Drug-loaded extracellular vesicles, preparation method, and application thereof

Abstract

The present invention relates to drug-loaded extracellular vesicles, and a preparation method and application thereof. The extracellular vesicles in the drug-loaded 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. The preparation method of the drug-loaded extracellular vesicles comprises incubating natural killer cells or extracellular vesicles of natural killer cells in a culture medium containing paclitaxel and / or urolithin A. By leveraging the tumor-targeting ability of the extracellular vesicles, the drug can precisely reach tumor tissue and increase the drug concentration within the tumor. The extracellular vesicles secreted by NK cells contain multiple immune components, which can exert immunomodulatory effects by targeting the immune system through paracrine pathways or through the circulatory system, while enhancing the safety of paclitaxel application and exerting a synergistic drug effect with paclitaxel.

Description

Technical Field

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

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Breast carcinoma (BC) is the uncontrolled proliferation of mammary epithelial cells in response to multiple carcinogens. In advanced stages, it can be life-threatening. Triple-negative breast cancer (TNBC), a subtype of breast cancer that lacks expression of estrogen and progesterone receptors, as well as human epidermal growth factor receptor, accounts for approximately 20% of breast cancer diagnoses worldwide. TNBC is primarily prevalent in young women and is characterized by high invasiveness, heterogeneity, and a poor prognosis.

[0004] Natural killer cells (NK cells) are cytotoxic lymphocytes of the innate immune system and are becoming a promising direction for clinical cancer research. Although NK cells have shown good effects in cancer treatment, they also have the following problems: (1) NK cells have a short survival time after being injected into the patient, which limits their therapeutic effect; (2) the microenvironment of solid tumors easily inhibits the function of NK cells, and NK cells have difficulty entering and surviving in the tumor; (3) there is a risk of cytokine storm; (4) the cost of cell expansion / transfer is high, and the storage conditions of live NK cells are demanding, making it difficult to ensure the homogeneity of different batches of cells.

[0005] Extracellular vesicles (EVs) are tiny membrane-bound vesicles actively secreted by cells, typically 30-150 nm in diameter. They have dual functions as disease treatment and drug delivery. EVs secreted by NK cells contain a variety of immune components, which can exert immunomodulatory effects through paracrine pathways or by targeting the immune system through the circulatory system. Therefore, NK cell-derived EVs could serve as an alternative therapy to NK cells.

[0006] Urolithin A (UA) is a natural small molecule compound produced by the intestinal flora through the metabolism of ellagitannins in the diet (such as those found in pomegranates and nuts). As a potent mitochondrial autophagy activator, UA 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 give it a protective effect against chronic inflammation and oxidative damage.

[0007] Paclitaxel (PTX) is a highly effective, low-toxic, broad-spectrum natural anticancer drug. As a diterpenoid alkaloid with anticancer activity, it can inhibit cancer cell proliferation 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, as well as lung cancers.

[0008] Currently, there are drug delivery schemes for extracellular vesicles and PTX, but how to further improve its therapeutic effect in TNBC is still an ongoing research topic for scientific researchers. Summary of the Invention

[0009] In response to the current state of the art, the present invention aims to provide drug-loaded extracellular vesicles, their preparation method, and applications. Extracellular vesicles derived from natural killer cells pretreated with urolithin A are prepared and used to deliver paclitaxel. This approach aims to leverage the synergistic effects of multiple drugs for the treatment of tumors, particularly TNBC, while also enhancing the safety of PTX applications.

[0010] In order to achieve the above object, the technical solution of the present invention is:

[0011] In a first aspect, a drug-loaded extracellular vesicle comprises an extracellular vesicle, wherein the extracellular vesicle is derived from natural killer cells and loaded with paclitaxel, and the extracellular vesicle or the natural killer cells are treated with urolithin A.

[0012] Optional, each 1×10 10 Each drug-loaded extracellular vesicle contains 10~20ng of paclitaxel to achieve a good therapeutic effect.

[0013] In a second aspect, the method for preparing the drug-loaded extracellular vesicles comprises incubating natural killer cells or extracellular vesicles of natural killer cells in a culture medium containing paclitaxel and / or urolithin A.

