Living cell coupled epigallocatechin gallate, platinum and carbon quantum dot co-doped prussian blue nano-enzyme lipid nanoparticles and preparation method thereof

Abstract

The invention belongs to the field of pharmaceutical preparations. The invention relates to living cell coupled epigallocatechin gallate, platinum and carbon quantum dot co-doped Prussian blue nano-enzyme lipid nanoparticles and a preparation method thereof. According to the prepared living cell coupled epigallocatechin gallate, platinum and carbon quantum dot co-doped Prussian blue nano-enzyme lipid nanoparticles, the stability and the inflammation targeting property of a medicine can be improved, the uric acid level of a hyperuricemia rat is remarkably reduced, and joint swelling of a gouty arthritis rat is relieved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and relates to living cell-coupled epigallocatechin gallate, platinum and carbon quantum dots co-doped Prussian blue nanozyme lipid nanoparticles (hereinafter referred to as: living cell-coupled EGCG / CP lipid nanoparticles in this patent) and a preparation method thereof. Background Art

[0002] Hyperuricemia is caused by purine metabolism disorder or reduced uric acid excretion, which leads to an imbalance between uric acid production and excretion in the body, causing a large amount of urate crystals to deposit in the joints and induce gouty arthritis. At present, nonsteroidal anti-inflammatory drugs, colchicine and glucocorticoids are mainly used in the clinical treatment of gouty arthritis, combined with long-term uric acid-lowering drug treatment. Epigallocatechin gallate (EGCG) is a polyphenol compound that can act as an antioxidant to remove reactive oxygen and reduce cytokine release to reduce inflammatory response, and can also inhibit uric acid production and promote uric acid excretion to reduce uric acid levels in the body. However, EGCG has the disadvantages of poor stability, poor targeting, and easy recognition and clearance by the immune system, which greatly limits its clinical application. Lipid nanoparticles can effectively encapsulate drugs inside, increase drug stability, prolong drug retention time in the body, and improve efficacy. Prussian blue nanozyme (CP) co-doped with platinum and carbon quantum dots has catalase-like and superoxide dismutase-like activities, which can effectively reduce the level of reactive oxygen in the inflammatory site; it also has uricase-like activity, which can reduce the level of uric acid; CP is also a photothermal reagent with high biocompatibility and excellent photothermal effect. Living cells have become an effective platform for drug delivery due to their good fluidity, stability and low immunogenicity. Among them, the immune cell delivery system can activate immune function and recruit cells to the inflammatory site, thereby improving the inflammatory targeting effect, improving the efficacy of EGCG and reducing toxic side effects.

[0003] After consulting patents and literature, there is no report on CP, no report on EGCG / CP-loaded lipid nanoparticles, no report on M0 macrophages, M2 macrophages, neutrophils and other living cells coupled to EGCG lipid nanoparticles, and no report on M0 macrophages, M2 macrophages, neutrophils and other living cells coupled to EGCG / CP lipid nanoparticles. The living cell coupled EGCG / CP lipid nanoparticles prepared by the present invention can not only increase the stability of EGCG, but also enhance the inflammatory targeting of EGCG, significantly reduce the uric acid level of hyperuricemia rats and reduce joint swelling of gouty arthritis rats. The living cell coupled EGCG / CP lipid nanoparticles of the present invention encapsulate EGCG inside the liposomes to improve the stability of EGCG. Living cells have immune escape properties and inflammation targeting properties, which can improve the targeting ability of EGCG to inflammatory sites. At the same time, CP exhibits good photothermal effect and enzyme-like activity. When used in combination with EGCG, it can not only reduce the uric acid level of rats with hyperuricemia, but also significantly reduce the joint swelling of rats with gouty arthritis. The present invention prepares living cell-coupled EGCG / CP lipid nanoparticles, which can be used for the treatment of hyperuricemia, gouty arthritis and related diseases. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide living cell-coupled EGCG / CP lipid nanoparticles and a preparation method thereof. Living cell-coupled EGCG / CP lipid nanoparticles overcome the shortcomings of EGCG such as poor stability, low bioavailability, and poor inflammation targeting, and can maintain a long blood circulation time, good biocompatibility, immune escape ability, and reduce toxic side effects. The living cell-coupled EGCG / CP lipid nanoparticles provided by the present invention can significantly inhibit the generation of uric acid, promote the excretion of uric acid, and can also significantly reduce joint swelling in rats with gouty arthritis. This study provides a new optional preparation for EGCG, which can be used for the treatment of hyperuricemia, gouty arthritis and related diseases.

