NK cell expansion composition, NK cell artificial extracellular vesicle and preparation method and application thereof

Abstract

The application belongs to the field of biological medicine and cell engineering technology, and particularly relates to an NK cell expansion composition, an NK cell artificial extracellular vesicle and a preparation method and application thereof. The NK cell expansion composition comprises a basic culture medium and IL-2, IL-12, nicotinamide and traditional Chinese medicine active small molecules such as curcumin and tanshinone IIA added therein. The composition can be used to expand high-purity NK cells with a purity greater than 95% and extremely high killing activity under serum-free conditions. Artificial extracellular vesicles (NK-aEVs) prepared by taking the NK cells as precursor cells have excellent selective elimination of senescent cell activity, and can eliminate senescent tumor cells induced by treatment, overcome tumor drug resistance and recurrence when combined with a chemotherapy drug; meanwhile, the NK-aEVs can significantly eliminate senescent cells and Aβ protein deposition in the brain and peripheral organs of natural / acute aging and Alzheimer's disease mice, significantly improve cognitive impairment and exercise tolerance and reshape the NK cell immune surveillance of the body.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and cell engineering technology, specifically relating to an NK cell expansion composition, artificial extracellular vesicles of NK cells, their preparation methods and applications. Background Technology

[0002] Cellular senescence is a state characterized by stable cell cycle arrest induced by stress-induced damage, accompanied by activation of anti-apoptotic pathways, metabolic reprogramming, and significant release of senescence-associated secretory phenotypes (SASPs). In cancer treatment, while radiotherapy and chemotherapy can effectively kill proliferating tumor cells, they also induce residual tumor cells to enter a therapy-induced senescence (TIS) state. These senescent tumor cells, although arrested in the cell cycle, still possess high metabolic activity and continuously reshape the microenvironment by secreting SASPs, promoting tumor invasion and metastasis, immune escape, relapse, and acquired drug resistance. Therefore, targeted elimination of senescent tumor cells has become an emerging strategy for reversing drug resistance and preventing relapse.

[0003] Besides tumors, excessive accumulation of cellular senescence also drives a variety of age-related diseases. In Alzheimer's disease (AD), neurons, astrocytes, and microglia all exhibit senescent characteristics. Senescent cells, along with Aβ deposition and tau hyperphosphorylation, mutually promote each other, accelerating cognitive decline. Clearing or regulating senescent cells provides a new direction for AD treatment and delaying brain aging.

[0004] Natural killer (NK) cells, as core effector cells of the innate immune system, can recognize and eliminate senescent cells and senescent tumor cells independently of the MHC. Preclinical studies have shown that adoptive transfer of NK cells can selectively eliminate residual senescent tumor cells after chemotherapy / radiotherapy; in the aging central nervous system, NK cells with senescence clearance function can reduce Aβ deposition and tau pathology. However, NK cells face challenges such as difficulty in expansion, high costs of preservation and transportation, impaired activity after cryogenic resuscitation, and low activity in clinical-grade serum-free expansion systems, which greatly limit their application. Furthermore, the blood-brain barrier further restricts the use of live NK cells in age-related brain diseases.

[0005] Artificial extracellular vesicles derived from natural killer cells (NK-aEVs) are nanoscale particles artificially prepared using NK cells as raw materials. They possess biological characteristics similar to natural exosomes, inheriting the anti-tumor and immunomodulatory properties of NK cells. They eliminate the risks of tumorigenesis and immune rejection associated with live cell infusion and can penetrate the blood-brain barrier. While exosomes derived from mesenchymal stem cells and other sources have been studied in the field of anti-aging, the role and application of NK-aEVs in anti-aging have not yet been reported, and related research remains in its infancy.

[0006] Therefore, there is an urgent need for a technology system that can efficiently expand highly active NK cells and thereby prepare NK-aEVs with clear anti-aging / anti-tumor / anti-AD activities, in order to solve the current bottlenecks in NK cell applications and provide a scalable and standardized upstream platform for aging-related diseases. Summary of the Invention

[0007] The primary objective of this invention is to provide a NK cell in vitro expansion culture medium composition that solves the technical problems of low NK cell expansion efficiency, insufficient cell purity, and poor killing activity in existing serum-free expansion systems.

[0008] The second objective of this invention is to provide a method for preparing NK cell artificial extracellular vesicles (NK-aEVs) based on the NK cells obtained by the above-mentioned amplification, and the obtained NK-aEVs product.

[0009] A third objective of this invention is to provide the application of the NK-aEVs in anti-tumor, anti-aging, and anti-Alzheimer's disease treatments.

[0010] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides an in vitro expansion culture medium composition for NK cells, comprising a basal culture medium and an amplification inducing factor added to the basal culture medium, wherein the amplification inducing factor comprises interleukin-2 (IL-2), interleukin-12 (IL-12), nicotinamide (NAM), and one or more small active molecules of traditional Chinese medicine selected from the group consisting of: curcumin, tanshinone IIA, quercetin, azadirachtin, hesperetin, chrysin, thymoquinone, resveratrol, astragaloside IV, sphondin, oxymatrine, methyl gallate, ursolic acid, and ingenol 3,20-dibenzoate.

[0012] In a preferred embodiment, the amplification inducing factors include IL-2, IL-12, nicotinamide, curcumin, and tanshinone IIA.

