Method for preparing nk cell-derived nanovesicles and uses thereof

Abstract

The application provides a preparation method of NK cell-derived nanovesicles and application thereof. Specifically, the application provides NK cell-derived nanovesicles (NK-NVs) which are similar to the characteristics and functions of NK cell-derived extracellular vesicles by an extrusion method / stress gradient membrane remodeling technology, and also provides a drug composition, a drug delivery system and corresponding use of the NK-NVs as a delivery carrier for delivering an antitumor drug. The preparation method of the application can mass-produce NK-NVs and kill various types of tumor cells. The NK-NVs have multiple advantages in drug delivery, so that they are expected to be a new choice for tumor immunotherapy, especially for tumor cells with drug resistance, and can significantly improve the sensitivity to chemotherapeutic drugs, and have a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a method for preparing NK cell-derived nanovesicles and their applications. Background Technology

[0002] Natural killer (NK) cells are cytotoxic lymphocytes that play a crucial role in the innate immune response, capable of eliminating viral-infected abnormal cells and tumor cells without recognizing specific antigens. As the body's first line of defense against tumors, NK cells have shown great potential in cancer treatment through adoptive infusion, achieving considerable efficacy in hematological malignancies. However, NK cell therapy is limited by its ability to reach the tumor site and the immunosuppression of the tumor microenvironment, leading to a reduced tumor-killing capacity. Furthermore, the low number of NK cells in peripheral blood or umbilical cord blood makes it difficult to obtain large-scale expansion of NK cells. Additionally, the application of NK cell therapy is constrained by storage and cell transport conditions.

[0003] NK cell-derived extracellular vesicles (NK-EVs) are nanoscale vesicles actively secreted by NK cells. They contain various bioactive substances such as proteins, nucleic acids, and lipids, and can selectively deliver these bioactive substances to recipient cells via body fluid circulation. NK-EVs possess antitumor activity due to carrying cytotoxic substances such as perforin, granzymes, and TNF-α, and exhibit unique biocompatibility and high permeability, attracting significant research interest in tumor immunotherapy. Compared to the limitations of NK cell therapy for tumors, NK-EVs can provide another option for cancer treatment. Furthermore, purified NK-EVs show higher safety in clinical treatment, can be used as a transport vehicle for other antitumor drugs, and offer advantages in storage and transportation, thus possessing immense application value in cancer therapy.

[0004] However, the advantages of NK-EVs are limited in application due to the time-consuming and labor-intensive nature of their conventional preparation and purification methods, which rely on large centrifugation equipment. Furthermore, NK-EVs prepared by different methods vary significantly in quality and functional performance, making standardization difficult. These limitations further hinder the widespread application of NK-EVs.

[0005] Therefore, there is an urgent need in this field to develop a method for the rapid and large-scale preparation of NK-EVs, thereby providing a solid foundation for the widespread application of NK-EVs. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing NK cell-derived nanovesicles (NK-NVs) and their applications.

[0007] In a first aspect of the present invention, a method for preparing NK cell-derived nanovesicles (NK-NVs) is provided, the method comprising the steps of:

[0008] (S1) Provides NK cells to be treated;

[0009] (S2) The NK cells to be processed were subjected to ultrasonic disruption.

[0010] (S3) Centrifuge the NK cells after ultrasonic disruption, discard large cell fragments or intact cells, and collect the supernatant.

[0011] (S4) Use a squeezer to push the supernatant collected in step (S3) through an 8-15 μm polycarbonate membrane for 7-13 cycles, and collect the liquid after squeezing.

[0012] (S5) Use a squeezer to push the squeezed liquid collected in step (S4) through a 3-7 μm polycarbonate membrane for 7-13 cycles, and collect the squeezed liquid;

[0013] (S6) Using a squeezer, push the squeezed liquid collected in step (S5) through a 0.22–3 μm polycarbonate membrane for 7–13 cycles, and collect the squeezed liquid; and

[0014] (S7) Centrifuge the squeezed liquid collected in step (S6) and collect the supernatant to obtain NK cell-derived nanovesicles (NK-NVs).

[0015] In another preferred embodiment, step (S1) further includes the step of: performing cell counting.

[0016] In another preferred embodiment, the number of NK cells to be treated in step (S1) is 10. 4 ~10 12 Cells, preferably 10 5 ~10 11 Cells, preferably 10 6 ~10 10 Cells, for example, 10 6 10 cells, 10 7 10 cells, 10 8 10 cells, 10 9 10 cells, 10 10 Each cell.

[0017] In another preferred embodiment, the ultrasonic conditions in step (S2) are 40% power, on for 5 seconds, off for 5 seconds, 3 minutes, and ice bath disruption.

[0018] In another preferred embodiment, the centrifugation conditions in step (S3) are 300g to 700g for 5 to 15 minutes, preferably 500g for 10 minutes.

[0019] In another preferred embodiment, the pore size of the polycarbonate membrane in step (S4) is 9–13 μm, more preferably 10–12 μm, and even more preferably 10 μm.

[0020] In another preferred embodiment, step (S4) involves extrusion for 8 to 12 cycles, more preferably 9 to 11 cycles, and even more preferably 10 cycles.