[0005] The living cell coupled EGCG / CP lipid nanoparticles provided by the present invention are characterized in that they include living cells and EGCG / CP lipid nanoparticles, wherein the CP includes the following components, and the mass ratio of the components is: 100-400 parts of potassium chloroplatinate, 200-800 parts of ascorbic acid, 25-100 parts of sodium citrate dihydrate, 190-760 parts of ammonium bicarbonate, 108-432 parts of ferric chloride hexahydrate, 211-844 parts of potassium hexacyanoferrate trihydrate, 20000-80000 parts of ultrapure water 1, 20000-80000 parts of ultrapure water 2, and 80000-120000 parts of ultrapure water 3; in the preparation method of the living cell coupled EGCG / CP lipid nanoparticles, the pH of the phosphate buffer 1 used is 5.5-7.4, and the pH of the phosphate buffer 2 is 7.4; in the living cell coupled EGCG / CP lipid nanoparticles, the mass ratio of the remaining components is: EGCG 10-40 parts, CP 5-20 parts, phospholipids 137-548 parts, cholesterol 42-170 parts, DSPE-PEG 2000 -Mal 60-240 parts, phosphate buffer 1 is 25000-80000 parts, phosphate buffer 2 is 10000-40000 parts; the living cells are one of M0 macrophages, M2 macrophages and neutrophils; the preparation method of living cell-coupled EGCG / CP lipid nanoparticles provided by the present invention comprises the following steps: (1) Preparation method of CP: dissolving potassium chloroplatinate and ascorbic acid in ultrapure water 1, heating at 70-90°C for reaction for 2-8 hours, centrifuging and collecting the product, and vacuum drying to obtain solid A; sodium citrate dihydrate and carbon Ammonium bicarbonate is dissolved in ultrapure water 2, heated at 180-200°C for reaction for 2-8 hours, dialyzed in the dark for 12-48 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated at 40-80°C for reaction for 0.5-2 hours, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 5.5-7.4 to obtain solution C; phospholipids, cholesterol and DSPE-PEG are taken. 2000-Mal, dissolved in one or more of dichloromethane, chloroform, anhydrous ethanol, methanol, acetone, ethyl acetate, and ether, and the organic solvent is removed by rotary evaporation to form a uniform lipid film, and solution C is added, hydrated in a water bath at 35-45°C for 1-3 hours, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modification of living cells: culturing and collecting living cells, the number of which is 1 million to 5 million, adding 1-2 mM tris(2-carboxyethyl)phosphine hydrochloride solution, incubating at 37°C for 10-40 minutes, centrifuging, collecting the precipitate and dispersing it in phosphate buffer 2 to obtain modified living cells; (4) mixing the EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) at a volume ratio of 1:5 to 1:20, incubating at 37°C for 20-60 minutes, centrifuging and collecting the precipitate to obtain living cell-coupled EGCG / CP lipid nanoparticles.

[0006] The living cell coupled EGCG / CP lipid nanoparticles provided by the present invention utilize the sulfhydryl groups on the surface of living cells to react with the maleimide on the surface of liposomes to form a preparation ( Figure 1 ). The living cell-coupled EGCG / CP lipid nanoparticles provided by the present invention improve the inflammatory targeting of EGCG. The living cells couple the lipid nanoparticles, giving the nanoparticles biological functions. The cell activity results of the living cell-coupled EGCG / CP lipid nanoparticles prepared by the present invention show that the cells in the living cell-coupled EGCG / CP lipid nanoparticles have good activity ( Figure 2 ), so that it retains the original biological characteristics of living cells. The reasons why living cells coupled to EGCG / CP lipid nanoparticles improve the drug's inflammatory targeting are analyzed as follows: (1) The living cells include M0 macrophages, M2 macrophages and neutrophils, which are important components of the immune system. The receptors and molecules on their cell surfaces can specifically recognize chemokines and signal molecules released from the inflammatory site, thereby recruiting cells to the inflammatory site; (2) The living cell-coupled lipid nanoparticles retain the activity and biological characteristics of the cells, making the lipid nanoparticles biomimetic and improving the drug's targeting to the inflammatory site.