[0014] Optionally, the preparation method includes a natural killer cell incubation method, comprising the steps of:

[0015] S11, taking natural killer cells and incubating them in a culture medium containing urolithin A and paclitaxel for a set time;

[0016] S12. The cell supernatant is collected, filtered through a filter membrane, and then concentrated. The supernatant is then treated by high-speed differential centrifugation and ultrahigh-speed centrifugation, and then resuspended to obtain the drug-loaded extracellular vesicles.

[0017] Optionally, in S11, the ratio of natural killer cells, paclitaxel, and urolithin A per ml of culture medium is (10 7 ~10 10 ) pieces: (0.01-10) μg: (2~20) μg, incubation time is 1~48h to obtain the set drug loading amount.

[0018] Optionally, in S12, the solution is filtered through a 0.20-0.30 μm filter membrane to prepare extracellular vesicles.

[0019] Optionally, in S12, the high-speed differential centrifugation method includes: centrifuging the concentrated cell supernatant at 300~500g for 10~20 minutes to collect the first supernatant; centrifuging the first supernatant at 2000~3000g for 10~20 minutes to collect the second supernatant; centrifuging the second supernatant at 12000~13000g for 30~60 minutes to collect the third supernatant; centrifuging the third supernatant at 120000~140000g for 70~90 minutes, and resuspending the precipitate with PBS buffer; after the resuspended precipitate, transferring it to a new ultracentrifuge tube, centrifuging it at 120000~140000g for 70~90 minutes, and resuspending the precipitate with PBS buffer, thereby collecting and obtaining the drug-loaded extracellular vesicles.

[0020] Optionally, the preparation method includes an extrusion method, comprising the steps of:

[0021] S21, taking natural killer cells, incubating them in a culture medium containing urolithin A and paclitaxel for a set time, centrifuging and resuspending them to collect the cells;

[0022] S22, squeeze the cells using filters in descending order;

[0023] S23, ultra-centrifuging the squeezed liquid and resuspending it to obtain the drug-loaded extracellular vesicles.

[0024] Optionally, in S21, the ratio of natural killer cells, paclitaxel and urolithin A per ml of culture medium is (10 7 ~10 10 ) pieces: (0.01-10) μg: (2~20) μg, incubation time is 1~48h.

[0025] Optional: In S21, resuspend the cells to (5-6) × 10 6 pcs / mL, used for the extrusion step in S22.

[0026] Optionally, in S22, a filter membrane with a specification of 0.2 to 1 μm is selected, and an extruder is used to extrude the membrane in order from coarse to fine, and each specification of the filter membrane is extruded 3 to 20 times to prepare extracellular vesicles of a set size.

[0027] Optionally, in S23, the ultrahigh-speed centrifugation method includes: centrifugation at 120,000-140,000 g for 70-90 min, discarding the supernatant, and resuspending the precipitate with PBS buffer, thereby collecting and obtaining the drug-loaded extracellular vesicles.

[0028] Optionally, the preparation method includes an extracellular vesicle incubation method, comprising the steps of:

[0029] S31, treating natural killer cells with urolithin A, taking the supernatant and filtering it through a filter membrane and concentrating it;

[0030] S32. treating the concentrated cell supernatant by high-speed differential centrifugation and ultra-high-speed centrifugation, and resuspending the cell supernatant to obtain extracellular vesicles derived from natural killer cells pretreated with urolithin A;

[0031] S33. The extracellular vesicles obtained in S32 are mixed with paclitaxel and incubated, and the mixture is resuspended after ultra-high-speed differential centrifugation to obtain the drug-loaded extracellular vesicles.

[0032] Optionally, in S31, the concentration of urolithin A is 2-20 μg / mL (molar concentration of about 10-80 μM); and filtered through a 0.20-0.30 μm filter membrane.

[0033] Optionally, 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 the resuspended 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 to generate extracellular vesicles pretreated with urolithin A.

[0034] Optionally, 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, incubated at room temperature for 1~48h, so that the extracellular vesicles pretreated with urolithin A can complete the drug loading process of paclitaxel.

[0035] Optionally, in S33, the ultra-high-speed differential centrifugation method includes: centrifuging the incubated liquid at 120,000~140,000g for 70~90min, discarding the supernatant, and resuspending the precipitate with PBS buffer; then centrifuging at 12,000~13,000g for 30~60min to collect a first supernatant; centrifuging the first supernatant at 12,000~13,000g for 30~60min to collect a second supernatant; the second supernatant contains the set concentration of the drug-loaded extracellular vesicles; thereby collecting and obtaining the drug-loaded extracellular vesicles.

