Application of exosome as irreversible ablation auxiliary preparation

Abstract

The invention discloses application of exosome as an irreversible ablation auxiliary preparation, and relates to the technical field of biology. According to the application of the exosome as the irreversible ablation auxiliary preparation, the exosome has a synergistic effect when acting on tumor cells in combination with microsecond pulses as the irreversible ablation auxiliary preparation, tumor cell apoptosis can be remarkably promoted, growth of the tumor cells can be inhibited, immune cell activation can be promoted, and the tumor cell apoptosis can be inhibited. Wide application prospects are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of exosomes as an auxiliary preparation for irreversible ablation. BACKGROUND

[0002] Exosomes are small vesicles with a diameter of about 30-150 nm secreted by living cells, with a typical lipid bilayer structure. Exosomes exist in cell culture supernatant, serum, plasma, saliva, urine, amniotic fluid and other biological fluids, carrying a variety of proteins, lipids, RNA and other important information. In recent years, with the continuous deepening of exosome research, its application has been involved in the fields of tumor treatment, medical basis and immunotherapy. NK cell-derived exosomes retain the immune function of the parent cell and can exert immune effects through the Fas / FasL pathway to kill tumor cells. They show good anti-tumor properties in vivo and in vitro. However, due to the complicated extraction process of exosomes and extremely low yield, how to effectively improve the disease treatment effect of exosomes is a difficult problem to be solved.

[0003] Chinese patent CN117402823A discloses the application of NK cell exosomes in tumor treatment. The preparation method of the NK cell exosomes comprises the following steps: 1) peripheral blood mononuclear cells are separated from the peripheral blood of a healthy subject; 2) the peripheral blood mononuclear cells separated in step 1) are sorted, and a cell suspension rich in NK cells is collected; 3) the cell suspension rich in NK cells obtained in step 2) is co-cultured with tumor cells in a culture medium for 7 days; the culture medium is selected from one of DMEM, RPMI 1640, F12K and B16F10-LUC; 4) after the co-culture is completed, CD3-CD56+CD16+ NK cells are further sorted by flow cytometry, and the cell supernatant is collected under the condition of 4°C and 2000xg; 5) the supernatant in step 4) is filtered by a filter membrane, and then subjected to ultrahigh-speed centrifugation under the condition of 4°C and 100000xg for 60 min; after the centrifugation is completed, the precipitate is collected; 6) the precipitate in step 5) is resuspended with 1xPBS, and then subjected to ultrahigh-speed centrifugation again under the condition of 4°C and 100000xg for 60 min; 7) the precipitate in step 6) is resuspended with 1xPBS, filtered by a filter membrane, and the filtrate collected is the NK cell exosomes. The prepared exosomes have good killing activity.

[0004] Chinese patent CN118987031A discloses the application of NK cell exosomes combined with NK cells in the treatment of triple-negative breast cancer. The scheme of using NK-Exos combined with NK cells to treat triple-negative breast cancer in this invention is proved to be able to significantly inhibit the growth of tumors in vivo and in vitro, and has safety in mice in vivo.

[0005] The application of exosomes as an auxiliary preparation for irreversible ablation has not been searched in the prior art, and the technical scheme of exosomes combined with irreversible ablation technology for treating tumors has not been disclosed. SUMMARY

[0006] The application aims to provide an application of exosomes as an auxiliary preparation for irreversible ablation.

[0007] To achieve the above application purposes, the technical scheme of the application is as follows. In one aspect, the application provides an application of exosomes as an auxiliary preparation for irreversible ablation.

[0008] Specifically, the exosomes include but are not limited to NK92 cell line-derived exosomes, umbilical cord blood NK cell-derived exosomes, immune cell-derived exosomes, stem cell-derived exosomes, epithelial cell-derived exosomes, neural cell-derived exosomes, endothelial cell-derived exosomes, and blood-derived exosomes.

[0009] Further, the immune cell-derived exosomes include B lymphocyte-derived exosomes, T lymphocyte-derived exosomes, dendritic cell-derived exosomes, and macrophage-derived exosomes.

[0010] Further, the stem cell-derived exosomes include mesenchymal stem cell-derived exosomes and hematopoietic stem cell-derived exosomes.

[0011] Further, the epithelial cell-derived exosomes include intestinal epithelial cell-derived exosomes, kidney epithelial cell-derived exosomes, and alveolar epithelial cell-derived exosomes.

[0012] Further, the exosomes include NK92 cell line-derived exosomes and umbilical cord blood NK cell-derived exosomes.

[0013] According to some embodiments of the application, the preparation method of the NK92 cell line-derived exosomes includes the following steps: culturing NK92 cells using NK92 cell complete medium, centrifuging, collecting supernatant, purifying exosomes through tangential flow filtration, and ultracentrifuging to obtain exosomes.

[0014] Specifically, the inoculation amount of the NK92 cells is 1-2×10^5 cells / mL, and is preferably 1.5×10^5 cells / mL.