[0007] The living cell-coupled EGCG / CP lipid nanoparticles provided by the present invention can significantly reduce the generation of reactive oxygen species in macrophages induced by monosodium uric acid crystals ( Figure 3). The level of reactive oxygen species in the living cells coupled with EGCG / CP lipid nanoparticles was 44.58% of that in the model group and 61.98% of that in the EGCG group, indicating that the living cells coupled with EGCG / CP lipid nanoparticles can significantly reduce the production of reactive oxygen species in macrophages induced by monosodium uric acid crystals, and its effect is stronger than that in the free drug group; in addition, after laser irradiation, the level of reactive oxygen species in the living cells coupled with EGCG / CP lipid nanoparticles was slightly higher, but still lower than that in the model group and the free drug group. The reasons why the living cells coupled with EGCG / CP lipid nanoparticles reduce reactive oxygen species may include: (1) EGCG has antioxidant activity, which can reduce the level of reactive oxygen species; (2) CP has catalase-like activity and superoxide dismutase-like activity, which can synergistically reduce the level of reactive oxygen species with EGCG.

[0008] The living cell-coupled EGCG / CP lipid nanoparticles provided by the present invention can promote uric acid excretion and reduce serum uric acid levels after in vivo administration to male SD rats with hyperuricemia model. The urine uric acid level of the living cell-coupled EGCG / CP lipid nanoparticles was 201.12% of that of the model group and 156.99% of that of the free drug group ( Figure 4 A). The serum uric acid level of living cells coupled with EGCG / CP lipid nanoparticles was close to that of the normal group, which was 24.96% of the model group and 39.10% of the free drug group ( Figure 4 B), indicating that living cell-coupled EGCG / CP lipid nanoparticles can significantly promote the excretion of uric acid in the body, reduce serum uric acid levels and the effect is stronger than that of free drugs.

[0009] The living cell-coupled EGCG / CP lipid nanoparticles provided by the present invention acted on male SD rats with gouty arthritis model, significantly reducing the degree of ankle joint swelling, and the joint swelling reduction speed was faster than that of the free drug group ( Figure 5 ). In addition, considering the photothermal effect of CP, the recovery of joints after near-infrared laser irradiation was investigated. Multiple laser irradiations were given at 1, 6, 24, 48, 72, and 96 hours after modeling. The recovery speed of joints was significantly improved compared with the non-laser group, and returned to normal in 96 hours.

[0010] The living cell coupled EGCG / CP lipid nanoparticle delivery system provided by the present invention has the characteristics of high stability, high biocompatibility and low toxicity, and provides an effective therapeutic means for the delivery of small molecule drugs for the treatment of related diseases such as hyperuricemia and gout. The living cell coupled EGCG / CP lipid nanoparticles, using M0 macrophages, M2 macrophages and neutrophils to couple EGCG / CP lipid nanoparticles, can enhance the stability of the drug, avoid phagocytosis and clearance of the drug, and improve inflammation targeting and biocompatibility. Compared with simple lipid nanoparticles, the living cell coupled lipid nanoparticle delivery carrier has better biological properties and stronger inflammation targeting.

