Preparation method and application method of NK cell preparation

Abstract

The invention belongs to the technical field of biology, and discloses a preparation method and an application method of an NK cell preparation, and the preparation method comprises the following steps: adding an NK cell liquid and an oxygen-carrying hemoglobin liposome into an oscillator, and carrying out constant temperature culture at 37 DEG C to obtain a cell mixed liquid; inputting the cell mixed solution and the gel solution into a microfluidic electronic injection device for injection, synchronously pumping an atomized cross-linking agent solution, and performing cross-linking in a reaction cavity to obtain gel particles; centrifuging the gel particles at 4 DEG C to obtain precipitate gel, and coating the precipitate gel with a casing material to obtain the NK cell preparation. According to the preparation method, the activity and function of the NK cells in hypoxia and slightly acidic environments of tumors are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a preparation method and application method of NK cell preparation. BACKGROUND

[0002] Natural killer (NK) cell preparation is a kind of cell therapy for tumor immunotherapy, which is usually composed of NK cells activated and expanded in vitro. The dosage form of NK cell preparation mainly includes cell suspension, freeze-dried powder and genetically modified NK cell preparation, among which the cell suspension is the most common preparation form. The treated NK cells are suspended in a suitable culture medium and applied to the patient in the form of intravenous infusion.

[0003] NK cells are an important part of the innate immune system and have a wide range of anti-tumor activity. Unlike T lymphocytes, NK cells can recognize and attack tumor cells, virus-infected cells and other abnormal cells without antigen presentation. Its main killing mechanism is as follows: one is to form holes on the target cell membrane by secreting perforin and granzyme, and then to induce apoptosis after entering the target cell; the other is to activate other immune cells by secreting cytokines (such as interferon gamma and tumor necrosis factor alpha) to promote the enhancement of immune response.

[0004] However, in the process of treating tumors by intravenous injection of NK cell suspension, the acidic environment in the tumor microenvironment is a challenge for NK cell therapy. In the tumor tissue, the proliferation rate of cancer cells exceeds the blood supply capacity, resulting in a lack of oxygen in the environment, which activates the anaerobic glycolysis process of cells, leading to the accumulation of a large amount of lactic acid, and thus the local environment of the tumor becomes acidic. The acidic environment can inhibit the function of NK cells. Studies have found that under low pH conditions, the cytotoxicity and migration ability of NK cells are significantly weakened. The acidic microenvironment not only damages the integrity of the NK cell membrane, but also affects the expression of its surface receptors and signal pathways, inhibiting its recognition and killing ability of tumor cells; in addition, the acidic environment also promotes the release of immunosuppressive factors (such as TGF-β and IL-10), further weakening the anti-tumor effect of NK cells. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a preparation method and application of NK cell preparation, aiming to solve the problem of decreased activity and function of NK cells in the hypoxic and slightly acidic environment of tumors.

[0006] In order to solve the above technical problems, a preparation method of NK cell preparation is provided, which comprises the following steps: S1, adding NK cell liquid and oxygen-carrying hemoglobin liposome into a shaker for constant temperature culture at 37℃ to obtain a cell mixture; S2, input the cell mixture and gel solution into the microfluidic electrospray device for spraying, and pump in the atomized crosslinking agent solution synchronously, crosslink in the reaction cavity, and obtain gel particles; S3, centrifuge the gel particles at 4℃ to obtain precipitated gel, and then coat the precipitated gel with intestinal coating material to obtain an NK cell preparation.

[0007] In some embodiments, the NK cell liquid in step S1 is obtained by culturing autologous plasma in a serum-free medium, wherein the NK cell liquid contains 1-2×10 6 cells / mL of NK cells, and the concentration of the autologous plasma in the serum-free medium is 8-10 wt%.