[0015] Specifically, the culture method of the NK92 cells is culturing at 35-40℃ for 46-50 h, and is preferably culturing at 37℃ for 48 h.

[0016] Specifically, the centrifugation is at 1600-2000 rpm for 4-6 min, and further preferably at 1800 rpm for 5 min.

[0017] Specifically, the tangential flow filtration purification of the exosomes comprises the following steps: pre-filtering the supernatant, pumping the supernatant into a TFF system liquid storage tank, setting the tangential flow rate to 200 mL / min and the transmembrane pressure to 3 psi, until the original supernatant volume is concentrated to 1 / 10-1 / 20 of the original volume. After the concentration is completed, the concentrated liquid is collected.

[0018] Specifically, the ultracentrifugation is at 90000-110000 xg for 1-2 h.

[0019] Further, the ultracentrifugation is at 100000 xg for 1.5 h.

[0020] According to some embodiments of the present application, the NK92 cell complete culture medium comprises: MEMa medium, sodium bicarbonate 2-3 g / L, myo-inositol 0.15-0.25 mM, folic acid 0.01-0.03 mM, beta-mercaptoethanol 0.05-0.15 mM, fetal bovine serum 11-14%, horse serum 11-14%, penicillin-streptomycin double antibody 0.5-1.5%, IL-2 15-25 ng / mL, IL-15 15-25 ng / mL, and water.

[0021] Further, the NK92 cell complete culture medium comprises: MEMa medium, sodium bicarbonate 2.2 g / L, myo-inositol 0.2 mM, folic acid 0.02 mM, beta-mercaptoethanol 0.1 mM, fetal bovine serum 12.5%, horse serum 12.5%, penicillin-streptomycin double antibody 1%, IL-2 20 ng / mL, IL-15 20 ng / mL, and water.

[0022] Specifically, the fetal bovine serum is exosome-free fetal bovine serum.

[0023] Specifically, the horse serum is exosome-free horse serum.

[0024] Specifically, the preparation method of the exosome-free fetal bovine serum and the exosome-free horse serum comprises: first, separately centrifuging the fetal bovine serum and the horse serum at 2-6℃ at 110000-130000 xg for 10-14 h, and then filtering the upper liquid 1-3 times using a 0.22 μm filter membrane to obtain the exosome-free fetal bovine serum and the exosome-free horse serum.

[0025] According to some embodiments of the present application, the preparation method of the umbilical cord blood NK cell-derived exosomes comprises the following steps: (1) Isolate umbilical cord blood mononuclear cells from umbilical cord blood, enrich umbilical cord blood NK cells, and co-culture umbilical cord blood NK cells with genetically engineered cells; (2) Resuspend umbilical cord blood NK cells, culture, centrifuge, and collect the supernatant; (3) Purify exosomes by tangential flow filtration and ultracentrifugation.

[0026] Specifically, in step (1), the separation is performed by density gradient centrifugation, and the conditions for density gradient centrifugation are: centrifugation at 350-450 ×g for 25-35 min at 20-25℃; the enrichment is performed using a human NK cell sorting kit.

[0027] Further, in step (1), the conditions for density gradient centrifugation are: centrifugation at 400 × g for 30 min at 22℃. Specifically, in step (1), the genetically engineered cells are genetically engineered K562-mbIL21 cells irradiated with γ rays.

[0028] Specifically, in step (1), the co-culture conditions are as follows: umbilical cord blood NK cells and genetically engineered cells are seeded at a 1:1 ratio and cultured at 35-40℃, 5% CO2, and >90% humidity for 6-8 days until the umbilical cord blood NK cells are in the logarithmic phase.

[0029] Further, in step (1), the co-culture conditions are as follows: umbilical cord blood NK cells and genetically engineered cells are seeded at a 1:1 ratio and cultured at 37°C, 5% CO2, and >90% humidity for 7 days until the umbilical cord blood NK cells are in the logarithmic phase.

[0030] Specifically, in step (2), the process of resuspending umbilical cord blood NK cells includes: resuspending the cells in umbilical cord blood NK cell culture medium until the cell concentration is 0.5-1.5 × 10⁻⁶ cells / day. 6 cells / mL; Furthermore, the cells were resuspended in umbilical cord blood NK cell culture medium to a cell concentration of 1.0 × 10⁻⁶. 6 cells / mL.

[0031] Furthermore, the umbilical cord blood NK cell culture medium comprises the following components at final concentrations: X-VIVO™ 15 serum-free medium, β-mercaptoethanol 0.05-0.15 mM, L-glutamine 1-3 mM, penicillin-streptomycin bispecific antibody 0.5-1.5%, sodium pyruvate 0.5-1.5%, IL-2 15-25 ng / mL, and IL-15 15-25 ng / mL; Furthermore, the umbilical cord blood NK cell culture medium comprises the following components at final concentrations: X-VIVO™ 15 serum-free medium, β-mercaptoethanol 0.1 mM, L-glutamine 2 mM, penicillin-streptomycin bispecific antibody 1%, sodium pyruvate 1%, IL-2 20 ng / mL and IL-15 20 ng / mL.