[0011] The present invention is different from the delivery carrier and preparation process of EGCG reported in the usual research. The delivery carriers of EGCG reported in the research include: conventional liposomes, nano micelles and self-assembled nanoparticles. The living cell-coupled EGCG / CP lipid nanoparticles in the present invention are a new bionic drug delivery system. At present, there are a small number of reports on living cells carrying drugs, such as macrophages hitchhiking interleukin 10 plasmid DNA delivery system to treat rheumatoid arthritis; red blood cells coupled to polylactic acid-glycolic acid copolymer and chitosan coated ivermectin nanoparticles to lung tissue to treat inflammation, etc. At present, there are no reports on EGCG / CP lipid nanoparticles, nor are there any reports on living cells such as M0 macrophages, M2 macrophages and neutrophils coupled to EGCG / CP lipid nanoparticles. The present invention couples living cells such as M0 macrophages, M2 macrophages and neutrophils with EGCG / CP lipid nanoparticles for the first time. This delivery system can increase the stability and inflammation targeting of the drug, significantly reduce the uric acid levels in hyperuricemia rats and alleviate joint swelling in gouty arthritis rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 FITC fluorescence image of living cells coupled with EGCG / CP lipid nanoparticles prepared by the present invention ( Figure 1 A) and fluorescence intensity graph ( Figure 1 B); scale bar: 10 μm.

[0013] Experimental conditions: EGCG was labeled with FITC to prepare EGCG / CP lipid nanoparticles, the modified living cells were coupled with EGCG / CP lipid nanoparticles, and the fluorescence and fluorescence intensity of the living cells coupled with EGCG / CP lipid nanoparticles were observed using a laser confocal microscope.

[0014] The results showed that compared with normal cells, living cells coupled with EGCG / CP lipid nanoparticles had obvious fluorescence and higher fluorescence intensity on the cell surface, indicating that the living cell surface provided by the present invention was successfully coupled with EGCG / CP lipid nanoparticles.

[0015] Figure 2 The figure shows the cell survival rate of living cells coupled with EGCG / CP lipid nanoparticles prepared by the present invention.

[0016] Experimental conditions: Live cells in the logarithmic growth phase and live cells coupled with EGCG / CP lipid nanoparticles were inoculated in a 96-well plate at a density of 5000 cells / well, cultured at 37°C, 5% CO2 for 24 hours, the culture medium was discarded, and MTT was added to determine the cell survival rate of each group.

[0017] The results showed that when EGCG / CP lipid nanoparticles were coupled to living cells, the cell viability was 95.96%, indicating that the cells still retained their viability during the preparation of living cell-coupled EGCG / CP lipid nanoparticles, and were able to retain the original biological properties of the cells, giving them a longer blood circulation time, good biocompatibility and immune escape ability, and enabling the drug to be targeted to the site of inflammation.

[0018] Figure 3 The in vitro active oxygen scavenging ability of the living cell-coupled EGCG / CP lipid nanoparticles prepared by the present invention. The data in the figure are expressed as mean ± standard deviation (n = 3), t test statistical analysis: compared with the normal group, ***p < 0.001, compared with the model group, ## p<0.01, ### p<0.001.

[0019] Experimental conditions: RAW264.7 macrophages in the logarithmic growth phase were collected and cultured at 1×10 4 The cells were inoculated at a density of 10 cells / well in a 6-well plate and cultured at 37°C and 5% CO2 for 24 hours. After 24 hours of monosodium uric acid crystal induction, the macrophages were washed three times with serum-free medium and incubated with medium containing EGCG, CP, EGCG / CP lipid nanoparticles and live cell-coupled EGCG / CP lipid nanoparticles for 24 hours. The laser group was irradiated with 808nm, 1W / cm 2 Laser irradiation for 5 minutes. Continue incubation for 24 hours. After incubation, add DCFH-DA working solution according to the instructions of the reactive oxygen species detection kit, and incubate at 37°C in the dark for 30 minutes. Wash the cells 2-3 times with serum-free medium and detect on a flow cytometer.

[0020] The results showed that living cells coupled with EGCG / CP lipid nanoparticles can significantly reduce the production of reactive oxygen species in macrophages induced by monosodium uric acid crystals. The living cells coupled with EGCG / CP lipid nanoparticles were 49.02% of the model group and 63.70% of EGCG, indicating that living cells coupled with EGCG / CP lipid nanoparticles can significantly reduce the production of reactive oxygen species in macrophages induced by monosodium uric acid crystals, and the effect is stronger than that of free drugs. After laser irradiation, the level of reactive oxygen species in the living cell coupled with EGCG / CP lipid nanoparticle group increased slightly, but was still much lower than that in the model group and free drug group.