[0021] In another preferred embodiment, the pore size of the polycarbonate membrane in step (S5) is 4 to 6 μm, preferably 5 μm.

[0022] In another preferred embodiment, step (S5) involves extrusion for 8 to 12 cycles, more preferably 9 to 11 cycles, and even more preferably 10 cycles.

[0023] In another preferred embodiment, the pore size of the polycarbonate membrane in step (S6) is 0.47 to 2 μm, preferably 1 μm.

[0024] In another preferred embodiment, step (S6) involves extrusion for 8 to 12 cycles, more preferably 9 to 11 cycles, and even more preferably 10 cycles.

[0025] In another preferred embodiment, the centrifugation conditions in step (S7) are 8000g to 12000g for 20 to 40 minutes, preferably 10000g for 30 minutes.

[0026] In another preferred embodiment, the method further includes the step of:

[0027] (S8) Add 2% trehalose to the NK cell-derived nanovesicles (NK-NVs) obtained in step (S7) and store at -80°C.

[0028] In another preferred embodiment, the method further includes the step of characterizing and / or functionally identifying the NK cell-derived nanovesicles (NK-NVs) obtained in step (S7).

[0029] In a second aspect of the invention, an NK cell-derived nanovesicle (NK-NVs) prepared by the method described in the first aspect of the invention is provided.

[0030] In a third aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0031] (Z1) NK cell-derived nanovesicles (NK-NVs) as described in the second aspect of the present invention; and

[0032] (Z2) Pharmaceutically acceptable carrier.

[0033] In a fourth aspect of the invention, a drug delivery system is provided, the drug delivery system comprising:

[0034] (Z1) NK cell-derived nanovesicles (NK-NVs) as described in the second aspect of the present invention; and

[0035] (Z2) Loaded drug.

[0036] In another preferred embodiment, the loaded drug is selected from the group consisting of: drugs (including but not limited to doxorubicin, gemcitabine, vincristine, cisplatin, cyclophosphamide, paclitaxel, metformin, sorafenib, etc.); nucleic acids (such as DNA, RNA, including but not limited to cDNA, miRNA, mRNA, siRNA, etc.); proteins (including but not limited to cytokines, insulin, etc.), or combinations thereof.

[0037] In another preferred embodiment, the loaded drug is doxorubicin.

[0038] In another preferred embodiment, the concentration of doxorubicin is 0.01–100 μg / mL, more preferably 0.1–50 μg / mL, even more preferably 1–25 μg / mL or 2.5–10 μg / mL, and most preferably 1.5 μg / mL or 5 μg / mL.

[0039] In a fifth aspect of the invention, a method for preparing a drug delivery system as described in the fourth aspect of the invention is provided, the method comprising the steps of:

[0040] (S1) Prepare a mixture of NK cell-derived nanovesicles (NK-NVs) as described in the second aspect of the present invention and loaded with a drug at a ratio of 1:1000 to 1000:1 (preferably 1:500 to 500:1, 1:200 to 200:1, 1:100 to 100:1, 1:10 to 10:1, 1:5 to 5:1; more preferably 1:3 to 3:1; most preferably 1:1);

[0041] (S2) Using an extruder, the mixture is extruded through a 100-300 nm (preferably 150-250 nm, more preferably 200 nm) polycarbonate membrane 10-30 times (preferably 15-25 times, more preferably 20 times), and the extruded liquid is collected; and

[0042] (S3) The squeezed liquid collected in step (S2) is centrifuged to collect the precipitate, thereby obtaining a drug-loaded system as described in the fourth aspect of the present invention.

[0043] In another preferred embodiment, the centrifugation conditions in step (S3) are 80,000g to 120,000g for 50 to 100 minutes, more preferably 90,000g to 110,000g for 60 to 80 minutes, and even more preferably 100,000g for 70 minutes.

[0044] In a sixth aspect of the invention, the use of NK cell-derived nanovesicles (NK-NVs) as described in the second aspect of the invention, a pharmaceutical composition as described in the third aspect of the invention, or a drug delivery system as described in the fourth aspect of the invention in the preparation of a medicament is provided.

[0045] In another preferred embodiment, the drug is a drug for the prevention and / or treatment of tumors.

[0046] In another preferred embodiment, the tumor includes: a solid tumor, a hematologic malignancy, or a combination thereof.

[0047] In another preferred embodiment, the tumor is selected from the group consisting of: ovarian cancer, pancreatic cancer, head and neck cancer, lung cancer (such as non-small cell lung cancer), kidney cancer, bladder cancer, colorectal cancer, melanoma, liver cancer, stomach cancer, prostate cancer, uterine cancer, cervical cancer, osteosarcoma, leukemia, lymphoma, myeloma, or combinations thereof.

[0048] In another preferred embodiment, the tumor is ovarian cancer and / or pancreatic cancer.

[0049] In a seventh aspect of the invention, a method for preventing and / or treating a condition is provided, the method comprising the steps of administering to a subject in need an NK cell-derived nanovesicle (NK-NVs) as described in a second aspect of the invention, a pharmaceutical composition as described in a third aspect of the invention, a drug delivery system as described in a fourth aspect of the invention, or a combination thereof.