[0032] Specifically, in step (2), the culture conditions are: 35-40℃, 5%CO2, and humidity >90% for 45-50 h; the centrifugation conditions are: 250-350 ×g for 4-6 min.

[0033] Further, in step (2), the culture conditions are: cultured at 37℃, 5% CO2, and humidity >90% for 48h; the centrifugation conditions are: centrifuged at 300 ×g for 5 min.

[0034] Specifically, the irreversible ablation includes, but is not limited to, thermal ablation, cryoablation, chemical ablation, and microsecond pulse ablation.

[0035] Furthermore, the thermal ablation includes, but is not limited to, microwave ablation, laser ablation, and radiofrequency ablation. Furthermore, the cryoablation includes, but is not limited to, argon-helium cryoablation.

[0036] Furthermore, the chemical ablation includes, but is not limited to, ethanol ablation and acetic acid ablation.

[0037] Furthermore, the irreversible ablation is described as microsecond pulse ablation.

[0038] According to some embodiments of the present invention, the conditions for the microsecond pulse are: 1-2 kV / cm, 3-5 mm, 500-700 V, 60-80 μs, 20-100 pulses, and 80-100 ppm.

[0039] Furthermore, the conditions for the microsecond pulse ablation are: 1.5 kV / cm, 4 mm, 600 V, 70 μs, 30 pulses, and 90 ppm.

[0040] Specifically, the concentration of the exosomes is 5-15 μg / mL; Furthermore, the concentration of the exosomes is 10 μg / mL.

[0041] Specifically, the irreversible ablation adjuvant is an antitumor agent.

[0042] Furthermore, the tumors mentioned include, but are not limited to, breast cancer, liver cancer, lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, esophageal cancer, gallbladder cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, and thyroid cancer.

[0043] Furthermore, the tumors mentioned are breast cancer and liver cancer.

[0044] Specifically, the combined effects of irreversible ablation adjuvants and irreversible ablation have the following benefits: (1) Promotes tumor cell apoptosis; (2) Inhibits tumor cell growth; (3) Promotes the activation of immune cells.

[0045] Furthermore, the tumor cells include, but are not limited to, MCF-7, 4T1, Huh7, and Hepa1-6.

[0046] Furthermore, promoting immune cell activation includes: 1) Promotes macrophage activation; 2) Increase IFNγ in the spleen + CD8 + The proportion of T cells; 3) Increase IFNγ in the spleen + The proportion of NK cells.

[0047] The beneficial effects of this invention are as follows: This invention provides the application of exosomes as an adjuvant agent for irreversible ablation. When exosomes are used as an adjuvant agent for irreversible ablation in combination with microsecond pulses to act on tumor cells, they have a synergistic effect. The combined effect of the two can significantly promote tumor cell apoptosis, inhibit tumor cell growth, and promote the activation of immune cells. Attached Figure Description

[0048] Figure 1 The particle size range of NK exosomes is 80 nm to 100 nm.

[0049] Figure 2 The NK exosome zeta potential is -45 mV to -15 mV.

[0050] Figure 3 Transmission electron microscopy of NK exosomes.

[0051] Figure 4 Identification of NK exosome marker proteins.

[0052] Figures 5-8 Results of apoptosis in MCF-7 cells.

[0053] Figures 9-12 Results of apoptosis in Huh7 cells.

[0054] Figures 13-16 The results show the activation of RAW264.7 cells for immune response.

[0055] Figure 17 This refers to the size of a triple-negative breast cancer tumor.

[0056] Figure 18 This refers to the weight of a triple-negative breast cancer tumor.

[0057] Figure 19 CD8+ in the spleen of triple-negative breast cancer mice + IFNγ positivity intensity of T cells.

[0058] Figure 20 This refers to the size of a hepatocellular carcinoma tumor.

[0059] Figure 21 This refers to the weight of a hepatocellular carcinoma tumor.

[0060] Figure 22 The positive intensity of IFNγ in NK cells of the spleen of mice with liver cancer. Detailed Implementation

[0061] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0062] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0063] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0064] The definition of standard chemical terms can be found in the reference "Animal Cell Culture Technology: Sun Yat-sen University Press, June 2018, 3rd Edition".

[0065] Unless otherwise stated, conventional methods within the scope of the art, such as flow cytometry for detecting cell viability and in vitro tumorigenicity, shall be used.

[0066] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0067] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0068] The term "cell line" as used in this article refers to a population of cells that can be passaged in vitro for a long period of time, usually derived from tumor or immortalized cells.

[0069] The term "differentiation" used in this article refers to the morphological and functional differences among cells within a multicellular organism, resulting from the specialization of survival behaviors after division. These differences manifest in different types of cells developing into different tissues and organs to perform different biological functions. Differentiation, as a survival skill of multicellular organisms, fully demonstrates the diversity of biological morphology and adaptability under natural selection. Differentiation differs from division, which typically refers to the reproduction of single cells in the primitive state of an organism; in multicellular organisms, both sexual and asexual reproduction occur through the division of reproductive cells to obtain a greater total number of cells.