[0021] Figure 4 The uric acid profile of hyperuricemia urine obtained by the present invention with living cells coupled with EGCG / CP lipid nanoparticles ( Figure 4 A) and serum uric acid chart ( Figure 4 B). The data in the figure are expressed as mean ± standard deviation (n = 6), t test statistical analysis: compared with the normal group, ***p < 0.001, compared with the model group, ###p<0.001.

[0022] Experimental conditions: 36 male SD rats were randomly divided into 6 groups, 6 rats in each group, namely normal group, model group, EGCG group, EGCG / CP lipid nanoparticle group, and living cell coupled EGCG / CP lipid nanoparticle group (no laser group, laser group). Except for the normal group, the other groups were given hypoxanthine by gavage at 500 mg / kg, and then potassium oxonate was injected intraperitoneally at 300 mg / kg to establish a hyperuricemia model for 7 consecutive days. Except for the normal group and the model group, each group was injected with the same dose of EGCG or EGCG-containing preparations through the tail vein every day. In the laser group, the ankle joint was irradiated with laser for 5 minutes (808nm, 1W / cm 2 ). After the treatment, urine and blood were collected, and the uric acid levels in urine and blood were tested using a uric acid kit.

[0023] The results showed that after the live cell-coupled EGCG / CP lipid nanoparticles were administered to the hyperuricemia model rats, the urine uric acid level increased and the blood uric acid level decreased significantly. The urine uric acid level of the live cell-coupled EGCG / CP lipid nanoparticles was 201.12% of that of the model group and 156.99% of that of the free drug group ( Figure 4 A). The blood uric acid level of living cells coupled with EGCG / CP lipid nanoparticles was close to that of the normal group, which was 24.96% of the model group and 39.10% of the free drug group ( Figure 4 B). This indicates that living cell-coupled EGCG / CP lipid nanoparticles can promote the excretion of uric acid in the body and reduce the abnormal increase of blood uric acid levels, and the effect is stronger than that of free drugs.

[0024] Figure 5 This is a graph of ankle swelling-time in gouty arthritis rats with living cell-coupled EGCG / CP lipid nanoparticles prepared in the present invention.

[0025] Experimental conditions: 36 male SD rats were randomly divided into 6 groups, 6 rats in each group, namely normal group, model group, EGCG group, EGCG / CP lipid nanoparticle group, and live cell coupled EGCG / CP lipid nanoparticle group (no laser group, laser group). Except for the normal group, the other groups were given hypoxanthine by gavage at 500 mg / kg, and then potassium oxonate was injected intraperitoneally at 300 mg / kg for 12 consecutive days. On the 8th day, except for the normal group and the model group, each group was subsequently injected with the same dose of EGCG or EGCG-containing preparations through the tail vein every day. After administration on the 8th day, a microcrystalline sodium urate suspension was injected into the ankle joint cavity from the inner side of the tibial tendon with a sterile syringe to establish a gouty arthritis rat model under hyperuricemia. The laser group was irradiated with laser for 5 minutes (808nm, 1W / cm 2The ankle diameters of each mouse in each group were measured at different time points.

[0026] The results showed that the live cell-coupled EGCG / CP lipid nanoparticles significantly reduced ankle joint swelling after in vivo administration, and the joint swelling was faster than the free drug group. At 96 hours, the model group had the most obvious joint swelling compared to the other groups. After multiple laser interval irradiations, the joint recovery rate was significantly improved compared to the non-laser group, and at 96 hours, the joint had returned to normal. DETAILED DESCRIPTION

[0027] 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.

[0028] Embodiment 1:

[0029] M0 macrophage-coupled EGCG / CP lipid nanoparticles include M0 macrophages and EGCG / CP lipid nanoparticles. The M0 macrophage-coupled EGCG / CP lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 100 parts of potassium chloroplatinate, 280 parts of ascorbic acid, 25 parts of sodium citrate dihydrate, 418 parts of ammonium bicarbonate, 302.4 parts of ferric chloride hexahydrate, 211 parts of potassium hexacyanoferrate trihydrate, 20,000 parts of ultrapure water 1, 45,000 parts of ultrapure water 2, and 80,000 parts of ultrapure water 3. The pH of phosphate buffer 1 in the M0 macrophage-coupled EGCG / CP lipid nanoparticles is 5.5, and the mass ratios of the other components are: 10 parts of EGCG, 5 parts of CP, 137 parts of phospholipids, 97.8 parts of cholesterol, DSPE-PEG 2000 -Mal 174 parts, phosphate buffer 1 is 25000 parts, phosphate buffer 2 is 10000 parts.

