Lipid nanoparticle for coupling and wrapping arginine deiminase, glucose oxidase and copper sulphide nano-enzyme in living cells and preparation method of lipid nanoparticle for coupling and wrapping arginine deiminase, glucose oxidase and copper sulphide nano-enzyme in living cells

Abstract

The invention belongs to the field of pharmaceutical preparations. The invention relates to lipid nanoparticles with arginine deiminase, glucose oxidase and copper sulphide nano-enzyme coupled and wrapped in living cells and a preparation method of the lipid nanoparticles with arginine deiminase, glucose oxidase and copper sulphide nano-enzyme coupled and wrapped in living cells. According to the lipid nanoparticles with the arginine deiminase, the glucose oxidase and the copper sulfide nano-enzyme coupled and wrapped in the living cells, the half-life period of enzyme drugs such as the arginine deiminase, the glucose oxidase and the copper sulfide nano-enzyme in body circulation can be prolonged, and the bioavailability of the enzyme drugs is improved; and the anti-cancer effect on cancers is enhanced by cooperating with photothermal therapy, photodynamic therapy and starvation therapy.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and relates to a living cell coupled lipid nanoparticle encapsulating arginine deiminase, glucose oxidase and copper sulfide nanozyme (hereinafter referred to as: living cell coupled complex enzyme lipid nanoparticle in this patent) and a preparation method thereof. Background Art

[0002] As a delivery carrier from nature, the application of cells has brought a new revolution to targeted tumor therapy, and cell delivery systems have become a hot topic in the current research of targeted delivery systems. Living cells such as red blood cells, neutrophils, lymphocytes and macrophages as a new generation of biological delivery systems can improve the defects and shortcomings of various previous drug delivery systems. Among them, macrophages, as a living cell drug carrier, can effectively deliver drugs to tumor sites and play an immunological role in killing tumor cells. Arginine deiminase can irreversibly hydrolyze L-arginine into L-citrulline and ammonia, and is a protein drug for the treatment of arginine auxotrophic cancer. Glucose oxidase can consume endogenous glucose to generate gluconic acid and hydrogen peroxide, consume glucose in tumors to cause tumor starvation, cut off nutrient supply, and the generation of gluconic acid aggravates the micro-acid environment of tumors and improves the catalytic efficiency of Fenton reaction. Nanozymes are nanomaterials with enzyme-like activity. Copper sulfide nanoparticles have become a research hotspot in the biomedical field due to their advantages such as strong near-infrared absorption, good biocompatibility and low cytotoxicity. The living cell-coupled complex enzyme lipid nanoparticles described in this article use living cells as carriers to deliver three complex enzymes to cancer tissues, increase the stability of enzyme drugs, prolong the efficacy time, increase drug bioavailability, reduce immunogenicity, improve anti-cancer effects, and reduce side effects.

[0003] The copper sulfide nanozyme described in this article is a copper sulfide nanoparticle with a composite coating of polydopamine, hyaluronic acid and folic acid, which can improve the water solubility of copper nanoparticles, have good dispersibility in water, and are easy to load into lipid nanoparticles. At the same time, the composite coating of polydopamine, hyaluronic acid and folic acid can improve the photothermal effect and photodynamic effect, generate a large amount of reactive oxygen, and induce oxidative stress death of cancer cells; the biological enzymes arginine deiminase and glucose oxidase can consume the nutrients arginine and glucose on which cancer cells depend for survival, exerting a starvation therapy effect; the combination of copper sulfide nanozymes and glucose oxidase can produce a cascade effect, promote the continuous generation of reactive oxygen and the continuous consumption of glucose oxidase, and improve the anti-cancer effect; living cells can actively bring lipid nanoparticles into the tumor microenvironment through surface chemokines and signal molecules, prevent the destruction of the mononuclear phagocytic system and lysosomes, enhance the stability of the drug in the body, increase the circulation time of lipid nanoparticles in the body, and improve the anti-cancer effect.

[0004] After searching patents and literature, there are no reports on copper sulfide nanoparticles with composite coatings of polydopamine, hyaluronic acid and folic acid, no reports on lipid nanoparticles encapsulating arginine deiminase, glucose oxidase and copper sulfide nanozymes, and no reports on one or more drugs in lipid nanoparticles modified with maleimide groups encapsulating arginine deiminase, glucose oxidase and copper sulfide nanozymes. At present, there are no reports on macrophages coupled with lipid nanoparticles encapsulating arginine deiminase, glucose oxidase and copper sulfide nanozymes, no reports on other immune cells coupled with nanoparticles encapsulating arginine deiminase, glucose oxidase and copper sulfide nanozymes, and no reports on other types of living cells coupled with nanoparticles encapsulating arginine deiminase, glucose oxidase and copper sulfide nanozymes. The lipid nanoparticles prepared by the present invention, which are coupled with living cells and encapsulate arginine deiminase, glucose oxidase and copper sulfide nanozymes, prolong the in vivo circulation half-life of enzyme drugs, improve the bioavailability of the complex enzyme, and are combined with photothermal therapy and photodynamic therapy. On the one hand, the high temperature generated by photothermal therapy can increase the uptake of copper sulfide nanozymes by tumor cells and enhance the effect of photodynamic therapy; on the other hand, photodynamic therapy can destroy tumor blood vessels, further aggravate the local hypoxia of the tumor, and promote the thermal damage of photothermal therapy to be more concentrated in the tumor tissue, thereby improving the therapeutic effect of the drug on cancer. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide lipid nanoparticles encapsulating arginine deiminase, glucose oxidase and copper sulfide nanozyme coupled with living cells and a preparation method thereof.

[0006] The living cell-coupled complex enzyme lipid nanoparticles prepared by the present invention overcome the shortcomings of enzyme drugs such as poor in vitro and in vivo stability, high immunogenicity, and easy allergic reactions, reduce toxic side effects, and cooperate with photothermal therapy and photodynamic therapy to produce a large amount of reactive oxygen, leading to stress death of cancer cells. This study provides a new optional preparation for enzyme drugs, which can be used for cancer treatment.