[0036] In a third aspect, the use of the drug-loaded extracellular vesicles described above in at least one of the following (a1) to (a3);

[0037] (a1) preparing a product for inhibiting breast cancer cell proliferation;

[0038] (a2) preparing products for inhibiting the growth and / or migration of breast cancer;

[0039] (a3) Preparation of products for treating breast cancer.

[0040] The beneficial effects of the present invention are:

[0041] 1. This invention addresses the shortcomings of natural killer cell therapy for breast cancer, including short cell survival, difficulty in entering and surviving within tumors, the risk of cytokine storms, and high costs of cell expansion and transport. By providing natural killer cell-derived extracellular vesicles loaded with urolithin A and paclitaxel, the invention addresses these shortcomings. Leveraging the tumor-targeting capabilities of the extracellular vesicles, the drugs can precisely reach tumor tissue, increasing drug concentrations within the tumor. The extracellular vesicles secreted by NK cells contain multiple immune components that can exert immunomodulatory effects by targeting the immune system through paracrine pathways or the circulatory system, while also enhancing the safety of paclitaxel and creating a synergistic effect with paclitaxel.

[0042] 2. Extracellular vesicles derived from natural killer cells treated with urolithin A contain more perforin 1 and granzyme B. The obtained extracellular vesicles have a significant inhibitory effect on triple-negative breast cancer cells and can inhibit tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0044] Figure 1 These are transmission electron micrographs from Example 4, wherein (A) shows a transmission electron micrograph of extracellular vesicles derived from natural killer cells pretreated with urolithin A, and (B) shows a transmission electron micrograph of extracellular vesicles derived from natural killer cells pretreated with urolithin A carrying PTX.

[0045] Figure 2 The particle size distribution diagrams in Example 4 are shown in (A) a particle size distribution diagram of natural killer cell-derived extracellular vesicles pretreated with urolithin A, and (B) a particle size distribution diagram of natural killer cell-derived extracellular vesicles pretreated with urolithin A carrying PTX.

[0046] Figure 3 This is a diagram showing the biomarker detection results in Example 4.

[0047] Figure 4 This is a graph showing the detection results of the granzyme B content in Example 4.

[0048] Figure 5 This is a graph showing the detection results of the perforin 1 content in Example 4.

[0049] Figure 6 This is a graph showing the detection results of the PTX content in Example 4.

[0050] Figure 7 This is a graph showing the inhibition of breast cancer cells by PTX in Example 5.

[0051] Figure 8 This is a graph showing the inhibitory effect of extracellular vesicles derived from natural killer cells pretreated with urolithin A in Example 5 on breast cancer cells.

[0052] Figure 9 This is a graph showing the inhibitory effect of PTX-carrying natural killer cell-derived extracellular vesicles on breast cancer cells in Example 5.

[0053] Figure 10 This is a graph showing the inhibitory effect of extracellular vesicles derived from natural killer cells pretreated with urolithin A carrying PTX on breast cancer cells in Example 5.

[0054] Figure 11 These are diagrams showing the therapeutic results of extracellular vesicles derived from natural killer cells pretreated with PTX-loaded urolithin A in breast cancer model mice in Example 5. (A) shows the in vivo bioimaging results of extracellular vesicles derived from natural killer cells pretreated with PTX-loaded urolithin A inhibiting mouse tumors. (B) shows the in vivo fluorescence intensity of extracellular vesicles derived from natural killer cells pretreated with PTX-loaded urolithin A inhibiting mouse tumors.

[0055] Figure 12 These are the results of enhancing PTX uptake by breast cancer cells in Example 5. (A) shows the uptake of PTX observed by confocal microscopy, (B) shows the fluorescence intensity of PTX after Cy5.5 labeling in cells detected by flow cytometry, and (C) shows a statistical graph of the fluorescence intensity of PTX after Cy5.5 labeling. DETAILED DESCRIPTION

[0056] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0057] 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 form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0058] The experimental methods in the following examples where specific conditions are not specified are generally based on conventional conditions. Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available.

