NK cell artificial extracellular vesicle loaded with small molecule compound as well as preparation method and application of NK cell artificial extracellular vesicle

Abstract

The invention discloses an NK cell artificial extracellular vesicle loaded with a small molecule compound and a preparation method and application of the NK cell artificial extracellular vesicle loaded with the small molecule compound, and the NK cell artificial extracellular vesicle loaded with the small molecule compound is prepared by taking NK cells and the small molecule compound as raw materials and adopting a high-pressure extrusion method and / or an ultrasonic cracking method. The small molecule compound has a chemical structure as shown in a formula I or a formula II: # imgabs0 #; wherein R1 is a saturated alkyl group or an unsaturated alkyl group; r2 is amido substituted by any one or two of alkyl, alkylene, alkyne, oxygen heterocyclic alkyl, alkoxy and hydroxyamido; and R3 and R4 are independently selected from hydrogen or deuterium. The NK cell artificial extracellular vesicle loaded with the small molecule compound can be prepared on a large scale, a remarkable synergistic effect is generated between the small molecule compound and the NK cell artificial extracellular vesicle, and the NK cell artificial extracellular vesicle is expected to be developed into an anti-tumor product, especially an anti-tumor immunotherapy product or a product for enhancing the anti-tumor immunotherapy effect.

Description

Technical Field

[0001] The present invention relates to an artificial extracellular vesicle of NK cells loaded with small molecule compounds and a preparation method and application thereof, belonging to the field of medical technology. Background Art

[0002] Extracellular vesicles (EVs) are membrane vesicles with a phospholipid bilayer structure that are secreted and released by cells into the extracellular matrix. They have the advantages of high physicochemical stability, high biocompatibility, biodegradability, low toxicity, and non-immunogenicity. They can be used as a natural nanocarrier to load drugs and have garnered widespread attention in the field of drug delivery in recent years. Currently, there are reports of using EVs derived from tumor cells or mesenchymal stem cells to deliver chemotherapy drugs (such as doxorubicin) or gene therapy molecules (such as siRNA), which can significantly reduce the biotoxicity of drugs while improving their tumor targeting. However, existing studies have also found that the use of natural EVs as drug carriers still faces many challenges. For example, the yield of natural EVs is very low, and large-scale preparation is difficult, which seriously restricts their industrialization as pharmaceuticals; the drug loading process is cumbersome and the drug loading efficiency is limited; tumor targeting still needs to be further improved; and in addition, it is impossible to effectively regulate anti-tumor immunity.

[0003] Natural killer (NK) cells (NK) account for 15% of circulating lymphocytes in humans and possess the ability to kill tumor cells. Many researchers consider them a promising target for cancer treatment. However, in clinical practice, the time-consuming process of preparing and amplifying fresh NK cells, coupled with the difficulty and high cost of transporting and supplying them over long distances, makes them unable to meet the enormous clinical demand. While ready-to-use NK cells offer a promising alternative to fresh NK cells for clinical application, they utilize fresh blood from healthy volunteers, extracting poly(mucosarcoma cell) (PMBC), and pre-amplifying these cells to produce high-purity NK cells. These cells are then cryopreserved in liquid nitrogen or other low-temperature environments for immediate resuscitation. However, ready-to-use NK cells face significant challenges in clinical application: cryopreservation can significantly damage cell viability. Numerous literature and clinical evidence demonstrate that NK cell function and activity are significantly reduced after cryopreservation and resuscitation, including decreased secretion of cytokines such as TNF-α, diminished proliferation and migration, and reduced cytotoxicity. These factors significantly limit the clinical application of ready-to-use NK cells. Summary of the Invention

[0004] In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide an artificial extracellular vesicle of NK cells loaded with small molecule compounds, which can be prepared on a large scale and can significantly enhance the anti-tumor immune activity of NK cells, as well as a preparation method and application thereof.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0006] An NK cell artificial extracellular vesicle loaded with a small molecule compound is prepared using NK cells and a small molecule compound as raw materials by high-pressure extrusion and / or ultrasonic lysis. The small molecule compound has a chemical structure shown in Formula I or Formula II below:

[0007]

[0008] Wherein: R1 is a saturated hydrocarbon group or an unsaturated hydrocarbon group; R2 is an amino group substituted by any one or two of an alkane group, an alkene group, an alkyne group, an oxacycloalkyl group, an alkoxy group, and a hydroxylamine group; R3 and R4 are independently selected from hydrogen or deuterium.

[0009] In a preferred embodiment, R1 is a C1-C4 alkane group, an allyl group, or a propargyl group, with methyl group or propargyl group being the most preferred.

[0010] In a preferred embodiment, R2 is an amino group substituted by any one or two of methyl, allyl, propargyl, 3-oxetanyl, methoxy, and hydroxylamine.

[0011] A method for preparing NK cell artificial extracellular vesicles loaded with small molecule compounds is a high-pressure extrusion method, which includes: first pressing 1×10 7 1 to 10 μg of the small molecule compound is added to each NK cell to prepare a cell suspension, and then the prepared cell suspension is subjected to a high-pressure extruder. At a pressure of 1 to 4 MPa, gradient filtration is performed on the prepared cell suspension using a 0.2 to 10 μm filter membrane in sequence, and each specification of filter membrane is extruded 3 to 10 times. Finally, the mixed cell suspension after extrusion is collected by gradient centrifugation.

[0012] A preferred solution is to sequentially use filter membranes of 8.0-10 μm, 1.0-5.0 μm, 0.4-0.8 μm, and 0.2-0.3 μm for gradient filter membrane extrusion, and extrude each specification of filter membrane 3-5 times.

[0013] A method for preparing NK cell artificial extracellular vesicles loaded with small molecule compounds is an ultrasonic lysis method, which includes: firstly pressing 1×10 7 1-10 μg of the small molecule compound is added to each NK cell to prepare a cell suspension, and then the prepared cell suspension is lysed using an ultrasonic lyser at 20-30 kHz, 800-1000 W power and 4-8° C. for 5-8 minutes. Finally, the mixed cell suspension after lysis is collected by gradient centrifugation.

[0014] In a preferred embodiment, the above-mentioned gradient centrifugation collection operation includes: first centrifuging at 300-500g for 5-10 minutes to collect a first supernatant; then centrifuging the first supernatant at 2000-3000g for 5-10 minutes to collect a second supernatant; and finally centrifuging the second supernatant at 100,000-120,000g for 70-90 minutes to collect a solid precipitate.

[0015] An application of NK cell artificial extracellular vesicles loaded with small molecule compounds, wherein the application refers to the preparation of an anti-tumor product using the NK cell artificial extracellular vesicles loaded with small molecule compounds as the sole active ingredient or one of the active ingredients.

[0016] In a preferred embodiment, the anti-tumor product is an anti-tumor immunotherapy product or a product for enhancing the effect of anti-tumor immunotherapy.

[0017] In a preferred embodiment, the mass content of the small molecule compound in the NK cell artificial extracellular vesicles loaded with the small molecule compound is 0.01% to 1%.

[0018] In a preferred embodiment, the average particle size of the NK cell artificial extracellular vesicles loaded with small molecule compounds is 50 to 200 nm, and the average Zeta potential value is -8 to -10 mV.

[0019] In a preferred embodiment, the small molecule compound is selected from any one of the following compounds:

[0020]

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The research results of the present invention show that the NK cell artificial extracellular vesicles loaded with small molecule compounds described in the present invention have a particle size similar to that of natural extracellular vesicles and good stability. Not only can they be prepared on a large scale, but also, relative to a single small molecule compound and a single NK cell artificial extracellular vesicle, they have stronger anti-tumor activity, especially anti-tumor immune activity. A significant synergistic effect is produced between the two, and they are expected to be developed into anti-tumor products, especially anti-tumor immunotherapy products or products for enhancing the effect of anti-tumor immunotherapy, with significant medicinal prospects and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the particle size distribution diagram of NK-aEVs (A) and RocA@NK-aEVs (B) prepared in Example 2;

[0024] Figure 2Graphs showing membrane potential detection of NK-aEVs (A) and RocA@NK-aEVs (B) prepared in Example 2;

[0025] Figure 3A 、 Figure 3B and Figure 3C The results show that when the dosage of RocA@NK-aEVs is 25 μg / mL, 50 μg / mL, and 100 μg / mL, the killing effect of RocA@NK-aEVs, single NK-aEVs, and single RocA on H1299-luc tumor cells is 24 h, 48 h, and 72 h, respectively.