[0008] In some embodiments, step S1 comprises: S1.1, add oxygen-carrying hemoglobin, dipalmitoyl phosphatidylcholine and cholesterol into a rotary evaporator containing a chloroform solution, then rotary evaporate at 40℃ and 200 rpm for 20-30 min to obtain monodisperse liposomes, wherein the molar ratio of hemoglobin, dipalmitoyl phosphatidylcholine and cholesterol is 5:4:1; S1.2, after dispersing the monodisperse liposomes with deionized water, add (2,3-dioleoyl-propyl)-trimethylammonium chloride and a targeting substance into a vortex mixer, vortex for 10-15 min, and filter to obtain oxygen-carrying hemoglobin liposomes, wherein the mass ratio of monodisperse liposomes, (2,3-dioleoyl-propyl)-trimethylammonium chloride and the targeting substance is 55-69:20-30:1-5, and the targeting substance includes at least one of folate polyethylene glycol phospholipid, transferrin polyethylene glycol phospholipid, and mannose polyethylene glycol phospholipid; S1.3, add the NK cell liquid, oxygen-carrying hemoglobin liposomes and antioxidants into a shaker for constant temperature culture at 37℃ for 20-40 min to obtain a cell mixture, wherein the cell ratio of the NK cell liquid to the oxygen-carrying hemoglobin liposomes is 1:100, the addition amount of the antioxidants is 0.02-0.05% of the mass of the oxygen-carrying hemoglobin liposomes, and the antioxidants include at least one of L-cysteine, water-soluble vitamin E derivative and tert-butyl hydroquinone.

[0009] In some embodiments, before step S1.1, it further comprises: placing the purified hemoglobin solution in an anaerobic environment with an oxygen concentration of less than 0.1%, and passing in nitrogen gas with a purity of 99.999% for 20-40 min to obtain deoxyhemoglobin, and then transferring into a shaker, and oscillating in an oscillation chamber with a volume ratio of O2 to CO2 of 95:5 at 37℃ for 1.5-2.5 h to obtain oxygen-carrying hemoglobin.

[0010] In some embodiments, the gel in the gel solution in step 2 comprises at least one of sodium alginate, chitosan, and high-methoxyl pectin, and the cross-linking agent in the cross-linking agent solution is calcium chloride and / or trisodium citrate.

[0011] In some embodiments, step S2 comprises: S2.1, synchronously pumping the cell mixture, the low-viscosity gel solution, and the atomized cross-linking agent solution containing 0.1M cross-linking agent into the reaction chamber of the microfluidic electrospinning device, and instantaneously reacting for 2-3s to obtain a loosely coated cell solution, wherein the flow rate ratio of the cell mixture, the low-viscosity gel solution, and the atomized cross-linking agent solution is 1:2:2, the average molecular weight of the gel in the low-viscosity gel solution is 80-90kDa, the concentration of the gel in the low-viscosity gel solution is 0.8-0.9wt%, and the electric field strength of the microfluidic electrospinning device is 3-4kV / cm and the alternating frequency is 1-3kHz; S2.2, synchronously pumping the loosely coated cell solution, the high-viscosity gel solution, and the atomized cross-linking agent solution containing 0.1M cross-linking agent into the reaction chamber of the microfluidic electrospinning device, and instantaneously reacting for 2-3s to obtain gel particles, wherein the flow rate ratio of the loosely coated cell solution, the high-viscosity gel solution, and the atomized cross-linking agent solution is 1:2:2, the average molecular weight of the gel in the high-viscosity gel solution is 240-260kDa, the concentration of the gel in the high-viscosity gel solution is 1.3-1.5wt%, and the electric field strength of the microfluidic electrospinning device is 4.5-5kV / cm and the alternating frequency is 0.8-1.2kHz.

[0012] In some embodiments, step S3 comprises: S3.1, adding the gel particles into a centrifuge, and performing 300g primary centrifugation for 4-6min at 4℃ to obtain a precipitate layer, resuspending the precipitate layer in a pre-cooled phosphate buffer solution at 4℃, and then performing 2000g secondary centrifugation for 10-12min to obtain precipitated gel, wherein the phosphate buffer solution contains 1mM EDTA and 0.5wt% serum albumin; S3.2, coating the precipitated gel with casing material to obtain an NK cell preparation, wherein the casing material comprises at least one of polyvinyl succinate, hydroxypropyl methylcellulose phthalate, and polymethyl methacrylate-ethyl acrylate copolymer, and the mass of the casing material is 3-8% of the mass of the precipitated gel.

[0013] In addition, an application method of the NK cell preparation is also provided, and the NK cell preparation can be applied to an animal model for tumor treatment by intravenous injection, wherein the NK cell preparation is prepared by the above-mentioned method for preparing an NK cell preparation.