[0050] In another preferred embodiment, the condition is a tumor.

[0051] In another preferred embodiment, the tumor includes: a solid tumor, a hematologic malignancy, or a combination thereof.

[0052] In another preferred embodiment, the tumor is selected from the group consisting of: ovarian cancer, pancreatic cancer, head and neck cancer, lung cancer (such as non-small cell lung cancer), kidney cancer, bladder cancer, colorectal cancer, melanoma, liver cancer, stomach cancer, prostate cancer, uterine cancer, cervical cancer, osteosarcoma, leukemia, lymphoma, myeloma, or combinations thereof.

[0053] In another preferred embodiment, the tumor is ovarian cancer and / or pancreatic cancer.

[0054] In another preferred embodiment, the subject is a human or a non-human mammal (such as a mouse, rat, rabbit, monkey, etc.).

[0055] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0056] Figure 1 The same number of NK cells (10) were shown. 10 The number of particles (in the case of) NK-EVs prepared by ultra-high speed centrifugation and NK-NVs prepared by extrusion / stress gradient film reshaping technology Figure 1 A) and total protein ( Figure 1 Comparison of B).

[0057] Figure 2 Transmission electron microscopy images of NK-NVs are shown, scale bar = 200 nm.

[0058] Figure 3 The particle size distribution of NK-NVs was shown using a nano-Coulter particle size analyzer.

[0059] Figure 4 The expression of extracellular vesicle (EV) markers (Calnexin, Alix, TSG101, and CD9), NK cell marker (CD56), and cytotoxic proteins (perforin and granzyme B) on NK-NVs was shown. Among them, NK cell lysate ( Figure 4 NK cells were used as a control.

[0060] Figure 5 The study demonstrated the cytotoxic effect of NK-NVs on human ovarian epithelial cells IOSE80.

[0061] Figure 6 The killing effects of NK-NVs and NK-EVs on different tumor cells were demonstrated.

[0062] Figure 7 The study compared the killing effects of NK-NVs on different tumor cells after co-incubation for 24 h and 48 h.

[0063] Figure 8 It shows different time points, Panc-1 ( Figure 8 A and Figure 8 B) and SKOV3 cells ( Figure 8 C and Figure 8D) Uptake of NK-NVs (Blue: DAPI-stained cell nuclei; Green: DiO-stained NK-NVs; Scale bar = 50 μm, local magnification scale bar = 20 μm; Uptake rate was obtained by calculating the proportion of green fluorescence).

[0064] Figure 9 The comparison of drug loading rates of NK-NVs-DOX prepared by different drug loading methods is shown.

[0065] Figure 10 The comparison of particle size and recovery yield of NK-NVs-DOX prepared by different drug loading methods is shown.

[0066] Figure 11 The drug loading rates of NK-NVs-DOX prepared at different temperatures and by different methods are compared.

[0067] Figure 12 The drug release rates of NK-NVs-DOX under different pH conditions are compared.

[0068] Figure 13 The study demonstrated the cytotoxic effects of NK-NVs-DOX prepared using different drug delivery methods on various tumor cells. Detailed Implementation

[0069] Through extensive and in-depth research and numerous screenings, the inventors have, for the first time, creatively discovered that NK-NVs prepared with specific polycarbonate membrane pore sizes and extrusion cycles (e.g., 9-13 μm polycarbonate membranes, 4-6 μm polycarbonate membranes, and 0.47-2 μm polycarbonate membranes, extruded for 9-11 cycles) exhibit superior technical performance. Utilizing the extrusion / stress gradient membrane remodeling technology of this invention, NK-NVs can be rapidly and massively prepared without relying on ultra-high-speed centrifuges. The prepared NK-NVs possess antitumor activity, and their advantages in drug delivery (e.g., good biocompatibility and the ability to be targeted for modification) make them a promising new option for tumor immunotherapy with broad application prospects. This invention was completed based on these findings.

[0070] the term

[0071] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0072] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0073] The term “administration” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0074] NK cell-derived nanovesicles (NK-NVs)

[0075] Recently, researchers have been trying to produce high-quality extracellular vesicles (EVs) using different physical or chemical methods, hoping to get closer to clinical translation in terms of scalability and efficiency.

[0076] The terms "extrusion method" and "stress gradient membrane remodeling technology" are used interchangeably, both referring to a general method for generating large quantities of nanovesicles (NVs) from living cells. Through mechanical extrusion, donor cells can release large amounts of cell-derived NVs in a short period of time. The formation of cell-derived NVs is due to the disruption of the cell membrane by shear or frictional forces, which remodels the lipid bilayer into vesicles within seconds. NK cell-derived NVs (NK-NVs) prepared by the extrusion method / stress gradient membrane remodeling technology share the same cell membrane components as the source NK cells, contain the contents of the source cells, and have similar characteristics and functions to NK-EVs, but the preparation method is simpler and yields higher quantities.