[0013] In a specific embodiment, the concentration of IL-2 in the culture medium composition is 50~1000 IU / mL; the concentration of IL-12 is 2~200 ng / mL; the concentration of NAM is 1~100 μM; the concentration of curcumin is 0.2~5 μM; and the concentration of tanshinone IIA is 2~20 μM.

[0014] In a specific implementation, the culture medium composition does not contain animal serum, including but not limited to fetal bovine serum (FBS), calf serum (FCS), horse serum, etc.

[0015] As used herein, basal culture medium refers to a liquid nutrient matrix capable of maintaining the basic survival and metabolism of cells. In a preferred embodiment of the invention, the basal culture medium is serum-free medium (SFM) or xeno-free medium (XFM). For example, the basal culture medium may include, but is not limited to, RPMI-1640, DMEM / F12, α-MEM, and other commercially available serum-free culture media.

[0016] Secondly, the present invention provides a method for in vitro expansion of NK cells, comprising: mixing peripheral blood mononuclear cells (PBMCs) with feeder cells, and culturing them in a serum-free culture medium composition for in vitro expansion of NK cells as described herein, to obtain NK cells with a purity >95%.

[0017] In a specific implementation scheme, the peripheral blood mononuclear cells are human peripheral blood mononuclear cells.

[0018] In a specific implementation plan, the feeder cells are engineered feeder cells, such as engineered K562 cells.

[0019] In a specific implementation plan, the serum-free culture is carried out for 10-21 days, preferably 14 days, with the medium replenished or changed regularly during the culture period.

[0020] In a specific implementation plan, the NK cells are CD3. - / CD56 + / CD16 + . cells.

[0021] Thirdly, the present invention provides an artificial extracellular vesicle for NK cells, which is prepared from NK cells obtained by the in vitro expansion method for NK cells as described herein.

[0022] In a preferred embodiment, the artificial extracellular vesicles of NK cells have a particle size of 50~200nm, a Zeta potential of -20~-5 mV, and express CD63, CD81 and CD9 protein markers on their surface.

[0023] In a specific implementation, the artificial extracellular vesicles of NK cells are prepared by high-pressure extrusion and / or ultrasonic lysis.

[0024] In a specific implementation, the high-pressure extrusion method uses a 0.2-10.0 μm filter membrane to perform gradient extrusion of the amplified NK cells, followed by gradient centrifugation; the ultrasonic lysis method involves lysing the amplified NK cells at 20-30 kHz, 800-1000 W, and 4-8℃ for 5-8 min, followed by gradient centrifugation.

[0025] In a specific implementation, the gradient extrusion includes extruding with filter membranes of 8.0 μm, 1.0 μm, 0.4 μm and 0.2 μm in sequence.

[0026] In a specific implementation plan, the gradient centrifugation is performed by sequentially centrifuging at 300g for 5-15 min, centrifuging at 2000g for 5-15 min, and ultracentrifuging at 100,000-150,000g for 80-120 min to collect the precipitate.

[0027] Fourthly, the present invention provides the use of NK cell artificial extracellular vesicles (NK-aEVs) as described herein in the preparation of antitumor products.

[0028] In a specific implementation plan, the anti-tumor product is used to eliminate senescent tumor cells induced by radiotherapy and chemotherapy, inhibit tumor growth, enhance the effect of chemotherapy, or be used in combination with chemotherapy drugs.

[0029] Fifthly, the present invention provides the use of NK cell artificial extracellular vesicles as described herein in the preparation of aging regulators or anti-aging products.

[0030] In specific implementations, the aging regulator or anti-aging product is used to improve natural aging, radiation-induced acute aging, or to remove senescent cells from tissues.

[0031] In a sixth aspect, the present invention provides the use of NK cell artificial extracellular vesicles as described herein in the preparation of products for the treatment or improvement of Alzheimer's disease.

[0032] In a seventh aspect, the present invention provides an antitumor, anti-aging, or anti-Alzheimer's disease product comprising an effective amount of artificial extracellular vesicles of NK cells as described herein.

[0033] In a specific implementation plan, the final concentration of the artificial extracellular vesicles for NK cells is 25~100 μg / mL.

[0034] Compared with the prior art, the present invention has the following significant advantages:

[0035] 1. Synergistic effect of active small molecules of traditional Chinese medicine: This invention is the first to discover that introducing specific active small molecules of traditional Chinese medicine, especially curcumin (Cur) and tanshinone IIA (TIIA), into the serum-free IL-2 / IL-12 / NAM amplification system can produce a significant synergistic effect, which not only greatly improves the cell yield and purity of NK cells (>95%), but also significantly enhances the toxicity of NK cells to tumor cells.

[0036] 2. Highly selective senescence clearance activity: NK-aEVs prepared by the specific amplification system of this invention exhibit excellent ability to target and clear senescent cells, and their killing efficiency against radiotherapy and chemotherapy-induced senescent tumor cells and senescent normal cells is significantly higher than that against non-senescent control cells.

[0037] 3. Synergistic effect of chemotherapy sensitization and anti-tumor: When NK-aEVs are used in combination with chemotherapy drugs such as etoposide, they can accurately eliminate senescent tumor cells caused by chemotherapy, overcome chemotherapy resistance and recurrence, significantly inhibit the growth of solid tumors, and increase the NK cell immune infiltration in the tumor site.