[0014] The beneficial effects of the present application are: NK cells are coated with oxygen-carrying hemoglobin liposomes, gel materials and enteric coating materials. During use, the enteric coating material can effectively protect NK cells from degradation by gastric acid, ensuring that they pass through the stomach smoothly and reach the intestine. In the alkaline environment of the intestine, the enteric coating material dissolves rapidly, releasing the gel-coated NK cells; the gel particles have good biocompatibility and can be absorbed by intestinal epithelial cells through endocytosis, enter the microvessels and eventually enter the veins, enter the systemic blood circulation, and enter the systemic blood circulation. At this time, the gel material interacts with enzymes and other substances in the blood, gradually degrading into low molecular weight substances, releasing NK cells wrapped in oxygen-carrying hemoglobin liposomes; the oxygen-carrying hemoglobin liposomes undergo ligand-receptor specific binding with specific receptors overexpressed on the surface of cancer cells through their surface-modified targeting substances, thereby achieving targeted recognition and positioning of NK cells. At the same time, the oxygen component in oxygen-carrying hemoglobin provides oxygen support for NK cells, effectively alleviating the hypoxic state in the tumor microenvironment and enhancing the biological activity and cell killing ability of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the process for preparing NK cell preparation in one embodiment. DETAILED DESCRIPTION

[0016] In the description of this application, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0017] Please refer to Figure 1 The present invention provides a method for preparing a NK cell preparation, comprising the following steps: S1. Add NK cell solution and oxygen-carrying hemoglobin liposomes to a shaker and culture at 37°C to obtain a cell mixture; The NK cell fluid in step S1 is obtained by culturing autologous plasma in a serum-free medium, wherein the NK cell fluid contains a cell density of 1-2×10 6 NK cells / mL, the concentration of autologous plasma in serum-free medium is 8~10wt% Oxygen-carrying hemoglobin liposomes can stably bind to the surface of NK cell membranes through hydrophobic interactions for coating. In the hypoxic microenvironment caused by rapid proliferation and imperfect angiogenesis in tumor tissues, oxygen-carrying hemoglobin liposomes can provide oxygen support for NK cells and enhance their oxygen supply, thereby effectively alleviating the hypoxic state in the tumor microenvironment and enhancing the biological activity and cell killing ability of NK cells.

[0018] Step S1 includes: The purified hemoglobin solution was placed in an anaerobic environment with an oxygen concentration below 0.1%, and nitrogen gas with a purity of 99.999% was introduced for 20-40 minutes to obtain deoxyhemoglobin. The solution was then transferred to an oscillator and incubated at 37°C for 1.5-2.5 hours in an oscillation chamber with an O2:CO2 volume ratio of 95:5 to obtain oxygen-carrying hemoglobin. The oxygen in oxygen-carrying hemoglobin is mainly bound to the iron ion center of the hemoglobin molecule in the form of molecular oxygen. When hemoglobin binds to oxygen, the iron ions inside the molecule undergo conformational changes, enabling it to stably bind and release oxygen, thereby providing oxygen support where needed; and during the preparation process, hemoglobin is placed in a low-oxygen environment and introduced with 99.999% pure nitrogen, which helps to effectively remove oxygen molecules from hemoglobin and convert it into deoxyhemoglobin, thereby creating good conditions for the subsequent oxygen-carrying process; incubation at 37°C with an oscillation in an environment with a volume ratio of oxygen to carbon dioxide of 95:5. This condition simulates the oxygen partial pressure and gas composition in the physiological environment, allowing hemoglobin to efficiently bind oxygen while maintaining its stability.

[0019] S1.1. Add oxygenated hemoglobin, dipalmitoylphosphatidylcholine, and cholesterol to a rotary evaporator containing chloroform solution, and then rotary evaporate at 40°C and 200 rpm for 20-30 min to obtain monodisperse liposomes in which the molar ratio of hemoglobin, dipalmitoylphosphatidylcholine, and cholesterol is 5:4:1. Rotary evaporation can effectively dissolve and mix lipid materials, while removing the chloroform solvent, making the composition of the liposomes more uniform and providing a suitable foundation for subsequent liposome preparation; dipalmitoylphosphatidylcholine, as the main phospholipid component, can form a bilayer lipid membrane, and the addition of cholesterol helps to enhance the stability of the membrane and optimize the physical properties of the liposomes, while hemoglobin is embedded in the lipid membrane.