[0026] Figure 4A 、 Figure 4B and Figure 4C The results show that when the dosage of RocA@NK-aEVs is 25 μg / mL, 50 μg / mL, and 100 μg / mL, the killing effect of RocA@NK-aEVs, single NK-aEVs, and single RocA on A549-luc tumor cells is 24 h, 48 h, and 72 h, respectively.

[0027] Figure 5A These are photos of tumor changes in mouse subcutaneous tumors in Example 5 on the 18th day after treatment with RocA@NK-aEVs, single NK-aEVs, and single RocA, respectively;

[0028] Figure 5B Figure 5 shows the changes in tumor weight of subcutaneous tumors in mice on day 18 after treatment with RocA@NK-aEVs, single NK-aEVs, and single RocA, respectively;

[0029] Figure 5C Figure 5 shows the changes in tumor volume of subcutaneous tumors in mice after 18 days of treatment with RocA@NK-aEVs, single NK-aEVs, and single RocA, respectively;

[0030] Figure 6A 、 Figure 6B and Figure 6C The effects of RocA@NK-aEVs, single NK-aEVs and single RocA on the levels of NK cells, CD8 + T cell levels and CD4 + Effects on T cell levels;

[0031] Figure 7 This demonstrates the toxic and side effects of RocA@NK-aEVs, single NK-aEVs, and single RocA on various organs of tumor-bearing mice investigated in Example 7;

[0032] Figure 8A 、 Figure 8B and Figure 8C These data respectively reflect the killing effects of each treatment group in Example 8 on tumor cells after 24 hours, 48 ​​hours and 72 hours of treatment. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0034] Example 1: Preparation of Compound 1-9

[0035] 1. Prepare compounds 1-5 according to the following synthetic route

[0036]

[0037] The specific preparation steps are as follows:

[0038] 1) Preparation of intermediate a-1

[0039] Weigh 200 mg (0.61 mmol) of 4′,5,7-trimethoxyflavonol (CAS No.: 93876-55-2, homemade, preparation reference: Wang Chun, Zhang Guolin, Xiong Wei. A one-pot aqueous synthesis method for 3-hydroxyflavone and its derivatives: CN109320488B[P]. 2020-08-18) and 494 mg (3.05 mmol) of methyl cinnamate (CAS No.: 103-26-4, purchased from Shaoyuan Technology (Shanghai) Co., Ltd.) in a quartz tube, add 20 mL of a mixture of anhydrous chloroform and trifluoroethanol (CAS No.: 75-89-8) The mixture was mixed (volume ratio of 7:3) and degassed with argon for 15 minutes. The reaction solution was placed in a -20°C refrigerator and the mixture was stirred under UV lamp for reaction. The reaction solution was monitored by LC-MS. After 33 hours of reaction, the solvent was evaporated under reduced pressure to obtain a yellow-brown oily solid. 10 mL of methanol solution was added, and 1.7 mL (0.50 mmol) of 0.3 M sodium methoxide methanol solution was added to the solution. The reaction solution was reacted at 60°C and monitored by LC-MS. After 45 minutes of reaction, the methanol solution was evaporated under reduced pressure. The crude reaction mixture was quenched with saturated sodium bicarbonate solution. 8 mL of saturated ammonium chloride solution and 4 mL of 1 M hydrochloric acid solution were added. The turbid reaction solution was extracted with ethyl acetate (6 mL × 2). The organic phase was washed with 10 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain a white solid, which was intermediate a-1, with a yield of 46.5%.

[0040] 1H NMR (400MHz, CDCl3) δ7.12-7.09(m,3H),6.97-6.90(m,4H),6.73-6.64(m,2H),6.35(d,J=2.0Hz,1H),6.11(d,J= 2.0Hz,1H),4.24(d,J=13.3Hz,1H),4.06(d,J=13.3Hz,1H),3.86(s,3H),3.82(s,3H),3.72(s,3H),3.66(s,3H). 13 C NMR (125MHz, CDCl3): δ203.3,167.2,164.9,161.0,158.9,158.6,135.4,129.1,128.0,127.9,127.7,12 7.1,125.4,113.2,112.2,106.1,99.3,92.9,89.9,88.5,56.4,55.7,55.6,55.1,55.0,52.9,52.0,51.5.

[0041] 2) Preparation of intermediate a-2

[0042] 100 mg (0.24 mmol) of compound a-2 was weighed into a reaction flask, 3 mL of acetonitrile was added, and 140 μL (2.4 mmol) of acetic acid and 380 mg (1.4 mmol) of (Me4N)BH(OAc)3 (CAS No.: s56553-60-7, purchased from Shaoyuan Technology (Shanghai) Co., Ltd.) were added in sequence at 0°C. The reaction solution was stirred at room temperature and monitored by LC-MS. After 20 h of reaction, the reaction mixture was quenched with saturated sodium bicarbonate solution, 8 mL of saturated ammonium chloride solution was added, and the reaction solution was extracted with ethyl acetate (15 mL×3). The organic phase was washed with 15 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain a white solid, which was intermediate a-2, with a yield of 70%.

[0043] 1 H NMR (400MHz, CDCl3): δ7.13–7.02(m,5H),6.86(dd,J=6.8,2.9Hz,2H),6.73–6.62(m,2H),6.27(d,J=1.9Hz,1H),6.11(d,J=1.9Hz ,1H),5.02(dd,J=6.6,1.5Hz,1H),4.30(d,J=14.2Hz,1H),3.92–3.87(m,1H),3.86(s,3H),3.81(s,3H),3.69(s,3H),3.64(s,3H). 13C NMR (125MHz, CDCl3): δ169.7,164.1,161.2,158.7,157.4,137.8,129.0×2,127.9×2,127.8×2,1 27.3,126.4,112.9×2,107.0,101.8,94.2,92.7,89.4,78.7,56.1,55.8,55.2,47.8,37.1,35.9.

[0044] 3) Preparation of intermediate a-3

[0045] 50 mg (0.1 mmol) of compound a-2 was dissolved in 4 mL of methanol, and 22 mg (0.39 mmol) of potassium hydroxide was added. The reaction mixture was heated at 44°C and monitored by LC-MS. After 8 h of reaction, the reaction mixture was quenched with 2 mL of 1 M hydrochloric acid solution and extracted with ethyl acetate (6 mL × 3). The organic phase was washed with 10 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain intermediate a-3 as a white solid with a yield of 96.5%.

[0046] 1 H NMR (400MHz, CDCl3): δ7.13-7.03(m,5H),6.92-6.85(m,2H),6.68(d,J=9.0Hz,2H),6.28(d,J=2.0Hz,1H),6.13(d,J=2.0H z,1H),5.06(d,J=6.6Hz,1H),4.26(d,J=14.0Hz,1H),3.90(dd,J=14.0,6.6Hz,1H),3.88(s,3H),3.84(s,3H),3.71(s,3H). 13 C NMR (125MHz, CDCl3): δ172.1,164.2,160.9,158.8,157.0,136.5,129.0×2,127.9×2,127.8×2 ,126.7,126.23,112.8×2,107.4,101.8,93.6,92.7,89.5,79.4,55.8,55.7,55.2,55.1,49.9.