[0007] The present invention provides a living cell-coupled complex enzyme lipid nanoparticle, which is characterized in that it comprises living cells and lipid nanoparticles encapsulating the complex enzyme, wherein the living cells include (1) M0 macrophages; (2) M1 macrophages; (3) red blood cells; (4) neutrophils; (5) tumor-infiltrating lymphocytes; (6) mesenchymal stem cells; (7) platelets; (8) cytotoxic T lymphocytes; (9) natural killer cells; (10) dendritic cells; and (11) endothelial cells. The copper sulfide nanozyme is a copper sulfide nanoparticle with a composite coating of polydopamine, hyaluronic acid and folic acid. The buffer 1 in the preparation method of copper sulfide nanoparticles includes sodium carbonate-sodium bicarbonate buffer, tris(hydroxymethylaminomethane)-hydrochloric acid buffer, boric acid-borax buffer, N,N-dihydroxyethylglycine-sodium hydroxide buffer, bis(2-hydroxyethylamino)tris(hydroxymethyl)methane-hydrochloride buffer, glycine-hydrochloride buffer, with a concentration of 50-150mM, a buffer pH of 7.5-9.5, and the mass ratios of the remaining components are: 45-180 parts of copper chloride dihydrate, 60-240 parts of sodium sulfide nonahydrate, 57-228 parts of sodium citrate, 5-20 parts of dopamine hydrochloride, 3-12 parts of hyaluronic acid, 3-12 parts of folic acid, 10000-40000 parts of buffer 1, and 80000-150000 parts of ultrapure water; the lipid encapsulated composite enzyme The content of arginine deiminase in the nanoparticles is 2.20-35.80 U / mL, the content of glucose oxidase is 2.50-40.00 U / mL, and the content of copper sulfide nanozyme is 0.05-0.80 mg / mL. The buffer 2 used includes sodium carbonate-sodium bicarbonate buffer, tris(hydroxymethylaminomethane)-hydrochloric acid buffer, boric acid-borax buffer, N,N-dihydroxyethylglycine-sodium hydroxide buffer, bis(2-hydroxyethylamino)tris(hydroxymethyl)methane-hydrochloride buffer, and glycine-hydrochloride buffer. Buffer 2 and buffer 3 are the same type of buffer in the same formula, with equal concentrations of 50-150 mM and the same pH of 5.5-8.5. The mass ratios of the remaining components are: 2-8 parts of copper sulfide nanozyme, 50-200 parts of lecithin, 25-100 parts of cholesterol, and DSPE-PEG2000-MAL 25-100 parts, vitamin E polyethylene glycol 1000 succinate (TPGS) 25-100 parts, buffer 2 is 10000-40000 parts, buffer 3 is 2500-10000 parts; the content of arginine deiminase in the living cell-coupled complex enzyme lipid nanoparticles is 2.20-35.80U / mL, the content of glucose oxidase is 2.50-40.00U / mL, and the content of copper sulfide nanozyme is 0.05-0.80 mg / mL, the lipid nanoparticles of living cell-coupled complex enzyme provided by the present invention, the preparation steps include: (1) Preparation method of copper sulfide nanozyme: copper chloride dihydrate and sodium citrate are stirred and dissolved in ultrapure water at 20-40°C to obtain solution A; sodium sulfide nonahydrate is added to solution A and stirred at 85-95°C for 20-60 minutes to obtain solution B; solution B is vacuum dried at 50-70°C for 20-36 hours to obtain solid C; dopamine hydrochloride is dissolved in pH A dopamine hydrochloride solution is formed in a buffer solution with a concentration of 8.0-9.5, and the solution is stirred magnetically at 20-30°C in the dark for 20-60 minutes to obtain a polydopamine solution D. Solid C, hyaluronic acid and folic acid are added to the solution D and stirred in the dark for 20-36 hours, centrifuged, and the precipitate is collected and dried to obtain a copper sulfide nanozyme. (2) A method for preparing lipid nanoparticles of arginine deiminase, copper sulfide nanozyme and glucose oxidase: arginine deiminase, copper sulfide nanozyme and glucose oxidase are dissolved in buffer solution 2 to obtain a solution F. Phospholipids, cholesterol, DSPE-PEG2000-MAL and TPGS are dissolved in one or more organic solvents such as dichloromethane, chloroform, anhydrous ethanol, methanol and ether, and ultrasonicated in the dark. The organic solvent is removed under reduced pressure to form a uniform film. Solution F is added and oscillated at 35-45°C for 1-3 hours, and then the film is filtered through 0.45 μm and 0.22 μm microporous membranes respectively. The operation is repeated 2-4 times to obtain lipid nanoparticles of arginine deiminase, copper sulfide nanozyme and glucose oxidase; (3) active M0 type macrophage Preparation method of macrophages: take M0 macrophages with a cell density of (2E+6) to (5E+6) cells / mL, culture them in a serum-free medium containing 1.0-2.0 mM tris(2-carboxyethyl)phosphine hydrochloride for 10-30 minutes, centrifuge, collect the precipitate, and obtain surface-modified live M0 macrophages; (4) Preparation method of live M1 macrophages: take M0 macrophages with a cell density of (2E+6) to (5E+6) cells / mL, incubate them in a drug-containing medium containing 100 ng / mL lipopolysaccharide for 2 After 4 hours, the cells were cultured in a serum-free medium containing 1.0-2.0 mM tri(2-carboxyethyl)phosphine hydrochloride for 10-30 minutes, centrifuged, and the precipitate was collected to obtain surface-modified live M1 macrophages. (5) Preparation method of other live cells: one of red blood cells, neutrophils, tumor-infiltrating lymphocytes, mesenchymal stem cells, endothelial cells, cytotoxic T lymphocytes, natural killer cells, platelets and dendritic cells was taken, with a cell density of (2E+6) to (5E+6) cells / mL, and cultured in a serum-free medium containing 1.0-2.0 mM tri(2-carboxyethyl)phosphine hydrochloride for 10-30 minutes, centrifuged, and the precipitate was collected to obtain surface-modified live M1 macrophages.0mM tri(2-carboxyethyl)phosphine hydrochloride serum-free medium is cultured for 10-30 minutes, centrifuged, and the precipitate is collected to obtain other living cells with surface modification; (6) Preparation method of living cell-coupled complex enzyme lipid nanoparticles: the lipid nanoparticles encapsulating the complex enzyme obtained in step (2) are mixed with the M0 type macrophages obtained in step (3), the M1 type macrophages obtained in step (4), or one of the various living cells obtained in step (5) at a volume ratio of 10:1 to 40:1, incubated at 37°C under shaking conditions for 20-40 minutes, centrifuged at 1000rpm for 2-5 minutes, the precipitate is collected, and buffer 3 is added to obtain living cell-coupled complex enzyme lipid nanoparticles. .

[0008] The average particle size of the lipid nanoparticles encapsulating the complex enzyme prepared by the present invention is less than 200 nm ( Figure 1 A), with the advantage of small particle size, lipid nanoparticles can easily penetrate the gap of tumor vascular endothelium, efficiently penetrate into tumor tissue, and are not easily recognized and cleared by the body's mononuclear phagocyte system, thereby prolonging the retention time in the blood circulation. The average potential of the lipid nanoparticles encapsulating the complex enzyme prepared by the present invention is -11.70mV ( Figure 1 B), while cells in the tumor site are usually positively charged and can be enriched in the tumor site by electrostatic attraction. The copper sulfide nanozyme provided by the present invention has good photothermal performance. As the concentration increases, its temperature value gradually increases, with a concentration-dependent trend, and can reach the required anti-cancer temperature of 42°C within 5 minutes ( Figure 2 ).

[0009] The living cell-coupled complex enzyme lipid nanoparticles provided by the present invention are attached to the cell surface by reducing the disulfide bonds on the surface of the living cells to sulfhydryl groups, and reacting with the maleimide groups in the lipid nanoparticles to undergo a nucleophilic addition reaction between maleimide and sulfhydryl groups to couple with the living cells. Figure 3 A), showing obvious green fluorescence on the cell surface, quantitative analysis can be used to calculate the average fluorescence intensity, which is about 250 times higher than that of uncoupled living cells ( Figure 3 B), coupling to the cell surface can prevent phagocytosis by macrophages. Chemokines and signaling molecules on the cell surface can bring lipid nanoparticles into the tumor microenvironment, thereby improving the targeting of the complex enzyme to the tumor microenvironment.

[0010] The living cells in the living cell-coupled complex enzyme lipid nanoparticles provided by the present invention have good activity, and the coupling can also better maintain the normal physiological function and biological function of the living cells ( Figure 4 ). Complex enzyme lipid nanoparticles can successfully bring lipid nanoparticles into the complex and special tumor microenvironment with the help of the physiological functions and migration abilities of living cells, and exert anti-tumor therapeutic effects.

[0011] The living cell-coupled complex enzyme lipid nanoparticles provided by the present invention can play a role in photodynamic therapy to generate a large amount of reactive oxygen species ( Figure 5 ). When living cells coupled with complex enzyme lipid nanoparticles combined with photothermal therapy produce a large amount of reactive oxygen species, the antioxidant defense system of tumor cells is more easily broken, thereby causing irreversible oxidative stress damage and ultimately leading to cell death. Normal cells, due to their relatively strong antioxidant capacity, can better tolerate the increase in this reactive oxygen level.