[0020] S1.2. Disperse the monodisperse liposomes with deionized water, add them together with (2,3-dioleoyl-propyl)-trimethylammonium chloride and a targeting substance into a vortex mixer, vortex mix for 10-15 minutes, and filter to obtain oxygen-carrying hemoglobin liposomes, wherein the mass ratio of the monodisperse liposomes to (2,3-dioleoyl-propyl)-trimethylammonium chloride to the targeting substance is 55-69:20-30:1-5, and the targeting substance comprises at least one of folic acid polyethylene glycol phospholipids, transferrin polyethylene glycol phospholipids, and mannose polyethylene glycol phospholipids; The combination of (2,3-dioleoyl-propyl)-trimethylammonium chloride and monodisperse liposomes can form a positively charged membrane layer on the surface of the liposomes, enhance the interaction between the liposomes and the cell membrane, help the cell uptake of the liposomes, improve the stability of the liposomes, reduce the aggregation between the liposomes, and ensure the uniformity of the liposomes; the targeting substance contains a hydrophobic phospholipid chain, which spontaneously inserts into the double-layer membrane of the liposomes through hydrophobic force during the mixing process, forms a stable anchor with the liposomes, and enables the liposomes to specifically combine with specific cell or tissue surface receptors and promote the uptake of the liposomes, so as to ensure that the therapeutic ingredients can accurately reach the target position, thereby improving the targeting of NK cells. At the same time, the introduction of the targeting substance can also improve the biocompatibility of the liposomes, reduce the clearance of the immune system, improve the half-life and stability of the liposomes in the body, and thus optimize the delivery efficiency of the drug.

[0021] S1.3, the NK cell liquid, the oxygen-carrying hemoglobin liposome and the antioxidant are added into the oscillator for constant temperature culture at 37℃ for 20-40min to obtain a cell mixture, wherein the cell ratio of the NK cell liquid to the oxygen-carrying hemoglobin liposome is 1:100, and the addition amount of the antioxidant is 0.02-0.05% of the mass of the oxygen-carrying hemoglobin liposome, and the antioxidant includes at least one of L-cysteine, water-soluble vitamin E derivative and tert-butyl hydroquinone; The cell ratio of the NK cell liquid to the oxygen-carrying hemoglobin liposome is 1:100, which ensures that each NK cell can obtain sufficient support from the oxygen-carrying hemoglobin liposome, thereby enhancing the function and activity of the NK cell, and at the same time ensuring that the cells do not compete excessively at a suitable density. The oxygen-carrying hemoglobin liposome can provide oxygen support for the NK cell, thereby improving its anti-tumor efficiency; the addition of the antioxidant can promote the survival and activity of the NK cell by removing active oxygen or reducing oxidative damage; and the antioxidant addition amount of 0.02-0.05% of the mass of the oxygen-carrying hemoglobin liposome can effectively prevent the oxidative stress reaction generated when the cells are exposed to a high-oxygen environment, and protect the cells from free radical damage.

[0022] S2, the cell mixture and the gel solution are input into a microfluidic electrospray device for spraying, and a misting crosslinking agent solution is pumped in synchronously, and crosslinking is carried out in a reaction chamber to obtain gel particles; The gel in the gel solution in step 2 includes at least one of sodium alginate, chitosan and high-methoxyl pectin, and the crosslinking agent in the crosslinking agent solution is calcium chloride and / or trisodium citrate; The gel particles have good biocompatibility, can be absorbed by intestinal epithelial cells through endocytosis, enter the microvessels and finally enter the venous vessels, and enter the systemic blood circulation; the crosslinking agent can form chemical bonds or physical networks between the constituent molecules of the gel, thereby connecting the gel molecules to each other to form a three-dimensional crosslinked structure, enhancing the stability of the gel particles, and at the same time, in the blood circulation, the gel particles are gradually degraded, the crosslinking agent interacts with enzymes, metal ions or other substances in the blood, prompting the gel to disintegrate or degrade into low molecular substances, helping to effectively release the encapsulated active substances.

