Droplet microsphere capsule as well as preparation method and application thereof

Abstract

The invention discloses a droplet microsphere capsule as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The droplet microsphere capsule has a core-shell structure; the surface layer of the core is an oil phase; the shell includes extracellular vesicles. Extracellular vesicles are adsorbed and assembled on the surface of the core, so that the uniformity and the stability of the droplet microsphere capsule are remarkably improved. The droplet microsphere capsule has good deformability, can carry medicine, a developing agent and other functional components, and can be effectively used for vascular embolism and treatment of cancer, trauma, gastrointestinal hemorrhage and other diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a droplet microsphere capsule and a preparation method and application thereof. BACKGROUND

[0002] Drug-loaded microspheres refer to drug-loaded particles formed by grafting or embedding drug molecules in biocompatible carrier materials through physical or chemical methods. Drug-loaded microspheres have various administration routes, are suitable for oral administration, local administration by intervention, and other administration modes, have excellent drug release and controlled release performance, good drug stability, and broad clinical application prospects. However, in the field of interventional embolization therapy, drug-loaded microsphere systems often load drugs through charge adsorption and physical adsorption, and can only load a few positively charged drugs. Moreover, most microsphere embolic agents are not visible and cannot carry a contrast agent, and a contrast agent needs to be used additionally to indirectly evaluate the embolization degree and embolization endpoint. Furthermore, drug-loaded microspheres with rigid structures are difficult to adapt to complex disease pathological microenvironments in vivo, have weak self-adapting deformation ability, are limited by the inherent particle size of the microspheres, have limited terminal blood vessel embolization capacity, often cause embolization defects, and are difficult to achieve deep embolization-chemotherapy and deep drug delivery. Therefore, it is very important to provide a new drug-loaded reagent. SUMMARY

[0003] The present application aims to at least solve one of the problems in the prior art. To this end, the present application provides a droplet microsphere capsule, which has good deformation ability and can carry functional components such as drugs and contrast agents, and can be effectively used for vascular embolization and treatment of various diseases such as cancer, trauma, and gastrointestinal bleeding.

[0004] The present application also provides a preparation method of the above-mentioned droplet microsphere capsule.

[0005] The present application also provides a reagent comprising the above-mentioned droplet microsphere capsule.

[0006] The present application also provides applications of the above-mentioned droplet microsphere capsule or reagent

[0007] According to the first aspect of the present application, a droplet microsphere capsule has a core-shell structure; the surface layer of the core is an oil phase; and the shell comprises an extracellular vesicle.

[0008] According to the droplet microsphere capsule of the present application, at least the following beneficial effects are achieved:

[0009] The extracellular vesicles are assembled on the surface of the core, which significantly improves the uniformity and stability of the droplet microsphere capsule. The droplet microsphere capsule of the embodiment has good deformation capacity, maintains stable spherical morphology without external force, and deforms without breaking under external force, which is beneficial to its reaching the terminal blood vessels and realizing the embolization of the terminal blood vessels. The droplet microsphere capsule can carry functional components such as drugs and imaging agents, and stably exists and slowly releases in artificial intestinal fluid, artificial gastric juice and mucin conditions, improves the delivery efficiency of the functional components, and has good stability; the storage condition of the droplet microsphere capsule is simple, and the droplet microsphere capsule can be effectively used for the embolization and treatment of various diseases such as cancer, trauma and gastrointestinal hemorrhage, has high flexibility, can be used for preparing drugs with the functions of visual embolization-chemotherapy or targeted therapy or immunotherapy of tumors, and has good clinical application prospect.

[0010] According to some embodiments of the present application, the core comprises at least one of an oil phase droplet, a water-in-oil droplet, and an oil-in-water-in-oil droplet.

[0011] According to some embodiments of the present application, the oil phase in the core is an oil or fat acceptable in food according to the pharmacopoeia. The oil or fat includes but is not limited to oil or fat for injection or oral use. The oil phase includes at least one of vegetable oil, animal oil, mineral oil, vegetable oil derivative, animal oil derivative, and mineral oil derivative.

[0012] According to some embodiments of the present application, the vegetable oil includes at least one of corn oil, rapeseed oil, peanut oil, olive oil, poppy seed oil, soybean oil, sunflower oil, walnut oil, sesame oil, flaxseed oil, tea seed oil, safflower oil, castor oil, cottonseed oil, and jojoba oil.

[0013] According to some embodiments of the present application, the animal oil includes at least one of fish oil and seal oil.

[0014] According to some embodiments of the present application, the mineral oil includes at least one of squalane, squalene, paraffin oil, and pristane.

[0015] According to some embodiments of the present application, the vegetable oil derivative includes an organic iodine compound combined with iodine. The organic iodine compound includes at least one of iodized oil and poppy ethiodized oil.

[0016] According to some embodiments of the present application, the extracellular vesicle includes at least one of plant-derived extracellular vesicle, animal-derived extracellular vesicle, microorganism-derived extracellular vesicle, and artificially synthesized extracellular vesicle.

[0017] According to some embodiments of the present application, the source of the plant-derived extracellular vesicle includes but is not limited to lemon, ginger, kiwi, grapefruit, strawberry, grape, orange, bitter gourd, ginseng, tangerine, atractylodes, or onion.

[0018] According to some embodiments of the present invention, the sources of the animal-derived extracellular vesicles include but are not limited to hepatocytes, mesenchymal stem cells or immune cells.

[0019] According to some embodiments of the present invention, the microbial-derived extracellular vesicles are derived from at least one of bacteria and fungi. The bacteria include, but are not limited to, Bacillus subtilis or Escherichia coli. The fungi include, but are not limited to, yeast.

[0020] According to some embodiments of the present invention, the extracellular vesicles are isolated from at least one of tissue homogenate, biological cell culture, tissue culture, and body fluid.

[0021] According to some embodiments of the present invention, the body fluid includes at least one of blood, lymph, saliva, urine, semen, sweat, tears, and milk.

[0022] According to some embodiments of the present invention, the method for isolating the extracellular vesicles includes at least one of ultracentrifugation, size exclusion chromatography, ultrafiltration, tangential flow filtration, membrane affinity kit, chromatography, immunomagnetic bead sorting, polymerization precipitation, immunoaffinity, microfluidics technology, and affinity chromatography.

[0023] According to some embodiments of the present invention, the ultracentrifugation includes differential ultracentrifugation and density gradient ultracentrifugation.