[0038] 4. Crossing the blood-brain barrier and reshaping systemic immunity: NK-aEVs have good nanoscale stability and the ability to penetrate the blood-brain barrier. They can effectively eliminate senescent cells and Aβ protein deposits in the brain of AD patients, significantly improve cognitive impairment and exercise endurance in AD and naturally aging animals, and increase the proportion of NK cells in the spleen of aging organisms, thus restoring immune surveillance function. Attached Figure Description

[0039] Figure 1 Flow cytometry (A) and cell amplification statistics (B) of NK cells obtained by amplification of different combinations of active small molecules from traditional Chinese medicine.

[0040] Figure 2 Comparison of the killing activity of NK cells obtained by amplification of different combinations of active small molecules of traditional Chinese medicine against A549 (A) and H1299 (B) tumor cells.

[0041] Figure 3 Flowchart (A) of NK-aEVs preparation, particle size distribution (B), zeta potential distribution (C), and nano-flow cytometry characterization (DF) of CD63 / CD81 / CD9 markers.

[0042] Figure 4 Comparison of the killing rates of NK-aEVs prepared from NK cells obtained by amplification of different combinations of active small molecules of traditional Chinese medicine against senescent tumor cells S-A549 (B) and S-H1299 (C) using a model of senescent cells induced by etoposide and X-ray (A).

[0043] Figure 5 Selective clearance activity of NK-aEVs against senescent tumor cells (A549(A), H1299(B)) and senescent epithelial cells (BEAS-2B(C), HaCaT(D)) at different time points.

[0044] Figure 6 Effects of NK-aEVs combined with etoposide on tumor material (A), tumor weight (B), growth curve (C), and SA-β-Gal staining (DE) of tumor tissue in tumor-bearing mice.

[0045] Figure 7 The effect of NK-aEVs on organ aging (SA-β-Gal staining, A) in acutely aged mice and the results of fatigue tests (BF) in water maze, forelimb grip, balance beam, and rotarod.

[0046] Figure 8 : The effect of NK-aEVs on alleviating weight loss in naturally aging mice (A) and clearing the SA-β-Gal aging marker in the liver and kidneys (B).

[0047] Figure 9The effect of NK-aEVs on the behavior of naturally aging mice: (AB) results of the water maze test, (CE) results of forelimb grip, balance beam and rotarod fatigue tests.

[0048] Figure 10 The reversal effect of NK-aEVs on body weight (A), senescent brain cells (B), and Aβ protein deposition (C) in AD mice.

[0049] Figure 11 The effect of NK-aEVs on the behavior of AD mice: (AB) results of water maze test, (CE) results of forelimb grip, balance beam and rotarod fatigue test.

[0050] Figure 12 Effects of NK-aEVs on the remodeling of the NK cell ratio in spleen / tumor tissue of tumor-bearing mice (A), acutely aged mice (B), naturally aged mice (C), and AD mice (D). Detailed Implementation

[0051] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0052] Example 1: Amplification of high-purity NK cells using small molecule combinations of traditional Chinese medicine

[0053] Fresh peripheral blood mononuclear cells (PBMCs) from healthy individuals were collected and engineered K562 feeder cells were added according to the suspension ratio. Serum-free medium (OptiVitro® NK cell serum-free basal medium P01) was used as the basal medium, and different combinations of amplification inducing factors as shown in Table 1 were added. The cells were cultured at 37 °C and 5% CO2 for 14 days. The medium was replenished or changed every 2 days.

[0054] On day 14, cells were collected and CD3 levels were detected by flow cytometry. - CD56 + CD16 + The purity of the NK cell population was assessed, and the number of NK cells obtained from the expansion was counted. The count was then calculated using the formula: Number of NK cells = Number × CD3. - % × CD56 + / CD16 + %, calculate the actual number of NK cells, and see the results. Figure 1 .

[0055] Figure 1 Flow cytometry results for A showed the purity of NK cells after 14 days of amplification using different amplification combinations. Figure 1The result of B is the number of NK cells obtained by each combination after 14 days of amplification.

[0056] Figure 1 The results showed that among the small molecule combinations of traditional Chinese medicine listed in Table 1, six combinations had higher amplification efficiency than the original amplification system, with the combination IL2+IL12+NAM+curcumin+tanshinone IIA showing the best effect.

[0057] Table 1

[0058]

[0059] Example 2: Detection of in vitro tumor-killing activity of NK cells obtained by amplification using small molecule combinations of traditional Chinese medicine

[0060] The NK cells obtained from each group in Example 1 were used as effector cells and co-incubated with target lung cancer A549 and H1299 cells at a 1:1 effector-target ratio for 24 h. Cell viability was detected using the CellTiter-Glo® Luminescent cell viability assay kit, and the killing efficiency was calculated using the following formula. The results are as follows: Figure 2 As shown:

[0061] Specific lysis (%) = [1-(Mean Mix -Mean NK ) / Mean Tumor ] × 100

[0062] Mean Mix : Average bioluminescence intensity value of the NK cell + tumor cell co-incubation group

[0063] Mean NK : Mean bioluminescence intensity value of the NK cell-only treatment group

[0064] Mean Tumor : Average bioluminescence intensity of the NK cell-only treatment group.