[0047] 4) Preparation of Compound 1

[0048] 10 mg (0.02 mmol) of compound a-3 was weighed into a reaction bottle and dissolved in 2 mL of DMF. 2.9 mg (0.03 mmol) of N, O-dimethylhydroxylamine hydrochloride (CAS No.: 6638-79-5, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) and 3.7 mg (0.03 mmol) of DMAP (CAS No.: 1122-58-3) were added to the solution in sequence. The reaction mixture was cooled to 0 ° C. 4.9 mg (0.03 mmol) of EDCI (CAS No.: 7084-11-9) was added in batches and stirred at 0 ° C for 30 min. 5 μL (0.04 mmol) of triethylamine (CAS No.: 121-44-8) was added to the reaction solution. After the reaction mixture was stirred at 0 ° C for 1 h, the reaction mixture was stirred at room temperature and the reaction was monitored by LC-MS. After the reaction was allowed to react for 10 h, the reaction mixture was washed with 0.5 mL of ethanol and the resulting mixture was lysed. The reaction mixture was quenched with 1 M hydrochloric acid solution and extracted with dichloromethane (6 mL × 2). The organic phase was washed with 5 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain compound 1 as a white solid with a yield of 75%.

[0049] 1 H NMR (400MHz, CDCl3): δ7.17-7.09(m,2H),7.07-7.00(m,3H),6.93-6.86(m,2H),6.74-6.67(m,2H),6.29(d,J=1.9Hz,1H),6.11(d,J=1.9Hz,1H) ,5.04(d,J=6.6Hz,1H),4.45(d,J=14.0Hz,1H),4.24(dd,J=13.9,6.6Hz ,1H),3.92(s,3H),3.86(s,3H),3.83(s,3H),3.71(s,3H),3.18(s,3H). 13 C NMR (125MHz, CDCl3): δ164.1,161.1,158.8,157.3,137.6,129.1×2,128.0×2,127.9×2,127.1,126 .5,112.9×2,108.0,102.0,94.2,92.8,89.6,79.5,77.4,77.2,76.9,62.1,55.8,55.3,47.4,32.6.

[0050] 5) Preparation of Compound 2

[0051] 10 mg (0.02 mmol) of compound a-3 was weighed into a reaction flask and dissolved in 2 mL of DMF. 2.6 mg (0.03 mmol) of dimethylamine hydrochloride (CAS No. 506-59-2, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) and 3.7 mg (0.03 mmol) of DMAP were added to the solution in sequence. The reaction mixture was cooled to 0°C and 4.9 mg (0.03 mmol) of EDCI was added portionwise. The mixture was stirred at 0°C for 30 min. 5 μL (0.04 mmol) of triethylamine was added to the reaction solution. The reaction mixture was stirred at 0°C for 1 h, then warmed to room temperature and stirred. The reaction was monitored by LC-MS. After 10 h of reaction, the reaction mixture was quenched with 0.5 mL of 1 M hydrochloric acid and extracted with dichloromethane (6 mL × 2). The organic phase was washed with 5 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The crude product was separated and purified by silica gel column chromatography to obtain compound 2 as a white solid with a yield of 75%.

[0052] 1 H NMR (400MHz, CDCl3) δ7.10(m,2H),7.02(m,3H),6.86(m,2H),6.68(m,2H),6.28(d,J=2.0Hz,1H),6.10(d,J=2.0Hz,1H),4.93(d,J= 6.4Hz,1H),4.55(d,J=13.5Hz,1H),4.05(dd,J=13.5,6.5Hz,1H),3.86(s,3H),3.83(s,3H),3.71(s,3H),3.31(s,3H),2.94(s,3H). 13 C NMR (125MHz, CDCl3) δ169.7,164.1,161.2,158.7,157.4,137.8,129.0,129.0,127.9,127.8,127.2, 126.4,112.9,112.9,107.7,101.8,94.2,92.6,89.4,78.7,56.1,55.8,55.2,53.9,37.1,35.9,31.0.

[0053] 6) Preparation of Compound 3

[0054] 10 mg (0.02 mmol) of compound a-3 was weighed into a reaction flask and dissolved in 2 mL of DMF. 2.2 mg (0.03 mmol) of acetamide oxime (CAS No.: 22059-22-9, purchased from Shaoyuan Technology (Shanghai) Co., Ltd.) and 3.7 mg (0.03 mmol) of DMAP were added to the solution in sequence. The reaction mixture was cooled to 0°C and 4.9 mg (0.03 mmol) of EDCI was added in batches. The mixture was stirred at 0°C for 30 min. 5 μL (0.04 mmol) of triethylamine was added to the reaction solution. After the reaction mixture was stirred at 0°C for 1 h, the reaction mixture was stirred at room temperature and monitored by LC-MS. After 12 h of reaction, the reaction mixture was quenched with 0.5 mL of 1 M hydrochloric acid solution and extracted with dichloromethane (6 mL × 2). The organic phase was washed with 5 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain compound 3 as a white solid with a yield of 61%.

[0055] 1 H NMR (400MHz, CDCl3): δ7.15-7.10(m,2H),7.08-7.02(m,3H),6.94-6.88(m, 2H),6.71-6.65(m,2H),6.28(d,J=1.9Hz,1H),6.12(d,J=2.0Hz,1H),5.12( dd,J=6.4,1.5Hz,1H),4.34(d,J=14.3Hz,1H),4.02(dd,J=14.2,6.3Hz,1H) ,3.87(s,3H),3.83(s,3H),3.78(d,J=1.4Hz,1H),3.71(s,3H),1.83(s,3H). 13 C NMR (125MHz, CDCl3): δ167.8,164.25,161.0,159.0,157.2,157.0,137.2,129.1×2,128.1×2,128.0× 2,126.8,126.4,112.9×2,107.9,101.9,93.6,92.8,89.6,79.9,56.0,55.9,55.3,54.9,50.5,16.9.

[0056] 7) Preparation of Compound 4

[0057] 10 mg (0.02 mmol) of compound a-3 was weighed into a reaction flask, 2 mL of DMF was added to dissolve it, 2.2 mg (0.03 mmol) of 3-aminooxetane (CAS No.: 21635-88-1, purchased from Shaoyuan Technology (Shanghai) Co., Ltd.) and 3.7 mg (0.03 mmol) of DMAP were added to the solution in sequence, the reaction mixture was cooled to 0 ° C, 4.9 mg (0.03 mmol) of EDCI was added in batches, and stirred at 0 ° C for 30 min. 5 μL (0.04 mmol) of triethylamine was added to the reaction solution. After the reaction mixture was stirred at 0 ° C for 1 h, the reaction mixture was stirred at room temperature and the reaction was monitored by LC-MS. After the reaction was allowed to react for 12 h, the reaction mixture was washed with 0.5 mL of EDCI. The reaction mixture was quenched with 1 M hydrochloric acid solution and extracted with dichloromethane (6 mL × 2). The organic phase was washed with 5 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain compound 4 as a white solid with a yield of 69%.

[0058] 1 H NMR (400MHz, CDCl3): δ7.17-7.11(m,2H),7.08(dd,J=5.2,1.9Hz,3H),6.96(dd,J=6.8,2.8Hz,2H),6.71- 6.63(m,2H),6.51(d,J=7.4Hz,1H),6.29(d,J=1.9Hz,1H),6.14(d,J=1.9Hz,1H),4.95(d,J=5.7Hz,1H),4 .88(dq,J=7.3,6.2Hz,1H),4.78(t,J=7.0Hz,1H),4.72(t,J=7.0Hz,1H),4.30(t,J=6.3Hz,1H),4.21(d,J =14.1Hz,1H),4.13(t,J=6.4Hz,1H),3.88(s,3H),3.84(s,3H),3.76(dd,J=14.0,5.7Hz,1H),3.70(s,3H). 13 CNMR (125MHz, CDCl3): δ170.1,164.3,161.1,156.0,157.3,136.3,129.2×2,128.4×2,128.1×2,127.2, 126.5,112.9×2,107.3,101.8,93.7,92.7,89.4,79.2,78.6,78.4,56.4,56.0,55.9,55.2,51.8,44.9.