[0059] Example 1

[0060] A drug-loaded extracellular vesicle is prepared by a natural killer cell incubation method, comprising the following steps:

[0061] S11. Take natural killer cells and incubate them in a culture medium containing urolithin A and paclitaxel for 48 hours. The ratio of natural killer cells, paclitaxel and urolithin A per milliliter of culture medium is 10. 10 Pieces: 0.5μg: 10μg.

[0062] S12. Take the cell supernatant, filter it through a 0.22μm filter membrane, and concentrate it using a tangential flow ultrafiltration system. Then, perform high-speed differential centrifugation and ultrahigh-speed centrifugation at 4°C, including: centrifuging the concentrated cell supernatant at 300g for 10 minutes, collecting the first supernatant, and discarding the precipitate; centrifuging the first supernatant at 2000g for 10 minutes, collecting the second supernatant, and discarding the precipitate; centrifuging the second supernatant at 12000g for 30 minutes, collecting the third supernatant, and discarding the precipitate; centrifuging the third supernatant at 120000g for 70 minutes, discarding the supernatant, and resuspending the precipitate with PBS buffer; after resuspending the precipitate, transfer it to a new ultracentrifuge tube, centrifuge it at 120000g for 70 minutes, discard the supernatant, and resuspend the precipitate with PBS buffer to obtain drug-loaded extracellular vesicles.

[0063] Among them, the tangential flow superconcentration system is the existing technology in this field, and the present invention does not make any technical improvement thereto.

[0064] Example 2

[0065] A drug-loaded extracellular vesicle is prepared by an extrusion method, comprising the following steps:

[0066] S21. Take natural killer cells and incubate them in a culture medium containing urolithin A and paclitaxel for 48 hours. The ratio of natural killer cells, paclitaxel and urolithin A per milliliter of culture medium is 10:1. 10 After centrifugation of the incubation liquid, resuspend the cells in PBS buffer until the number of cells is 5×10 6 pieces / mL.

[0067] S22. Use an extruder to squeeze the resuspended cells through filter membranes of 1 μm, 0.4 μm, and 0.2 μm, respectively. Perform 11 squeezes for each filter membrane specification.

[0068] S23. The squeezed liquid was subjected to ultrahigh-speed centrifugation at 120,000 g for 70 min at 4° C., and then the supernatant was discarded and the precipitate was resuspended in PBS buffer to obtain the drug-loaded extracellular vesicles.

[0069] Example 3

[0070] A drug-loaded extracellular vesicle is prepared by an extracellular vesicle incubation method, comprising the following steps:

[0071] S31. Urolithin A was added to the natural killer cell culture process to a final concentration of 9.12 μg / mL. The culture was continued for 48 h. The cell supernatant was collected and filtered through a 0.22 μm filter membrane and concentrated using a tangential flow ultrafiltration system.

[0072] S32. The concentrated cell supernatant is treated by high-speed differential centrifugation and ultra-high-speed centrifugation at 4°C, including: centrifuging at 300g for 10 minutes, collecting a first supernatant, and discarding the precipitate; centrifuging the first supernatant at 2000g for 10 minutes, collecting a second supernatant, and discarding the precipitate; centrifuging the second supernatant at 12000g for 30 minutes, discarding the precipitate, and collecting a third supernatant; centrifuging the third supernatant at 120000g for 70 minutes, discarding the supernatant, and resuspending the precipitate with PBS buffer; after the resuspended precipitate, transferring the precipitate to a new ultracentrifuge tube, centrifuging at 120000g for 70 minutes, and resuspending the precipitate with PBS buffer to obtain extracellular vesicles derived from natural killer cells pretreated with urolithin A.

[0073] S33, the extracellular vesicles obtained in S32 were mixed with PBS buffer containing PTX, and incubated at room temperature for 24 h. The ratio of extracellular vesicles to PTX per ml of culture medium was 10 10After the incubation, the mixed liquid was centrifuged at 120,000 g for 70 min at 4°C, the supernatant was discarded, and the precipitate was resuspended with PBS buffer. After the resuspended precipitate, it was transferred to a new ultracentrifuge tube and centrifuged at 13,000 g for 30 min. The first supernatant was collected and the precipitate was discarded. The first supernatant was centrifuged at 13,000 g for 30 min, the second supernatant was collected, and the precipitate was discarded to obtain drug-loaded extracellular vesicles.