[0012] The living cell-coupled complex enzyme lipid nanoparticles provided by the present invention can effectively kill cancer cells by combining photothermal therapy and photodynamic therapy ( Figure 6 ). Compared with the normal group, the proportion of dead cells in the live cell-coupled complex enzyme lipid nanoparticle group was 83%, and the proportion of dead cells after combined laser irradiation reached 95%, indicating that the live cell-coupled complex enzyme lipid nanoparticle combined with photothermal therapy can effectively kill cancer cells and exert an anti-tumor effect. The reasons why this system exerts an anti-cancer effect may include: (1) There are many chemokines and signaling molecules on the surface of living cells, which can accurately bring lipid nanoparticles to the tumor microenvironment, increase the concentration of complex enzymes in the local tumor, and then increase the production of reactive oxygen species; (2) Arginine deiminase and glucose oxidase can respectively exert starvation therapy effects, consume arginine and glucose required by cancer cells, and improve the anti-cancer effect; (3) The dual protection of living cells and lipid nanoparticles prevents the complex enzyme from being hydrolyzed by proteases and cleared by phagocytes; (4) The copper sulfide nanozyme and glucose oxidase are used together to form a cascade reaction, which promotes the production of a large amount of reactive oxygen species and the consumption of glucose, causing cancer cells to die due to stress.

[0013] The present invention is different from the delivery carriers and preparation processes of arginine deiminase, glucose oxidase and copper sulfide nanozymes reported in the usual research. The delivery carriers of enzyme drugs such as arginine deiminase are reported in the research and reports of conventional liposomes, nano micelles, etc. The lipid nanoparticles of macrophage-coupled complex enzymes in the present invention are a novel living cell delivery system for biomimetic drugs. At present, there are no reports of copper sulfide nanozymes with a composite coating of polydopamine, hyaluronic acid and folic acid, no research reports of nanoparticles of complex enzymes of arginine deiminase, glucose oxidase and copper sulfide nanozymes, and no reports of one or more substances in lipid nanoparticles of macrophage-coupled complex enzymes of arginine deiminase, glucose oxidase and copper sulfide nanozymes. The present invention prepares copper sulfide nanoparticles with a composite coating of polydopamine, hyaluronic acid and folic acid for the first time, and the present invention encapsulates arginine deiminase, glucose oxidase and copper sulfide nanozymes in lipid nanoparticles for the first time. The present invention is the first to couple living cells with maleimide-functionalized lipid nanoparticles containing arginine deiminase, glucose oxidase and copper sulfide nanozymes as a composite enzyme through a maleimide-thiol nucleophilic addition reaction. The preparation can improve the stability of the preparation, prolong the circulation time in the body, enhance tumor targeting, and can improve the efficacy and reduce toxic side effects when combined with photothermal therapy and photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The particle size of the lipid nanoparticles encapsulating the complex enzyme prepared by the present invention is ( Figure 1 A) and potential ( Figure 1 B)Fig.

[0015] Experimental conditions: Malvern laser particle size analyzer was used to measure the particle size and potential of lipid nanoparticles encapsulating complex enzymes.

[0016] The results showed that the average particle size of lipid nanoparticles encapsulating the complex enzyme was 121.70 nm and the average potential was -11.70 mV.

[0017] Figure 2 The photothermal performance of the copper sulfide nanozyme prepared by the present invention in vitro ( Figure 2 ).

[0018] Test conditions: 808nm infrared instrument (2W / cm 2 ) The copper sulfide nanozyme prepared by the present invention was irradiated with light for 10 minutes at different copper sulfide nanozyme concentrations (75, 100, 150, 200, 250 μg / mL), and the temperature rise of the liquid was measured at the set time point.

[0019] The results showed that the photothermal effect of copper sulfide nanozymes is concentration-dependent. When the concentration is 200 μg / mL and the power is 2.0w, the temperature can rise to 42°C within 5 minutes, exerting a good photothermal therapeutic effect and can be used for cancer treatment.

[0020] Figure 3 This is a laser confocal image of the living cell-coupled complex enzyme lipid nanoparticles prepared by the present invention ( Figure 3 A) and fluorescence intensity diagram ( Figure 3 B).

[0021] Experimental conditions: The living cell-coupled complex enzyme lipid nanoparticles were prepared after the nanoparticles were labeled with a fluorescent agent. The images of the living cells alone and the living cell-coupled complex enzyme lipid nanoparticles were observed and photographed using a laser confocal microscope, and the average fluorescence intensity was calculated by quantitative analysis.

[0022] The results showed that the laser confocal image showed that the living cell-coupled complex enzyme lipid nanoparticles showed obvious green fluorescence on the surface of living cells. Compared with living cells alone, the living cell-coupled complex enzyme lipid nanoparticles can attach to the cell surface by coupling with living cells and show obvious green fluorescence on the cell surface ( Figure 3 A), and the average fluorescence intensity can be calculated by quantitative analysis, which is about 250 times higher than that of uncoupled living cells ( Figure 3 B), indicating that the living cells provided by the present invention are successfully coupled with the complex enzyme lipid nanoparticles.

[0023] Figure 4 The invention discloses the influence of the living cell-coupled complex enzyme lipid nanoparticles prepared in the present invention on the activity of cells.

[0024] Experimental conditions: Live cells in the logarithmic growth phase and live cells coupled to lipid nanoparticles encapsulating complex enzymes were inoculated at 5E+3 cells / well in a 96-well plate, and high-glucose culture medium containing 10% fetal bovine serum was added. The cells were cultured at 37°C and 5% carbon dioxide for 24 hours. The culture medium was discarded and MTT reagent was added to detect cell viability.

[0025] The results showed that compared with uncoupled living cells, the viability of cells in living cell-coupled complex enzyme lipid nanoparticles was 95.55%, indicating that coupling lipid nanoparticles encapsulating complex enzymes to the surface of living cells through surface modification will not affect the viability of the cells, but can retain the cell viability and biological characteristics of living cells, giving them good biocompatibility and immune escape ability, and the lipid nanoparticles can be targeted to the tumor microenvironment by hitchhiking to exert an anti-cancer effect.

[0026] Figure 5 The invention relates to the ability of the living cell-coupled complex enzyme lipid nanoparticles to produce active oxygen.

[0027] Experimental conditions: NCl-H1688 cells in the logarithmic growth phase were seeded at 5E+3 cells / well in a 6-well plate, and 1640 medium containing 10% fetal bovine serum was added. The cells were cultured at 37°C and 5% carbon dioxide for 24 hours to adhere to the wall. The culture medium containing arginine deiminase, glucose oxidase, lipid nanoparticles encapsulating complex enzymes, and live cell-coupled complex enzyme lipid nanoparticles was added to each group and incubated for 24 hours. The live cell-coupled complex enzyme lipid nanoparticles + laser group was exposed to 808nm near-infrared light (2.0W / cm 2 After the incubation, the cells were incubated with reactive oxygen species detection reagent at 37°C in the dark for 30 minutes, and the samples from each well were collected and the fluorescence intensity was detected by flow cytometry.

[0028] The results showed that the living cell-coupled complex enzyme lipid nanoparticles prepared by the present invention combined with photothermal therapy can effectively produce a large amount of reactive oxygen. Compared with the normal group, the reactive oxygen produced by the living cell-coupled complex enzyme lipid nanoparticle group was 5 times that of the normal group and 3 times that of the free enzyme drug group; the living cell-coupled complex enzyme lipid nanoparticles + laser group was 6 times that of the normal group and 4 times that of the free enzyme drug group, indicating that the macrophage-coupled complex enzyme-encapsulated lipid nanoparticles combined with photothermal therapy produced a large amount of reactive oxygen, which can cause oxidative stress response in tumor cells, thereby killing tumor cells.