[0059] 8) Preparation of Compound 5

[0060] 10 mg (0.02 mmol) of compound a-3 was weighed into a reaction flask and dissolved in 2 mL of DMF. 2.0 μL (0.03 mmol) of propargylamine (CAS No.: 2450-71-7, purchased from Shaoyuan Technology (Shanghai) Co., Ltd.) and 3.7 mg (0.03 mmol) of DMAP were added to the solution in sequence. The reaction mixture was cooled to 0°C and 4.9 mg (0.03 mmol) of EDCI was added in batches. The mixture was stirred at 0°C for 30 min. 5 μL (0.04 mmol) of triethylamine was added to the reaction solution. After the reaction mixture was stirred at 0°C for 1 h, the reaction mixture was stirred at room temperature and monitored by LC-MS. After 12 h of reaction, the reaction mixture was quenched with 0.5 mL of 1 M hydrochloric acid solution and extracted with dichloromethane (6 mL × 2). The organic phase was washed with 5 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain compound 5 as a white solid with a yield of 81%.

[0061] 1 H NMR (400MHz, CDCl3): δ7.16-7.05(m,5H),6.98(dd,J=7.5,2.0Hz,2H),6.68-6.62(m,2H ),6.28(d,J=2.0Hz,1H),6.12(d,J=1.9Hz,1H),4.94(dd,J=5.6,1.9Hz,1H),4.25(d,J=1 4.1Hz,1H),3.99(ddd,J=17.6,5.3,2.6Hz,1H),3.87(s,3H),3.84(s,3H),3.79(dd,J=1 4.1,5.6Hz,1H),3.70(s,3H),3.54(d,J=1.0Hz,1H),2.11(t,J=2.6Hz,1H),1.91(s,1H). 13 CNMR (125MHz, CDCl3): δ170.1,164.3,161.3,158.9,157.4,136.4,129.2×2,128.4×2,128.0×2,127.0, 126.6,112.8×2,107.2,101.9,93.6,92.6,89.3,79.4,79.1,77.4,56.3,55.9,55.8,55.3,51.9,29.4.

[0062] 2. Prepare compound 6 according to the following synthetic route

[0063]

[0064] Weigh 200 mg (0.57 mmol) of flavonol a-4 (self-made, reference: Wang Chun, Zhang Guolin, Xiong Wei. A one-pot aqueous synthesis method of 3-hydroxyflavone and its derivatives: CN109320488B[P]. 2020-08-18) and 299 mg (1.71 mmol) of cinnamic acid dimethylamide (CAS No.: 77302-27-3, purchased from RR A quartz tube (produced by Sigma-Aldrich Scientific Company) was added with 30 mL of trifluoroethanol, the air in the system was replaced with argon, and the quartz tube was placed under a UV lamp (wavelength, 370 nm) and stirred at room temperature for reaction, monitored by LC-MS. After 48 h of reaction, the reaction solution was concentrated under reduced pressure, then dissolved in 20 mL of methanol, and 30.78 mg (0.57 mmol) of solid sodium methoxide (CAS No.: 124-41-4) was added to the solution. The reaction was stirred at 60°C for reaction, monitored by LC-MS. After 45 min of reaction, the methanol was evaporated under reduced pressure, and dilute hydrochloric acid was added dropwise to adjust the pH to neutral. The reaction solution was extracted with dichloromethane (6 mL × 3), and the retained organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated and purified by C-18 column chromatography to obtain intermediate a-5 in a 40% yield.

[0065] 120 mg (0.228 mmol) of intermediate a-5 was weighed into a 100 mL reaction flask, 15 mL of acetonitrile was added, 134 μL (2.3 mmol) of acetic acid and 624 mg (2.3 mmol) of (Me4N)BH(OAc)3 were added in sequence, and the reaction was stirred at room temperature and monitored by LC-MS. After 10 h of reaction, the reaction was terminated, the acetonitrile was evaporated under reduced pressure, the pH was adjusted to neutral with saturated sodium bicarbonate solution, and the mixture was extracted with dichloromethane (15 mL × 3). The organic phase was retained and washed with 15 mL of saturated saline solution, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated and purified by HPLC to obtain compound 6 as a white amorphous powder with a yield of 41.1%.

[0066] 1H NMR (400MHz, CDCl3) δ7.15(m,2H),7.08(m,3H),6.99(m,2H),6.66(m,2H),6.35(d,J=2.0Hz,1H),6.20(d,J =2.0Hz,1H),5.85(t,J=5.7Hz,1H),5.63(ddt,J=17.2,10.5,5.3Hz,1H),4.95(d,J=5.5Hz,1H),4.92(dq,J =10.4,1.5Hz,1H),4.86(dq,J=17.3,1.7Hz,1H),4.72(d,J=2.4Hz,2H),4.24(d,J=14.2Hz,1H),3.87(s,3H ),3.80(dd,J=14.2,5.5Hz,1H),3.77(m,2H),3.70(s,3H),3.59(s,1H),2.58(t,J=2.4Hz,1H),1.91(s,1H). 13 C NMR (126MHz, CDCl3) δ170.1,162.0,161.0,158.9,157.5,136.4,133.9,129.2×2,128.5×2,128.1×2,127.1,1 26.6,115.9,112.8×2,108.3,102.1,93.6,93.3,90.4,79.3,78.3,76.1,56.3,56.3,56.0,55.3,52.2,41.8.

[0067] 3. Prepare compounds 7-9 according to the following synthetic route

[0068]

[0069] (1) Preparation of Compound 7

[0070] 3.52 g (10.71 mmol) of 4′,5,7-trimethoxyflavonol (CAS No.: 93876-55-2) and 5.63 g (32.12 mmol) of cinnamic acid dimethylamide (CAS No.: 17431-39-9, purchased from Sigma-Aldrich) were weighed into a quartz tube, 200 mL of trifluoroethanol was added, and the air in the system was replaced with argon. The quartz tube was placed under an ultraviolet lamp (wavelength of 370 nm) and stirred at room temperature for reaction, monitored by LC-MS. After 48 h of reaction, the reaction solution was purified by HPLC. The mixture was concentrated under reduced pressure, and then 20 mL of methanol was added to dissolve it. 24.84 mg (0.46 mmol) of solid sodium methoxide was added to the solution, and the mixture was stirred under reflux at 60°C and monitored by LC-MS. After 45 min of reaction, the methanol was evaporated under reduced pressure, and dilute hydrochloric acid was added dropwise to adjust the pH to neutral. The reaction solution was extracted with dichloromethane (10 mL × 3), and the retained organic phase was washed with 8 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated and purified by C18 column chromatography to obtain white needle-shaped crystals as intermediate a-6 in a yield of 50%.

[0071] 120 mg (0.238 mmol) of intermediate a-6 was weighed into a 100 mL reaction flask, 15 mL of anhydrous acetonitrile was added, 134 μL (2.3 mmol) of acetic acid and 627 mg (2.38 mmol) of (Me4N)BH(OAc)3 were added in sequence, and the reaction was stirred at room temperature and monitored by LC-MS. After 10 h of reaction, the reaction was terminated, the acetonitrile was evaporated under reduced pressure, the pH was adjusted to neutral with saturated sodium bicarbonate solution, and the mixture was extracted with dichloromethane (15 mL × 3). The organic phase was retained and washed with 15 mL of saturated saline solution, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was separated and purified by diol column to obtain compound 2 with a yield of 95%.

[0072] 10 mg of compound 2 was placed in a 10 mL dry reaction flask, the air was replaced with argon, and then anhydrous THF was added to dissolve it. After stirring on an ice bath for 10 minutes, 50 μL of lithium aluminum tetrahydride tetrahydrofuran solution was carefully added to the reaction flask. The mixture was stirred on an ice bath overnight for 16 hours. After the reaction was completed, the reaction solution was diluted with ether, the aluminum solid was removed by filtration, and the mixture was washed once with saturated NaHCO3. The mixture was extracted with ether, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by HPLC preparative column to obtain compound 7 as a white powder with a yield of 57%.