[0065] Figure 2 The results of A showed that among the 18 active small molecule combinations of traditional Chinese medicine, when the target cells were A549, 11 active small molecule combinations of traditional Chinese medicine had higher killing efficiency than the control group, while the killing efficiency of 3 active small molecule combinations of traditional Chinese medicine decreased. Figure 2The results of study A showed that when the target cells were H1299, seven combinations of active small molecules from traditional Chinese medicine (TCM) exhibited higher killing efficiency than the control group, while eight combinations showed decreased killing efficiency. Among the two target cell types, the NK cells amplified using the combination of IL2+IL12+NAM+curcumin+tanshinone IIA showed the strongest killing activity. Based on the NK cell amplification results, it is suggested that the combination of IL2+IL12+NAM+curcumin+tanshinone IIA can obtain the largest number and strongest activity of NK cells. Subsequently, we will use the combination of IL2+IL12+NAM+curcumin+tanshinone IIA as an amplification method to amplify NK cells from PBMCs and use them for the subsequent preparation of NK cell artificial extracellular vesicles (NK-aEVs).

[0066] Example 3: Physical preparation and characterization of NK cell artificial extracellular vesicles (NK-aEVs)

[0067] NK cells obtained after 14 days of expansion using the IL2+IL12+NAM+curcumin+tanshinone IIA combination were collected, washed 2-3 times with PBS, and then re-prepared with PBS to form cells at a density of 1×10⁶. 6 Cell suspensions of NK cells / mL were extruded using a high-pressure extruder under nitrogen pressure of 1–4 MPa, sequentially through 8.0 μM, 1.0 μM, 0.4 μM, and 0.2 μM filters, with each filter size extruded three times to obtain particles of uniform size; alternatively, an ultrasonic lysis apparatus was used to lyse the cells at 20 kHz, 800 W power, and 4°C for 5 min (0.5 s run, 0.5 s stop). After preparation, the cells were centrifuged at 300g for 10 min to remove large cell clumps, and the supernatant was centrifuged at 2000g for 10 min to remove cell debris. Finally, the supernatant was centrifuged at 100,000g for 90 min to collect artificial extracellular vesicles of NK cells. A simplified procedure is as follows: Figure 3 As shown in Figure A.

[0068] The particle size, drug carrier particle stability, and exosome surface markers of the prepared NK-aEVs were analyzed using an NTA, NanoCoulter Particle Analyzer, and NanoFCM nanoflow cytometer. The results are as follows: Figure 3 As shown.

[0069] Figure 3 B represents the particle size analysis results of NK cell artificial extracellular vesicles (NK-aEVs). Data from three independent replicate experiments showed that the average particle size of NK-aEVs was 96.5 ± 42.7 nM, which is similar to the particle size characteristics of natural exosomes. Figure 3C represents the particle stability test results of NK-aEVs. The average Zeta potential ranges from -5 to -15 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 is considered suitable for use as a stable drug carrier, suggesting that it has the potential to carry drugs, similar to natural exosomes. Figure 3 DF, as detected by nanoflow cytometry, shows that NK-aEVs express natural exosome marker proteins CD63, CD81, and CD9 on their surface, exhibiting surface markers similar to those of natural exosomes.

[0070] Example 4: The effect of NK-aEVs on the in vitro elimination of senescent tumor cells and senescent epithelial cells

[0071] We constructed two cell senescence models in vitro, simulating radiotherapy and chemotherapy respectively, and used a β-galactosidase staining kit to detect cell senescence. The specific methods were as follows: (1) Etoposide-induced cell senescence model: 500 nM etoposide was added to the cell culture and cultured continuously for 10 days to successfully induce cell senescence. (2) X-ray-induced cell senescence model: When the cells reached a confluence of 70%-80%, they were irradiated with 10 Gy of X-rays using an irradiator. After irradiation, they were cultured continuously for 10 days to successfully induce cell senescence. The model construction results are shown in […]. Figure 4 A.

[0072] Figure 4 Results A showed that both senescence models successfully induced senescence in lung cancer cell lines A549 and H1299, bronchial epithelial cells BEAS-2B, and human keratinocytes HaCaT.

[0073] Based on the successful establishment of a tumor cell senescence model, we first used the Cell Counting Kit-8 to determine the ability of NK cells amplified from the following combinations of active small molecules from traditional Chinese medicine, which were then prepared by extrusion and / or sonication to clear senescent cells using NK-aEVs: IL2 group, IL2+IL12+NAM group, IL2+IL12+NAM+curcumin combination, IL2+IL12+NAM+tanshinone IIA combination, and IL2+IL12+NAM+curcumin+tanshinone IIA combination. Specific experimental conditions were: A549 cells, H1299 cells, and senescent-A549 cells (S-A549) and senescent-H1299 cells (S-H1299) induced from these two cell lines, at a concentration of 5 × 10⁻⁶ cells / year. 4Cells were seeded at a density of 100 μg / mL in 96-well plates and allowed to adhere overnight. Then, NK-aEVs prepared from each amplification group were added at a dose of 100 μg / mL. After further culturing for 24 h, absorbance (OD) values ​​were measured, and the cell killing rate was calculated using the following formula: Killing rate (%) = [1 - (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%. The results are shown below. Figure 4 As shown in B and C.