[0024] According to some embodiments of the present invention, the droplet microsphere capsule includes a functional component. The functional component can be loaded into the core and / or shell. For example, the functional component can be loaded into the extracellular vesicles through direct incubation, ultrasound, electroporation, chemical coupling, etc.; or the functional component can be dissolved in an aqueous or oily phase by utilizing its solubility, thereby loading the core with the functional component.

[0025] According to some embodiments of the present invention, the functional component includes at least one of a drug and a developer.

[0026] According to some embodiments of the present invention, the drug includes at least one of a compound drug and a protein drug.

[0027] According to some embodiments of the present invention, the compound drug includes at least one of a chemotherapy drug, a tyrosine kinase inhibitor, and a hemostatic drug.

[0028] According to some embodiments of the present invention, the tyrosine kinase inhibitor includes at least one of vatalanib, dasatinib, imatinib, N-desmethylimatinib, sorafenib, lapatinib, pazopanib, crizotinib, apatinib, gefitinib, ibrutinib, dasatinib, nilotinib, ruxolitinib, axitinib, osimertinib, erlotinib, ponatinib, sunitinib, and nilotinib.

[0029] According to some embodiments of the application, the chemotherapy drug comprises at least one of a platinum drug, an anthracycline drug, a taxane drug, an anti-pyrimidine drug, a vinca alkaloid drug, a camptothecin drug, a nitrosourea drug, an anti-folate drug.

[0030] According to some embodiments of the application, the platinum drug comprises at least one of cisplatin, carboplatin, oxaliplatin, lobaplatin, nedaplatin, oxaliplatin.

[0031] According to some embodiments of the application, the anthracycline drug comprises at least one of doxorubicin, epirubicin, pirarubicin, mitoxantrone.

[0032] According to some embodiments of the application, the taxane drug comprises at least one of paclitaxel, docetaxel.

[0033] According to some embodiments of the application, the anti-pyrimidine drug comprises at least one of fluorouracil, gemcitabine, capecitabine, xeloda, furtulon, floxuridine, uft.

[0034] According to some embodiments of the application, the vinca alkaloid drug comprises at least one of vinblastine, vincristine, vindesine, vinorelbine.

[0035] According to some embodiments of the application, the camptothecin drug comprises at least one of irinotecan, topotecan, rubitecan.

[0036] According to some embodiments of the application, the nitrosourea drug comprises at least one of carmustine, lomustine, semustine, nimustine.

[0037] According to some embodiments of the application, the anti-folate drug comprises at least one of methotrexate, pemetrexed, raltitrexed, leucovorin.

[0038] According to some embodiments of the application, the hemostatic drug comprises at least one of aminocaproic acid, aminomethylbenzoic acid, tranexamic acid, etamsylate, carboxymethylcellulose, vitamin K3, sodium bisulfite, menadiol and menadiol amine.

[0039] According to some embodiments of the application, the protein drug comprises at least one of an immune checkpoint inhibitor, a cytokine, thrombin.

[0040] According to some embodiments of the application, the immune checkpoint inhibitor comprises at least one of a PD-1 monoclonal antibody, a PD-L1 monoclonal antibody.

[0041] According to some embodiments of the application, the PD-1 monoclonal antibody comprises at least one of nivolumab, camrelizumab.

[0042] According to some embodiments of the application, the PD-1 mAb comprises at least one of atezolizumab, durvalumab.

[0043] According to some embodiments of the application, the cytokine comprises at least one of interleukin-6, gamma-interferon.

[0044] According to some embodiments of the application, the contrast agent comprises at least one of iodized oil, iophendylate, sodium iodide, diatrizoate, iohexol, barium sulfate, iodixanol, ioversol, iopromide, iobitridol, iotrol.

[0045] According to some embodiments of the application, the particle size of the droplet microsphere capsule is 20-1500 μm. For example, it can be 25 μm, 50 μm, 75 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 325 μm, 350 μm, 375 μm, 400 μm, 425 μm, 450 μm, 475 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1100 μm, 1200 μm, 1300 μm, 1400 μm or 1500 μm.

[0046] According to some embodiments of the application, the particle size of the extracellular vesicle is 40-1000 nm. For example, it can be 40 nm, 60 nm, 80 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm or 1000 nm.

[0047] The preparation method of the droplet microsphere capsule according to the first aspect of the second aspect embodiments of the application comprises the following steps:

[0048] S1, mixing the preparation raw materials of the core to obtain solution A; the preparation raw materials comprise an oil phase;

[0049] S2, preparing a mixture of the solution A and an extracellular vesicle solution, and homogenously stirring to obtain the droplet microsphere capsule.

[0050] According to the preparation method of the embodiments of the application, at least the following beneficial effects are achieved:

[0051] The preparation method of the embodiment has simple process and easy-to-control experimental conditions. By using solution A and extracellular vesicles as basic building units, and by using homogenizing stirring technology to make the extracellular vesicles adsorbed and assembled on the oil-water interface, a stable droplet microsphere capsule is constructed.

[0052] By controlling the time, temperature, speed, oil-water ratio and extracellular vesicle concentration of shearing stirring, a series of droplet microsphere capsules with different particle sizes can be synthesized.

[0053] The preparation and use method, post-processing method and storage are simple and easy to implement.

[0054] According to some embodiments of the present application, the solution A is at least one of an oil phase, a water-in-oil emulsion and a water-in-oil-in-oil emulsion.

[0055] According to some embodiments of the present application, when the solution A is a water-in-oil emulsion, the solution A is prepared by an emulsification method.

[0056] According to some embodiments of the present application, when the solution A is a water-in-oil-in-oil emulsion, the solution A is prepared by a double emulsification method.

[0057] According to some embodiments of the present application, the emulsification method comprises the following steps:

[0058] A mixture of a first water phase and a first oil phase is prepared, and ultrasonic treatment is performed, so as to obtain the solution A; the volume of the first water phase is less than that of the first oil phase.

[0059] According to some embodiments of the present application, the preparation raw material of the core comprises a functional component.

[0060] When the functional component comprises a water-soluble component, the emulsification method comprises: dissolving the water-soluble component in the first water phase.

[0061] When the functional component comprises an oil-soluble component, the emulsification method comprises: dissolving the oil-soluble component in the first oil phase.

[0062] According to some embodiments of the present application, in the emulsification method, the volume ratio of the first water phase to the first oil phase is 1:(2-40). For example, it can be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39 or 1:40.

[0063] According to some embodiments of the present application, the double emulsification method comprises the following steps:

[0064] A1, preparing a mixture of the second aqueous phase and the second oil phase, ultrasonic treatment, to obtain an oil-in-water emulsion;

[0065] The volume of the second aqueous phase is greater than that of the second oil phase;

[0066] A2, preparing a mixture of the oil-in-water emulsion and the third oil phase, to obtain the solution A;

[0067] The volume of the third oil phase is greater than that of the oil-in-water emulsion.