[0029] Figure 6 The invention discloses the ability of the living cell-coupled complex enzyme lipid nanoparticles to kill cancer cells.

[0030] Experimental conditions: NCl-H1688 cells in the logarithmic growth phase were seeded at 5E+3 cells / well in a 6-well plate, and 1640 medium containing 10% fetal bovine serum was added. The cells were cultured at 37°C and 5% carbon dioxide for 24 hours to adhere to the wall. The culture medium containing arginine deiminase, glucose oxidase, lipid nanoparticles encapsulating complex enzymes, and live cell-coupled complex enzyme lipid nanoparticles was added to each group and incubated for 24 hours. The live cell-coupled complex enzyme lipid nanoparticles + laser group was exposed to 808nm near-infrared light (2.0W / cm 2 After the incubation, the cells were incubated with cell activity and cytotoxicity detection reagents at 37°C in the dark for 30 minutes, and samples from each well were collected and the fluorescence intensity was detected by flow cytometry.

[0031] The results showed that the living cell-coupled complex enzyme lipid nanoparticles prepared by the present invention can effectively kill cancer cells. Compared with the normal group, the proportion of dead cells in the glucose oxidase group was 20%, the proportion of dead cells in the arginine deiminase group was 22%, and the proportion of dead cells in the living cell-coupled complex enzyme lipid nanoparticle group was 83%. After combined laser irradiation, the proportion of dead cells reached 95%, indicating that the living cell-coupled complex enzyme lipid nanoparticles combined with photothermal therapy can effectively kill cancer cells and play an anti-tumor effect. DETAILED DESCRIPTION

[0032] In order to further illustrate the present invention and its advantages, the following specific examples are given. It should be understood that these examples are only for specific illustration and are not intended to limit the scope of the present invention.

[0033] Embodiment 1:

[0034] The M0 type macrophage coupled complex enzyme lipid nanoparticles include M0 type macrophages and lipid nanoparticles encapsulating complex enzymes. In the copper sulfide nanozyme, buffer 1 is 100mM bis(2-hydroxyethylamino)tris(hydroxymethyl)methane-hydrochloride buffer with a pH of 7.5, and the mass ratios of the remaining components are: 68 parts of copper chloride dihydrate, 85 parts of sodium sulfide nonahydrate, 155 parts of sodium citrate, 8 parts of dopamine hydrochloride, 5 parts of hyaluronic acid, 7 parts of folic acid, 12000 parts of buffer 1, and 85000 parts of ultrapure water. The content of arginine deiminase in the preparation is 13.42U / mL, the content of glucose oxidase is 14.10U / mL, the content of copper sulfide nanozyme is 0.25mg / mL, buffer 2 and buffer 3 are both 100mM bis(2-hydroxyethylamino)tris(hydroxymethyl)methane-hydrochloride buffer with a pH of 5.5, and the mass ratios of the remaining components are: 3 parts of copper sulfide nanozyme, 78 parts of lecithin, 28 parts of cholesterol, 42 parts of DSPE-PEG2000-MAL, 32 parts of TPGS, 12000 parts of buffer 2, and 2500 parts of buffer 3.

[0035] Preparation method: (1) Preparation method of copper sulfide nanozyme: copper chloride dihydrate and sodium citrate are stirred and dissolved in ultrapure water at 30°C to obtain solution A; sodium sulfide nonahydrate is added to solution A and stirred at 85°C for 20 minutes to obtain solution B; solution B is vacuum dried at 50°C for 20 hours to obtain solid C; dopamine hydrochloride is dissolved in 100mM, pH A dopamine hydrochloride solution was formed in a 7.5 bis(2-hydroxyethylamino)tris(hydroxymethyl)methane-hydrochloride buffer solution, and the solution was stirred magnetically at 20°C in the dark for 20 minutes to obtain a polydopamine solution D. Solid C, hyaluronic acid and folic acid were added to the solution D and stirred in the dark for 20 hours, centrifuged, and the precipitate was collected and dried to obtain a copper sulfide nanozyme. (2) Preparation method of lipid nanoparticles encapsulating complex enzymes: arginine deiminase, copper sulfide nanozyme and glucose oxidase were dissolved in 50 mM bis(2-hydroxyethylamino)tris(hydroxymethyl)methane-hydrochloride buffer solution with a pH of 5.5 to obtain a solution F; lecithin, cholesterol, DSPE-PEG2000-MAL and TPGS were dissolved in 20 mL chloroform, and the solution was stirred in the dark for 20 minutes under reduced pressure to form a uniform film, and then the solution F was added. After constant temperature oscillation at 35°C for 1 hour, the solution was passed through 0.45 μm and 0. .22μm microporous filter membrane, repeat the operation twice to obtain lipid nanoparticles encapsulating complex enzymes; (3) M0 macrophage preparation method: take M0 macrophages with a cell density of (2E+6) cells / mL, culture them in a serum-free medium containing 1.0mM tris(2-carboxyethyl)phosphine hydrochloride for 10 minutes, centrifuge, collect the precipitate, and obtain surface-modified M0 macrophages; (4) M0 macrophage-coupled complex enzyme lipid nanoparticle preparation method: the lipid nanoparticles encapsulating complex enzymes obtained in step (2) and the M0 macrophages obtained in step (3) are mixed at a volume ratio of 40:1, incubated at 37°C for 20 minutes under shaking conditions, centrifuged at 1000rpm for 2 minutes, collected the precipitate, added 50mM bis(2-hydroxyethylamino)tris(hydroxymethyl)methane-hydrochloride buffer with a pH of 5.5, and obtained M0 macrophage-coupled complex enzyme lipid nanoparticles.

[0036] Embodiment 2:

[0037] The erythrocyte-coupled complex enzyme lipid nanoparticles include erythrocytes and lipid nanoparticles encapsulating the complex enzyme. The buffer 1 in the copper sulfide nanozyme is 50 mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 9.0, and the mass ratios of the remaining components are: 125 parts of copper chloride dihydrate, 150 parts of sodium sulfide nonahydrate, 80 parts of sodium citrate, 12 parts of dopamine hydrochloride, 8 parts of hyaluronic acid, 4 parts of folic acid, 18,000 parts of buffer 1, and 92,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 33.65U / mL, the content of glucose oxidase is 39.00U / mL, and the content of copper sulfide nanozyme is 0.05mg / mL. The buffer 2 and buffer 3 used are both 50mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 7.5. The mass ratios of the remaining components are: 2 parts of copper sulfide nanozyme, 16 parts of arginine deiminase, 15 parts of glucose oxidase, 180 parts of lecithin, 33 parts of cholesterol, 61 parts of DSPE-PEG2000-MAL, 73 parts of TPGS, 38000 parts of buffer 2, and 3180 parts of buffer 3.

[0038] The preparation method is basically the same as that in Example 1, including the following different steps: in step (1), the preparation conditions are changed to "dissolve cupric chloride dihydrate and sodium citrate in ultrapure water with stirring at 20°C", "stir at 80°C for 30 minutes", "vacuum dry at 60°C for 36 hours", "dissolve dopamine hydrochloride in 50 mM N,N-dihydroxyethylglycine-sodium hydroxide buffer solution with a pH of 9.0", "magnetically stir at 25°C in the dark for 25 minutes", "stir in the dark for 25 hours"; in step (2), the conditions are changed to "dissolve in N,N-dihydroxyethylglycine-sodium hydroxide buffer solution to obtain solution F "," dissolved in 20 mL of methanol," "oscillated at 40 ° C for 2 hours," "repeat the operation twice"; in step (3), change to "red blood cells," "take the cell density to (3E+6) cells / mL," "1.5 mM tris(2-carboxyethyl)phosphine hydrochloride," "culture for 20 minutes"; in step (4), change to "with the red blood cells obtained in step (3)," "mixed at a volume ratio of 18:1," "incubated at 37 ° C for 20 minutes," "centrifuged for 2 minutes," and "add 50 mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 9.0."