[0030] Preparation method: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated at 70°C for 2 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated at 180°C for 2 hours, dialyzed in the dark for 12 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated at 40°C for 2 hours, centrifuged, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 5.5 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in dichloromethane, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a water bath at 35°C for 1 hour, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modified M0 macrophages: culture and collect M0 macrophages (the number is 1 million), add 1 mM tris(2-carboxyethyl)phosphine hydrochloride solution, incubate at 37°C for 10 minutes, centrifuge, collect the precipitate and disperse it in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were evenly mixed in a volume ratio of 1:5, incubated at 37°C for 20 minutes, centrifuged and the precipitate was collected to obtain M0 macrophage-coupled EGCG / CP lipid nanoparticles.

[0031] Embodiment 2:

[0032] M2 macrophage-coupled EGCG / CP lipid nanoparticles include M2 ​​macrophages and EGCG / CP lipid nanoparticles. The M2 macrophage-coupled EGCG / CP lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 240 parts of potassium chloroplatinate, 640 parts of ascorbic acid, 30 parts of sodium citrate dihydrate, 190 parts of ammonium bicarbonate, 129.3 parts of ferric chloride hexahydrate, 253.2 parts of potassium hexacyanoferrate trihydrate, 30,000 parts of ultrapure water 1, 20,000 parts of ultrapure water 2, and 112,000 parts of ultrapure water 3. In the M2 macrophage-coupled EGCG / CP lipid nanoparticles, the pH of phosphate buffer 1 is 6.0, and the mass ratios of the remaining components are: 12 parts of EGCG, 6 parts of CP, 356.2 parts of phospholipids, 42 parts of cholesterol, and DSPE-PEG. 2000 -Mal 60 parts, phosphate buffer 1 is 60000 parts, and phosphate buffer 2 is 16000 parts.

[0033] Preparation methods: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated to 80°C for 4 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated to 180°C for 3 hours, dialyzed in the dark for 12 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated to 50°C for 2 hours, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 6.0 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in chloroform, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a water bath at 35°C for 1.5 hours, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modified M2 macrophages: M0 macrophages (a total of 4.4 million) were cultured and collected, 10 ng / mL of IL-4 and 1 μM of lactic acid were added to induce for 24 hours to obtain M2 macrophages, 1.5 mM tris(2-carboxyethyl)phosphine hydrochloride solution was added, incubated at 37°C for 20 minutes, centrifuged, the precipitate was collected and dispersed in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were evenly mixed at a volume ratio of 1:6, incubated at 37°C for 30 minutes, centrifuged and the precipitate was collected to obtain M2 macrophage-coupled EGCG / CP lipid nanoparticles.

[0034] Embodiment 3:

[0035] Neutrophil-coupled EGCG / CP lipid nanoparticles include neutrophils and EGCG / CP lipid nanoparticles. Neutrophil-coupled EGCG / CP lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 300 parts of potassium chloroplatinate, 200 parts of ascorbic acid, 35 parts of sodium citrate dihydrate, 790 parts of ammonium bicarbonate, 108 parts of ferric chloride hexahydrate, 295.4 parts of potassium hexacyanoferrate trihydrate, 35,000 parts of ultrapure water 1, 55,000 parts of ultrapure water 2, and 84,000 parts of ultrapure water 3. The pH of phosphate buffer 1 in neutrophil-coupled EGCG / CP lipid nanoparticles is 6.5, and the mass ratios of the remaining components are: 14 parts of EGCG, 7 parts of CP, 548 parts of phospholipids, 51 parts of cholesterol, and DSPE-PEG. 2000 -Mal 96 parts, phosphate buffer 1 35000 parts, phosphate buffer 2 29000 parts.