[0068] According to some embodiments of the present application, the raw material for preparing the core comprises a functional component;

[0069] When the functional component comprises a water-soluble component, the double emulsification method comprises dissolving the water-soluble component in the second aqueous phase;

[0070] And / or, when the functional component comprises an oil-soluble component, the double emulsification method comprises dissolving the oil-soluble component in the second oil phase and / or the third oil phase.

[0071] According to some embodiments of the present application, the temperature of the ultrasonic treatment is 0-60℃. For example, it can be 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃ or 60℃.

[0072] According to some embodiments of the present application, the time of the ultrasonic treatment is 1min-100min. For example, it can be 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, 65min, 70min, 75min, 80min, 85min, 90min, 95min or 100min.

[0073] According to some embodiments of the present application, the power of the ultrasonic treatment is 20w-1000w. For example, it can be 20w, 30w, 40w, 50w, 60w, 70w, 80w, 90w, 100w, 200w, 300w, 400w, 500w, 600w, 700w, 800w, 900w or 1000w.

[0074] According to some embodiments of the application, the rotation speed of the homogenizing stirring is between 100 rpm and 10000 rpm. For example, it can be 100 rpm, 500 rpm, 1000 rpm, 15000 rpm, 2000 rpm, 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm, 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm or 10000 rpm.

[0075] According to some embodiments of the application, the time of the homogenizing stirring is between 0.5 h and 50 h. For example, it can be 0.5 h, 0.6 h, 0.7 h, 0.8 h, 1 h, 5 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h or 50 h.

[0076] According to some embodiments of the application, the temperature of the homogenizing stirring is between 20 °C and 60 °C. For example, it can be 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C.

[0077] According to some embodiments of the application, the concentration of the extracellular vesicles in the extracellular vesicles solution is between 0.1 mg / mL and 1000 mg / mL. For example, it can be 0.1 mg / mL, 1 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, 600 mg / mL, 700 mg / mL, 800 mg / mL, 900 mg / mL or 1000 mg / mL.

[0078] According to some embodiments of the application, the volume ratio of the solution A to the extracellular vesicles solution is 1: (0.001-1000).

[0079] According to some embodiments of the application, the volume ratio of the solution A to the extracellular vesicles solution is 1: (1-100). For example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95 or 1:100.

[0080] According to some embodiments of the present application, the extracellular vesicle solution comprises an aqueous extracellular vesicle solution.

[0081] According to some embodiments of the present application, the preparation raw material of the core comprises a functional component.

[0082] When the functional component comprises a drug, the drug is added in an amount of 0.1 mg to 100 mg. For example, the drug can be added in an amount of 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg.

[0083] According to some embodiments of the present application, the preparation method further comprises a homogenizing stirring post-treatment. The homogenizing stirring post-treatment comprises at least one of separating a liquid phase and washing.

[0084] According to some embodiments of the third aspect of the present application, a reagent comprises the droplet microsphere capsule of the first aspect of the present application. Since the reagent adopts all the technical solutions of the droplet microsphere capsule of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments.

[0085] According to some embodiments of the present application, the reagent further comprises other pharmaceutically acceptable excipients.

[0086] According to some embodiments of the present application, the excipients comprise at least one of a solvent, a dye, and a lubricant.

[0087] According to some embodiments of the fourth aspect of the present application, the droplet microsphere capsule of the first aspect of the present application or the reagent of the third aspect of the present application is used in any one of A1) to A3):

[0088] A1) preparing an embolization agent;

[0089] A2) preparing a contrast agent;

[0090] A3) preparing a drug for treating a tumor.

[0091] According to some embodiments of the present application, the tumor comprises at least one of liver cancer, colorectal cancer, kidney cancer, uterine fibroids, lung cancer, prostate cancer, ovarian cancer, and breast cancer. Those skilled in the art can also select other target diseases based on the loaded functional component.

[0092] According to some embodiments of the present application, the administration mode of the drug comprises at least one of embolization and oral administration.

[0093] Other features and advantages of the present application will be set forth in the following description, and in part will be apparent from the description, or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0094] Figure 1 Results of characterization of the lemon extracellular vesicles prepared in Preparation Example 1; a: transmission electron microscope (TEM) image (scale bar: 200 nm), b: particle size distribution graph;

[0095] Figure 2 Results of characterization of the droplet microspheres capsules of different particle sizes in Examples 1-3; a, b, c: optical microscope images of the droplet microspheres capsules of Examples 1-3 (scale bar: 200 μm), d, e, f: particle size distribution graphs of the droplet microspheres capsules of Examples 1-3;

[0096] Figure 3 Confocal image of the droplet microspheres capsules prepared in Example 7;

[0097] Figure 4 Results of stability detection of the droplet microspheres capsules prepared in Example 8 in artificial gastric juice, artificial intestinal juice, and mucin solution; a: optical microscope image (scale bar: 50 μm), b: particle size change curve;

[0098] Figure 5 Results of sustained-release performance detection of the droplet microspheres capsules prepared in Example 8 in different solutions;

[0099] Figure 6 Drug concentration change curves in the plasma of different groups of rats in Test Example 6;

[0100] Figure 7 Results of cytotoxicity detection of different drugs on N1S1 hepatoma cells in Test Example 7; *** indicates that there is a very significant difference (p < 0.001);

[0101] Figure 8 Results of plugging performance test of the droplet microspheres capsules of Example 7 in a capillary glass tube;

[0102] Figure 9 Imaging image of the droplet microspheres capsules of Example 7 under CT;

[0103] Figure 10 Results of plugging performance test of the droplet microspheres capsules of Example 7 in a SD rat liver decellularization model;

[0104] Figure 11 Representative images of abdominal magnetic resonance (MR) before and after the droplet microspheres capsules of Example 7 were used for interventional therapy of a rat hepatoma model; wherein, the part circled by the dashed line shows the schematic tumor position;

[0105] Figure 12 Results of tumor pathological examination after the droplet microspheres of Example 7 were used to intervene in a rat liver cancer model;

[0106] Figure 13 Results of immunohistochemical examination of different treatment groups in Test Example 10. DETAILED DESCRIPTION

[0107] The concept and technical effects of the present application will be described below in conjunction with examples, so as to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0108] Unless otherwise specified, the examples in the examples were carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used were conventional products that can be obtained by commercial purchase.