[0039] Embodiment 3:

[0040] M1 macrophage-coupled complex enzyme lipid nanoparticles include red blood cells and lipid nanoparticles encapsulating complex enzymes. Buffer 1 in the copper sulfide nanozyme is a 160mM glycine-hydrochloride buffer with a pH of 8.0, and the mass ratios of the remaining components are: 72 parts of copper chloride dihydrate, 90 parts of sodium sulfide nonahydrate, 70 parts of sodium citrate, 15 parts of dopamine hydrochloride, 7 parts of hyaluronic acid, 5 parts of folic acid, 23,000 parts of buffer 1, and 115,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 7.34U / mL, the content of glucose oxidase is 8.60U / mL, the content of copper sulfide nanozyme is 0.18mg / mL, buffer 2 and buffer 3 are both 50mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 6.0, and the mass ratios of the remaining components are: 5 parts of copper sulfide nanozyme, 23 parts of arginine deiminase, 24 parts of glucose oxidase, 120 parts of lecithin, 45 parts of cholesterol, 55 parts of DSPE-PEG2000-MAL, 58 parts of TPGS, 28000 parts of buffer 2, and 5900 parts of buffer 3.

[0041] The preparation method is basically the same as Example 1, including the following different steps: in step (1), the preparation conditions are changed to "dissolve copper chloride dihydrate and sodium citrate in ultrapure water at 40°C with stirring", "stir at 90°C for 40 minutes", "vacuum drying at 55°C for 24 hours", "dissolve dopamine hydrochloride in 160mM glycine-hydrochloride buffer with a pH of 8.0", "magnetically stir at 30°C in the dark for 40 minutes", "stir in the dark for 24 hours"; in step (2), it is changed to "dissolve in 50mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 6.0 to obtain solution F", "dissolve in 20mL chloroform", "oscillate at a constant temperature of 45°C for 2 hours", "repeat the operation 4 times"; in step (3), it is changed to "M1 macrophages",

[0042] "The cell density is (2E+6) cells / mL", "1.0 mM tris(2-carboxyethyl)phosphine hydrochloride", "culture for 10 minutes"; in step (4), change to "with the M1 macrophages obtained in step (3)", "mix at a volume ratio of 25:1", "incubate at 37°C for 20 minutes", "centrifuge for 3 minutes", "add 160 mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 6.0".

[0043] Embodiment 4:

[0044] Neutrophil-coupled complex enzyme lipid nanoparticles include neutrophils and lipid nanoparticles encapsulating complex enzymes. Buffer 1 in the copper sulfide nanozyme is a 70mM sodium carbonate-sodium bicarbonate buffer with a pH of 9.5, and the mass ratios of the remaining components are: 150 parts of copper chloride dihydrate, 180 parts of sodium sulfide nonahydrate, 180 parts of sodium citrate, 6 parts of dopamine hydrochloride, 4 parts of hyaluronic acid, 8 parts of folic acid, 15,000 parts of buffer 1, and 128,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 7.70U / mL, the content of glucose oxidase is 3.30U / mL, the content of copper sulfide nanozyme is 0.19mg / mL, buffer 2 and buffer 3 are both 70mM sodium carbonate-sodium bicarbonate buffer with a pH of 7.0, and the mass ratios of the remaining components are: 7 parts of copper sulfide nanozyme, 31 parts of arginine deiminase, 12 parts of glucose oxidase, 155 parts of lecithin, 56 parts of cholesterol, 78 parts of DSPE-PEG2000-MAL, 65 parts of TPGS, 36000 parts of buffer 2, and 6580 parts of buffer 3.

[0045] The preparation method is basically the same as that in Example 1, including the following different steps: in step (1), the preparation conditions are changed to "dissolve copper chloride dihydrate and sodium citrate in ultrapure water with stirring at 35°C", "stir at 95°C for 25 minutes", "vacuum dry at 65°C for 30 hours", "dissolve dopamine hydrochloride in 70mM sodium carbonate-sodium bicarbonate buffer with a pH of 9.5", "magnetically stir at 25°C in the dark for 40 minutes", "stir in the dark for 30 hours"; in step (2), the conditions are changed to "dissolve in 70mM sodium carbonate-sodium bicarbonate buffer with a pH of 7.0 to obtain a solution F", "dissolve in 20 mL of ether", "keep at 37°C for 2 hours", "repeat the operation 3 times"; in step (3), change to "neutrophils", "take the cell density to (4E+6) cells / mL", "2.0 mM tris(2-carboxyethyl)phosphine hydrochloride", "culture for 10 minutes"; in step (4), change to "with the neutrophils obtained in step (3)", "mix at a volume ratio of 15:1", "incubate at 37°C for 20 minutes", "centrifuge for 5 minutes", "add 70 mM sodium carbonate-sodium bicarbonate buffer with a pH of 7.0".

[0046] Embodiment 5:

[0047] Dendritic cell-coupled complex enzyme lipid nanoparticles include dendritic cells and lipid nanoparticles encapsulating complex enzymes. Buffer 1 in the copper sulfide nanozyme is a 90mM boric acid-borax buffer with a pH of 8.1, and the mass ratios of the remaining components are: 50 parts of copper chloride dihydrate, 70 parts of sodium sulfide nonahydrate, 95 parts of sodium citrate, 18 parts of dopamine hydrochloride, 9 parts of hyaluronic acid, 3 parts of folic acid, 28,000 parts of buffer 1, and 133,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 2.69U / mL, the content of glucose oxidase is 7.80U / mL, the content of copper sulfide nanozyme is 0.15mg / mL, buffer 2 and buffer 3 are both 90mM boric acid-borax buffer with a pH of 7.5, and the mass ratios of the remaining components are: 6 parts of copper sulfide nanozyme, 12 parts of arginine deiminase, 31 parts of glucose oxidase, 90 parts of lecithin, 72 parts of cholesterol, 33 parts of DSPE-PEG2000-MAL, 41 parts of TPGS, 40,000 parts of buffer 2, and 7,260 parts of buffer 3.

[0048] The preparation method is basically the same as that in Example 1, including the following different steps: in step (1), the preparation conditions are changed to "dissolve copper chloride dihydrate and sodium citrate in ultrapure water with stirring at 25° C.", "stir at 85° C. for 40 minutes", "vacuum dry at 70° C. for 24 hours", "dissolve dopamine hydrochloride in 90 mM boric acid-borax buffer with a pH of 8.1", "magnetically stir at 20° C. in the dark for 40 minutes", "stir in the dark for 36 hours"; in step (2), the conditions are changed to "dissolve in 90 mM boric acid-borax buffer with a pH of 7.5 to obtain solution F". "dissolved in 20 mL of anhydrous ethanol", "oscillated at 35°C for 3 hours", "repeated the operation 3 times"; in step (3), changed to "dendritic cells", "take the cell density to (5E+6) cells / mL", "1.0 mM tris(2-carboxyethyl)phosphine hydrochloride", "cultured for 20 minutes"; in step (4), changed to "with the dendritic cells obtained in step (3)", "mixed at a volume ratio of 30:1", "incubated at 37°C for 30 minutes", "centrifuged for 5 minutes", "added 90 mM boric acid-borax buffer with a pH of 7.5".