[0073] 1H NMR (400MHz, CDCl3) δ7.21-7.15(m,2H),7.12-7.07(m,3H),6.84(dd,J=6.6,3.0Hz,2H ),6.69(d,J=8.5Hz,2H),6.23(d,J=2.0Hz,1H),6.13(d,J=2.0Hz,1H),5.35(t,J=4.9H z,2H),4.97(d,J=5.0Hz,1H),3.90(s,3H),3.82(s,3H),3.72(s,3H),3.62(d,J=13.1H z,1H),3.29(q,J=12.0Hz,2H),2.70(s,6H),2.52(d,J=10.9Hz,1H),2.04-1.98(m,3H).

[0074] (2) Preparation of Compound 8

[0075] Take 200 mg of intermediate a-6, dissolve it in 1 mL of deuterated acetone (CAS number: 666-52-4), add 4 mL of heavy water, react at 50°C for 8 hours, spin dry the deuterated acetone and heavy water, repeat the reaction three times, and detect by LC-MS that the reaction is complete. The sample is allowed to stand in deuterated acetone. The precipitated white needle-shaped crystals are intermediate a-7, with a yield of 80%.

[0076] 60 mg of intermediate a-7 was dissolved in 8 mL of anhydrous acetonitrile, and 63 μL of deuterated acetic acid (10 eq, CAS number: 758-12-3) and 50 mg of sodium deuterated borohydride (CAS number: 15681-89-7) were added sequentially. The reaction was carried out at room temperature for 8 h. After the reaction was completed, the mixture was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and purified by diol column to obtain compound 8 as a white powder with a yield of 85%.

[0077] 1 H NMR (600MHz, CDCl3) δ7.15-7.08(m,2H),7.03(dt,J=12.1,6.7Hz,3H),6.86(d,J=7.3Hz,2H),6.68(d,J=8.6Hz,2H),6.28(d,J= 1.9Hz,1H),6.11(d,J=1.9Hz,1H),5.30(s,1H),4.55(s,1H),3.86(s,3H),3.83(s,3H),3.71(s,3H),3.31(s,3H),2.94(s,3H).

[0078] (3) Preparation of Compound 9

[0079] 60 mg of intermediate a-7 was dissolved in 8 mL of anhydrous acetonitrile, and 63 μL of deuterated acetic acid (10 eq) and 50 mg of sodium borohydride (CAS No.: 16940-66-2) were added sequentially. The reaction was allowed to proceed at room temperature for 8 h. After the reaction was completed, the mixture was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and purified on a diol column to obtain compound 9 as a white powder with a yield of 85%.

[0080] 1 H NMR (600MHz, CDCl3) δ7.15-7.08(m,2H),7.03(dt,J=12.1,6.6Hz,3H),6.86(d,J=7.3Hz,2H),6.71-6.66(m,2H),6.28(d,J=1.9Hz,1 H), 6.10 (d, J = 2.0Hz, 1H), 5.29 (s, 1H), 4.92 (s, 1H), 4.55 (s, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.70 (s, 3H), 3.31 (s, 3H), 2.94 (s, 3H).

[0081] The chiral separation method of the enantiomers of the obtained compound 1-9 is: 70% n-hexane / 30% isopropanol, flow rate 10 mL / min, detection wavelength 214 nm, chiral column model: UniChiral CNZ-5H (5 μm, 10×250 mm).

[0082] Example 2: Preparation of RocA@NK-aEVs and blank control NK-aEVs

[0083] After 14 days of expansion of PBMC from healthy individuals, NK cells with a purity of >85% were obtained. The cells were collected and washed 2-3 times with PBS, and then reconstituted with PBS to a concentration of 1×10 6 1×10 cells / mL of cell suspension, and then 7 1 μg or 10 μg of compound 2 (abbreviated as RocA) was added to each NK cell to prepare a cell suspension;

[0084] Each cell suspension was extruded using a high-pressure extruder under 1-4 MPa nitrogen pressure using gradient membrane extrusion using 8.0 μm, 1.0 μm, 0.4 μm, and 0.2 μm filters, with each membrane size extruded three times. Alternatively, cells were lysed using an ultrasonic lyser at 20 kHz, 800 W, and 4°C for 5 min (0.5 s on, 0.5 s off).

[0085] The mixed cell suspension prepared above was first centrifuged at 300g for 10 minutes to collect the first supernatant, then the collected first supernatant was centrifuged at 2000g for 10 minutes to collect the second supernatant, and finally the collected second supernatant was centrifuged at 100,000g for 90 minutes to collect the solid precipitate. The collected solid precipitate is the NK cell artificial extracellular vesicles loaded with compound 2 (abbreviated as RocA), abbreviated as: RocA@NK-aEVs.

[0086] The preparation of blank control (NK cell artificial extracellular vesicles without small molecule compounds, abbreviated as: NK-aEVs) was the same as the preparation of RocA@NK-aEVs described above, with the only difference being that no small molecule compounds were added to the prepared cell suspension, and only 1×10 7 cells / mL NK cell suspension.

[0087] Equal amounts of the prepared NK-aEVs and RocA@NK-aEVs were lysed using RIPA lysis buffer (containing 1% PMSF) and centrifuged at 13,000 rpm for 5 minutes. The supernatant was collected. An equal amount of acetonitrile was added to the supernatant, vortexed, and centrifuged at 13,000 rpm for 5 minutes. The supernatant was collected for analysis. The NK-aEVs group served as a blank control, and the RocA@NK-aEVs group served as the test sample.

[0088] Weigh 5.52 mg of the standard compound 2 and add methanol to a 50 mL volumetric flask. Sonicate and vortex to mix well for analysis. Set the injection volume of the standard solution to 0.1, 0.2, 0.5, 1, and 2 μL using a liquid chromatography-mass spectrometer. Measure the peak area according to the chromatographic conditions and plot a standard curve with injection volume (μL) as the horizontal axis and peak area as the vertical axis.

[0089] The chromatographic conditions are:

[0090] Phase A: 0.02% formic acid aqueous solution (pH ≈ 2.5); Phase B: acetonitrile; Flow rate setting: 0.5 mL / min, column temperature: 40°C, detection wavelength: 200 nm; The injection time and ratio of phase A and phase B are as follows:

[0091] Time (minutes) A％ B％ 0 70 30 10 20 80 11 0 100 13 0 100 13.5 70 30 15 70 30

[0092] Inject 20 μL of blank control sample and test sample solution respectively, substitute the respective test data into the standard curve to obtain the corresponding injection volume, and subtract the injection volume of blank control sample obtained from the injection volume of test sample to calculate the actual content of the entrapped compound.

[0093] The content of compound 2 in the sample under test was measured at 200 nm according to the external standard method as follows:

[0094] (1) 1×10 7 Detection of RocA content in RocA@NK-aEVs prepared with a ratio of 1 μg NK cells + 1 μg RocA:

[0095] Blank control sample: RT: 5.2405.2240.034μL Ch1 200nm;

[0096] Sample 1: RT: 5.240, 5.156, 0.041 μL, Ch1 200 nm.

[0097] Standard sample concentration: 5.52 mg / 50 mL = 110.4 μg / mL, actual injection volume: 20 μL, detection injection volume: 0.041 μL - 0.034 μL = 0.007 μL.

[0098] According to the formula: (test injection volume / actual injection volume)*standard sample concentration=test sample concentration, the test sample concentration can be calculated as 0.03864 μg / mL, that is, the final calculation is: every 100 μg RocA@NK-aEVs contains 0.016 μg RocA.

[0099] (2) 1×10 7 Detection of RocA content in RocA@NK-aEVs prepared with a ratio of 10 μg NK cells + 10 μg RocA:

[0100] Blank control sample: RT: 5.2405.2240.013μL Ch1 200nm;

[0101] Sample 1: RT: 5.240, 5.156, 0.146 μL, Ch1 200 nm.

[0102] Standard sample concentration: 5.52 mg / 50 mL = 110.4 μg / mL, actual injection volume: 10 μL, detection injection volume: 0.146 μL - 0.013 μL = 0.033 μL.

[0103] According to the formula: (test injection volume / actual injection volume)*standard sample concentration=test sample concentration, the test sample concentration can be calculated as 1.46832 μg / mL, that is, the final calculation is: every 100 μg RocA@NK-aEVs contains 0.12 μg RocA.