[0023] In an embodiment, when the gel is sodium alginate or high methoxyl pectin, the crosslinking agent used is calcium chloride; when the gel is chitosan, the crosslinking agent used is trisodium citrate.

[0024] Step S2 comprises: S2.1, synchronously pump the cell mixture, low-viscosity gel solution, and atomized crosslinking solution containing 0.1M crosslinking agent into the reaction chamber of the microfluidic electrospray device, and instantaneously react for 2-3s to obtain a loose cell-coated solution, wherein the flow rate ratio of the cell mixture, low-viscosity gel solution, and atomized calcium chloride solution is 1:2:2, the average molecular weight of the gel in the low-viscosity gel solution is 80-90kDa, the concentration of the gel in the low-viscosity gel solution is 0.8-0.9wt%, the electric field strength of the microfluidic electrospray device is 3-4kV / cm, and the alternating frequency is 1-3kHz; the technical principle and use method of the microsphere preparation instrument are known to those skilled in the art, and will not be described here.

[0025] The low-viscosity gel solution with a gel concentration of 0.7-0.9wt% can maintain the flowability of the gel solution, facilitate the addition of the atomized crosslinking agent solution, and thus rapidly gelate to form a loose coating layer in a short time, ensuring the protection of the cells while not hindering the metabolic activity of the cells; the 0.1M crosslinking agent in the crosslinking solution can ensure that the gel is fully crosslinked to form a uniform porous structure, while controlling the time of the instantaneous reaction, which can avoid excessive penetration of the crosslinking agent into the inner layer of the liposome, thereby destroying the stability of the inner layer of the liposome; the electric field strength helps to enhance the charge and electrostatic force of the droplets, thereby promoting the atomization of the droplets and the uniformity of the cell coating layer, allowing the cell solution to be quickly coated and form a loose structure in a short time, and at the same time, the alternating frequency further optimizes the stability and size distribution of the droplets by adjusting the oscillation frequency of the electric field, avoiding excessive aggregation or excessively large droplets, and ensuring the integrity of the cells.

[0026] S2.2, pumping the loose cell coating liquid, high-viscosity gel solution, and atomized cross-linking liquid containing 0.1M cross-linking agent into the reaction chamber of the microfluidic electrospray device at the same time, and then performing instantaneous reaction for 2-3s to obtain gel particles, wherein the flow rate ratio of the loose cell coating liquid, the high-viscosity gel solution, and the atomized cross-linking liquid containing 0.1M cross-linking agent is 1:2:2, the average molecular weight of the gel in the high-viscosity gel solution is 240-260kDa, the concentration of the gel in the high-viscosity gel solution is 1.3-1.5wt%, and the electric field strength of the microfluidic electrospray device is 4.5-5kV / cm and the alternating frequency is 0.8-1.2kHz; The gel material with a molecular weight of 240-260kDa makes the solution thereof have strong viscosity, can form a thick and stable coating layer, and thus makes the coating film more tough, avoids damage of the cells under external environmental changes, and does not hinder the activity or metabolism of the cells during the coating process.