[0074] In Examples 1 to 3 above, the culture supernatant was derived from the culture supernatant obtained by amplifying and culturing human umbilical cord blood NK cells, and the culture and amplification method of Chinese Patent Application No. 2023111277604 (Preparation and Application of Extracellular Vesicles of NK Cells) was adopted. This technical solution did not provide any improvement compared to the above.

[0075] Examples 1 to 3 can all prepare drug-loaded extracellular vesicles loaded with urolithin A and paclitaxel, and the size range of the drug-loaded extracellular vesicles obtained in each example is consistent with the drug loading amount. The following examples are performed using the drug-loaded extracellular vesicles prepared in Example 3.

[0076] Example 4

[0077] Identify the obtained drug-loaded extracellular vesicles, including:

[0078] The morphology of the extracellular vesicles derived from natural killer cells pretreated with urolithin A (obtained from step S32 in Example 3, referred to as urolithin A-treated extracellular vesicles) and the extracellular vesicles derived from natural killer cells pretreated with urolithin A and loaded with PTX (drug-loaded extracellular vesicles) obtained in Example 3 were observed under TEM (transmission electron microscopy). 20 μL of each type of extracellular vesicle was added dropwise to a copper grid and dried with filter paper after 1 minute. A drop of 1% (m / m, mass percentage) uranyl acetate solution was then added and dried with filter paper after 1 minute. After drying, the mixture was observed and photographed under TEM. The observation results are shown in Figure 2. Figure 1 As shown, Figure 1 (A) in the figure represents extracellular vesicles treated with urolithin A. Figure 1 (B) in the figure shows the final drug-loaded extracellular vesicles. Both have a typical saucer-like shape with a depression in the middle, which meets the morphological standards of extracellular vesicles.

[0079] The particle size of urolithin A-treated extracellular vesicles and the final drug-loaded extracellular vesicles was measured by nanoparticle tracking analyzer (NTA): the extracellular vesicles treated with urolithin A were diluted with ultrapure water to 1×10 7 The particle size was measured using a nanoparticle tracking analyzer. Figure 2 As shown, Figure 2 (A) in the figure shows the particle size of extracellular vesicles treated with urolithin A. Figure 2(B) in the figure indicates the particle size of drug-loaded extracellular vesicles, which are all around 100 nm.

[0080] Western blotting was used to analyze the specific proteins in urolithin A-treated and drug-loaded extracellular vesicles. The two extracellular vesicles were separated and purified, and a 5-fold concentration of loading buffer was added. The samples were boiled at 100°C for 5 min and cooled to room temperature. After separation by 12% (mass concentration) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins in the gel were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA), sealed with 5% skim milk powder, and reacted at room temperature for 1 minute. h, incubated with rabbit anti-human CD63 monoclonal antibody (diluted in antibody diluent at a volume ratio of 1:1000), rabbit anti-human CD81 monoclonal antibody (diluted in antibody diluent at a volume ratio of 1:1000), rabbit anti-human TSG101 monoclonal antibody (diluted in antibody diluent at a volume ratio of 1:1000), and rabbit anti-human Calnexin monoclonal antibody (diluted in antibody diluent at a volume ratio of 1:1000) at 4°C overnight; washed with Tris-buffered Saline with Tween-20 (TBST) (5 min × 3 times), then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody at room temperature for 2 hours, and washed with TBST (5 min × 5 times); finally, developed using an ultrasensitive chemiluminescence (ECL) kit, as shown in the following figure. Figure 3 As shown, both the extracellular vesicles treated with urolithin A and the final drug-loaded extracellular vesicles expressed CD63, CD81, and TSG101, and lacked the expression of Calnexin, meeting the identification criteria for extracellular vesicles.

[0081] Granzyme and perforin content detection: After ultrasonic disruption of natural killer cell-derived extracellular vesicles, urolithin A-treated extracellular vesicles and drug-loaded extracellular vesicles, ELISA kits were used for detection. The results were as follows: Figure 4 and Figure 5 shown. Figure 4 The content of granzyme B in extracellular vesicles treated with urolithin A and drug-loaded extracellular vesicles derived from natural killer cells increased by 52.01% and 15.12%, respectively. Figure 5 The content of perforin-1 in extracellular vesicles treated with urolithin A and drug-loaded extracellular vesicles was increased by 116.07% and 90.57%, respectively, compared with that in extracellular vesicles derived from natural killer cells.