[0109] In the description of the present application, if first, second, third, etc. are described, it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features or the sequence of indicated technical features.

[0110] “and / or” is used to indicate that one or both of the described cases can occur, for example, A and / or B includes (A and B) and (A or B).

[0111] Unless otherwise specified, “room temperature” and “normal temperature” in the present application both mean (25±5) °C.

[0112] Unless otherwise specified, “about” in the present application means ±5%.

[0113] In this context, “extracellular vesicle” refers to a nanovesicle consisting of a bilayer lipid membrane with a size of tens of nanometers to hundreds of nanometers.

[0114] Unless otherwise specified, the iodized oil used in the examples was purchased from Hengrui Medicine, Jiangsu, China Drug Code H20163348; doxorubicin was purchased from Shanghai Aladdin Biochemical, Item No.: D107159.

[0115] Preparation Example 1

[0116] This example provides a preparation method of lemon extracellular vesicles, the steps are as follows:

[0117] Fresh lime was washed and peeled, and the pulp was squeezed to obtain juice. The juice was filtered through medical gauze and a filter screen to remove larger fruit residues, and the clear fresh lemon juice was obtained. Then, the juice was transferred to a 250 mL centrifuge bottle, and the following centrifugal treatment was carried out in sequence: 500g centrifugation for 10 minutes, 2000g centrifugation for 20 minutes, 5000g centrifugation for 30 minutes, and 10000g centrifugation for 1 hour, to gradually remove smaller fruit residues. The clear supernatant was collected and transferred to a 50 mL centrifuge tube, and 100000g ultrahigh speed centrifugation was performed for 1 hour. The supernatant was discarded, and the precipitate was resuspended in sterile PBS. An 8% sucrose solution was added to the bottom of the ultracentrifuge tube, followed by the addition of 30%, 45% and 60% sucrose solutions to form a concentration gradient. The precipitate suspension was slowly injected into the centrifuge tube, and ultracentrifugation was performed at 150000g for 2 hours. Three bands were observed, and the second band was the extracellular vesicle (EV). The second band was collected and resuspended in PBS, and 100000g centrifugation was performed for 1 hour to remove excess sucrose in the resuspension. The obtained lemon extracellular vesicles were resuspended in PBS and divided into 1.5 mL EP tubes for long-term storage at -80°C.

[0118] Example 1

[0119] This example provides a droplet microsphere capsule, and the preparation method is as follows:

[0120] 50 μL of water and 1.5 mL of iodized oil were mixed in a 5 mL centrifuge tube, and ultrasonic treatment was performed under an ice bath at a power of 60w for 5 min to obtain a primary emulsion. The primary emulsion was transferred to a 50 mL centrifuge tube, and 3 mL of PBS and 600 μg of lemon extracellular vesicles were sequentially added to the 50 mL centrifuge tube. Shearing was performed at a homogenization speed of 8000 rpm for 45 min, and the reaction was completed. After standing at room temperature for 10 min, the supernatant was aspirated, and 5 mL of pure water was backfilled along the wall of the container, and continued to stand for 10 min. The above steps were repeated 5 times, and the lower precipitate after standing was the droplet microsphere capsule. The droplet microsphere capsule can be stored at room temperature or 4°C.

[0121] Example 2

[0122] This example provides a droplet microsphere capsule, and the preparation method is as follows:

[0123] Take 50 μL water and 1.5 mL iodized oil, mix in a 5 mL centrifuge tube, ultrasonic treatment for 5 min under ice bath at 60 w power, get the primary emulsion. Transfer the primary emulsion to a 50 ml centrifuge tube, add 3 mL PBS and 400 μg lemon extracellular vesicles in the 50 mL centrifuge tube in turn, shear at a homogeneous speed of 8000 rpm for 45 min, after the reaction is completed, stand for 10 min at room temperature. Absorb the supernatant, backfill into 5 mL pure water along the container wall, continue to stand for 10 min. Repeat for 5 times, the lower precipitate after standing is the droplet microsphere capsule. The droplet microsphere capsule can be stored at room temperature or 4°C.

[0124] Example 3

[0125] This example provides a droplet microsphere capsule, the preparation method is as follows:

[0126] Take 50 μL water and 1.5 mL iodized oil, mix in a 5 mL centrifuge tube, ultrasonic treatment for 5 min under ice bath at 60 w power, get the primary emulsion. Transfer the primary emulsion to a 50 ml centrifuge tube, add 3 mL PBS and 400 μg lemon extracellular vesicles in the 50 mL centrifuge tube in turn, shear at a homogeneous speed of 8000 rpm for 45 min, after the reaction is completed, stand for 10 min at room temperature. Absorb the supernatant, backfill into 5 mL pure water along the container wall, continue to stand for 10 min. Repeat for 5 times, the lower precipitate after standing is the droplet microsphere capsule. The droplet microsphere capsule can be stored at room temperature or 4°C.

[0127] Example 4

[0128] This example provides a droplet microsphere capsule, the preparation method is as follows:

[0129] Disperse 0.5 mg doxorubicin into 1.5 mL iodized oil, ultrasonic treatment for 5 min under ice bath at 60 w power, get the primary emulsion. Transfer the primary emulsion to a 50 ml centrifuge tube, add 3 mL PBS and 600 μg lemon extracellular vesicles in the 50 mL centrifuge tube in turn, shear at a homogeneous speed of 8000 rpm for 45 min, after the reaction is completed, stand for 10 min at room temperature, 0.33 μg doxorubicin per μg droplet. Absorb the supernatant, backfill into 5 mL pure water along the container wall, continue to stand for 10 min. Repeat for 5 times, the lower precipitate after standing is the droplet microsphere capsule. The droplet microsphere capsule can be stored at room temperature or 4°C.

[0130] Example 5

[0131] This example provides a droplet microsphere capsule, the preparation method is as follows:

[0132] 0.5 mg doxorubicin was dispersed into 1.5 mL iodinated oil, and an initial emulsion was obtained by ultrasonic treatment at 60 w power under ice bath for 5 min. The initial emulsion was transferred into a 50 ml centrifuge tube, 3 mL PBS and 400 μg lemon extracellular vesicles were sequentially added into the 50 mL centrifuge tube, and shearing was performed at a homogenization speed of 8000 rpm for 45 min. After reaction completion, the mixture was allowed to stand at room temperature for 10 min. Each μg of the droplet contained 0.28 μg of doxorubicin. The supernatant was aspirated, and 5 mL pure water was backfilled along the wall of the container, and the mixture was allowed to stand for another 10 min. The above process was repeated for 5 times. The lower precipitate after standing was the droplet microsphere capsule. The droplet microsphere capsule could be stored at room temperature or 4 °C.