[0049] Embodiment 6:

[0050] Cytotoxic T lymphocyte coupled complex enzyme lipid nanoparticles, including cytotoxic T lymphocytes and lipid nanoparticles encapsulating complex enzymes. Buffer 1 in the copper sulfide nanozyme is a 120mM glycine-hydrochloride buffer with a pH of 7.6, and the mass ratios of the remaining components are: 100 parts of copper chloride dihydrate, 165 parts of sodium sulfide nonahydrate, 160 parts of sodium citrate, 11 parts of dopamine hydrochloride, 3 parts of hyaluronic acid, 9 parts of folic acid, 25,000 parts of buffer 1, and 110,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 12.17U / mL, the content of glucose oxidase is 23.60U / mL, the content of copper sulfide nanozyme is 0.43mg / mL, buffer 2 and buffer 3 are both 120mM glycine-hydrochloride buffer with a pH of 6.0, and the mass ratios of the remaining components are: 6 parts of copper sulfide nanozyme, 19 parts of arginine deiminase, 33 parts of glucose oxidase, 160 parts of lecithin, 90 parts of cholesterol, 28 parts of DSPE-PEG2000-MAL, 91 parts of TPGS, 14000 parts of buffer 2, and 7940 parts of buffer 3.

[0051] The preparation method is basically the same as that in Example 1, including the following different steps: in step (1), the preparation conditions are changed to "dissolve cupric chloride dihydrate and sodium citrate in ultrapure water at 20°C with stirring", "stir at 95°C for 30 minutes", "vacuum dry at 60°C for 36 hours", "dissolve dopamine hydrochloride in 120 mM glycine-hydrochloride buffer with a pH of 7.6", "magnetically stir at 25°C in the dark for 40 minutes", "stir in the dark for 25 hours"; in step (2), the conditions are changed to "dissolve in 120 mM glycine-hydrochloride buffer with a pH of 6.0 to obtain solution F", "dissolve "dissolved in 20 mL of anhydrous ethanol", "oscillated at 40°C for 2 hours", "repeated the operation 3 times"; in step (3), change to "cytotoxic T lymphocytes", "take the cell density to (4E+6) cells / mL", "2.0 mM tris(2-carboxyethyl)phosphine hydrochloride", "cultured for 30 minutes"; in step (4), change to "with the cytotoxic T lymphocytes obtained in step (3)", "mixed at a volume ratio of 32:1", "incubated at 37°C for 40 minutes", "centrifuged for 5 minutes", "added 120 mM glycine-hydrochloride buffer with a pH of 6.0".

[0052] Embodiment 7:

[0053] Mesenchymal stem cell-coupled complex enzyme lipid nanoparticles, including mesenchymal stem cells and lipid nanoparticles encapsulating complex enzymes. Buffer 1 in the copper sulfide nanozyme is a 140mM glycine-hydrochloride buffer with a pH of 8.3, and the mass ratios of the remaining components are: 130 parts of copper chloride dihydrate, 135 parts of sodium sulfide nonahydrate, 130 parts of sodium citrate, 7 parts of dopamine hydrochloride, 3 parts of hyaluronic acid, 9 parts of folic acid, 32,000 parts of buffer 1, and 98,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 9.67U / mL, the content of glucose oxidase is 10.40U / mL, the content of copper sulfide nanozyme is 0.32mg / mL, buffer 2 and buffer 3 are both 140mM glycine-hydrochloride buffer with a pH of 8.5, and the mass ratios of the remaining components are: 8 parts of copper sulfide nanozyme, 27 parts of arginine deiminase, 26 parts of glucose oxidase, 65 parts of lecithin, 81 parts of cholesterol, 85 parts of DSPE-PEG2000-MAL, 88 parts of TPGS, 25000 parts of buffer 2, and 8620 parts of buffer 3.

[0054] The preparation method is basically the same as Example 1, including the following different steps: in step (1), the preparation conditions are changed to "dissolve cupric chloride dihydrate and sodium citrate in ultrapure water with stirring at 25°C", "stir at 85°C for 40 minutes", "vacuum dry at 70°C for 36 hours", "dissolve dopamine hydrochloride in 140 mM glycine-hydrochloride buffer with a pH of 8.3", "magnetically stir at 25°C in the dark for 40 minutes", "stir in the dark for 36 hours"; in step (2), the conditions are changed to "dissolve in 140 mM glycine-hydrochloride buffer with a pH of 8.5 to obtain solution F ”, “dissolved in 20 mL of ether”, “oscillated at 45°C for 2 hours”, “repeat the operation 3 times”; in step (3), change to “mesenchymal stem cells”, “take the cell density to (3E+6) cells / mL”, “1.0 mM tris(2-carboxyethyl)phosphine hydrochloride”, “culture for 30 minutes”; in step (4), change to “with the mesenchymal stem cells obtained in step (3)”, “mixed at a volume ratio of 14:1”, “incubated at 37°C for 30 minutes”, “centrifuged for 3 minutes”, “add 140 mM glycine-hydrochloride buffer with a pH of 8.5”.

[0055] Embodiment 8:

[0056] The natural killer cell-coupled complex enzyme lipid nanoparticles include natural killer cells and lipid nanoparticles encapsulating complex enzymes. In the copper sulfide nanozyme, buffer 1 is 80 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer with a pH of 8.7, and the mass ratios of the remaining components are: 85 parts of copper chloride dihydrate, 100 parts of sodium sulfide nonahydrate, 105 parts of sodium citrate, 14 parts of dopamine hydrochloride, 5 parts of hyaluronic acid, 7 parts of folic acid, 16,000 parts of buffer 1, and 105,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 18.97U / mL, the content of glucose oxidase is 11.90U / mL, the content of copper sulfide nanozyme is 0.31mg / mL, buffer 2 and buffer 3 are both 80mM tris(hydroxymethylaminomethane)-hydrochloride buffer with a pH of 6.5, and the mass ratios of the remaining components are: 5 parts of copper sulfide nanozyme, 34 parts of arginine deiminase, 19 parts of glucose oxidase, 130 parts of lecithin, 67 parts of cholesterol, 92 parts of DSPE-PEG2000-MAL, 38 parts of TPGS, 16000 parts of buffer 2, and 9300 parts of buffer 3.

[0057] The preparation method is basically the same as that in Example 1, and includes the following different steps: in step (1), the preparation conditions are changed to "dissolve cupric chloride dihydrate and sodium citrate in ultrapure water at 30°C with stirring", "stir at 85°C for 20 minutes", "vacuum dry at 50°C for 20 hours", "dissolve dopamine hydrochloride in 80 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer solution with a pH of 8.7",

[0058] "Magnetic stirring at 20°C in the dark for 20 minutes", "Stirring in the dark for 20 hours"; in step (2), change to "dissolve in 80 mM Tris-HCl buffer at pH 6.5 to obtain solution F", "dissolve in 20 mL of dichloromethane", "oscillate at 35°C for 1 hour", "repeat the operation 3 times"; in step (3), change to "natural killer cells",

[0059] "The cell density is (5E+6) cells / mL", "1.0 mM tris(2-carboxyethyl)phosphine hydrochloride", "culture for 20 minutes"; in step (4), change to "with the natural killer cells obtained in step (3)", "mix at a volume ratio of 36:1", "incubate at 37°C for 20 minutes", "centrifuge for 3 minutes", "add 80 mM tris(2-carboxyethyl)phosphine hydrochloride buffer, pH 6.5".