[0082] Detection of PTX content in drug-loaded extracellular vesicles: 50 μL of drug-loaded extracellular vesicle concentrated stock solution was taken, 50 μL of acetonitrile solution was added and ultrasonicated for 30 minutes, then docetaxel solution was added as internal standard substance, and the PTX concentration was detected by liquid chromatography-mass spectrometry after filtering through a 0.22 μm filter membrane. The results are as follows Figure 6 As shown in the figure, the peaks representing the internal standard and the PTX in the drug-loaded extracellular vesicles obtained by LC-MS / MS are shown. The peak value of the drug-loaded extracellular vesicles is 3.52×10 4 , the absolute concentration of PTX can be calculated to be per 1×10 10 Each drug-loaded extracellular vesicle contained 13.11 ng of PTX.

[0083] Example 5

[0084] The inhibitory effect of the prepared drug-loaded extracellular vesicles on breast cancer cells is detected, including the following contents.

[0085] The inhibition of breast cancer cells by drug-loaded extracellular vesicles was tested using a CCK-8 assay kit (Dojindo). The inhibition rates of PTX, urolithin A-treated extracellular vesicles, natural killer cell-derived extracellular vesicles carrying only PTX (the PTX loading amount was the same as that of the drug-loaded extracellular vesicles prepared in Example 3), and the drug-loaded extracellular vesicle preparation on 4T1 cells were determined. 4T1 cells (1×10 4 Cells were seeded into 96-well culture plates (100 μL / well) and incubated for 24 hours. The culture medium was then replaced with 100 μL of serum-free DMEM containing different concentrations of PTX, urolithin A-treated extracellular vesicles, or drug-loaded extracellular vesicles. DMEM-treated cells served as a control group. After 48 hours of incubation, 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. The absorbance was then measured at a wavelength of 450 nm using a spectrophotometer. All experiments were repeated three times, and the inhibition rate is expressed as a percentage relative to the control group. The results are shown in Table 1. Figure 7 、 Figure 8 and Figure 9 As shown, Figure 7 The IC50 of PTX for 4T1 cells is 8.02 μg / mL. Figure 8 The IC50 of extracellular vesicles treated with urolithin A against 4T1 cells was 1.37×10 9 Particles / mL (particles / mL). Figure 9 The IC50 of NK cell-derived extracellular vesicles carrying only PTX against 4T1 cells was 8.65×10 8 Particles / mL (particles / mL). Figure 10The IC50 of drug-loaded extracellular vesicles containing urolithin A and PTX on 4T1 cells was 1.93×10 8 The synergistic index of extracellular vesicles treated with urolithin A and extracellular vesicles derived from natural killer cells carrying only PTX was calculated to be 0.36, indicating that extracellular vesicles treated with urolithin A and extracellular vesicles derived from natural killer cells carrying only PTX exerted a strong synergistic effect.

[0086] The therapeutic effect of urolithin A-treated extracellular vesicles on breast cancer tumor model mice: Balb / C female mice were subcutaneously injected with 4T1-Luc cells (1×10 6 The breast cancer tumor model was established by dividing the mice into 4 groups and injecting: PBS buffer (control group), urolithin A-treated extracellular vesicles (1×10 10 / mL), natural killer cell-derived extracellular vesicles carrying only PTX (1×10 10 / mL), and drug-loaded extracellular vesicles loaded with urolithin A and PTX (1×10 10 During the treatment period, IVIS® imaging system was used to perform tumor fluorescence emission imaging at different time points (day 1, day 4, day 7, day 10, day 13) to observe the development of the tumor. The results are as follows: Figure 11 As shown, Figure 11 (A) indicates that 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 (B) shows that drug-loaded extracellular vesicles carrying both PTX and urolithin A can significantly reduce the fluorescence intensity of breast cancer tumors in vivo.