[0036] Preparation methods: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated to 80°C for 2 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated to 180°C for 4 hours, dialyzed in the dark for 24 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated to 50°C for 1.5 hours, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 6.5 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in anhydrous ethanol, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a water bath at 35°C for 3 hours, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for neutrophil modification: culturing and collecting neutrophils (the number is 2.4 million), adding 1 mM tris(2-carboxyethyl)phosphine hydrochloride solution, incubating at 37°C for 30 minutes, centrifuging, collecting the precipitate and dispersing it in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were evenly mixed in a volume ratio of 1:7, incubated at 37°C for 40 minutes, centrifuged and the precipitate was collected to obtain neutrophil-coupled EGCG / CP lipid nanoparticles.

[0037] Embodiment 4:

[0038] M2 macrophage-coupled EGCG / CP lipid nanoparticles include M2 ​​macrophages and EGCG / CP lipid nanoparticles. The M2 macrophage-coupled EGCG / CP lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 120 parts of potassium chloroplatinate, 240 parts of ascorbic acid, 40 parts of sodium citrate dihydrate, 532 parts of ammonium bicarbonate, 432 parts of ferric chloride hexahydrate, 675.2 parts of potassium hexacyanoferrate trihydrate, 60,000 parts of ultrapure water 1, 35,000 parts of ultrapure water 2, and 96,000 parts of ultrapure water 3. The pH of the phosphate buffer in the M2 macrophage-coupled EGCG / CP lipid nanoparticles is 5.5, and the mass ratios of the other components are: 16 parts of EGCG, 8 parts of CP, 164.4 parts of phospholipids, 136.2 parts of cholesterol, DSPE-PEG 2000 -Mal 72 parts, phosphate buffer 1 30000 parts, phosphate buffer 2 14000 parts.

[0039] Preparation methods: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated to 80°C for reaction for 3 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated to 180°C for reaction for 5 hours, dialyzed in the dark for 48 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated to 50°C for reaction for 1 hour, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 5.5 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in methanol, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a water bath at 36°C for 1 hour, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modified M2 macrophages: M0 macrophages (amounting to 1.2 million) were cultured and collected, 10 ng / mL of IL-4 and 1 μM of lactic acid were added to induce for 24 hours to obtain M2 macrophages, 1 mM tris(2-carboxyethyl)phosphine hydrochloride solution was added, incubated at 37°C for 40 minutes, centrifuged, the precipitate was collected and dispersed in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were mixed evenly at a volume ratio of 1:8, incubated at 37°C for 50 minutes, centrifuged and the precipitate was collected to obtain M2 macrophage-coupled EGCG / CP lipid nanoparticles.

[0040] Embodiment 5:

[0041] M0 macrophage-coupled EGCG / CP lipid nanoparticles include M0 macrophages and EGCG / CP lipid nanoparticles. The M0 macrophage-coupled EGCG / CP lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 400 parts of potassium chloroplatinate, 600 parts of ascorbic acid, 45 parts of sodium citrate dihydrate, 228 parts of ammonium bicarbonate, 151.2 parts of ferric chloride hexahydrate, 506.4 parts of potassium hexacyanoferrate trihydrate, 40,000 parts of ultrapure water 1, 65,000 parts of ultrapure water 2, and 88,000 parts of ultrapure water 3. The pH of phosphate buffer 1 in the M0 macrophage-coupled EGCG / CP lipid nanoparticles is 6.5, and the mass ratios of the other components are: 18 parts of EGCG, 9 parts of CP, 191.8 parts of phospholipids, 59.5 parts of cholesterol, DSPE-PEG 2000 -Mal2 10 parts, phosphate buffer 1 45000 parts, phosphate buffer 2 35000 parts.

[0042] Preparation methods: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated to 80°C for 4 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated to 180°C for 6 hours, dialyzed in the dark for 12 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated to 60°C for 1.5 hours, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 5.5 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in acetone, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a water bath at 37°C for 2 hours, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modified M0 macrophages: culturing and collecting M0 macrophages (the number is 3.2 million), adding 1 mM tris(2-carboxyethyl)phosphine hydrochloride solution, incubating at 37°C for 20 minutes, centrifuging, collecting the precipitate and dispersing it in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were evenly mixed in a volume ratio of 1:9, incubated at 37°C for 30 minutes, centrifuged and the precipitate was collected to obtain M0 macrophage-coupled EGCG / CP lipid nanoparticles.