[0077] In the technical process of preparing NK-NVs using the extrusion / stress gradient membrane remodeling technique, parameters such as the pore size of the polycarbonate membrane or nanoporous membrane used for mechanical extrusion and the number of extrusion cycles have a significant impact on the size, uniformity, and stability of the nanovesicles. In this study, through extensive screening and verification, the inventors discovered that NK-NVs prepared with specific polycarbonate membrane pore sizes and extrusion cycles (e.g., 9-13 μm polycarbonate membranes extruded for 9-11 cycles, 4-6 μm polycarbonate membranes extruded for 9-11 cycles, and 0.47-2 μm polycarbonate membranes extruded for 9-11 cycles; screening data for other polycarbonate membrane pore sizes and extrusion cycles are not shown due to space limitations) exhibit better technical performance.

[0078] Compared with NK-EVs prepared by ultracentrifugation, the preparation of NK-NVs using the extrusion / stress gradient membrane remodeling technique is more time-saving, labor-saving, and yields higher quantities. The number of NK-NV particles and protein yield can be increased by approximately 402.18 and 325.76 times, respectively, for the same cell number. Transmission electron microscopy analysis showed that NK-NVs have a clear lipid bilayer membrane structure and a cup-shaped morphology. The average particle size of NK-NVs, as detected by a nanocoulter particle size analyzer, is approximately 78 ± 29.8 nm, similar to NK-EVs. NK-NVs also express the EV marker proteins Alix, TSG101, and CD9, while the EV-negative marker Calnexin was only detected in NK92MI cell lysates. Western blot analysis confirmed the presence of the NK cell marker CD56 in NK-NVs, and they also express NK cell cytotoxic proteins (granzyme B and perforin). CCK-8 experiments demonstrated that NK-NVs exhibited cytotoxic effects against tumor cell lines from various sources (e.g., ovarian and pancreatic cancer). Similar to NK-EVs, NK-NVs are also an excellent tool for drug delivery. The inventors further evaluated the drug delivery capabilities of NK-NVs. Doxorubicin (DOX) was loaded into NK-NVs (NK-NVs-DOX) using different drug loading methods, including co-incubation, sonication, extrusion, and electroporation. The drug loading, encapsulation efficiency, particle size, and tumor-killing effect of NK-NVs-DOX were comprehensively evaluated, and the extrusion method was determined to be the optimal method for preparing NK-NVs-DOX. After NK-NVs were loaded with DOX, they enhanced the sensitivity of various tumor cells to DOX, especially significantly improving the sensitivity of chemotherapy-resistant tumor cells to chemotherapeutic drugs.

[0079] Therapeutic applications

[0080] This invention provides NK-NVs prepared by extrusion / stress gradient film remodeling technology, and the therapeutic applications of pharmaceutical compositions and drug delivery systems containing NK-NVs.

[0081] On the one hand, the NK-NVs of the present invention have almost no effect on the activity of normal cells and exhibit good safety. On the other hand, when co-incubated with various tumor cells (especially for 48 hours), the NK-NVs of the present invention show a similar killing effect on tumor cells as NK-EVs. The antitumor activity of the NK-NVs of the present invention is based on the uptake and internalization of NK-NVs by tumor cells, and this process is not limited to the type of tumor cell. Therefore, based on the disclosure of the present invention, those skilled in the art can understand that, based on a similar internalization process, various types of tumor cells can effectively uptake NK-NVs, thereby exerting a killing effect on tumor cells. In other words, based on the disclosure of the present invention, those skilled in the art can reasonably expect that the NK-NVs of the present invention can effectively kill various tumors / tumor cells.

[0082] The NK-NVs of the present invention can be formulated into pharmaceutical compositions with pharmaceutically acceptable carriers, excipients, diluents, etc.

[0083] The NK-NVs of this invention can also serve as an excellent tool for drug delivery, used for loading drugs, i.e., the drug loading system of this invention. When used to load the same drug, such as doxorubicin (DOX), also known as adriamycin, the drug loading system (NK-NVs-drug, such as NK-NVs-DOX) prepared by extrusion method shows advantages in drug loading rate, particle size, and recovery rate compared to co-incubation, electroporation, and sonication methods. After being loaded with drugs, the NK-NVs of this invention can enhance the sensitivity of various tumor cells to drugs, especially drug-resistant (e.g., chemotherapy-resistant) tumor cells, significantly improving their sensitivity to drugs (e.g., chemotherapy drugs).

[0084] It is worth noting that the drug delivery system of the present invention exhibits high stability, with minimal change in drug loading rate at both 4°C and -80°C. Furthermore, the drug release rate of the drug delivery system of the present invention is significantly higher in a pH 5.5 buffer than in a pH 7.4 buffer. Since pH 7.4 is similar to the blood environment, this indicates that the drug delivery system of the present invention possesses sufficient stability in the body fluid circulation, allowing it to retain more of the loaded drug before reaching the target organ (tumor site). Simultaneously, the higher drug release rate of the drug delivery system of the present invention at pH 5.5, given the acidic conditions of the tumor microenvironment, suggests that the drug delivery system of the present invention can release more loaded drug in the tumor microenvironment, increasing the effective drug concentration entering the tumor tissue. It should be noted that the above mechanisms of action depend on the properties of the drug delivery system itself and are not limited to tumor type. Therefore, the drug delivery system of the present invention can achieve the above effects for various types of tumors, namely, releasing more loaded drug in the tumor microenvironment and increasing the effective drug concentration entering the tumor tissue.