[0027] In an embodiment, in order to judge the coating state of the gel material, the following formula is designed: ; wherein E is the actual electric field strength, E0 is the electric field strength reference value (kV / cm), the electric field strength is gradually increased through the electrophoresis experiment, and the E value when the gel first deforms is recorded as E0. In the present embodiment, when the gel is sodium alginate, chitosan, and high-methoxyl pectin, respectively, E0 is 3.5kV / cm, 4.0kV / cm, and 3.2kV / cm, respectively; C is the gel concentration (wt%), β is the nonlinear influence of concentration on conductivity or polarization intensity (dimensionless), different concentration gradient gel samples are prepared, and the conductivity thereof is measured, the corresponding experimental data of concentration and electrical response parameters are recorded, the slope relationship of the linear fitting in the double logarithmic coordinate system is obtained, when the error of the linear fitting is the smallest, the obtained slope is β, in the present embodiment, β=0.7; f is the actual alternating frequency, f0 is the alternating frequency reference value (kHz), different frequency alternating electric fields are applied, and the f value when the droplet resonance amplitude is the largest is recorded as f0. When the gel is sodium alginate, chitosan, and high-methoxyl pectin, respectively, f0 is 1.5kHz, 2.8kHz, and 0.9kHz, respectively; ΔM w is the molecular weight difference coefficient, dimensionless, the gel sample to be tested is subjected to gel permeation chromatography (GPC) analysis, a standard product with a known molecular weight is injected into the GPC system at the same time, the corresponding relationship between the retention time and the molecular weight is recorded, the standard curve is fitted to obtain the weight average molecular weight, and then the weight average molecular weight is divided by the reference value 100kDa to obtain normalization processing, in the present embodiment, when the low-viscosity gel material is used, the value range of ΔM w is 0.8-0.9, and when the high-viscosity gel material is used, the value range of ΔM wthe value range of a is 2.4-2.6; a is a material correction coefficient, dimensionless, the corresponding relationship between the measured parameters and the standard parameters is recorded by performing performance tests on the material to be tested and the standard sample under the same conditions for at least 20 groups, the calibration curve is fitted to obtain the reference value, and then the actual measurement value is divided by the reference value, and finally the material correction coefficient a is calculated. In this embodiment, when the gel is sodium alginate, chitosan, and high-methoxyl pectin, respectively, a is 0.12, 0.18, and 0.15, respectively. GEI is a gel wrapping state index, when GEI>2.1, it is over-wrapped state, when 0.8≤GEI≤2.1, it is ideal wrapping state, and when GEI<0.8, it is not completely wrapped state.

[0028] In an embodiment, when sodium alginate is used as the gel material, in step S2.1, E=3.5kV / cm, E0=3.5kV / cm, f=1.05kHz, f0=1.5kHz, ΔM w =0.85, a=0.12, C=0.85, GEI=0.92 can be obtained by calculation; in step S2.2, E=4.8kV / cm, E0=3.5kV / cm, f=1.1kHz, f0=1.5kHz, ΔM w =2.5, a=0.12, C=1.4, GEI=1.82 can be obtained by calculation, so the gel reaches the ideal wrapping state.

[0029] S3, centrifuge the gel particles at 4°C to obtain precipitated gel, and then coat the precipitated gel with enteric coating material to obtain the NK cell preparation.

[0030] By centrifuging the gel particles at 4°C, impurities, uncoated gel materials and other unnecessary solution components in the gel particles are removed, ensuring that the finally obtained cell preparation is more pure. At the same time, centrifugation is carried out in a low-temperature environment of 4°C, which can slow down cell metabolism and reduce heat accumulation caused by centrifugation, thereby reducing damage to cells caused by excessive temperature or low temperature. In the stomach and intestinal environment, the pH of the stomach juice is generally <5.0, while the pH of the intestinal tract is >7.0. In the stomach environment, the gel material will react with gastric acid, causing the gel particles to dissolve or degrade prematurely, exposing the internal NK cells prematurely and affecting their subsequent absorption and targeting functions. At the same time, the degradation products of the gel particles released in the stomach will also stimulate the stomach wall, causing stomach pain or gastrointestinal discomfort and other side effects. Therefore, the use of enteric coating material to coat the surface of the gel layer can protect the gel particles, allowing the gel particles to remain stable in the stomach juice with a pH <5.0, protecting the gel particles from gastric acid degradation, and then rapidly dissolving the enteric coating material to release the gel particles when they reach the alkaline environment of the intestinal tract. The gel particles are then absorbed through the intestinal mucosa into the venous blood vessels for circulation, releasing and delivering NK cells to the cancer cell area, thereby improving the accuracy of treatment.

[0031] Step S3 comprises: S3.1, adding the gel particles into a centrifuge, centrifuging at 300g for 4-6min at 4℃ to obtain a precipitate layer, resuspending in a pre-cooled phosphate buffer at 4℃, and then centrifuging at 2000g for 10-12min to obtain precipitated gel, wherein the phosphate buffer contains 1mM EDTA and 0.5wt% serum albumin; 4℃ is to slow down the rate of cell metabolism, reduce the activity of enzymes, reduce the changes or degradation that may occur in the cells during processing, so as to ensure that the cell components can remain stable; and the first centrifugation can remove 90% of free liposomes and Ca 2+ , to realize the separation of free liposomes and cells, and the second centrifugation can collect the liposome aggregates that are not fully separated and the calcium ions adsorbed on the cell membrane surface.