[0104] It can be seen from this that increasing the compound content in the prepared cell suspension can proportionally increase the drug loading capacity of NK-aEVs, indicating that the preparation method of NK-aEVs-loaded drugs is feasible.

[0105] The NK-aEVs and RocA@NK-aEVs prepared in this example were analyzed for particle size using a NanoCoμLter counter. The results are shown in Figure 2. Figure 1 As shown. Figure 1 As shown in the figure, the average particle size of NK-aEVs is about 80 nm, and the particle size is very uniform; the particle size of RocA@NK-aEVs is similar to that of NK-aEVs, with an average particle size of about 81 nm; this shows that the RocA@NK-aEVs prepared by the extrusion method or ultrasonic lysis method in the present invention have similar particle sizes to NK-aEVs, with an average value of less than 200 nm, which is similar to the particle size level characteristics of natural exosomes.

[0106] The stability of NK-aEVs and RocA@NK-aEVs prepared in this example was analyzed using a NanoCoμLter counter instrument. The results are shown in Figure 2. Figure 2 As shown. Figure 2 As shown in the figure, the Zeta potential value of NK-aEVs is around -9.21 mV, and the Zeta potential value of RocA@NK-aEVs is around -8.24 mV. This shows that the RocA@NK-aEVs prepared by the extrusion method or ultrasonic lysis method in the present invention have similar Zeta potential levels as NK-aEVs, with an average potential of around -8 to -10 mV. The absolute value of the Zeta potential reflects the stability of the nanoparticles as drug carriers. Generally, an average potential between -20 and -5 mV is considered to have good stability.

[0107] Example 3: Investigating the killing effect of RocA@NK-aEVs on H1299-luc tumor cells

[0108] The bioluminescence method was used to detect the killing effect of RocA@NK-aEVs (0.016μg@100μg) on ​​H1299-luc tumor cells expressing Luciferase fluorescence.

[0109] 3×10 NK cells were obtained after 14 days of expansion. 3 3×10 H1299-luc cells / well in logarithmic growth phase 3 100 cells / well were plated simultaneously in a 96-well plate at a 1:1 effector-target ratio, and then different concentrations of NK-aEVs, RocA@NK-aEVs, and RocA were added and co-incubated in a 37°C incubator. After 24 hours of co-incubation, substrate was added for color development, and the fluorescence intensity was measured using a microplate reader. The fluorescence intensity differences between the groups were compared, and the killing efficiency was calculated. The results are shown in Figure 2. Figure 3A 、 3B and 3C.

[0110] Figure 3A 、 3B The killing effects of NK-aEVs, RocA@NK-aEVs and equal concentrations of RocA on H1299-luc tumor cells at doses of 25 μg / mL, 50 μg / mL and 100 μg / mL, respectively, were observed after 24 h, 48 h and 72 h. Figure 3A It can be seen that at a dose of 25μg / mL, at 24h, the level of RocA@NK-aEVs killing H1299-luc cells was 20% higher than that of the Ctrl group (control group), about 5% higher than the NK-aEVs group alone, and nearly 14% higher than the RocA group alone; at 48h, it was 50% higher than the Ctrl group, about 35% higher than the NK-aEVs group alone, and nearly 17% higher than the RocA group alone; at 72h, it was 73% higher than the Ctrl group, about 30% higher than the NK-aEVs group alone, and about 20% higher than the RocA group alone. Figure 3B It can be seen that at a dose of 50 μg / mL, at 24 hours, the level of RocA@NK-aEVs killing H1299-luc cells was 30% higher than that of the Ctrl group (control group), about 15% higher than the NK-aEVs group alone, and about 20% higher than the RocA group alone; at 48 hours, it was 60% higher than the Ctrl group, about 45% higher than the NK-aEVs group alone, and about 46% higher than the RocA group alone; at 72 hours, it was 77% higher than the Ctrl group, about 30% higher than the NK-aEVs group alone, and about 7% higher than the RocA group alone. Figure 3C As shown in Table 1, at a dose of 100 μg / mL, RocA@NK-aEVs demonstrated a 60% higher level of killing H1299-luc cells than the Ctrl group (control group), approximately 40% higher than the NK-aEVs group alone, and approximately 36% higher than the RocA group alone at 24 hours. At 48 hours, the level of killing H1299-luc cells by RocA@NK-aEVs was 76% higher than the Ctrl group, approximately 53% higher than the NK-aEVs group alone, and approximately 30% higher than the RocA group alone. At 72 hours, the level of killing H1299-luc cells by RocA@NK-aEVs was 86% higher than the Ctrl group, approximately 25% higher than the NK-aEVs group alone, and approximately 10% higher than the RocA group alone. Specific test data are shown in Table 1.

[0111] Table 1 Killing effect of each group on H1299-luc tumor cells

[0112]

[0113] Note: The data in Table 1 were analyzed using one-way ANOVA. **** indicates comparison with the Ctrl group, p < 0.0001.### Y indicates p < 0.001 compared with the group using RocA alone; #### Y indicates p < 0.0001 compared with the group using RocA alone; ▲▲▲▲ Y indicates that compared with the group using NK-aEVs alone, p < 0.0001.

[0114] The synergistic effect of RocA@NK-aEVs (0.016μg@100μg) was calculated based on the Loewe isobologram model. A ) and drug B (dose d B ) are used together to achieve effect E, while the dose required to achieve effect E when using drug A and drug B alone is D A and D B , Combination Index CI = d A / D A +d B / D B The CI value is used to determine whether the two interact synergistically: CI < 1 indicates a synergistic effect; CI = 1 indicates an additive effect; and CI > 1 indicates antagonism. Calculations show that the CI values ​​of RocA@NK-aEVs are less than 1 compared to NK-aEVs and RocA alone, indicating that the RocA@NK-aEVs obtained by encapsulating the NK-aEV compound RocA in the present invention produce a significant synergistic effect. Detailed calculation results are shown in Table 2.

[0115] Table 2 Synergistic effect calculation results

[0116]

[0117] Example 4: Investigating the killing effect of RocA@NK-aEVs on A549-luc tumor cells

[0118] The bioluminescence method was used to detect the killing effect of RocA@NK-aEVs (0.016μg@100μg) on ​​A549-luc tumor cells expressing Luciferase fluorescence.

[0119] 3×10 NK cells were obtained after 14 days of expansion. 3 3×10 A549-luc cells / well in the logarithmic growth phase 3Each well was plated simultaneously in a 96-well plate at a 1:1 effector-target ratio, and then different concentrations of NK-aEVs, RocA@NK-aEVs, and RocA were added and co-incubated in a 37°C incubator. After 24 hours of co-incubation, substrate was added for color development, and the fluorescence intensity was measured using a microplate reader. The fluorescence intensity differences between the groups were compared, and the killing efficiency was calculated. The results are shown in Figure 2. Figure 4A 、 4B and 4C.

[0120] Figure 4A 、 4B The killing effects of NK-aEVs, RocA@NK-aEVs and equal concentrations of RocA on A549-luc tumor cells at doses of 25 μg / mL, 50 μg / mL and 100 μg / mL, respectively, were observed after 24 h, 48 h and 72 h. Figure 4A It can be seen that at a dose of 25μg / mL, at 24h, the level of RocA@NK-aEVs killing A549-luc cells was 30% higher than that of the Ctrl group (control group), about 23% higher than the NK-aEVs group alone, and nearly 9% higher than the RocA group alone; at 48h, it was 46% higher than the Ctrl group, about 30% higher than the NK-aEVs group alone, and nearly 17% higher than the RocA group alone; at 72h, it was 75% higher than the Ctrl group, about 54% higher than the NK-aEVs group alone, and nearly 14% higher than the RocA group alone. Figure 4B It can be seen that at a dose of 50 μg / mL, at 24 hours, the level of RocA@NK-aEVs killing A549-luc cells was 56% higher than that of the Ctrl group (control group), about 49% higher than the NK-aEVs group alone, and about 30% higher than the RocA group alone; at 48 hours, it was 64% higher than the Ctrl group, about 45% higher than the NK-aEVs group alone, and about 33% higher than the RocA group alone; at 72 hours, it was 86% higher than the Ctrl group, about 55% higher than the NK-aEVs group alone, and about 26% higher than the RocA group alone. Figure 4C As can be seen, at a dose of 100 μg / mL, at 24 hours, RocA@NK-aEVs demonstrated a 60% higher level of killing A549-luc cells than the Ctrl group (control group), approximately 50% higher than the NK-aEVs group alone, and approximately 30% higher than the RocA group alone. At 48 hours, the killing rate was 75% higher than the Ctrl group, approximately 52% higher than the NK-aEVs group alone, and approximately 20% higher than the RocA group alone. At 72 hours, the killing rate was 93% higher than the Ctrl group, approximately 57% higher than the NK-aEVs group alone, and approximately 17% higher than the RocA group alone. Specific test data are shown in Table 3.