[0085] Main advantages of the invention

[0086] 1. Compared with NK-EVs prepared by ultracentrifugation, the extrusion / stress gradient membrane remodeling technology of the present invention is more time-saving and labor-saving for preparing NK-NVs, does not rely on ultracentrifuges and has a higher yield. The number of NK-NV particles and the protein yield prepared under the same cell number can be increased by about 402.18 and 325.76 times, respectively.

[0087] 2. Transmission electron microscopy analysis showed that the NK-NVs prepared in this invention have a clear lipid bilayer membrane structure and cup-shaped morphology, with an average particle size of about 78±29.8 nm, similar to NK-EVs.

[0088] 3. The NK-NVs prepared in this invention also express the marker proteins Alix, TSG101, and CD9 of EVs, as well as the NK cell marker CD56, and express cytotoxic proteins of NK cells (granzyme B and perforin). Furthermore, NK-NVs exhibit killing effects against tumor cell lines from different sources (e.g., ovarian cancer and pancreatic cancer).

[0089] 4. The NK-NVs of the present invention are a good tool for drug delivery. After encapsulating doxorubicin, they can enhance the sensitivity of various tumor cells to doxorubicin, especially for chemotherapy-resistant tumor cells, and can significantly improve their sensitivity to chemotherapy drugs.

[0090] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0091] Example 1: Preparation method of NK cell-derived nanovesicles

[0092] 1. Collect NK92 cells

[0093] Collect NK92 cells and transfer them to centrifuge tubes. Centrifuge at 350g for 5 min, discard the supernatant, resuspend in 5 mL of physiological saline, and perform cell counting (generally 10⁻⁶). 8 (cells).

[0094] 2. Ultrasonic cell disruption

[0095] (1) Centrifuge the counted cells at 350g for 5 minutes and discard the supernatant. Resuspend the cells in 4mL of physiological saline.

[0096] (2) NK92 cells were disrupted using an ultrasonic disruptor under the following conditions: power: 40%, on for 5 seconds, off for 5 seconds, 3 minutes, ice bath disruption. All cells were disrupted.

[0097] (3) Centrifuge at 500g for 10 minutes, discard large cell fragments or intact cells, and collect the supernatant.

[0098] 3. Preparation of nanovesicles

[0099] (1) Use a squeezer to push the supernatant through a 10μm polycarbonate membrane for 10 cycles and collect the squeezed liquid.

[0100] (2) Use a squeezer to push the squeezed liquid collected in the previous step through a 5μm polycarbonate membrane for 10 cycles and collect the squeezed liquid.

[0101] (3) Use a squeezer to push the squeezed liquid collected in the previous step through a 1μm polycarbonate membrane for 10 cycles and collect the squeezed liquid.

[0102] (4) Collect the squeezed liquid, centrifuge at 10,000g for 30min, collect the supernatant, add 2% trehalose, and store at -80℃.

[0103] Example 2: Preparation of NK-EVs by Ultra-high Speed ​​Centrifugation

[0104] (1) NK92 cells were cultured in conditioned medium containing 5% exosome-free FBS for 48 h. The supernatant was centrifuged at 300 g and 2000 g for 10 min at each time to remove cells and large vesicles.

[0105] (2) Centrifuge the supernatant obtained in the previous step at 10000g for 30min and at 100000g for 70min twice, and collect the precipitate, i.e. NK-EVs.

[0106] (3) Resuspend NK-EVs in PBS, add 2% trehalose, and store at -80℃.

[0107] Example 3: Characterization of NK-NVs

[0108] 1. Determination of particle number and total protein:

[0109] The particle number and total protein content of NK-NVs prepared by extrusion / stress gradient membrane remodeling technology and NK-EVs prepared by ultra-high speed centrifugation were determined, respectively.

[0110] The particle size distribution and number of NK-NVs were measured using a nano-Coulter particle size analyzer. The total protein concentration was measured using the BCA protein assay.

[0111] like Figure 1 As shown, the data for the NK-NVs group respectively show the particle number of NK-NVs prepared by extrusion / stress gradient film reshaping technology ( Figure 1 A) and total protein ( Figure 1 B); the data for the NK-EVs group show the particle number of NK-EVs prepared by ultra-high speed centrifugation ( Figure 1 A) and total protein ( Figure 1 B).

[0112] according to Figure 1 A comparison of data from the NK-NVs group and the NK-EVs group shows that the 10... 10 Each NK cell can produce 4.11 × 10 14 ±5.09×10 12 The sample yielded 16.17 ± 0.26 mg of protein, representing a 402.18-fold increase in particle yield and an approximately 325.76-fold increase in protein yield compared to exosomes prepared by ultracentrifugation from cell culture medium (i.e., NK-EVs prepared by ultracentrifugation). These results indicate that the extrusion / stress gradient membrane remodeling technique can yield NK-NVs with significantly improved particle and protein yields compared to ultracentrifugation.

[0113] 2. Morphological observation of NK-NVs using transmission electron microscopy:

[0114] NK-NVs prepared by extrusion / stress gradient film reshaping technology were observed under a transmission electron microscope. For example... Figure 2 As shown in the transmission electron microscope images, similar to NK-EVs, NK-NVs have a clear lipid bilayer membrane structure and a cup-shaped morphology.