[0032] S3.2, adding the precipitated gel into a coating pan, and using an ethanol solution with a casing material concentration of 10-15% to spray and dry at a speed of 10-30rpm to obtain the NK cell preparation, wherein the casing material includes at least one of polyvinyl succinate, hydroxypropyl methylcellulose phthalate, and polymethyl methacrylate-ethyl acrylate copolymer, and the mass of the casing material is 3-8% of the mass of the precipitated gel; Controlling the mass of the casing material to be 3-8% of the mass of the precipitated gel helps to improve the stability of the NK cell preparation, effectively protects the cells from losing activity due to changes in temperature, pH, etc., thereby maintaining the activity and function of the NK cells; by drying, the solvent can be effectively evaporated, and the wet film coated on the surface of the precipitated gel is converted into a continuous casing film.

[0033] An application method of an NK cell preparation is provided herein, which can be applied to an animal model for tumor treatment by intravenous injection, wherein the NK cell preparation is prepared by the above-mentioned method for preparing an NK cell preparation; The NK cells are coated by the oxygen-carrying hemoglobin liposome, the gel material and the enteric material, in the use process, the enteric material can effectively protect the NK cells from gastric acid degradation, ensure that it passes through the stomach smoothly and reaches the intestine, in the alkaline environment of the intestine, the enteric material is quickly dissolved, and the gel-coated NK cells are released; the gel particles have good biocompatibility, can be absorbed by intestinal epithelial cells through endocytosis, enter the microvessels and finally enter the venous vessels, enter the systemic blood circulation, enter the systemic blood circulation, at this time, the gel material interacts with enzymes and other substances in the blood, and is gradually degraded into low molecular weight substances, and the NK cells wrapped by the oxygen-carrying hemoglobin liposome are released; the oxygen-carrying hemoglobin liposome realizes the specific binding of ligand-receptor through the specific binding of the targeting material on the surface of the oxygen-carrying hemoglobin liposome and the specific receptors overexpressed on the surface of the cancer cells, realizes the targeted recognition and positioning of the NK cells, at the same time, the oxygen component in the oxygen-carrying hemoglobin provides oxygen support for the NK cells, effectively relieves the hypoxic state in the tumor microenvironment, and enhances the biological activity and cell killing ability of the NK cells.

[0034] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for preparing a NK cell preparation, characterized in that: The preparation method comprises the following steps: S1. Add NK cell solution and oxygen-carrying hemoglobin liposomes to a shaker and culture at 37°C to obtain a cell mixture; S2, inputting the cell mixture and gel solution into a microfluidic electrospray device for spraying, and simultaneously pumping in an atomized crosslinker solution to perform crosslinking in the reaction chamber to obtain gel particles; S3. Centrifuge the gel particles at 4° C. to obtain a precipitated gel, and then coat the precipitated gel with an enteric coating material to obtain a NK cell preparation.

2. The method for preparing a NK cell preparation according to claim 1, wherein: The NK cell fluid in step S1 is obtained by culturing autologous plasma in a serum-free medium, wherein the NK cell fluid contains a cell density of 1-2×10 6 NK cells / mL, and the concentration of autologous plasma in serum-free culture medium is 8~10wt%.

3. The method for preparing a NK cell preparation according to claim 1 or 2, characterized in that: Step S1 includes: S1.

1. Add oxygenated hemoglobin, dipalmitoylphosphatidylcholine, and cholesterol to a rotary evaporator containing chloroform solution, and then rotary evaporate at 40°C and 200 rpm for 20-30 min to obtain monodisperse liposomes in which the molar ratio of hemoglobin, dipalmitoylphosphatidylcholine, and cholesterol is 5:4:

1. S1.

2. Disperse the monodisperse liposomes with deionized water, add them together with (2,3-dioleoyl-propyl)-trimethylammonium chloride and a targeting substance into a vortex mixer, vortex mix for 10-15 minutes, and filter to obtain oxygen-carrying hemoglobin liposomes, wherein the mass ratio of the monodisperse liposomes to (2,3-dioleoyl-propyl)-trimethylammonium chloride to the targeting substance is 55-69:20-30:1-5, and the targeting substance comprises at least one of folic acid polyethylene glycol phospholipids, transferrin polyethylene glycol phospholipids, and mannose polyethylene glycol phospholipids; S1.