[0074] Figure 4 The results of B showed that the clearance efficiency of NK-aEVs prepared from NK cells amplified by each amplification combination against senescent-A549 cells (S-A549) was significantly higher than that of non-senescent normal A549 cells at 24 h. Furthermore, the clearance efficiency of NK-aEVs prepared from NK cells amplified by the combination of IL2+IL12+NAM+curcumin+tanshinone IIA was significantly higher than that of other amplification combinations of active small molecules of traditional Chinese medicine. Figure 4 The results of C also showed that the clearance efficiency of NK-aEVs prepared from NK cells amplified by each amplification combination was significantly higher than that of non-senescent normal H1299 cells. Similarly, the clearance efficiency of NK-aEVs prepared from NK cells amplified by the combination of IL2+IL12+NAM+curcumin+tanshinone IIA was the best.

[0075] Next, we further investigated the ability of NK-aEVs prepared from NK cells amplified using the Cell Counting Kit-8 to eliminate senescent cells in four cell lines (A549, H1299, BEAS-2B, and HaCaT). The specific experimental conditions were: A549 cells, H1299 cells, BEAS-2B cells, and HaCaT cells; and senescent-A549 cells (S-A549), senescent-H1299 cells (S-H1299), senescent-BEAS-2B cells (S-BEAS-2B), and senescent-HaCaT cells (S-HaCaT) induced from these four cell lines, at a concentration of 5 × 10⁻⁶ cells per cell line. 4Cells were seeded at a density of 100 μg / mL in 96-well plates and allowed to adhere overnight. Then, NK-aEVs prepared from NK cells amplified using the combination of IL2+IL12+NAM+curcumin+tanshinone IIA were added. After further culture for 24 h, 48 h, and 72 h, the absorbance (OD value) was measured, and the cell killing rate was calculated using the following formula: Killing rate (%) = [1 - (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%. The results are shown below. Figure 5 As shown.

[0076] Figure 5 The results showed that the clearance efficiency of 100 μg / mL NK-aEVs against senescent-A549 cells (S-A549) was significantly higher than that of non-senescent normal A549 cells at 24 h, 48 h, and 72 h. Figure 5 The results of B showed that the clearance efficiency of NK-aEVs against senescent-H1299 cells (S-H1299) was significantly higher than that of non-senescent normal A549 cells at 24 h, 48 h, and 72 h. Figure 5 The results of C showed that NK-aEVs were significantly more efficient at clearing senescent BEAS-2B cells (S-BEAS-2B) than non-senescent normal BEAS-2B cells at 24 h, 48 h, and 72 h; similarly, Figure 5 The results showed that NK-aEVs significantly improved the clearance efficiency of senescent-HaCaT cells (S-HaCaT) at 24 h, 48 h, and 72 h compared with non-senescent normal HaCaT cells.

[0077] Example 5: The effect of NK-aEVs in clearing senescent tumor cells in tumor-bearing mice and inhibiting subcutaneous tumor growth in mice

[0078] A mouse model of subcutaneous lung cancer tumor was established by subcutaneously inoculating LLC tumor cells into the back of C57BL / 6 mice. The inoculation amount of LLC tumor cells was 5 x 10⁶ cells / year. 5Five mice were assigned to each group. Treatment began on day 3 of modeling. Mice in the Ctrl group (control group) received the same volume of drug solution; mice in the NK-aEVs treatment group received 100 μg / mouse of NK-aEVs via tail vein injection three times a week; mice in the etoposide (ETO) treatment group received 4 mg / kg of ETO via intraperitoneal injection twice a week; mice in the etoposide and NK-aEVs combination treatment group received both 100 μg / mouse of NK-aEVs and 4 mg / kg of ETO. Tumor growth was assessed every two days. Mice were sacrificed on day 21, tumor size was measured and photographed, and frozen sections of tumor tissue were prepared for β-galactosidase staining to assess the senescence induced by etoposide chemotherapy and the clearance effect of NK-aEVs on senescent tumor cells. Results are as follows: Figure 6 As shown.

[0079] Figure 6 A and B are photographs of mouse tumors and their weights on day 21. Figure 6 C represents monitoring tumor growth in mice during treatment. Figure 6 D represents the staining of β-galactosidase in tumor tissue from tumor-bearing mice. Figure 6 E represents the statistical results after quantifying the staining of β-galactosidase using software.

[0080] Figure 6 AC results showed that, based on both tumor volume changes during treatment and tumor weight on day 21, etoposide and NK-aEVs inhibited the growth of subcutaneous tumors in mice to varying degrees, and the differences were statistically significant. The combined treatment group of etoposide and NK-aEVs showed a stronger tumor-suppressive effect than either treatment alone. Figure 6 D and Figure 6 The results of E clearly show that etoposide chemotherapy induces tumor senescence, while NK-aEVs alone do not induce tumor cell senescence; when ETO and NK-aEVs are used in combination, NK-aEVs can significantly eliminate senescent tumor cells induced by ETO treatment.

[0081] Example 6: The effect of NK-aEVs on aging amelioration in irradiated acutely aged mice

[0082] Five-week-old male C57BL / 6J mice weighing 18-20g were selected and subjected to whole-body X-ray irradiation with a dose of 5 Gy using an irradiator. This time point was recorded as day 0. On day 30, the irradiated mice were randomly divided into three groups: control group (Ctrl group, without whole-body irradiation), whole-body irradiation group (WBI group, receiving only whole-body X-ray irradiation), and whole-body irradiation + NK-aEVs treatment group (WBI+NK-aEVs group, receiving whole-body irradiation and NK-aEVs treatment). The treatment frequency and dosage of the WBI+NK-aEVs group were as follows: 100 μg / mouse of NK-aEVs was injected via the tail vein 3 times a week. The other two groups of mice were injected with an equal volume of physiological saline via the tail vein during the same period. On day 60, after 30 days of treatment, the mice underwent behavioral tests, including forelimb grip strength test, balance beam test, rotarod fatigue test, and water maze test. Mice were euthanized at the end of the testing period, and their livers and kidneys were collected for β-galactosidase staining analysis. The results are as follows: Figure 7 As shown.