[0115] 3. Average particle size of NK-NVs was determined using a nano-Coulter particle size analyzer:

[0116] The particle size distribution and number of NK-NVs were measured using a nano-Coulter particle size analyzer. The samples were diluted to 200 μL and used in nanoporous chips with a diameter of 60–200 nm.

[0117] like Figure 3 As shown, the average particle size of NK-NVs prepared using the nanocoulter extrusion / stress gradient film remodeling technique is approximately 78 ± 29.8 nm.

[0118] 4. Detection of protein biomarkers for NK-NVs:

[0119] Total protein concentration was measured using the BCA protein assay.

[0120] like Figure 4As shown, Western blot analysis identified characteristic proteins of NK-NVs, confirming the presence of typical EV marker proteins Alix, TSG101, and CD9 in NK-NVs, while the negative marker Calnexin was only detected in NK92MI cell lysates. Furthermore, NK-NVs also contained NK cell markers CD56 and cytotoxic proteins perforin and granzyme B.

[0121] In summary, based on measurements of concentration, size, morphology, and protein labeling, the NK-NVs prepared from NK92 cells in this invention have advantages such as high quality and large yield.

[0122] Example 4: Cytotoxic effects of NK-NVs

[0123] Human ovarian epithelial cells IOSE80 were used at a density of 2 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates and co-cultured with different concentrations of NK-NVs (10, 20, 40, 60, 80, 100 μg / mL) at 37°C for 24 hours. 10 μL of CCK-8 reagent (Meilun Biotechnology, China, catalog number: MA0218) was added to the cultured cells, and after incubation at 37°C for 2 hours, the optical density (OD) was measured at 450 nm. Three replicates were performed, and three independent experiments were conducted. Cell viability was calculated using the following formula:

[0124] Cell viability % = (OD 实验组 –OD 空白组 ) / (OD 对照组 –OD 空白组 )×100%

[0125] like Figure 5 As shown in the CCK-8 experiment, 100 μg / mL of NK-NVs had almost no effect on the cell viability of human ovarian epithelial cells IOSE80, demonstrating that the NK-NVs prepared in this invention have good safety.

[0126] Ovarian cancer cells (SKOV3, COC1 / DDP, A2780) and pancreatic cancer cells (Panc-1) were cultured at 2 × 10⁻⁶ cells per well. 4 Cells were seeded in 96-well plates and co-cultured with different concentrations of NK-NVs (10, 20, 40, 60, 80, 100 μg / mL) at 37°C for 24 or 48 hours. 10 μL of CCK-8 reagent was added to the cultured cells, and after incubation at 37°C for 2 hours, the optical density (ODs) was measured at 450 nm.

[0127] Three replicates per well, with three independent experiments. The formula for calculating cell viability is as follows:

[0128] Cell viability % = (OD 实验组 –OD 空白组 ) / (OD 对照组 –OD 空白组 )×100%

[0129] like Figure 6 As shown, after co-incubation with human ovarian cancer cell lines (COC1 / DDP, A2780, and SKOV3) and human pancreatic cancer cell line (Panc-1) for 24 h, NK-EVs and NK-NVs at a concentration of 60 μg / mL showed significant killing effects on COC1 / DDP, SKOV3, and Panc-1 cells, but the killing effect on A2780 cells was lower.

[0130] like Figure 7 As shown, when the co-incubation time of NK-NVs with tumor cells was extended to 48 hours, the cell activity of tumor cells was significantly reduced, and the killing effect on A2780 was significantly enhanced.

[0131] Example 5: Tumor cell uptake of NK-NVs

[0132] NK-NVs were labeled with DiO, and then centrifuged using a 100 kDa ultrafiltration tube to remove free dye. SKOV3 and Panc-1 cells were co-cultured with DiO-labeled NK-NVs, washed with PBS, fixed with 4% paraformaldehyde, and the cell nuclei were stained with 10 μg / mL DAPI. All images were acquired using laser scanning confocal microscopy (Nikon, AX), and the fluorescence intensity of intracellular NK-NVs (DiO dye labeled, green) was analyzed using Image J 1.53e software.

[0133] like Figure 8 As shown in the confocal microscopy images, NK-NVs (labeled with DiO dye, green) entered Panc-1 cells and SKOV3 cells (labeled with DAPI dye in the nucleus, blue). At 3 hours, the uptake efficiency of NK-NVs by tumor cells was lowest, with internalization gradually increasing over time. At 24 hours, a large amount of NK-NVs was observed to have been taken up by tumor cells. These results indicate that the uptake of NK-NVs by tumor cells is not limited to any particular type of tumor cell; based on a similar internalization process, various types of tumor cells can effectively take up NK-NVs.

[0134] Example 6: Preparation of NK-NVs-DOX by Different Methods

[0135] Doxorubicin (DOX) was diluted with physiological saline for later experimental use. The concentration of DOX was analyzed using a Synergy H1 (BioTek Instruments) microplate reader, and the absorbance of the DOX solution at 480 nm was measured. A standard curve was plotted based on the absorbance data. The concentration of DOX for subsequent experiments was calculated based on the standard curve. In the drug loading experiment, NK-NVs and DOX were prepared as a mixture at a 1:1 concentration ratio.