[0121] Table 3 Killing effect of each group on A549-luc tumor cells

[0122]

[0123] Note: The data in Table 3 were analyzed using one-way ANOVA. * indicates that compared with the Ctrl group, p < 0.05; *** indicates that compared with the Ctrl group, p < 0.001; **** indicates that compared with the Ctrl group, p < 0.0001; # Y indicates p < 0.05 compared with the group using RocA alone; ### Y indicates p < 0.001 compared with the group using RocA alone; #### Y indicates p < 0.0001 compared with the group using RocA alone; ▲▲▲▲ Y indicates that compared with the group using NK-aEVs alone, p < 0.0001.

[0124] The Loewe isobol model method described in Example 3 was also used to calculate the synergistic effect of RocA@NK-aEVs. The calculation showed that compared with using NK-aEVs and RocA alone, the CI values ​​of RocA@NK-aEVs were less than 1, indicating that the present invention enables NK-aEVs to encapsulate the compound RocA to obtain RocA@NK-aEVs with a significant synergistic effect. The detailed calculation results are shown in Table 4.

[0125] Table 4 Synergistic effect calculation results

[0126]

[0127] Example 5: Investigating the inhibitory effect of RocA@NK-aEVs on subcutaneous tumor growth in mice

[0128] LLC tumor cells were inoculated subcutaneously on the back of C57BL / 6 mice to establish a mouse lung cancer subcutaneous tumor model. The LLC tumor cell inoculation dose was 5×10 5 Each group had 4 mice.

[0129] Treatment began on the third day after modeling. The Ctrl group (control group) was given the same volume of drug solvent. The NK-aEVs group was given an intratumoral injection of 100 μg of NK-aEVs. The RocA group was given an intraperitoneal injection of RocA 0.12 μg. The RocA@NK-aEVs group was given an intratumoral injection of 100 μg of RocA@NK-aEVs (each 100 μg of RocA@NK-aEVs contained 0.12 μg of RocA). The drugs were administered once every three days, and the tumor growth and body weight changes of the mice were detected. The test results are shown in Figure 2. Figure 5A、 5B and 5C.

[0130] Figure 5A The following are photos of the changes in subcutaneous tumors of mice on the 18th day after the action of RocA@NK-aEVs, single NK-aEVs, and single RocA. Figure 5A It can be seen that: on day 18, the tumors of half of the mice in the RocA@NK-aEVs treatment group completely regressed; Figure 5B Figure 5 is a graph showing the changes in tumor weight of subcutaneous tumors in mice on day 18 after treatment with RocA@NK-aEVs, single NK-aEVs, and single RocA. Figure 5B It can be seen that the RocA treatment group alone had no significant effect on mouse tumor growth; the mean tumor weight of the NK-aEVs treatment group alone was about 0.56g, nearly 2 times lower than the mean of 1.2425g in the Ctrl control group, while the mean tumor weight of the RocA@NK-aEVs treatment group was about 0.067g, nearly 20 times lower than that of the Ctrl control group, nearly 8 times lower than that of the NK-aEVs treatment group alone, and about 14 times lower than that of the RocA treatment group alone. Figure 5C Figure 5 is a graph showing the changes in tumor volume of subcutaneous tumors in mice after 18 days of treatment with RocA@NK-aEVs, single NK-aEVs, and single RocA. Figure 5C As can be seen, the tumor volume in the NK-aEVs-only treatment group was approximately three times lower than that in the Ctrl group. However, the tumor volume in the RocA@NK-aEVs-treated group was only one-fifth of that in the NK-aEVs-only treatment group, and was also far smaller than that in the RocA-only treatment group, at nearly 15 times lower. Furthermore, by day 18, two mice in the RocA@NK-aEVs-treated group had complete tumor regression, and no recurrence was observed until the end of the experiment, demonstrating that RocA@NK-aEVs have a significant therapeutic effect on tumors. Specific test data are shown in Table 5.

[0131] Table 5 Inhibitory effect of each group on subcutaneous tumor growth in mice

[0132]

[0133] The data in Table 5 were analyzed using one-way ANOVA, * indicates that compared with the Ctrl group, p < 0.05; ** indicates that compared with the Ctrl group, p < 0.01; **** indicates that compared with the Ctrl group, p < 0.0001; # Y indicates p < 0.05 compared with the group using RocA alone; ### Y indicates p < 0.001 compared with the group using RocA alone; #### Y indicates p < 0.0001 compared with the group using RocA alone;▲ Y indicates that compared with the group using NK-aEVs alone, p < 0.05.

[0134] The Q value was calculated using the King's formula to determine whether RocA@NK-aEVs has a synergistic effect. The King's formula calculation method is: Q = E A+B / E A +E B -(E A ×E B ), where: E A+B is the combined drug inhibition rate, E A and E B The inhibition rates of drugs A and B, respectively, are shown when used alone. If Q < 1, the two drugs are antagonistic; if Q = 1, the two drugs are additive; and if Q > 1, they exhibit a synergistic effect. Based on the data in Table 6, Q = 1.47, which is greater than 1, indicating that the RocA@NK-aEVs described in this invention exhibit significant synergistic effects.

[0135] Table 6 Synergistic effect calculation results

[0136] Group Tumor weight Tumor inhibition rate relative to Ctrl group (%) Ctrl 1.2425 0 RocA (0.12 μg) 0.9925 20.12 NK-aEVs (100 μg) 0.56 54.93 RocA@NK-aEVs (0.12μg@100μg) 0.067 94.6

[0137] Example 6: Investigating the effect of RocA@NK-aEVs on the remodeling of the immune microenvironment in tumor-bearing mice

[0138] The tumors of the mice in each treatment group in Example 5 were prepared into single cell suspensions, and the red blood cells in the samples were lysed and removed using red blood cell lysis buffer, and 5×10 6 cells were fluorescently stained with flow cytometry antibodies, and the antibody color combination was CD4 + T cells (CD3 + / CD4 + ), CD8 + T cells (CD3 + / CD8 + ), NK cells (CD3 - / NKp46 + After staining, flow cytometry was used to analyze the CD4 + T cells, CD8 + T cell and NK cell clustering were tested, and the results were as follows Figure 6A 、 6B and 6C.

[0139] Figure 6A The results show that RocA@NK-aEVs, single NK-aEVs and single RocA have an impact on the level of NK cells in tumors of tumor-bearing mice. Figure 6AThe NK cell levels in the tumors of mice treated with RocA@NK-aEVs were nearly 4 times higher than those in the Ctrl group, nearly 2 times higher than those in the RocA-only treatment group, and about 1.6 times higher than those in the NK-aEVs-only treatment group. Figure 6B The results showed that RocA@NK-aEVs, single NK-aEVs and single RocA could effectively inhibit the expression of CD8 + The impact of T cell levels, Figure 6B Visible: CD8 in tumors of mice treated with RocA@NK-aEVs + The T cell level was significantly higher than that in the Ctrl group, and nearly 1.5- to 2-fold higher than that in the RocA treatment group alone, and also higher than that in the NK-aEVs treatment group alone; Figure 6C The results showed that RocA@NK-aEVs, single NK-aEVs and single RocA could effectively inhibit the expression of CD4 + The impact of T cell levels, Figure 6C Visible: CD4 in tumors of mice treated with RocA@NK-aEVs + The effect on T cell levels was not significant. This example demonstrates that RocA@NK-aEVs can reshape the tumor immune microenvironment, enhancing the efficacy of anti-tumor immunotherapy. The effect was superior to that seen in the RocA and NK-aEV treatment groups alone, demonstrating a reversal of the suppressive tumor microenvironment. Specific test data are shown in Table 7.