[0070] The term "passage" used in this article refers to a cell population capable of continuous proliferation in an in vitro environment, primarily achieved through the transfer of culture samples to new containers. The culture process involves three typical phases: a free phase, an exponential growth phase, and a stationary phase. The logarithmic growth phase exhibits the best cell viability and is suitable for experimental manipulation. Based on growth characteristics, cells can be classified as adherent or non-adherent, the latter being common in special types such as tumor cells. After successful primary cell culture, as the culture time increases and cells continue to divide, contact inhibition occurs between cells, slowing or even halting growth. Furthermore, insufficient nutrients and the accumulation of metabolites can also hinder growth or lead to poisoning. Passage culture is one of the routine methods for preserving tissue culture and is fundamental to almost all cell biology experiments. Once cells have reached confluence in a culture flask, they need to be diluted and divided into multiple flasks to continue cell growth. Passage culture can obtain a large number of cells for experimental needs.

[0071] The term "exosome" as used in this article refers to extracellular vesicles with a diameter of approximately 30-150 nm (sometimes exceeding this range), actively secreted by cells, carrying bioactive substances such as proteins and nucleic acids (e.g., RNA, DNA), and playing a crucial role in intercellular communication, disease development, and treatment. Exosomes possess a lipid bilayer structure and contain a variety of components. These include proteins (such as transmembrane proteins, CD9, CD63), and proteins related to signaling pathways; nucleic acids (such as mRNA, microRNA, non-coding RNA), and small amounts of DNA; and metabolites such as lipids and carbohydrates. The components within exosomes can reflect the physiological or pathological state of the cell from which they originate.

[0072] The term "serum-free medium" as used in this article refers to a synthetic culture medium that can maintain cell growth and reproduction for a relatively long period of time in vitro without the addition of serum. The basic components of a serum-free medium generally include two main parts: the basal medium and the added components. Cells used in biopharmaceutical and vaccine production mostly exhibit adherent or facultative adherent growth when cultured in vitro; however, when grown in serum-free medium, the cells often grow in suspension.

[0073] Example 1: Preparation of exosomes derived from NK92 cell line 1. Methods to increase NK92 exosome production 1.1 Prepare the basal culture medium for NK92 cells, which consists of: sterile ultrapure water, MEMα powder (Gibco, 1 L / packet), sodium bicarbonate (2.2 g / L), inositol (0.2 mM), folic acid (0.02 mM), β-mercaptoethanol (0.1 mM), exosome-free fetal bovine serum (12.5%), exosome-free horse serum (12.5%), penicillin-streptomycin bispecific antibody (1%), and interleukin-2 (IL-2, 20 ng / mL).

[0074] 1.2 NK92 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were stimulated in the following manner, including: (1) control group (NK92 cells were cultured in basal medium for 48 hours without the addition of other substances); (2) interleukin-15 (IL-15) stimulation group (20 ng / mL IL-15 was added to NK92 cells in basal medium and cultured for 48 hours); (3) phorbol ester / ionomycin stimulation group (NK92 cells were cultured in basal medium for 42 hours, and after 42 hours, 50 ng / mL phorbol 12-myristate 13-acetate (PMA) and 1 μg / mL ionomycin were added and cultured for another 6 hours).

[0075] 1.3 Acquisition and detection of NK92 exosomes: NK92 cell suspensions with a cell density of 3 × 10^5 cells / mL after different treatments were collected and centrifuged at 1800 rpm for 5 minutes to obtain the corresponding supernatants. The three supernatants were then ultracentrifuged separately as follows: pre-treated supernatant, centrifuged at 350 g for 5 min to remove cells, followed by centrifugation at 2000 g for 10 min to remove cell debris, and centrifugation at 10000 g for 30 min to remove organelles and cell debris. The mixture was then transferred to thin-walled centrifuge tubes (UltraClear; 344058), balanced with an ultracentrifuge sleeve (SW 32 TI; 19U5791; 33.7 mL), and centrifuged using an ultracentrifuge (Beckman Coulter Optima XPN-100) set to RCF 100000 g, 90 minutes, 4℃, acceleration 4, deceleration 4. After 90 minutes, the supernatant was discarded, and the NK exosomes were at the bottom of the ultrafiltration tube. The NK exosomes were resuspended with PBS buffer and then filtered once in a clean bench using a 0.22 μm filter (BIOFIL). Quantification was performed using a BCA kit (Beyotime).

[0076] 1.4 Experimental Results IL-15 stimulation increased NK92 cell exosome production by 4.64-fold, and the NK92 cells exhibited better condition, higher aggregation rate, and sustained proliferation after IL-15 stimulation. Phorbolone / ionomycin stimulation increased NK92 cell exosome production by 6.17-fold, but significantly affected NK92 cell viability. Therefore, considering the combined effects of different stimuli on exosome production and cell viability, subsequent experiments added IL-15 to the basal NK92 cell culture medium to create a complete NK92 cell culture medium.