[0087] Effect of urolithin A-treated extracellular vesicles on PTX uptake by breast cancer cells: 1×10 5 4T1 cells were seeded in a 20 mm confocal microplate. After 24 h of culture, the culture medium was replaced with serum-free DMEM containing cy5.5-labeled PTX (100 ng / mL) and drug-loaded extracellular vesicles (wherein the concentration of cy5.5-labeled PTX was 100 ng / mL) and incubated for 0.5 h. The cells treated with DMEM served as the control group. After the incubation, the cells were fixed with 500 μL paraformaldehyde for 25 min and stained with 400 μL DAPI for 10 min. After the staining, the cells were detected by confocal microscopy. The results are shown in Figure 2. Figure 12 As shown in (A), the accumulation of PTX in breast cancer cells was significantly increased after PTX was encapsulated in extracellular vesicles treated with urolithin A compared with PTX alone.

[0088] 2×10 5 4T1 cells were seeded in a six-well plate and cultured for 24 hours. The medium was replaced with serum-free DMEM containing cy5.5-labeled PTX (100 ng / mL) and drug-loaded extracellular vesicles (where the concentration of cy5.5-labeled PTX was 100 ng / mL) and incubated for 0.5 hours. Cells treated with DMEM served as the control group. After the incubation, the cells were collected and analyzed by flow cytometry. The fluorescence intensity of cy5.5 represents the PTX concentration in the cells. The results are shown in Figure 2. Figure 12 (B) shows that compared with PTX alone, the fluorescence intensity of PTX-cy5.5 in breast cancer cells was significantly increased after PTX was encapsulated in extracellular vesicles derived from natural killer cells pretreated with urolithin A. Figure 12 (C) shows that the mean fluorescence intensity of PTX-cy5.5 in breast cancer cells was significantly increased after PTX was encapsulated in extracellular vesicles pretreated with urolithin A compared with PTX alone.

[0089] Based on the above content, the present invention encapsulates the broad-spectrum anticancer drug PTX into extracellular vesicles derived from natural killer cells pretreated with urolithin A to prepare a new type of PTX-loaded extracellular vesicles derived from natural killer cells pretreated with urolithin A, which can exert a synergistic drug effect in treating 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 containing the PTX of the present invention have a better inhibitory effect on breast cancer cells and can be used to prepare drugs for treating breast cancer.

[0090] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An application of drug-loaded extracellular vesicles, characterized in that: The drug-loaded extracellular vesicles include extracellular vesicles, the extracellular vesicles are derived from natural killer cells and loaded with paclitaxel, and the natural killer cells are treated with urolithin A; The application includes at least one of the following applications (a1) to (a3); (a1) preparing a drug for inhibiting the proliferation of triple-negative breast cancer cells; (a2) preparing drugs for inhibiting the growth and / or migration of triple-negative breast cancer; (a3) Prepare drugs for treating triple-negative breast cancer.

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

3. The use of the drug-loaded extracellular vesicles according to claim 1, characterized in that: The preparation method includes an incubation method of natural killer cell-derived extracellular vesicles, 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 ultrahigh-speed centrifugation to obtain extracellular vesicles derived from natural killer cells pretreated with urolithin A after resuspending the cell supernatant; S33. The extracellular vesicles obtained in S32 are mixed with paclitaxel and incubated, and the mixture is resuspended after ultra-high-speed differential centrifugation to obtain the drug-loaded extracellular vesicles.

4. The use of the drug-loaded extracellular vesicles according to claim 3, characterized in that: In S31, the concentration of urolithin A was 2-20 μg / mL and filtered through a 0.20-0.30 μm filter membrane; Alternatively, in S32, the high-speed differential centrifugation method includes: centrifuging the concentrated cell supernatant at 300-500 g for 10-20 minutes to collect a first supernatant; centrifuging the first supernatant at 2000-3000 g for 10-20 minutes to collect a second supernatant; centrifuging the second supernatant at 12000-13000 g for 30-60 minutes to collect a third supernatant; centrifuging the third supernatant at 120000-140000 g for 70-90 minutes, and resuspending the precipitate with PBS buffer; after the resuspended precipitate, transferring it to a new ultracentrifuge tube, centrifuging it at 120000-140000 g for 70-90 minutes, and resuspending the precipitate with PBS buffer; Alternatively, 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; Alternatively, in S33, the ultrahigh-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 a first supernatant; centrifuging the first supernatant at 12,000-13,000 g for 30-60 min to collect a second supernatant; the second supernatant contains the drug-loaded extracellular vesicles at a set concentration.

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