[0133] Example 6

[0134] The present example provides a droplet microsphere capsule, and the preparation method is as follows:

[0135] 0.5 mg doxorubicin was dispersed into 1.5 mL iodinated oil, and an initial emulsion was obtained by ultrasonic treatment at 60 w power under ice bath for 5 min. The initial emulsion was transferred into a 50 ml centrifuge tube, 3 mL PBS and 200 μg lemon extracellular vesicles were sequentially added into the 50 mL centrifuge tube, and shearing was performed at a homogenization speed of 8000 rpm for 45 min. After reaction completion, the mixture was allowed to stand at room temperature for 10 min. Each μg of the droplet contained 0.28 μg of doxorubicin. The supernatant was aspirated, and 5 mL pure water was backfilled along the wall of the container, and the mixture was allowed to stand for another 10 min. The above process was repeated for 5 times. The lower precipitate after standing was the droplet microsphere capsule. The droplet microsphere capsule could be stored at room temperature or 4 °C.

[0136] Example 7

[0137] The present example provides a droplet microsphere capsule, and the preparation method is as follows:

[0138] 0.5 mg doxorubicin was dispersed into 1.5 mL iodinated oil, and an initial emulsion was obtained by ultrasonic treatment at 60 w power under ice bath for 5 min. The initial emulsion was transferred into a 50 ml centrifuge tube, 3 mL PBS (containing 3 mg of FITC-labeled anti-PD-L1 antibody and 400 μg of lemon extracellular vesicles) was sequentially added into the 50 mL centrifuge tube, and shearing was performed at a homogenization speed of 8000 rpm at room temperature for 45 min. After reaction completion, the mixture was allowed to stand at 4 °C for 10 min. The supernatant was aspirated, and 5 mL pure water was backfilled along the wall of the container, and the mixture was allowed to stand for another 10 min. The above process was repeated for 5 times. The lower precipitate after standing was the droplet microsphere capsule. The droplet microsphere capsule was stored at 4 °C. Each μg of the droplet microsphere capsule contained 0.28 μg of doxorubicin and 2.25 μg of anti-PD-L1 antibody.

[0139] The preparation method of the FITC-labeled anti-PD-L1 antibody is as follows:

[0140] Take 1 mg of anti-PD-L1 antibody into a test tube, add sterile PBS buffer with pH 9, so that the total volume of liquid is 1 mL. Add 1 μL of FITC fluorescent dye, mix well, then stir on a magnetic stirrer at 4°C for 1 hour. After the reaction is completed, transfer the mixture to a dialysis bag and dialyze at 4°C for 24 hours. Collect the dialyzed liquid to obtain the FITC-labeled anti-PD-L1 monoclonal antibody solution. The whole operation process needs to be strictly protected from light, and the final product needs to be stored in the dark.

[0141] Example 8

[0142] This example provides a droplet microsphere capsule, and the preparation method is as follows:

[0143] Disperse 0.5 mL of PBS solution (containing 75 mg of capecitabine) into 1.5 mL of rice oil (food grade, Yumi Mountain rice oil), and ultrasonically treat under an ice bath at a power of 60 w for 5 min to obtain a primary emulsion. Transfer the primary emulsion to a 50 mL centrifuge tube, and sequentially add 3 mL of PBS solution containing 300 μg of lemon extracellular vesicles into the 50 mL centrifuge tube. Shear at a uniform speed of 8000 rpm at room temperature for 60 min. After the reaction is completed, stand at 4°C for 10 min. Suck out the supernatant, and backfill into 5 mL of pure water along the wall of the container, and continue to stand for 10 min. Repeat for 5 times. The supernatant after standing is the droplet microsphere capsule. The droplet microsphere capsule is stored at 4°C.

[0144] Test Example 1

[0145] The lemon extracellular vesicles obtained by separation and purification in Preparation Example 1 were characterized by transmission electron microscopy and dynamic light scattering (DLS).

[0146] The results are shown in Figure 1 .

[0147] The lemon-derived extracellular vesicles exhibit a typical tea tray-like structure, similar to the morphology of most fruit-derived extracellular vesicles. They have a clear lipid bilayer membrane structure and a smooth surface with varying sizes. DLS detection shows that the average hydration particle size of the lemon-derived extracellular vesicles is 210.35 ± 4.18 nm.

[0148] Test Example 2

[0149] The size and morphology of the droplet microsphere capsules prepared in Examples 1-3 were observed by optical microscopy, and the particle size distribution was statistically analyzed using Image J software.

[0150] The results are shown in Figure 2 , Table 1.

[0151] Table 1

[0152] Group Example 1 Example 2 Example 3 Particle size (pm) 100±20 150±30 230±42

[0153] The liquid droplet microsphere capsule prepared by the present application has a spherical structure and good size distribution. The particle size of the liquid droplet microsphere capsule of Examples 1-3 is mainly distributed in 75-350 μm.

[0154] By regulating the concentration of extracellular vesicles, microsphere liquid droplet microsphere capsules with different particle size distributions can be obtained. The higher the concentration of extracellular vesicles, the smaller the particle size of the microsphere liquid droplet microsphere capsule.

[0155] Test Example 3

[0156] The distribution of each fluorescent label in the liquid droplet microsphere capsule prepared in Example 4 was detected by laser scanning confocal microscope.

[0157] The results are shown in Figure 3 .

[0158] The green fluorescent labeled anti-PD-L1 monoclonal antibody is distributed on the surface of the liquid droplet microsphere capsule, and the doxorubicin is inside the liquid droplet microsphere capsule. This shows that the anti-PD-L1 monoclonal antibody is distributed at the oil-water interface due to its amphiphilic characteristics, and the doxorubicin is encapsulated in the oil phase core.

[0159] Test Example 4 (encapsulation efficiency test)

[0160] In the preparation of the liquid droplet microsphere capsule in Examples 4-6, after the reaction was completed and the sample was placed at 4℃ for 10 min, 2 mL of supernatant was taken, and the amount of doxorubicin in the supernatant was measured under ultraviolet spectrometer. The mass of the unloaded doxorubicin was calculated. The encapsulation efficiency of doxorubicin was calculated (encapsulation efficiency (%) = 1 - mass of unloaded doxorubicin / total theoretical doxorubicin input mass).

[0161] The test results are shown in Table 2.