[0060] Embodiment 9:

[0061] Platelet-coupled complex enzyme lipid nanoparticles, comprising platelets and lipid nanoparticles encapsulating complex enzymes. Buffer 1 in the copper sulfide nanozyme is a 110 mM sodium carbonate-sodium bicarbonate buffer with a pH of 7.8, and the mass ratio of the remaining components is: 160 parts of copper chloride dihydrate, 190 parts of sodium sulfide nonahydrate, 200 parts of sodium citrate, 9 parts of dopamine hydrochloride, 4 parts of hyaluronic acid, 8 parts of folic acid, 19,000 parts of buffer 1, and 140,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 8.95U / mL, the content of glucose oxidase is 12.70U / mL, the content of copper sulfide nanozyme is 0.18mg / mL, buffer 2 and buffer 3 are both 110mM sodium carbonate-sodium bicarbonate buffer with a pH of 7.0, and the mass ratios of the remaining components are: 4 parts of copper sulfide nanozyme, 22 parts of arginine deiminase, 28 parts of glucose oxidase, 80 parts of lecithin, 40 parts of cholesterol, 70 parts of DSPE-PEG2000-MAL, 45 parts of TPGS, 22000 parts of buffer 2, and 9980 parts of buffer 3.

[0062] The preparation method is basically the same as that in Example 1, and includes the following different steps: in step (1), the preparation conditions are changed to "dissolve cupric chloride dihydrate and sodium citrate in ultrapure water at 40°C with stirring", "stir at 90°C for 40 minutes", "vacuum drying at 65°C for 36 hours", "dissolve dopamine hydrochloride in 110 mM sodium carbonate-sodium bicarbonate buffer with a pH of 7.8", "magnetically stir at 30°C in the dark for 25 minutes", "stir in the dark for 24 hours"; in step (2), the conditions are changed to "dissolve in 110 mM sodium carbonate-sodium bicarbonate buffer with a pH of 7.8". 0 sodium carbonate-sodium bicarbonate buffer to obtain solution F", "dissolve in 20 mL methanol", "oscillate at 45°C for 2 hours", "repeat the operation 3 times"; in step (3), change to "platelets", "take the cell density to (3E+6) cells / mL", "1.5 mM tris(2-carboxyethyl)phosphine hydrochloride", "culture for 20 minutes"; in step (4), change to "with the platelets obtained in step (3)", "mix at a volume ratio of 12:1", "incubate at 37°C for 20 minutes", "centrifuge for 3 minutes",

[0063] "Add 110 mM sodium carbonate-bicarbonate buffer, pH 7.0".

[0064] Embodiment 10:

[0065] M1 macrophage-coupled complex enzyme lipid nanoparticles include M1 macrophages and lipid nanoparticles encapsulating complex enzymes. Buffer 1 in the copper sulfide nanozyme is 150 Mm tris(hydroxymethylaminomethane)-hydrochloride buffer with a pH of 8.5, and the mass ratios of the remaining components are: 90 parts of copper chloride dihydrate, 120 parts of sodium sulfide nonahydrate, 114 parts of sodium citrate, 10 parts of dopamine hydrochloride, 6 parts of hyaluronic acid, 6 parts of folic acid, 20,000 parts of buffer 1, and 100,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 8.95U / mL, the content of glucose oxidase is 10U / mL, the content of copper sulfide nanozyme is 0.2mg / mL, buffer 2 and buffer 3 are both 150mM tris(hydroxymethylaminomethane)-hydrochloride buffer with a pH of 6.5, and the mass ratios of the remaining components are: 4 parts of copper sulfide nanozyme, 20 parts of arginine deiminase, 20 parts of glucose oxidase, 100 parts of lecithin, 50 parts of cholesterol, 50 parts of DSPE-PEG2000-MAL, 50 parts of TPGS, 20,000 parts of buffer 2, and 5,000 parts of buffer 3.

[0066] The preparation method is basically the same as that in Example 1, and includes the following different steps: in step (1), the preparation conditions are changed to "dissolve cupric chloride dihydrate and sodium citrate in ultrapure water at 25° C. with stirring", "stir at 90° C. for 40 minutes", "vacuum dry at 60° C. for 24 hours", "dissolve dopamine hydrochloride in 150 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer solution at pH 8.5",

[0067] "Magnetic stirring at 25°C in the dark for 30 minutes", "Stirring in the dark for 24 hours"; in step (2), change to "dissolved in 150 mM tris(2-hydroxymethyl)aminomethane-hydrochloride buffer with a pH of 6.5 to obtain solution F", "dissolved in 20 mL of dichloromethane", "oscillated at a constant temperature of 35-45°C for 2 hours", "repeat the operation 3 times"; in step (3), change to "M1 macrophages", "take a cell density of (2E+6) cells / mL", "1.5 mM tris(2-carboxyethyl)phosphine hydrochloride", "culture for 20 minutes"; in step (4), change to "with the M1 macrophages obtained in step (3)", "mixed at a volume ratio of 20:1",

[0068] “Incubate at 37°C for 20 minutes”, “Centrifuge for 3 minutes”, “Add 150 mM Tris-HCl buffer, pH 6.5”.

[0069] Embodiment 11:

[0070] Endothelial cell-coupled complex enzyme lipid nanoparticles include endothelial cells and lipid nanoparticles encapsulating complex enzymes. Buffer 1 in the copper sulfide nanozyme is a 60Mm boric acid-borax buffer with a pH of 9.3, and the mass ratios of the remaining components are: 75 parts of copper chloride dihydrate, 65 parts of sodium sulfide nonahydrate, 65 parts of sodium citrate, 16 parts of dopamine hydrochloride, 8 parts of hyaluronic acid, 4 parts of folic acid, 35,000 parts of buffer 1, and 145,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 10.02U / mL, the content of glucose oxidase is 4.10U / mL, the content of copper sulfide nanozyme is 0.09mg / mL, buffer 2 and buffer 3 are both 60mM boric acid-borax buffer with a pH of 7.5, and the mass ratios of the remaining components are: 3 parts of copper sulfide nanozyme, 38 parts of arginine deiminase, 14 parts of glucose oxidase, 140 parts of lecithin, 60 parts of cholesterol, 36 parts of DSPE-PEG2000-MAL, 62 parts of TPGS, 34000 parts of buffer 2, and 10000 parts of buffer 3.

[0071] The preparation method is basically the same as that in Example 1, including the following different steps: in step (1), the preparation conditions are changed to "dissolve cupric chloride dihydrate and sodium citrate in ultrapure water at 35°C with stirring", "stir at 90°C for 50 minutes", "vacuum dry at 55°C for 24 hours", "dissolve dopamine hydrochloride in 60 mM boric acid-borax buffer with a pH of 9.3", "magnetically stir at 25°C in the dark for 50 minutes", "stir in the dark for 20 hours"; in step (2), the conditions are changed to "dissolve in 60 mM boric acid-borax buffer with a pH of 7.5 acid-borax buffer to obtain solution F", "dissolve in 20 mL of chloroform", "oscillate at 40°C for 1 hour", "repeat the operation twice"; in step (3), change to "endothelial cells", "take the cell density to (4E+6) cells / mL", "2.0 mM tris(2-carboxyethyl)phosphine hydrochloride", "culture for 10 minutes"; in step (4), change to "with the endothelial cells obtained in step (3)", "mix at a volume ratio of 28:1", "incubate at 37°C for 30 minutes", "centrifuge for 5 minutes",

[0072] "Add 60 mM boric acid-borax buffer, pH 7.5".

[0073] Embodiment 12:

[0074] Tumor-infiltrating lymphocyte-coupled complex enzyme lipid nanoparticles, including tumor-infiltrating lymphocytes and lipid nanoparticles encapsulating complex enzymes. Buffer 1 in the copper sulfide nanozyme is 130 mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 9.1, and the mass ratios of the remaining components are: 110 parts of copper chloride dihydrate, 210 parts of sodium sulfide nonahydrate, 195 parts of sodium citrate, 13 parts of dopamine hydrochloride, 7 parts of hyaluronic acid, 5 parts of folic acid, 13,000 parts of buffer 1, and 120,000 parts of ultrapure water. The content of arginine deiminase in the preparation is 4.21U / mL, the content of glucose oxidase is 11.30U / mL, the content of copper sulfide nanozyme is 0.22mg / mL, buffer 2 and buffer 3 are both 130mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 8.5, and the mass ratios of the remaining components are: 7 parts of copper sulfide nanozyme, 15 parts of arginine deiminase, 36 parts of glucose oxidase, 110 parts of lecithin, 78 parts of cholesterol, 68 parts of DSPE-PEG2000-MAL, 77 parts of TPGS, 32000 parts of buffer 2, and 4540 parts of buffer 3.