[0136] (1) Co-incubation method: Incubate the mixture at 37°C for 1 hour.

[0137] (2) Electroporation method: The mixture is pulsed at 450V and 120ms using an electroporator.

[0138] (3) Extrusion method: The mixture is extruded through a 200nm polycarbonate membrane 20 times using an extruder.

[0139] (4) Ultrasonic method: Use an ultrasonic disruptor, 30% amplitude, 20s on / off, 2min ice bath ultrasound.

[0140] After loading, the mixtures obtained in (1)-(4) were centrifuged at 100,000g for 70 min, and the precipitate, i.e., NK-NVs-DOX, was collected and resuspended in 50 μL PBS for subsequent experimental analysis. The supernatant (containing free DOX) was collected and the absorbance of the supernatant was detected at a wavelength of 480 nm using an ELISA reader. The concentration of unloaded DOX was calculated, and the drug loading rate was calculated according to the formula:

[0141] Drug loading rate % = (Total drug mass – Unloaded drug mass) / Total mass of NK-NVs × 100%

[0142] like Figure 9 As shown, the drug loading rates of NK-NVs-DOX prepared by different drug loading methods are compared. Among them, the drug loading rates of NK-NVs-DOX prepared by electroporation and extrusion are the highest, while the drug loading rate of NK-NVs-DOX prepared by co-incubation is the lowest.

[0143] The particle size distribution and number of NK-NVs-DOX were measured using a nano-Coulter particle size analyzer. The sample was diluted to 200 μL and nanoporous chips with diameters of 60–200 nm and 150–500 nm were used. The recovery rate was calculated by dividing the number of recovered NK-NVs-DOX particles by the number of added NK-NVs particles.

[0144] like Figure 10As shown, the particle size and recovery yield of NK-NVs-DOX prepared by different drug loading methods are compared. In terms of particle size, the particle size of NK-NVs-DOX prepared by different methods is not significantly different, all being less than 100 nm; in terms of recovery yield, the NK-NVs-DOX prepared by extrusion method has a higher recovery rate, while the recovery rate of electroporation method is the lowest.

[0145] After comprehensively comparing drug loading rate, particle size, and recovery rate, extrusion loading is considered the optimal preparation method.

[0146] Example 7: Stability evaluation of NK-NVs-DOX under different conditions

[0147] 100 μg / mL NK-NVs-DOX prepared by different methods were stored at 4℃ or -80℃ for 28 days. Samples were taken at different time points (1, 3, 5, 7, 14, 28 days) to detect the drug loading rate of NK-NVs-DOX and analyze the effect of different temperatures on the drug loading rate of NK-NVs-DOX.

[0148] 100 μg / mL NK-NVs-DOX was dispersed in dialysis bags containing PBS (molecular weight cutoff: 8000-14000). The dialysis bags were immersed in centrifuge tubes (50 mL) containing 20 mL of PBS (pH 5.5 and pH 7.4), ensuring the bags were completely covered. The tubes were then clamped on a rotary shaker and rotated at 70 rpm in a 37°C oven, protected from light. Samples were collected at different time points (0, 0.5, 1, 2, 3, 4, 5, 6, 12, 24, and 48 h), and replenished with an equal volume of PBS to maintain the conditions. The DOX release from the samples was measured at 480 nm using a microplate reader. All experiments were performed in triplicate.

[0149] like Figure 11 As shown, within 28 days, regardless of the method used to prepare NK-NVs-DOX, the drug loading rate did not change significantly under conditions of 4℃ and -80℃, indicating that NK-NVs-DOX has high stability.

[0150] like Figure 12 As shown, NK-NVs-DOX exhibited rapid initial release within 0.5 hours at pH 5.5 and pH 7.4 and 37°C. However, the release rate of DOX in the pH 5.5 buffer (30.45±3.12) was significantly higher than that in the pH 7.4 buffer (19.82±3.21). Subsequently, the release of DOX increased slowly over time, reaching a plateau at 12 hours, at which point the release rate in the pH 5.5 buffer was 86.18±3.12, approximately 30% higher than the release rate in the pH 7.4 buffer (50.91±3.93).

[0151] Since the pH value of 7.4 is similar to that of the blood environment, it suggests that NK-NVs-DOX has sufficient stability in the body fluid circulation, allowing it to retain more DOX before reaching the target organ. Simultaneously, NK-NVs-DOX exhibits a higher drug release rate at pH 5.5. Due to the acidic conditions of the tumor microenvironment, this result enhances the release of more DOX within the tumor microenvironment, increasing the effective drug concentration entering tumor tissue. It should be noted that the above mechanism of action depends on the properties of NK-NVs-DOX itself and is not limited to tumor type. Therefore, NK-NVs-DOX can achieve the above effects against other types of tumors, namely, releasing more DOX in the tumor microenvironment and increasing the effective drug concentration entering tumor tissue.