[0162] Table 2

[0163] Group Example 4 Example 5 Example 6 Encapsulation efficiency 78.2±2.67％ 67.7±4.18％ 54.4±5.26％

[0164] The particle sizes of the liquid droplet microsphere capsules of Examples 4, 5, and 6 are similar to those of Examples 1, 2, and 3, respectively.

[0165] Test Example 5 (stability and release test)

[0166] At room temperature, 5 mL of PBS containing 1 mg / mL of the liquid droplet microsphere capsules prepared in Example 8 was placed into 100 mL of artificial gastric fluid (Solarbio, Catalog No. A7920), artificial intestinal fluid (Solarbio, Catalog No. A1790), mucin solution (Mucin purchased from Macklin, Catalog No. M885077, mucin dissolved in PBS at a concentration of 0.8 (w / v)%), and PBS. A control group was maintained without the liquid droplet microsphere capsules. Each solution was shaken on a shaker at 60 rpm. The supernatant was collected at different time points for capecitabine concentration measurement, and then the solution was backfilled with an equal volume of liquid. After 72 hours, the morphological changes of the liquid droplet microsphere capsules were observed using an optical microscope.

[0167] The results are as follows Figure 4 、 Figure 5 shown.

[0168] In a 72-hour release test, the release of capecitabine-loaded droplet microsphere capsules in artificial gastric and intestinal fluids was less than 20%, demonstrating that the droplet microsphere capsules significantly protect the drug, allowing for slow release and avoiding adverse reactions associated with sudden drug release. Even after 72 hours, the droplet microsphere capsules maintained excellent stability and dispersibility in artificial gastric and intestinal fluids, as well as in mucin solutions. The capsules maintained a regular, spherical morphology, exhibited no microsphere rupture, and exhibited no significant change in particle size.

[0169] Test Example 6 (Long-term effect test)

[0170] SD rats (female, 200 g, 9 rats) were randomly divided into three groups. The treatment methods of each group were as follows:

[0171] (1) Capecitabine group: Oral administration of 2 mL of PBS solution containing 20 mg of capecitabine;

[0172] (2) Extracellular vesicle-capecitabine group (EV-CAP): Oral administration of 2 mL of extracellular vesicle-capecitabine solution containing 20 mg of capecitabine; wherein, the synthesis method of extracellular vesicle-capecitabine is as follows: 20 mg of capecitabine is dissolved in 1 mL of PBS solution, the capecitabine solution is placed in an eggplant-shaped bottle, 400 μg of extracellular vesicles prepared in Preparation Example 1 (quantified by protein), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) are added, and the mixture is stirred at room temperature for 10 hours. After the reaction is completed, the mixture is centrifuged twice at 130,000 g for 1 hour in an ultracentrifuge, the supernatant is carefully discarded after centrifugation, and 1 mL of PBS solution is added to fully resuspend the mixture to obtain the extracellular vesicle-capecitabine solution.

[0173] (3) Droplet microspheres capsule group (EVOC) loaded with capecitabine: 2 mL of PBS solution containing 20 mg of capecitabine prepared by the droplet microspheres capsule of Example 8 was orally administered by gavage.

[0174] The rat orbital blood was collected at 0 h, 1 h, 2 h, 3 h, 6 h, 9 h, 12 h, 24 h, 48 h, 72 h, 96 h after gavage administration, and the plasma was separated to detect the concentration of capecitabine in the rat plasma.

[0175] The results are shown in Table 1. Figure 6

[0176] The results show that the drug concentration in the plasma of rats in the capecitabine group and the EV-CAP group reached the peak value at 1 h and 3 h after gavage administration, respectively. With the passage of time, the drug concentration in the plasma decreased rapidly. The drug concentration in the plasma of the EVOC group reached the peak value at 6 h, which greatly delayed the circulation time of the drug in the blood. After calculation, the elimination half-life of the capecitabine group, the EV-CAP group and the EVOC group was 6.74 h, 8.07 h and 15.80 h, respectively. The elimination half-life of the EVOC group was significantly longer than that of the capecitabine group and the EV-CAP group. The area under the curve (AUC) of the EVOC group was 7.93 times that of the capecitabine group and 2.35 times that of the EV-CAP group. This shows that the oral administration of the drug wrapped in the droplet microspheres capsule has a longer blood circulation time, which can significantly improve the bioavailability of oral administration of the drug and is beneficial to the inhibition of tumors.

[0177] Test Example 7 (cytotoxicity test)

[0178] ​N1S1 hepatoma cells were inoculated in 96-well plates at 5000 cells / well and incubated in a cell incubator for about 12 hours until the cells adhered, and then the culture medium in the 96-well plates was removed. The free doxorubicin group was treated with 200 μL of DMEM medium containing 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL and 1 μg / mL of doxorubicin, respectively; the droplet microsphere capsule (loaded with doxorubicin) group was treated with 200 μL of DMEM medium containing the droplet microsphere capsules prepared in Example 5 (so that the final concentration of doxorubicin was 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL and 1 μg / mL, respectively); the droplet microsphere capsule (loaded with doxorubicin + PD-L1 inhibitor) group was treated with 200 μL of DMEM medium containing the droplet microsphere capsules prepared in Example 7 (so that the final concentration of doxorubicin was 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL and 1 μg / mL, respectively), and the cells without any drug were used as a blank control group; the wells without any drug and cells were used as a zero control group. Three replicates were set for each concentration, and the experiment was repeated three times. After the plates were incubated in the cell incubator for 48 hours, the culture medium was replaced with 100 μL of complete culture medium (DMEM medium containing 10% FBS) containing 10% CCK-8, and the incubation was continued for 3 hours. The 96-well plates were taken out of the cell incubator, and the color of the solution in each well was observed. Then the absorbance at 450 nm was measured by a multifunctional enzyme label instrument, and the cell survival rate was calculated (cell survival rate (%) = (absorbance of drug group - absorbance of zero control group) / (absorbance of blank control group - absorbance of zero control group)).

[0179] The results are shown in Table 1. Figure 7

[0180] At the same concentration of doxorubicin, the killing effect of the droplet microsphere capsule carrying doxorubicin on tumor cells was more significant than that of free doxorubicin. This indicates that the droplet microsphere capsule can efficiently deliver small molecule drugs as a carrier.

[0181] Test Example 8 (embolization performance test)

[0182] 1. According to conventional operations in the art, a capillary glass tube model was constructed for simulating the embolization effect in vivo, and the inner diameter of the capillary glass tube was 1 mm. 0.2 mL of the droplet microsphere capsules in Example 7 were slowly injected into the capillary glass tube by a peristaltic pump. The accumulation of the droplet microsphere capsules in the capillary glass tube was observed by an optical microscope.