[0077] 1.5 Complete culture medium for NK92 cells, consisting of: sterile ultrapure water, MEMα powder (Gibco, 1 L / packet), sodium bicarbonate (2.2 g / L), inositol (0.2 mM), folic acid (0.02 mM), β-mercaptoethanol (0.1 mM), exosome-free fetal bovine serum (12.5%), exosome-free horse serum (12.5%), penicillin-streptomycin bispecific antibody (1%), interleukin-2 (IL-2, 20 ng / mL), and interleukin-15 (IL-15, 20 ng / mL).

[0078] Table 1. Exosome production and cell state of NK cells after different treatments

[0079] 2. Preparation of NK92 exosomes 2.1 Exosome-free fetal bovine serum (FBS) and exosome-free horse serum were obtained by ultracentrifugation. The procedure was as follows: FBS and horse serum were thawed at 4°C 12 hours in advance, then transferred to thick-walled centrifuge tubes (OPEN TOP; 355631). After balancing with an ultracentrifuge sleeve (SW 32 TI; 19U5791; 33.7 mL), the ultracentrifuge (Beckman Coulter Optima XPN-100) was set to RCF 120000 g, 12 hours, 4°C, acceleration 4, deceleration 4, and centrifugation began. After 12 hours, approximately 90% of the upper layer of FBS and horse serum was transferred to separate 50 mL centrifuge tubes using a pipette, and the remaining approximately 10% of serum was discarded. Exosome-free FBS and horse serum were filtered twice using a 0.22 μm filter (BIOFIL) in a clean bench to remove bacteria.

[0080] 2.2 Obtaining NK92 supernatant: The steps are as follows: Add 20 mL of the prepared NK92 complete culture medium to each T75 culture flask, seed NK92 cells, adjust the cell density to 1.5 × 10^5 cells / mL, and culture NK92 cells in a 37℃, 5% CO2 incubator for 48 h. Then transfer the culture medium and cells to centrifuge tubes, centrifuge at 1800 rpm for 5 minutes, collect the supernatant, and store it in a -80℃ freezer. Resuspend the NK92 cells in 1 mL of NK92 complete culture medium, add it to a T75 culture flask, add 19 mL of fresh culture medium, and continue culturing.

[0081] 2.3 Purification of NK92 exosomes by tangential flow filtration (TFF) was performed as follows: The supernatant collected in the previous step was thawed at 4°C and pre-filtered once with a 0.22 µm or 0.45 µm filter to remove any remaining small particles and prevent clogging of the TFF membrane. The TFF system (EXOPi-M1, purchased from Shenzhen Aitai Biomedical Technology Co., Ltd.) and the filter membrane were then rinsed and equilibrated with PBS (pH 7.4). The supernatant was pumped into the TFF system reservoir. The peristaltic pump was turned on, and the tangential flow rate was set to 200 mL / min and the transmembrane pressure to 3 psi, allowing the liquid to circulate on the membrane surface. The filtrate outlet was opened to allow the filtrate (containing impurities smaller than the molecular weight cutoff) to flow out, while PBS was added to the reservoir to maintain volume. This process was continued until the original supernatant volume was concentrated to 1 / 10–1 / 20 of its original volume. After concentration, close the filtrate outlet or direct it to a waste collection bottle. Add sufficient PBS to the reservoir, open the filtrate outlet, and allow the liquid to continue circulating and flowing out through the filter membrane. Continue washing until the accumulated filtrate volume reaches the volume of added PBS (i.e., 5-10 volumes of washing are completed), or confirm that the residue has been sufficiently removed by conductivity monitoring. After washing, close the filtrate outlet and allow the system to recirculate for a few minutes. Switch the system to "full circulation" mode and run it for a few minutes to recover substances from the membrane surface. Carefully collect the concentrate in the reservoir, and flush the system tubing and membrane / hollow fiber column interior with a small amount of PBS, incorporating the flushing solution into the concentrate.

[0082] 2.4 Obtaining NK92 exosomes by ultracentrifugation: The concentrated solution obtained in the above steps was transferred to a thin-walled centrifuge tube (Ultra Clear; 344058), and balanced with an ultracentrifugation sleeve (SW 32 TI; 19U5791; 33.7ML). The ultracentrifuge (Beckman Coulter Optima XPN-100) was set to RCF 100000 g, 90 minutes, 4°C, acceleration 4, deceleration 4, and centrifugation began. After 90 minutes, the supernatant was discarded, leaving the NK exosomes at the bottom of the ultracentrifugation tube. The NK exosomes were resuspended in PBS buffer and then filtered once using a 0.22 μm filter (BIOFIL) in a clean bench. Quantification was performed using a BCA kit (Beyotime). After quantification, the molecule was aliquoted into EP tubes and stored at -80°C.