[0083] Figure 7 A shows the staining results of β-galactosidase in mouse liver and kidney. Figure 7 B and C represent the results of the water maze experiment. Figure 7 D represents the results of the forelimb gripping test. Figure 7 E represents the result of the balance beam experiment. Figure 7 F represents the results of the rotor fatigue test.

[0084] Figure 7 Results showed that irradiation induced acute aging in mice, with a significant increase in senescent cells in the liver and kidneys. NK-aEVs treatment effectively improved the senescent cells in the liver and kidneys of mice and cleared senescent cells. The water maze is a classic behavioral experiment for evaluating the spatial learning and memory abilities of mice. Figure 7 Results B showed that after acute aging, mice exhibited disordered swimming trajectories, were unable to effectively locate the hidden platform after training, and had increased total swimming distance, prolonged exercise duration, and increased platform latency. After NK-aEV treatment, mice were able to successfully locate and board the platform within a limited time, significantly shortening the total swimming distance, exercise time, and escape latency, indicating that the platform targeting ability was improved. Figure 7 The results showed that in experiments without hidden platforms to assess long-term memory retention, acutely aged mice exhibited disordered swimming paths, rarely crossed the original platform quadrant, had the lowest platform crossing frequency, the shortest swimming distance, and the shortest dwell time in the target quadrant. In contrast, mice treated with NK-aEVs showed frequent crossings of the target area, significantly increasing platform crossing frequency, swimming distance in the target quadrant, and dwell time. These results indicate that NK-aEV treatment effectively enhances the long-term spatial memory retention of acutely aged mice. Figure 7The results of D showed that after acute aging, the grip strength of the forelimb decreased, and the grip strength was significantly restored after NK-aEVs treatment; Figure 7 The results from E showed that after irradiation-induced acute aging in mice, the time it took for mice to cross the balance beam was significantly prolonged, and the crossing speed was significantly accelerated after NK-aEVs treatment. Figure 7 The results showed that in the rotundus experiment, the time interval between falls from the rotundus in acutely aging mice was shortened, indicating that the mice were more prone to falling. NK-aEVs treatment significantly prolonged the time mice spent on the rotundus. All these results demonstrate that NK-aEVs treatment can significantly improve irradiation-induced acute aging in mice.

[0085] Example 7: The aging-improving effect of NK-aEVs on naturally aging mice

[0086] Male mice of strain 129 were bred in a specific pathogen-free (SPF) environment and routinely housed in an SPF-grade barrier environment. Day 0 of the experiment was recorded as the day the mice reached 17 months of age. The mice were randomly divided into two groups: a control group (Ctrl group) and an NK-aEVs treatment group; treatment began immediately. Mice in the NK-aEVs treatment group received a tail vein injection of NK-aEVs at a dose of 100 μg / mouse, administered weekly. Mice in the control group received an equal volume of physiological saline via the same administration method. Weight loss was monitored throughout the experiment. The experiment ended on day 90. Before euthanasia, behavioral tests were performed on the mice, including forelimb grip strength test, balance beam test, rotarod fatigue test, and water maze test. Mice were euthanized at the end of the tests, and their livers and kidneys were collected for β-galactosidase staining analysis. Results are as follows: Figure 8 and Figure 9 As shown.

[0087] Figure 8 A represents the weight loss of naturally aging mice during treatment. Figure 8 B shows the staining results of β-galactosidase in mouse liver and kidney.

[0088] Figure 8 Results showed that naturally aging mice experienced a gradual decrease in body weight with increasing age, while NK-aEVs treatment effectively slowed down this weight loss. Figure 8 Results B showed that staining for β-galactosidase revealed a significant increase in senescent cells in the liver and kidneys of aged mice, while NK-aEVs treatment effectively improved the senescent cells in the liver and kidneys of mice and cleared senescent cells.

[0089] Figure 9 A and B are the results of the water maze experiment. Figure 9 These are the results of the forelimb gripping test. Figure 9 D represents the result of the balance beam experiment. Figure 9E represents the results of the rotor fatigue test.

[0090] Figure 9 The results of A showed that, in the water maze experiment, naturally aged mice treated with NK-aEVs, compared with untreated mice, had an increased total swimming distance in the quadrant where the platform was located and a shorter latency to enter the platform quadrant after training, indicating that their ability to find the platform was improved and their spatial memory was enhanced. Figure 9 The results of B showed that in the experiment without hidden platforms to assess long-term memory retention, acutely aged mice rarely crossed the original platform quadrant, had the lowest platform crossing frequency, and the shortest swimming distance. However, mice treated with NK-aEVs showed that they frequently crossed the target area and significantly increased the platform crossing frequency. These results indicate that NK-aEV treatment effectively enhances the long-term spatial memory retention of acutely aged mice. Figure 9 The results of C showed that the forelimb grip strength of aged mice decreased, and the grip strength was significantly restored and the score improved after NK-aEVs treatment; Figure 9 The results of D showed that the time taken for aging mice to cross the balance beam was significantly prolonged, while the crossing speed was significantly shortened after NK-aEVs treatment, indicating that the mice were able to cross the balance beam faster. Figure 8 The results from E showed that in the rotundus experiment, the time interval between falls from the rotundus in aging mice was shortened, indicating that the mice were more prone to falling. NK-aEVs treatment significantly prolonged the time mice spent on the rotundus.