3. Add NK cell fluid, oxygen-carrying hemoglobin liposomes, and antioxidants to an oscillator and culture at a constant temperature of 37°C for 20-40 minutes to obtain a cell mixture, wherein the cell ratio of NK cell fluid to oxygen-carrying hemoglobin liposomes is 1:100, and the amount of antioxidant added is 0.02-0.05% of the mass of oxygen-carrying hemoglobin liposomes. The antioxidant includes at least one of L-cysteine, a water-soluble vitamin E derivative, and tert-butylhydroquinone.

4. The method for preparing a NK cell preparation according to claim 3, wherein: Before step S1.1, the following steps are also included: The purified hemoglobin solution was placed in an anaerobic environment with an oxygen concentration below 0.1%, and nitrogen gas with a purity of 99.999% was introduced for a reaction of 20 to 40 minutes to obtain deoxyhemoglobin. The solution was then transferred to an oscillator and incubated at 37°C for 1.5 to 2.5 hours in an oscillation chamber with a volume ratio of O2:CO2 of 95:5 to obtain oxygen-carrying hemoglobin.

5. The method for preparing a NK cell preparation according to claim 1, wherein: The gel in the gel solution in step 2 includes at least one of sodium alginate, chitosan, and high methoxy pectin, and the crosslinking agent in the crosslinking agent solution is calcium chloride and / or trisodium citrate.

6. The method for preparing a NK cell preparation according to claim 1, wherein: Step S2 includes: S2.

1. Simultaneously pump a cell mixture, a low-viscosity gel solution, and an atomized crosslinker solution containing 0.1 M crosslinker into the reaction chamber of a microfluidic electrospray device for a transient reaction of 2-3 seconds to obtain a loosely coated cell solution, wherein the flow rate ratio of the cell mixture, the low-viscosity gel solution, and the atomized crosslinker solution is 1:2:2, the average molecular weight of the gel in the low-viscosity gel solution is 80-90 kDa, and the concentration of the gel in the low-viscosity gel solution is 0.8-0.9 wt%. The electric field strength of the microfluidic electrospray device is 3-4 kV / cm, and the alternating frequency is 1-3 kHz. S2.

2. The loosely coated cell fluid, high viscosity gel solution, and atomized crosslinker solution containing 0.1M crosslinker are synchronously pumped into the reaction chamber of the microfluidic electrospray device, and then reacted instantaneously for 2~3s to obtain gel particles, wherein the flow rate ratio of the loosely coated cell fluid, high viscosity gel solution, and atomized crosslinker solution is 1:2:2, the average molecular weight of the gel in the high viscosity gel solution is 240~260kDa, the concentration of the gel in the high viscosity gel solution is 1.3~1.5wt%, the electric field strength of the microfluidic electrospray device is 4.5~5kV / cm, and the alternating frequency is 0.8~1.2kHz.

7. The method for preparing a NK cell preparation according to claim 1, characterized in that: Step S3 includes: S3.

1. Add the gel particles to a centrifuge and centrifuge once at 300g for 4-6 minutes at 4°C to obtain a precipitate. Resuspend the pellet in 4°C precooled phosphate buffer, and then centrifuge again at 2000g for 10-12 minutes to obtain a precipitated gel. The phosphate buffer contains 1 mM EDTA and 0.5 wt% serum albumin. S3.

2. Add the precipitated gel to the coating pan, spray it with an ethanol solution having an enteric coating material concentration of 10-15% at a rotation speed of 10-30 rpm, and dry it to obtain an NK cell preparation, wherein the enteric coating material includes at least one of polyvinyl alcohol succinate, hydroxypropyl methylcellulose phthalate, and polymethacrylic acid-ethyl acrylate copolymer, and the mass of the enteric coating material is 3-8% of the mass of the precipitated gel.

8. A method for using a NK cell preparation, characterized in that: The NK cell preparation can be used in animal models for tumor treatment, wherein the NK cell preparation is prepared by the preparation method of an NK cell preparation according to any one of claims 1-7.

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