[0083] Example 2: Preparation of exosomes derived from umbilical cord blood NK cells 1. Prepare the culture medium for Umbilical Cord Blood Natural Killer Cells (UCB NKCells) with the following composition: X-VIVO™ 15 serum-free medium (Lonza, #04-418Q), β-mercaptoethanol (0.1 mM), L-glutamine (2 mM), penicillin-streptomycin (1%), sodium pyruvate (1%), interleukin-2 (IL-2, 20 ng / mL), and interleukin-15 (IL-15, 20 ng / mL).

[0084] 2. Obtaining the supernatant of umbilical cord blood NK cells: Umbilical cord blood mononuclear cells were isolated from the cord blood by density gradient centrifugation (using Ficoll-PaquePLUS, 400 × g, 22℃, 30 min). Umbilical cord blood NK cells were enriched using a human NK cell sorting kit (Miltenyi, 130-092-657). The umbilical cord blood NK cells were seeded at a 1:1 ratio with genetically engineered K562-mbIL21 cells (feeder cells, purchased from Wuhan Pronosei Life Science Technology Co., Ltd.) irradiated with γ-rays (100 Gy) into culture flasks and cultured in an incubator (37℃, 5% CO2, humidity >90%). The medium was changed at half volume every 72 hours. The first passage was performed on day 7, followed by continued expansion of the umbilical cord blood NK cells. When the umbilical cord blood NK cells were in the logarithmic growth phase, the cells were resuspended in the above-mentioned medium to a cell concentration of 1*10^6 cells / mL. The cell suspension was then transferred to a T75 culture flask and incubated for another 48 hours. After incubation, the cell suspension was transferred to a centrifuge tube, centrifuged at 300 g for 5 minutes, and the supernatant was carefully collected and stored at -80°C.

[0085] 3. Purify UCB NK exosomes by tangential flow filtration, following the same steps as shown in 2.3.

[0086] 4. Obtain UCB NK exosomes by ultracentrifugation, the steps of which are the same as those shown in 2.4.

[0087] Example 3 Characterization of NK exosomes The characterization of NK exosomes was performed using the following steps: the particle size range of NK exosomes was determined using a nanoparticle tracking analyzer (brand: Malvern Panaco, signal: NanoSight NS300), ranging from 80 nm to 100 nm. Figure 1 The zeta potentials of NK exosomes were determined using a particle size analyzer (Brand: Malvern Panaco, Model: Zetasizer Pro), ranging from -45 mV to -15 mV. Figure 2Its morphology was observed using a transmission electron microscope (brand: JEOL, model: JEM-1400Flash). Figure 3 NK cell exosome markers, including CD63, TSG-101, CD9, and Calnexin, were identified using Western blotting. The specific steps were as follows: NK cells and NK exosomes were lysed using RIPA lysis buffer, followed by BCA quantification of protein concentration. Samples were prepared according to the protein concentration, with the addition of appropriate amounts of ultrapure water and loading buffer to achieve a final sample concentration of 1 μg / μL. The samples were heated at 70°C and 300 rpm for 10 min. After heating, 15 μg of sample was added to a 10% polyacrylamide gel, followed by running at 80V for 30 min and then at 120V for 90 min. After gel running, the gel was transferred at 330mA for 90 min. The gel was then blocked with 5% milk at room temperature for 1 h on a shaker at 60 rpm. The membrane was washed three times with TBST at 90 rpm for 3 minutes each time. Finally, the membrane was incubated with primary antibody at 4°C and 60 rpm for 14 h. Wash the membrane three times with TBST at 90 rpm for 10 minutes each time. After washing, incubate with secondary antibody at room temperature for 90 minutes at 60 rpm. Wash the membrane three times with TBST at 90 rpm for 10 minutes each time. Finally, add developing buffer and develop using an immunoblotting imaging system (Brand: Baygene, Model: BG-gdsAUTO 730). The positive markers for exosomes were CD63, TSG-101, and CD9, and the negative marker was Calnexin. NK exosomes met the criterion of 3 positive and 1 negative. Figure 4 ).

[0088] Example 4: NK exosomes as a microsecond pulse synergist can promote tumor cell apoptosis. 1. Experimental Methods: Tumor cells were digested and prepared into a cell suspension of 1×10^6 cells / mL. 200 μL of the cell suspension was transferred to electrode cups spaced 4 mm apart and processed using the following parameters (Table 2). The cell suspension after pulsed incubation was seeded into 24-well plates with or without exosomes and incubated for 3 h. After 3 h, the supernatant was transferred to a 1.5 mL EP tube, washed once with 500 μL PBS, and digested with 200 μL of EDTA-free trypsin for 1.5 min. Digestion was then stopped, and the cell suspension was transferred to a 1.5 mL EP tube and centrifuged at 1500 rpm for 5 min. 1 mL PBS was added for washing, and the cells were centrifuged at 1500 rpm for 5 min. Annexin V / PI flow cytometry antibody was then added for staining for 15 min. After staining, 400 μL of binding buffer was added to stop the staining. Tumor cell apoptosis was detected using flow cytometry.