[0140] Table 7 Effects of each group on the remodeling of the immune microenvironment in tumor-bearing mice

[0141]

[0142] The data in Table 7 were analyzed using one-way ANOVA, * indicates that compared with the Ctrl group, p < 0.05; ** indicates that compared with the Ctrl group, p < 0.01; # Y indicates p < 0.05 compared with the group using RocA alone; ▲ Y indicates that compared with the group using NK-aEVs alone, p < 0.05.

[0143] Example 7: Investigating the in vivo toxicity of RocA@NK-aEVs in tumor-bearing mice

[0144] The hearts, livers, lungs, kidneys, and brains of the mice in each treatment group in Example 5 were removed, fixed with 4% paraformaldehyde, and then embedded in paraffin. The paraffin blocks were sectioned and stained with HE to observe the in vivo toxicity of each drug group. The detailed results are shown in Table 5. Figure 7 shown.

[0145] Depend on Figure 7 As shown, there were no abnormalities in the staining results of the heart, liver, lung, kidney and brain of each group of mice, indicating that various treatment methods including RocA@NK-aEVs had no obvious toxic side effects on tumor-bearing mice.

[0146] Example 8: Investigating the killing effect of NK-aEVs loaded with other compounds on tumor cells

[0147] Use Figure 2 The preparation method shown is used to prepare NK cell artificial extracellular vesicles encapsulating compounds 5 / 6 / 7 / 8 / 9, which are abbreviated as: compound 5@NK-aEVs, compound 6@NK-aEVs, compound 7@NK-aEVs, compound 8@NK-aEVs, and compound 9@NK-aEVs, respectively.

[0148] ATP bioluminescence method was used to detect the killing effect of each group on H1299 tumor cells: 3×10 NK cells were taken after 14 days of expansion. 3 The cells were plated into 96-well plates at a 1:1 effector-target ratio with H1299 cells. RocA@NK-aEVs, compound 5@NK-aEVs, compound 6@NK-aEVs, compound 7@NK-aEVs, compound 8@NK-aEVs, and compound 9@NK-aEVs were added at the same time. The colorimetric substrate in the ATP activity detection kit was added after 24 h, 48 h, and 72 h of co-incubation, and the fluorescence expression was detected after 10 minutes of incubation. The killing efficiency of H1299 tumor cells was calculated. The results are shown in the figure. Figure 8A 、 8B and 8C.

[0149] Figure 8A This shows the killing effect of each treatment group with the same drug loading amount on H1299 tumor cells after 24 hours. Figure 8B This shows the killing effect of each treatment group with the same drug loading amount on H1299 tumor cells after 48 hours. Figure 8C This shows the killing effect of each treatment group with the same drug loading amount on H1299 tumor cells after 72 hours of action; Figure 8A 、 8BThe results shown in Figure 8C show that at a dosage of 100 μg / mL, the same drug loading amount of compound 5@NK-aEVs, compound 6@NK-aEVs, compound 7@NK-aEVs, compound 8@NK-aEVs, and compound 9@NK-aEVs treatment groups had significantly higher killing activities against H1299 tumor cells at 24 h, 48 h, and 72 h than the RocA@NK-aEVs treatment group, among which the killing activity of compound 5@NK-aEVs group was significantly higher than that of RocA@NK-aEVs group. The killing activity of compound 6@NK-aEVs was 10%-20% higher than that of the RocA@NK-aEVs group; the killing activity of compound 7@NK-aEVs was approximately 20% higher than that of the RocA@NK-aEVs group; the killing activity of compound 8@NK-aEVs was approximately 30%-50% higher than that of the RocA@NK-aEVs group; and the killing activity of compound 9@NK-aEVs was approximately 30%-40% higher than that of the RocA@NK-aEVs group. Specific test data are shown in Table 8.

[0150] Table 8 Killing effect of each treatment group on tumor cells

[0151]

[0152] The data in Table 8 were analyzed using one-way ANOVA, **** indicates comparison with the Ctrl group, p < 0.0001; ### Y indicates p < 0.001 compared with the NK-aEVs alone group; #### Y indicates that compared with the group using NK-aEVs alone, p < 0.0001.

[0153] Finally, it is necessary to explain here that the above embodiments are only used to further illustrate the technical solution of the present invention in detail and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above content of the present invention all fall within the scope of protection of the present invention.

Claims

1. An artificial extracellular vesicle for NK cells loaded with a small molecule compound, characterized in that: The method is prepared by using NK cells and small molecule compounds as raw materials through high-pressure extrusion and / or ultrasonic lysis. The small molecule compound has a chemical structure shown in the following formula I or formula II: ； Wherein: R1 is a saturated hydrocarbon group or an unsaturated hydrocarbon group; R2 is an amino group substituted by any one or two of an alkane group, an alkene group, an alkyne group, an oxacycloalkyl group, an alkoxy group, and a hydroxylamine group; R3 and R4 are independently selected from hydrogen or deuterium.

2. The NK cell artificial extracellular vesicles loaded with small molecule compounds according to claim 1, characterized in that: The R1 is a C1-C4 alkyl group, an allyl group, or a propargyl group.

3. The NK cell artificial extracellular vesicles loaded with small molecule compounds according to claim 1, characterized in that: The R2 is an amino group substituted by any one or two of methyl, allyl, propargyl, 3-oxetanyl, methoxy, and hydroxylamine.

4. A method for preparing NK cell artificial extracellular vesicles loaded with small molecule compounds according to any one of claims 1 to 3, characterized in that: It is a high pressure extrusion method. The specific operation includes: first press every 1×10 7 1 to 10 μg of the small molecule compound is added to each NK cell to prepare a cell suspension, and then the prepared cell suspension is subjected to a high-pressure extruder. At a pressure of 1 to 4 MPa, gradient filtration is performed on the prepared cell suspension using a 0.2 to 10 μm filter membrane in sequence, and each specification of filter membrane is extruded 3 to 10 times. Finally, the mixed cell suspension after extrusion is collected by gradient centrifugation.

5. A method for preparing NK cell artificial extracellular vesicles loaded with small molecule compounds according to any one of claims 1 to 3, characterized in that: The ultrasonic lysis method is as follows: first press 1×10 7 1-10 μg of the small molecule compound is added to each NK cell to prepare a cell suspension, and then the prepared cell suspension is lysed using an ultrasonic lyser at 20-30 kHz, 800-1000 W power, and 4-8°C for 5-8 minutes. Finally, the mixed cell suspension after lysis is collected by gradient centrifugation.

6. The preparation method according to claim 5 or 6, characterized in that: The gradient centrifugation collection operation includes: first centrifuging at 300-500 g for 5-10 minutes to collect a first supernatant; then centrifuging the first supernatant at 2000-3000 g for 5-10 minutes to collect a second supernatant; and finally centrifuging the second supernatant at 100,000-120,000 g for 70-90 minutes to collect a solid precipitate.

7. A use of the artificial extracellular vesicles for NK cells loaded with small molecule compounds according to any one of claims 1 to 3, characterized in that: The application refers to the preparation of an anti-tumor product using the NK cell artificial extracellular vesicles loaded with small molecule compounds as the sole active ingredient or one of the active ingredients.

8. The use according to claim 7, characterized in that: The anti-tumor product is an anti-tumor immunotherapy product or a product used to enhance the effect of anti-tumor immunotherapy.

9. The use according to claim 7, characterized in that: In the NK cell artificial extracellular vesicles loaded with small molecule compounds, the mass content of the small molecule compounds is 0.01% to 1%.

10. The use according to claim 7, characterized in that The small molecule compound is selected from any one of the following compounds:

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