[0091] All of the above results demonstrate that NK-aEVs treatment can significantly improve the aging phenotype in naturally aging mice and has a definite effect on improving aging.

[0092] Example 8: Therapeutic effect of NK-aEVs on Alzheimer's disease mice

[0093] 5xFAD APP / PS1 double transgenic mice carrying three FAD mutations in the human APP gene (K670N / M671L, I716V, V717I) and two FAD mutations in the human PSEN1 gene (M146L, L286V) were selected as Alzheimer's disease model mice. Experiments began at 7 months of age, with mice divided into two groups: the AD group and the AD+NK-aEVs group. Normal 7-month-old C57BL / 6J mice served as a control, designated as the Ctrl group. Mice in the AD+NK-aEVs group were treated once daily with a tail vein injection of 100 μg / mouse of NK-aEVs. Mice in the Ctrl and AD groups received the same volume of saline via the same administration method. The experiment ended one month after treatment. Mouse body weight was measured at the start and end points to assess the weight gain of AD mice before and after NK-aEVs treatment. Before euthanasia, mice underwent behavioral tests, including forelimb grip strength, balance beam, rotarod fatigue, and water maze tests. After the behavioral tests, mice were euthanized, and brain tissue was collected for β-galactosidase staining and Aβ protein immunofluorescence staining analysis. Results are as follows: Figure 10 , Figure 11 As shown.

[0094] Figure 10 A represents the weight gain of normal mice and AD mice in each group during treatment; Figure 10 B shows the staining results of β-galactosidase in mouse brain tissue; Figure 10 C represents the deposition of Aβ protein in mouse brain tissue.

[0095] Figure 10 The results of A showed that AD mice gained weight slowly compared to normal mice, and that NK-aEVs treatment could effectively increase the weight gain of AD mice. Figure 10 The results of B showed that the cells in the brain tissue of AD mice were significantly senescent, and NK-aEVs treatment for one month could effectively reverse the senescence of brain tissue and clear senescent brain cells. Figure 10 The results of C showed that significant Aβ protein deposition occurred in the brain tissue of AD mice, and NK-aEVs treatment could significantly reverse the deposition level of Aβ protein.

[0096] Figure 11 A and B are the results of the water maze experiment. Figure 11 C represents the results of the forelimb gripping test. Figure 11 D represents the result of the balance beam experiment. Figure 11 E represents the results of the rotor fatigue test.

[0097] Figure 11The results of A showed that in the water maze experiment, AD mice treated with NK-aEVs had increased total swimming distance and time in the quadrant where the platform was located, and a shortened latency to enter the platform quadrant, indicating that their ability to find the platform was improved and their spatial learning and memory abilities were increased. Figure 11 The results of B showed that, in the absence of a platform, NK-aEVs treatment could effectively improve the long-term spatial memory retention ability of AD, mainly by increasing the number of times AD crossed the original platform quadrant, increasing the movement distance and time in the target quadrant, increasing the number of times the target quadrant was entered, and shortening the latency time to enter the target quadrant. Figure 11 The results of C showed that AD mice had decreased forelimb grip strength, and NK-aEVs treatment restored AD mice's forelimb grip strength to some extent. Figure 11 The results of D showed that NK-aEVs treatment shortened the time AD mice took to cross the balance beam to some extent. Figure 11 The results from E showed that NK-aEVs treatment significantly prolonged the time AD mice spent on the rotarod in the rotarod experiment. These results demonstrate that NK-aEVs treatment can significantly improve the learning, cognitive, and memory abilities of AD mice, enhance their limb motor skills, and has a definite therapeutic effect on AD.

[0098] Example 9: Immunomodulatory effects of NK-aEVs on tumor-bearing mice

[0099] Spleens from various model mice in Examples 5-7 and tumors from tumor-bearing mice were prepared into single-cell suspensions. Red blood cells were lysed and removed from the samples using red blood cell lysis buffer. Then, 5 × 10⁶ cells were collected. 6 NK cells were stained with fluorescent antibody using flow cytometry. The NK cell antibody was labeled with CD3. - / NKp46 + After staining, flow cytometry was used to detect NK cell populations, and the results are as follows: Figure 12 As shown.

[0100] Figure 12 A represents the ratio of NK cells in the spleen and tumor in tumor-bearing mice in combination with NK-aEVs and etoposide. Figure 12 B represents the proportion of NK cells in the spleen of irradiated acutely aged mice. Figure 12 C represents the proportion of NK cells in the spleen of naturally aging mice. Figure 12 D represents the proportion of NK cells in the spleen of Alzheimer's disease mice.