[0089] Table 2 Treatment parameters for different groups

[0090] 2. The synergistic effect of NK exosomes and microsecond pulsed ablation in promoting apoptosis in the estrogen receptor-positive / progesterone receptor-positive breast cancer cell line MCF-7 is as follows: Figures 5-8 As shown, PEF$NK92-EV and NK92-EV or PEF alone were highly significant (****p<0.0001, ****p<0.0001), and PEF$UCB-NK-EV and UCB-NK-EV or PEF alone were significant (****p<0.0001, **p<0.01).

[0091] 3. The results of synergistic promotion of apoptosis in the hepatocellular carcinoma cell line Huh7 by NK exosomes and microsecond pulse ablation are as follows: Figures 9-12 As shown, PEF$NK92-EV was significantly different from NK92-EV or PEF alone (****p<0.0001, *p<0.05), and PEF$UCB-NK-EV was significantly different from UCB-NK-EV or PEF alone (****p<0.0001, ***p<0.001).

[0092] Example 5: NK exosomes as a microsecond pulse synergist can promote immune cell activation. Experimental methods: (1) Seeding RAW264.7 cells: Digest RAW264.7 cells, count them, and prepare a cell suspension of 5×10^4 cells / mL. Then seed the suspension into 6-well plates, adding 2 mL of cell suspension to each well.

[0093] (2) Collection of Huh7 supernatant: Huh7 cells were digested and prepared into a cell suspension of 1×10^6 cells / mL. 200 μL of the cell suspension (2×10^5 cells) was transferred to an electrode cup with a spacing of 4 mm. Pulsed ablation was performed using parameters of 1.5 kV / cm, 4 mm, 600 V, 70 μs, 30 pulses, and 90 ppm. Approximately 2 mL of the cell suspension after the pulse was collected and incubated in a 37℃ incubator for 3 h to allow for the full release of immunogenic substances. After 3 h, the cells were centrifuged at 300xg for 5 min, and then filtered through a 0.45 μm filter membrane to remove cells and cell debris, obtaining the immunogenic substances released by Huh7 cells after the pulse.

[0094] (3) Drug treatment: After RAW264.7 cells were seeded for 24 hours, they were treated as follows.

[0095] (4) Flow cytometry detection of RAW264.7 cell immune activation: After 12 h of drug treatment, RAW264.7 cells were digested, centrifuged at 300 x g for 5 min, and the supernatant was discarded. The cells were then washed once with 1 mL PBS, centrifuged at 300 x g for 5 min, and the supernatant was discarded. 150 μL of fixative was added, and the cells were fixed at room temperature for 20 min, then centrifuged at 300 x g for 5 min, and the supernatant was discarded. Next, 150 μL of permeabilization buffer was added, and the cells were centrifuged at 300 x g for 5 min. Under light-protected conditions, 100 μL of APC-Cy7-MHC-II flow cytometry antibody prepared with permeabilization buffer was added, and the cells were stained at room temperature for 15 min. After staining, the cells were centrifuged at 300 x g for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL PBS. Finally, the expression of MHC-II in RAW264.7 cells was detected using flow cytometry.

[0096] Table 3 Treatment parameters for different groups

[0097] Experimental results are as follows Figures 13-16 As shown, NK exosomes and microsecond pulsed ablation synergistically promoted the activation of RAW264.7 macrophages and enhanced their antigen presentation function. Specifically, PEF$NK92-EV showed significant differences compared to NK92-EV or PEF alone (***p<0.001, *p<0.05). PEF$UCB-NK-EV also showed significant differences compared to UCB-NK or PEF alone (****p<0.0001, *p<0.05).

[0098] Example 6: Immune activation effect of NK exosomes as a microsecond pulse synergist in solid tumor-bearing mice. 1. The effect of NK92 exosomes combined with microsecond pulse ablation in triple-negative breast cancer (TNBC) orthotopic tumor mice: A mouse model of triple-negative breast cancer orthotopic tumor was established on day 0. The specific steps were as follows: 4T1 cells were digested, resuspended in PBS, and the cell suspension was adjusted to 6 × 10^7 cells / mL. The cell suspension was then mixed with matrix gel at a volume ratio of 1:1. Six-week-old female Balb / c mice (n=5 per group) were anesthetized, and the fourth pair of nipples was located and the skin was prepared. Using a 1 mL insulin syringe, the mixed 4T1 suspension was drawn up and injected into the right nipple of the fourth pair of nipples. Each mouse received 100 μL of suspension (3 × 10^6 4T1 cells). After injection, the mice were allowed to recover. On day 6, the tumor grew to approximately 150 mm. 3 Mice were randomly assigned to groups and subjected to pulse ablation (parameter settings: 1.5 kV / cm, 3.33 mm, 70 μs, 90 pulses, 90 ppm). Two days later, NK92 exosomes were administered (dose: 25 μg exosomes / g mouse body weight), for a total of three administrations, each one day apart. Mice were sacrificed on day 15, and tumor and spleen tissues were collected.

[0099] The size of the tumor tissue was photographed and its weight was measured using an analytical balance. Spleen tissue was stained by flow cytometry, and the immune activation of mice under different treatments was analyzed using flow cytometry.