[0152] Example 8: Cytotoxic effects of NK-NVs-DOX prepared by different drug loading methods on tumor cells

[0153] To evaluate the tumor cell killing effect of NK-NVs-DOX prepared by different loading strategies, SKOV3, COC1 / DDP, A2780, and Panc-1 cells were loaded at 2 × 10⁶ cells per well. 4 Cell viability was seeded into 96-well plates. Equal amounts of 10 μg / mL NK-NVs, 1.5 μg / mL DOX, and 1.5 μg / mL NK-NVs-DOX (DOX concentration 1.5 μg / mL) were added to the tumor cells, and the cells were co-cultured at 37°C for 24 hours. 10 μL of CCK-8 reagent (Meilun Biotechnology, China, catalog number: MA0218) was added to the cultured cells, and after incubation at 37°C for 2 hours, the optical density (OD) was measured at 450 nm. Three replicates were performed, and three independent experiments were conducted. The formula for calculating cell viability is as follows:

[0154] Cell viability % = (OD 实验组 –OD 空白组 ) / (OD 对照组 –OD 空白组 )×100%

[0155] like Figure 13 As shown, the killing effect of NK-NVs-DOX on tumor cancer cells (SKOV3, A2780, COC1 / DDP, and Panc-1) was determined using the CCK-8 assay. The results showed that NK-NVs-DOX prepared by different methods all exhibited killing effects on all tumor cells, and these effects were higher than those of free DOX and the NK-NVs group. Among these, NK-NVs-DOX prepared by the extrusion method and the electroporation method showed the highest tumor-killing effects.

[0156] Furthermore, as shown in Table 1, compared with the individual NK-NVs and free DOX groups, the NK-NVs-DOX prepared by the extrusion method showed higher killing effects against all four types of tumor cells, especially chemotherapy-resistant COC1 / DDP cells, than the sum of the two. This indicates that NK-NVs-DOX may make tumor cells more sensitive to the effects of DOX, producing a synergistic effect. Moreover, among the NK-NVs-DOX prepared by different drug loading methods, the NK-NVs-DOX prepared by the extrusion method exhibited the strongest killing effect on tumor cells.

[0157] Table 1

[0158]

[0159]

[0160] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing NK cell-derived nanovesicles (NK-NVs), characterized in that, The method includes the following steps: (S1) Provides NK cells to be treated; (S2) The NK cells to be processed were subjected to ultrasonic disruption. (S3) Centrifuge the NK cells after ultrasonic disruption, discard large cell fragments or intact cells, and collect the supernatant. (S4) Use a squeezer to push the supernatant collected in step (S3) through an 8-15 μm polycarbonate membrane for 7-13 cycles, and collect the liquid after squeezing. (S5) Use a squeezer to push the squeezed liquid collected in step (S4) through a 3-7 μm polycarbonate membrane for 7-13 cycles, and collect the squeezed liquid; (S6) Using a squeezer, push the squeezed liquid collected in step (S5) through a 0.22–3 μm polycarbonate membrane for 7–13 cycles, and collect the squeezed liquid; and (S7) Centrifuge the squeezed liquid collected in step (S6) and collect the supernatant to obtain NK cell-derived nanovesicles (NK-NVs).

2. The preparation method according to claim 1, characterized in that, In step (S2), the ultrasonic conditions are 40% power, on for 5 seconds, off for 5 seconds, 3 minutes, and ice bath disruption.

3. The preparation method according to claim 1, characterized in that, In step (S4), the pore size of the polycarbonate membrane is 9-13 μm, preferably 10-12 μm, and more preferably 10 μm.

4. The preparation method according to claim 1, characterized in that, In step (S5), the pore size of the polycarbonate membrane is 4-6 μm, preferably 5 μm.

5. The preparation method according to claim 1, characterized in that, In step (S6), the pore size of the polycarbonate membrane is 0.47–2 μm, preferably 1 μm.

6. An NK cell-derived nanovesicle (NK-NVs) prepared by the method of any one of claims 1-5.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (Z1) NK cell-derived nanovesicles (NK-NVs) as described in claim 6; and (Z2) Pharmaceutically acceptable carrier.

8. A drug delivery system, characterized in that, The drug delivery system includes: (Z1) NK cell-derived nanovesicles (NK-NVs) as described in claim 6; and (Z2) Loaded drug.

9. A method for preparing the drug delivery system as described in claim 8, characterized in that, The method includes the following steps: (S1) Prepare a mixture of NK cell-derived nanovesicles (NK-NVs) as described in claim 6 and loaded with a drug at a ratio of 1:1000 to 1000:1 (preferably 1:500 to 500:1, 1:200 to 200:1, 1:10 to 100:1, 1:10 to 10:1, 1:5 to 5:1; more preferably 1:3 to 3:1; most preferably 1:1); (S2) Using an extruder, the mixture is extruded through a 100-300 nm (preferably 150-250 nm, more preferably 200 nm) polycarbonate membrane 10-30 times (preferably 15-25 times, more preferably 20 times), and the extruded liquid is collected. and (S3) The squeezed liquid collected in step (S2) is centrifuged to collect the precipitate, thereby obtaining the drug-loaded system as described in claim 8.

10. Use of an NK cell-derived nanovesicle (NK-NVs) as described in claim 6, a pharmaceutical composition as described in claim 7, or a drug delivery system as described in claim 8 in the preparation of a medicament.

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