[0075] The preparation method is basically the same as that in Example 1, including the following different steps: in step (1), the preparation conditions are changed to "dissolve cupric chloride dihydrate and sodium citrate in ultrapure water with stirring at 35°C", "stir at 95°C for 60 minutes", "vacuum dry at 60°C for 25 hours", "dissolve dopamine hydrochloride in 130 mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 9.1", "magnetically stir at 25°C in the dark for 40 minutes", "stir in the dark for 36 hours"; in step (2), the conditions are changed to "dissolve in 130 mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 8.5 to obtain a solution Liquid F", "dissolved in 20 mL of dichloromethane", "oscillated at 40°C for 2 hours", "repeated the operation 3 times"; in step (3), change to "tumor infiltrating lymphocytes", "take the cell density to (5E+6) cells / mL", "2.0 mM tris(2-carboxyethyl)phosphine hydrochloride", "culture for 20 minutes"; in step (4), change to "with the tumor infiltrating lymphocytes obtained in step (3)", "mixed at a volume ratio of 22:1", "incubated at 37°C for 30 minutes", "centrifuged for 3 minutes", "add 130 mM N,N-dihydroxyethylglycine-sodium hydroxide buffer with a pH of 8.5".

Claims

1. A living cell-coupled lipid nanoparticle encapsulating arginine deiminase, glucose oxidase and copper sulfide nanozyme and a preparation method thereof, characterized in that: include: living cells and lipid nanoparticles encapsulating arginine deiminase, glucose oxidase, and copper sulfide nanozymes; The living cells include one of M0 macrophages, M1 macrophages, erythrocytes, neutrophils, tumor infiltrating lymphocytes, mesenchymal stem cells, endothelial cells, cytotoxic T lymphocytes, natural killer cells, platelets and dendritic cells; The copper sulfide nanozyme is a copper sulfide nanoparticle with a composite coating of polydopamine, hyaluronic acid and folic acid, and includes the following components: the pH of buffer 1 is 5.5-8.5, and the mass ratio of the remaining components is: Buffer 1 includes one of sodium carbonate-sodium bicarbonate buffer, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, boric acid-borax buffer, N,N-dihydroxyethylglycine-sodium hydroxide buffer, bis(2-hydroxyethylamino)tris(hydroxymethyl)methane-hydrochloride buffer, and glycine-hydrochloride buffer; The lipid nanoparticles encapsulating arginine deiminase, glucose oxidase and copper sulfide nanozyme are hereinafter referred to as lipid nanoparticles encapsulating complex enzymes, wherein the content of arginine deiminase is 2.20-35.80 U / mL, the content of glucose oxidase is 2.50-40.00 U / mL, buffer 2 and buffer 3 are the same type of buffer in the same formula, the concentration is equal to 50-150 mM, the pH is the same as 5.5-8.5, and the mass fraction ratio of the remaining components is: Buffer 2 and buffer 3 include one of sodium carbonate-sodium bicarbonate buffer, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, boric acid-borax buffer, N,N-dihydroxyethylglycine-sodium hydroxide buffer, bis(2-hydroxyethylamino)tris(hydroxymethyl)methane-hydrochloride buffer, and glycine-hydrochloride buffer; The preparation method of living cell-coupled lipid nanoparticles encapsulating arginine deiminase, glucose oxidase and copper sulfide nanozyme (hereinafter referred to as living cell-coupled complex enzyme lipid nanoparticles) comprises the following steps: (1) Preparation method of copper sulfide nanozyme: copper chloride dihydrate and sodium citrate are stirred and dissolved in ultrapure water at 20-40°C to obtain solution A; sodium sulfide nonahydrate is added to solution A and stirred at 85-95°C for 20-60 minutes to obtain solution B; solution B is vacuum dried at 50-70°C for 20-36 hours to obtain solid C; dopamine hydrochloride is dissolved in pH 5. A dopamine hydrochloride solution is formed in a buffer solution with a concentration of 8.0-9.5, and the solution is stirred magnetically at 20-30° C. in the dark for 20-60 minutes to obtain a polydopamine solution D. Solid C, hyaluronic acid and folic acid are added to the solution D and stirred in the dark for 20-36 hours, centrifuged, and the precipitate is collected and dried to obtain a copper sulfide nanozyme. (2) A method for preparing lipid nanoparticles encapsulating a complex enzyme: arginine deiminase, copper sulfide nanozyme and glucose oxidase are dissolved in buffer solution 2 to obtain a solution F; lecithin, cholesterol, DSPE-PEG 2000-MAL and vitamin E polyethylene glycol 1000 succinate were dissolved in one or more organic solvents such as dichloromethane, chloroform, anhydrous ethanol, methanol, and ether, and then protected from light and ultrasonicated. The organic solvent was removed under reduced pressure to form a uniform film. Solution F was added and oscillated at 35-45°C for 1-3 hours, and then passed through 0.45 μm and 0.22 μm microporous membranes respectively. The operation was repeated 2-4 times to obtain lipid nanoparticles of arginine deiminase, copper sulfide nanozyme, and glucose oxidase; (3) active M0 macrophages Preparation method of cells: take M0 macrophages with a cell density of (2E+6) to (5E+6) cells / mL, culture them in a serum-free medium containing 1.0-2.0 mM tris(2-carboxyethyl)phosphine hydrochloride for 10-30 minutes, centrifuge, collect the precipitate, and obtain surface-modified live M0 macrophages; (4) Preparation method of live M1 macrophages: take M0 macrophages with a cell density of (2E+6) to (5E+6) cells / mL, culture them in a drug-containing medium containing 100 ng / mL lipopolysaccharide for 24 After one hour, the cells are cultured in a serum-free medium containing 1.0-2.0 mM tri(2-carboxyethyl)phosphine hydrochloride for 10-30 minutes, centrifuged, and the precipitate is collected to obtain surface-modified live M1 macrophages. (5) Preparation method of other live cells: one of red blood cells, neutrophils, tumor-infiltrating lymphocytes, mesenchymal stem cells, endothelial cells, cytotoxic T lymphocytes, natural killer cells, platelets and dendritic cells is taken, with a cell density of (2E+6) to (5E+6) cells / mL, and the cells are cultured in a serum-free medium containing 1.0-2.0 mM tri(2-carboxyethyl)phosphine hydrochloride for 10-30 minutes, centrifuged, and the precipitate is collected to obtain surface-modified live M1 macrophages.0mM tris(2-carboxyethyl)phosphine hydrochloride serum-free medium is cultured for 10-30 minutes, centrifuged, and the precipitate is collected to obtain other living cells with surface modification; (6) Preparation method of living cell-coupled complex enzyme lipid nanoparticles: the lipid nanoparticles encapsulating the complex enzyme obtained in step (2) are mixed with the M0 macrophages obtained in step (3), the M1 macrophages obtained in step (4), or one of the various living cells obtained in step (5) at a volume ratio of 10:1 to 40:1, incubated at 37°C for 20-40 minutes under shaking conditions, centrifuged at 1000rpm for 2-5 minutes, the precipitate is collected, and buffer 3 is added to obtain living cell-coupled complex enzyme lipid nanoparticles. .

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