[0100] Experimental results: Compared with the PBS group, pulsed ablation alone, or NK exosomes, pulsed ablation combined with NK92 exosomes significantly inhibited tumor growth in mice with triple-negative breast cancer in situ, and the effect was the best. Figures 17-18 Furthermore, pulsed ablation combined with NK92 exosomes significantly increased IFNγ levels in the spleen of mice. + CD8 + The proportion of T cells, thereby maximizing the activation of the mouse's immune system ( Figure 19 ).

[0101] 2. The role of NK exosomes combined with pulsed ablation in the formation of subcutaneous hepatocellular carcinoma (HCC) tumors in mice: A mouse model of subcutaneous HCC tumors was established on day 0. The specific steps were as follows: Hepa1-6 cells were digested, resuspended in PBS, and the cell suspension was adjusted to 4 × 10^7 cells / mL. The cell suspension was then mixed with matrix gel at a 1:1 volume ratio. Six-week-old C57BL / 6 mice (5 mice per group, half male and half female) were anesthetized, and the skin near the hind limb thigh on the right back was located. Using a 1 mL insulin syringe, 100 μL of the well-mixed Hepa1-6 suspension (2 × 10^6 Hepa1-6 cells) was subcutaneously injected into each mouse. Mice were allowed to recover after injection. On day 14, the tumor had grown to approximately 300 mm. 3 Mice were randomly grouped and subjected to pulse ablation (parameter settings: 1.5kV / cm, 3.33 mm, 70 μs, 90 pulses, 90 PPM). Two days later, exosomes were administered (dose: 25 μg exosomes / g mouse body weight). The administration was repeated three times, with one day between each administration. On day 23, the mice were sacrificed, and tumor tissue and spleen tissue were collected.

[0102] The size of the tumor tissue was photographed and its weight was measured using an analytical balance. Spleen tissue was stained by flow cytometry, and the immune activation of mice under different treatments was analyzed using flow cytometry.

[0103] Experimental results: Compared with the PBS group, pulsed ablation alone, or NK exosomes, pulsed ablation combined with NK92 exosomes significantly inhibited tumor growth in mice with subcutaneous stem cell carcinoma, and the effect was the best. Figures 20-21 Furthermore, pulsed ablation combined with NK92 exosomes significantly increased IFNγ levels in the spleen of mice. + The proportion of NK cells, thereby maximizing the activation of the mouse's immune system ( Figure 22 ).

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of exosomes as an auxiliary preparation for irreversible ablation.

2. Use according to claim 1, characterized in that, The exosomes include NK92 cell line-derived exosomes, umbilical cord blood NK cell-derived exosomes, immune cell-derived exosomes, stem cell-derived exosomes, epithelial cell-derived exosomes, neural cell-derived exosomes, endothelial cell-derived exosomes, and blood-derived exosomes.

3. Use according to claim 2, characterized in that, The exosomes include NK92 cell line-derived exosomes and umbilical cord blood NK cell-derived exosomes.

4. Use according to claim 3, characterized in that, The preparation method of the NK92 cell line-derived exosomes comprises the following steps: culturing NK cells using NK92 cell complete medium, centrifuging, collecting supernatant, purifying exosomes by tangential flow filtration, and ultracentrifuging to obtain exosomes.

5. Use according to claim 4, characterized in that, The NK92 cell complete medium comprises MEMα medium, sodium bicarbonate 2-3 g / L, myo-inositol 0.15-0.25 mM, folic acid 0.01-0.03 mM, β-mercaptoethanol 0.05-0.15 mM, fetal bovine serum 11-14%, horse serum 11-14%, penicillin-streptomycin double antibody 0.5-1.5%, IL-2 15-25 ng / mL, IL-15 15-25 ng / mL, and water.

6. Use according to claim 3, characterized in that, The preparation method of the umbilical cord blood NK cell-derived exosomes comprises the following steps: (1) isolating umbilical cord blood mononuclear cells from umbilical cord blood, enriching umbilical cord blood NK cells, and culturing the umbilical cord blood NK cells together with genetically engineered cells; (2) resuspending the umbilical cord blood NK cells, culturing, centrifuging, and collecting supernatant; (3) purifying exosomes by tangential flow filtration, and ultracentrifuging to obtain the exosomes.

7. Use according to claim 1, characterized in that, The irreversible ablation includes thermal ablation, cryoablation, chemical ablation, and microsecond pulse ablation.

8. Use according to claim 7, characterized in that, The microsecond pulse has the following conditions: 1-2 kV / cm, 3-5 mm, 500-700 V, 60-80 μs, 20-100 pulses, and 80-100 ppm.

9. The use according to claim 1, characterized in that, The concentration of the exosomes is 5-15 μg / mL.

10. The use according to claim 1, characterized in that, The auxiliary preparation for irreversible ablation is an antitumor preparation, and the tumors include breast cancer, liver cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, esophageal cancer, gallbladder cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, renal cancer, bladder cancer, and thyroid cancer.

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