[0101] Figure 12The results showed that when combined with the chemotherapy drug etoposide to treat tumors, both NK-aEVs alone and in combination with etoposide significantly increased the level of NK cells in the spleen of mice and also increased the level of NK cell infiltration in the tumor. Figure 12 The results of B showed that irradiation-induced acute aging led to a significant decrease in the level of NK cells in the spleen of mice, and NK-aEVs treatment could restore the level of NK cells in the spleen and improve the immune status of aging mice. Figure 12 The results also showed that the proportion of NK cells in the spleen of normally aging mice was extremely low, corresponding to the low immune function of aged mice. NK-aEVs treatment significantly increased the level of NK cells in the spleen. Figure 12 The results showed that the NK cell level in the spleen of Alzheimer's disease mice was significantly lower than that in normal mice, and the NK cell level was significantly increased after treatment with NK-aEVs. These results collectively confirm that treatment with NK-aEVs can significantly restore the NK cell level in the spleen of aging and age-related Alzheimer's disease mice, and improve the immune function of aging mice.

[0102] In summary, the method described in this application, which uses a combination of traditional Chinese medicine active small molecules and amplification to amplify high-yield, high-purity NK cells from PBMCs, and then uses extrusion and / or ultrasound methods to prepare NK cells into natural killer cell-derived artificial extracellular vesicles (NK-aEVs) with a particle size of 50-200 nM, exhibits excellent stability. NK-aEVs can effectively eliminate senescent cells in vitro, including senescent tumor cells and senescent epithelial cells. In tumor-bearing mice, NK-aEVs, combined with etoposide, can more effectively control tumor growth and effectively eliminate senescent tumor cells induced by etoposide treatment. In naturally aging mice and irradiated acutely aging mice, it can effectively improve the aging state. In Alzheimer's disease mice, it can also effectively improve cognitive abilities. Simultaneously, it can increase the proportion of NK cells in the spleen of mice and enhance the immune function of aging mice. Therefore, the NK cell artificial extracellular vesicle anti-aging preparation obtained by using the combined amplification method of active small molecules of traditional Chinese medicine to amplify NK cells described in this application has strong clinical application prospects and value in the fields of anti-tumor and anti-aging, and is expected to be developed into a related senescent cell scavenger and aging regulator for anti-tumor, anti-aging, and anti-Alzheimer's disease.

[0103] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A composition for in vitro expansion culture medium of NK cells, characterized in that, The medium includes a basal culture medium and amplification inducing factors added to the basal culture medium. The amplification inducing factors include IL-2, IL-12, nicotinamide, and one or more small active molecules of traditional Chinese medicine selected from the group consisting of curcumin, tanshinone IIA, quercetin, azadirachtin, hesperidin, thymol, resveratrol, astragaloside IV, bufotenol, oxymatrine, methyl gallate, and ursolic acid 3,20-dibenzoic acid euphorbia terpenoid.

2. The culture medium composition according to claim 1, characterized in that, The amplification inducing factors include IL-2, IL-12, nicotinamide, curcumin, and tanshinone IIA; Preferably, in the culture medium composition, the concentration of IL-2 is 50~1000 IU / mL; the concentration of IL-12 is 2~200 ng / mL; the concentration of NAM is 1~100 μM; the concentration of curcumin is 0.2~5 μM; and the concentration of tanshinone IIA is 2~20 μM.

3. The culture medium composition according to claim 1 or 2, characterized in that, The culture medium composition does not contain animal serum.

4. A method for in vitro expansion of NK cells, characterized in that, Includes the following steps: Peripheral blood mononuclear cells (PBMCs) are mixed with feeder cells and cultured in serum-free medium in any one of the NK cell in vitro expansion culture media compositions according to any one of claims 1-3 to obtain NK cells with a purity >95%.

5. An artificial extracellular vesicle for NK cells, characterized in that, Prepared from NK cells obtained by the in vitro expansion method according to claim 4; Preferably, the artificial extracellular vesicles of NK cells have a particle size of 50~200nm, a Zeta potential of -20~-5 mV, and express CD63, CD81 and CD9 protein markers on their surface.

6. The NK cell artificial extracellular vesicle according to claim 5, characterized in that, The artificial extracellular vesicles of NK cells were prepared by high-pressure extrusion and / or ultrasonic lysis. Preferably, the high-pressure extrusion method uses a 0.2~10.0 μm filter membrane to perform gradient extrusion of the amplified NK cells, followed by gradient centrifugation; the ultrasonic lysis method involves lysing the amplified NK cells at 20~30 kHz, 800~1000 W, and 4~8℃ for 5~8 min, followed by gradient centrifugation. Preferably, the gradient centrifugation is performed by sequentially centrifuging at 300g for 5-15 min, at 2000g for 5-15 min, and at 100,000-150,000g for 80-120 min to collect the precipitate.

7. The use of the NK cell artificial extracellular vesicles according to claim 5 or 6 in the preparation of antitumor products; Preferably, the antitumor product is used to eliminate senescent tumor cells induced by radiotherapy and chemotherapy, inhibit tumor growth, enhance the effect of chemotherapy, or be used in combination with chemotherapy drugs.

8. The use of the NK cell artificial extracellular vesicles according to claim 5 or 6 in the preparation of aging regulators or anti-aging products; Preferably, the aging regulator or anti-aging product is used to improve natural aging, radiation-induced acute aging, or to remove senescent cells in tissues.

9. Use of the NK cell artificial extracellular vesicles according to claim 5 or 6 in the preparation of products for the treatment or improvement of Alzheimer's disease.

10. A product for treating tumors, aging, or Alzheimer's disease, characterized in that, Contains an effective amount of the NK cell artificial extracellular vesicles according to claim 5 or 6; Preferably, the final concentration of the NK cell artificial extracellular vesicles used is 25~100 μg / mL.

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