging the incubated liquid at 120,000-140,000 g for 70-90 min, discarding the supernatant, and resuspending the precipitate with PBS buffer; then centrifuging at 12,000-13,000 g for 30-60 min, collecting a first supernatant; centrifuging the first supernatant at 12,000-13,000 g for 30-60 min, and collecting a second supernatant; the second supernatant contains the drug-loaded extracellular vesicles at a set concentration.

10. A use of the drug-loaded extracellular vesicles according to any one of claims 1 to 2, characterized in that: The application includes at least one of the following (a1) to (a3); (a1) preparing products for inhibiting breast cancer cell proliferation; (a2) preparing products for inhibiting the growth and / or migration of breast cancer; (a3) Preparing products for treating breast cancer.

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