[0183] The results are shown in Table 2. Figure 8

[0184] ​​The droplet microsphere capsules are tightly packed in the capillary glass tube, and there is no obvious gap between the microspheres. The droplet microsphere capsules are ellipsoidal under the action of the packing pressure. The droplet microsphere capsules of Example 7 maintain a stable spherical shape without external force; and when an external force in different directions is applied, the droplet microsphere capsules can be deformed and will not be broken. Based on this, in the actual embolization of blood vessels, when the diameter of the blood vessel is much larger than the diameter of the droplet microsphere capsule, the droplet microsphere capsule can maintain a spherical shape in the blood vessel and achieve embolization of large-sized blood vessels through dense packing. When the diameter of the blood vessel is smaller than the size of the droplet microsphere capsule, the droplet microsphere capsule can deform and move to the distal end, achieving embolization of the distal end terminal blood vessels.

[0185] 2. Fresh liver was obtained by dissecting the abdomen of euthanized rats. The portal vein was found and ligated at the distal end, a large indwelling needle was inserted, and a suture was used to fix it. After loosening the distal suture, the liver was carefully separated. The fresh liver with the indwelling needle was placed in a 10 cm cell culture dish and frozen in a -80℃ refrigerator for 12 hours to fully destroy the cell structure. After freezing, the liver was taken out and completely thawed at room temperature. After thawing, the indwelling needle was connected to a peristaltic pump, and perfusion treatment began: first, distilled water was perfused for about 30 minutes to flush the blood cells. Then, 4% Triton X-100 solution was perfused for 3 hours to increase the permeability of the liver. Subsequently, a 0.5% SDS solution was used to continuously perfuse at a flow rate of 7 mL / min for 10 hours until the liver gradually became translucent. Then, 1% SDS solution was used to continue perfusion at a flow rate of 5 mL / min for 3 hours until the liver was completely transparent and presented a jelly-like state. Finally, deionized water was used to perfuse at a flow rate of 5 mL / min for 30 minutes to completely remove the residual SDS solution during the decellularization process. The whole perfusion process needs to be continuous to ensure that no air enters, thereby preparing the SD rat liver decellularization model. 0.5 mL of the droplet microsphere capsules of Example 7 were slowly injected into the liver, and the injection was stopped when the microspheres reached the terminal blood vessels under naked eye observation. The actual distribution of the droplet microsphere capsules in the blood vessels was evaluated by inverted microscope, CT, and 3D reconstruction.

[0186] The results are shown in Figure 10 .

[0187] The droplet microsphere capsules filled the entire blood vessel network under the action of the syringe pushing force, including the peripheral blood vessels. The decellularized liver was placed under the CT device for scanning, and the scanning results showed that the droplet microsphere capsules were densely packed inside the blood vessels. Due to its good X-ray opacity, the image results can clearly reflect the blood vessel embolization and the specific distribution position of the embolic agent (droplet microsphere capsules). Observation of the decellularized model by inverted microscope showed that effective embolization was achieved from the main artery to the peripheral artery and the small capillary network.

[0188] During the injection process, the droplet microsphere capsule exhibits significant viscoelastic deformation under external force, and presents various morphologies such as ellipses, triangles or fingers. This deformation capacity enables the droplet microsphere capsule to penetrate into smaller blood vessels of the next level, thereby achieving a deeper embolization effect. This shows that the droplet microsphere capsule of the application has excellent viscoelastic deformation performance.

[0189] Test Example 9 (treatment effect on liver cancer)

[0190] (1) Construction of N1S1 rat liver cancer model:

[0191] Male healthy SD rats (average weight 350-400g) were raised in the SPF level animal room of the experimental animal center, and the diet and mental state of the rats were monitored daily. After a 5-day adaptation period, the rats were ensured to adapt to the environment, and the tumor model construction could be started.

[0192] The N1S1 cells were recovered in advance and cultured for one week. Before tumor inoculation, the logarithmic growth phase N1S1 cells were digested, washed twice with high-pressure sterilized PBS buffer, resuspended, and adjusted to a cell concentration of 5×10 6 / mL, and stored on ice. The tumor inoculation operation was completed as soon as possible within 30 minutes to ensure that the cell viability was in the best state.

[0193] The rats were subjected to water and food deprivation treatment 12 hours before the operation. According to the rat body weight, 0.5 mL of 1% sodium pentobarbital anesthetic was injected into the abdominal cavity at a dose of 100 g. Within about 5 minutes after injection, the rats were observed to gradually fall asleep and their breathing and heart rate were stable, indicating that they were in an anesthetized state. Then the rat abdominal hair was removed using depilatory cream and a hair clipper, and the residual depilatory cream was removed with a wet cotton ball to prevent skin irritation during the open abdominal surgery. The anesthetized rat was placed on a sterile drape, and a heating pad was placed under the sterile sheet to maintain body temperature. The depilation area was disinfected with an alcohol cotton ball, and a sterile scalpel was used to make a surgical incision of about 5 cm along the white line of the abdomen to expose the rat liver lobe. A 29G syringe was used to slowly inject the cell suspension into the left liver lobe, and the needle was removed after the injection was completed. A cotton ball was used to press for 1 minute to prevent the cell suspension from spilling out. Before suturing the wound, an appropriate amount of penicillin was injected into the abdominal cavity to prevent infection. Then, sterile instruments were used for layer-by-layer suturing. After the suturing was completed, erythromycin ointment was applied to the wound to keep it clean and prevent infection. The rat with tumor inoculation was placed on a heating pad for warming, and its state was closely observed. After it completely woke up, it was returned to the animal room for further feeding.

[0194] (2) After 10 days of tumor implantation, 6 tumor-bearing rats were randomly divided into 2 groups. The tumor-bearing rats were fasted for 12 hours the night before surgery, and then anesthetized with 1% sodium pentobarbital by intraperitoneal injection according to the body weight of the rats. After the rats entered the anesthetic state, they were fixed on the operating table in a supine position for skin disinfection. After abdominal white line laparotomy, the liver lobes were pulled up to fully expose the common hepatic artery, proper hepatic artery and gastroduodenal artery. The common hepatic artery was first clamped with an artery clamp, and then the distal gastroduodenal artery was ligated with a suture, leaving a certain length of suture. Then a microcatheter with an inner diameter of 250 μm and an outer diameter of 350 μm was inserted into the proper hepatic artery through the gastroduodenal artery. After the intubation was completed, the rats were placed on the operating table. Under the guidance of DSA, 250 μL of the liquid droplet microsphere capsules prepared in Example 7 (loaded with doxorubicin and PD-L1 inhibitor, containing about 10,000 liquid droplet microsphere capsules) were injected through the microcatheter to target embolize the intrahepatic tumor, and after the injection was completed, the microcatheter was removed, the proximal end of the gastroduodenal artery puncture site was ligated, and then the abdomen was closed with double-layer suture. The control group was treated with the same puncture and injection of the same volume of PBS on the rat liver cancer model. The abdominal MR of the rats was taken before embolization, on the 7th day after embolization and on the 14th day after embolization, and the tumor size was recorded. The rats in the treatment group were sacrificed on the 14th day after the operation, and the liver tumor part was taken for HE staining and compared with the control group of liver cancer rats without treatment.

[0195] The results are shown in Figure 11 , Figure 12 .

[0196] After the rat liver cancer model was treated with the liquid droplet microsphere capsules of Example 7, the liver tumor volume of the rats decreased significantly after 14 days, with a decrease of 95.5%. N1S1 liver cancer is a poorly differentiated hepatocellular carcinoma, with polygonal cancer cells, abundant cytoplasm, large and darkly stained nuclei, and cancer cells arranged in trabecular form. After treatment, the pathological section showed that the liver cancer cells in the treatment group were necrotic, the tumor cell structure was lost, the nuclei were shed, and the staining was homogeneous and powdery. This shows that the liquid droplet microsphere capsules prepared in Example 7 have excellent treatment effect on tumor-bearing rats, and can achieve embolization-chemotherapy-immunotherapy combined treatment.

[0197] Test Example 10 (treatment effect on colon cancer)

[0198] C57BL / 6J mice (female, body weight 18-20 g) were injected with 5 x 10 6MC38-luc cells, to construct a colon tumor-bearing mouse model. After two weeks, the model was successfully constructed, and 15 mice with colon cancer models were randomly divided into three groups. The free capecitabine group of mice was orally gavaged with 100 μL of PBS solution containing 10 mg / mL capecitabine; the droplet microspheres capsule group of mice was orally gavaged with 100 μL of PBS solution containing 10 mg / mL of the droplet microspheres capsule prepared in Example 8; the PBS group of mice was gavaged with an equal amount of PBS as a control group. The first administration time was recorded as day 0, and the mice were administered once a day, and after two weeks of continuous administration, the mice were euthanized, and the expression level of Ki67 and the positive level of CD34 at the tumor site were detected by immunohistochemistry.

[0199] The results are shown in Table 1. Figure 13

[0200] After oral treatment with the droplet microspheres capsule loaded with capecitabine in Example 8, the expression level of Ki67 at the tumor site decreased significantly, and the number of CD34-positive lumens also decreased significantly. This indicates that the droplet microspheres capsule has excellent therapeutic effect on tumor-bearing mice, can efficiently kill colorectal cancer cells, and effectively inhibit the progression and metastasis of colorectal cancer.

[0201] The above embodiments of the present application are described in detail in combination with examples, but the present application is not limited to the above examples, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the purpose of the present application.​

Claims

1. A liquid drop microsphere capsule, characterized in that: The droplet microsphere capsule has a core-shell structure; the surface layer of the core is an oil phase; and the shell includes extracellular vesicles.

2. The liquid drop microsphere capsule according to claim 1, characterized in that The core comprises at least one of an oil phase droplet, a water-in-oil droplet, and an oil-in-water-in-oil droplet; and / or, the oil phase comprises at least one of plant oil, animal oil, mineral oil, plant oil derivatives, animal oil derivatives, and mineral oil derivatives; And / or, the extracellular vesicles include at least one of plant-derived extracellular vesicles, animal-derived extracellular vesicles, microbial-derived extracellular vesicles, and artificially synthesized extracellular vesicles.

3. The liquid drop microsphere capsule according to claim 1, characterized in that The droplet microsphere capsule includes functional components; the functional components include at least one of a drug and a developer.

4. The liquid drop microsphere capsule according to claim 3, characterized in that The drug includes at least one of a compound drug and a protein drug; Preferably, the compound drug includes at least one of a chemotherapy drug, a tyrosine kinase inhibitor, and a hemostatic drug; Preferably, the tyrosine kinase inhibitor comprises at least one of vatalanib, dasatinib, imatinib, N-desmethylimatinib, sorafenib, lapatinib, pazopanib, crizotinib, apatinib, gefitinib, ibrutinib, dasatinib, nilotinib, ruxolitinib, axitinib, osimertinib, erlotinib, ponatinib, sunitinib, and nilotinib; Preferably, the chemotherapy drugs include at least one of platinum drugs, anthracycline drugs, taxane drugs, antipyrimidine drugs, vinblastine drugs, camptothecin drugs, nitrosoureas, and antifolate drugs; Preferably, the protein drug includes at least one of an immune checkpoint inhibitor and a cytokine.

5. The liquid drop microsphere capsule according to claim 1, characterized in that The particle size of the droplet microsphere capsule is 20 μm-1500 μm.

6. The method for preparing the liquid drop microsphere capsule according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Mixing the raw materials for preparing the core to obtain solution A; the raw materials for preparing the core include an oil phase; S2. Prepare a mixture of the solution A and the extracellular vesicle solution, and stir the mixture homogenously to obtain the liquid drop microsphere capsule.

7. The preparation method according to claim 6, characterized in that The solution A is at least one of an oil phase, a water-in-oil emulsion, and an oil-in-water-in-oil emulsion; Preferably, when the solution A is a water-in-oil emulsion, the solution A is prepared by an emulsification method; Preferably, when the solution A is an oil-in-water-in-oil emulsion, the solution A is prepared by a double emulsification method.

8. The preparation method according to claim 7, characterized in that The extracellular vesicle concentration in the extracellular vesicle solution is 0.1 mg / mL-1000 mg / mL; and / or, the volume ratio of the solution A to the extracellular vesicle solution is 1:(1-100); And / or, the rotation speed of the homogenizing stirring is 100 rpm-10000 rpm; And / or, the homogenizing and stirring time is 0.5h-50h; And / or, the temperature of the homogenizing and stirring is 20°C-60°C.

9. A reagent, characterized in that The reagent comprises the liquid drop microsphere capsule according to any one of claims 1 to 5.

10. Use of the liquid drop microsphere capsule according to any one of claims 1 to 5 or the reagent according to claim 9 in any one of A1) to A3): A1) preparing embolic agents; A2) preparing a contrast agent; A3) Preparation of drugs for treating tumors.

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