[0043] Embodiment 6:

[0044] M0 macrophage-coupled EGCG / CP lipid nanoparticles include M0 macrophages and EGCG / CP lipid nanoparticles. The M0 macrophage-coupled EGCG / CP lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 180 parts of potassium chloroplatinate, 480 parts of ascorbic acid, 55 parts of sodium citrate dihydrate, 266 parts of ammonium bicarbonate, 237.5 parts of ferric chloride hexahydrate, 337.6 parts of potassium hexacyanoferrate trihydrate, 70,000 parts of ultrapure water 1, 75,000 parts of ultrapure water 2, and 118,000 parts of ultrapure water 3. The pH of phosphate buffer 1 in the M0 macrophage-coupled EGCG / CP lipid nanoparticles is 7.0, and the mass ratios of the remaining components are: 23 parts of EGCG, 11.5 parts of CP, 438.4 parts of phospholipids, 76.5 parts of cholesterol, DSPE-PEG 2000 -Mal 84 parts, phosphate buffer 1 40000 parts, phosphate buffer 2 26000 parts.

[0045] Preparation methods: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated to 80°C for reaction for 8 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated to 180°C for reaction for 8 hours, dialyzed in the dark for 12 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated to 60°C for reaction for 2 hours, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 6.8 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in chloroform, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a water bath at 35°C for 1 hour, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modified M0 macrophages: culturing and collecting M0 macrophages (the number is 3.6 million), adding 1.5 mM tris(2-carboxyethyl)phosphine hydrochloride solution, incubating at 37°C for 30 minutes, centrifuging, collecting the precipitate and dispersing it in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were evenly mixed in a volume ratio of 1:12, incubated at 37°C for 40 minutes, centrifuged and the precipitate was collected to obtain M0 macrophage-coupled EGCG / CP lipid nanoparticles.

[0046] Embodiment 7:

[0047] M2 macrophage-coupled EGCG / CP lipid nanoparticles include M2 ​​macrophages and EGCG / CP lipid nanoparticles. The M2 macrophage-coupled EGCG lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 140 parts of potassium chloroplatinate, 800 parts of ascorbic acid, 70 parts of sodium citrate dihydrate, 304 parts of ammonium bicarbonate, 345.6 parts of ferric chloride hexahydrate, 844 parts of potassium hexacyanoferrate trihydrate, 80,000 parts of ultrapure water 1, 40,000 parts of ultrapure water 2, and 116,000 parts of ultrapure water 3. The phosphate buffer 1 in the M2 macrophage-coupled EGCG / CP lipid nanoparticles has a pH of 5.5, and the mass ratios of the remaining components are: 29 parts of EGCG, 14.5 parts of CP, 479.5 parts of phospholipids, 123.4 parts of cholesterol, and DSPE-PEG. 2000 -Mal 108 parts, phosphate buffer 1 80000 parts, phosphate buffer 2 23000 parts.

[0048] Preparation method: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated at 90°C for reaction for 3 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated at 190°C for reaction for 4 hours, dialyzed in the dark for 48 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated at 70°C for reaction for 1 hour, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 5.5 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in chloroform, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a water bath at 37°C for 2 hours, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modified M2 macrophages: M0 macrophages (4 million in number) were cultured and collected, 10 ng / mL of IL-4 and 1 μM of lactic acid were added to induce for 24 hours to obtain M2 macrophages, 2 mM tris(2-carboxyethyl)phosphine hydrochloride solution was added, incubated at 37°C for 20 minutes, centrifuged, the precipitate was collected and dispersed in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were mixed evenly at a volume ratio of 1:15, incubated at 37°C for 30 minutes, centrifuged and the precipitate was collected to obtain M2 macrophage-coupled EGCG / CP lipid nanoparticles.

[0049] Embodiment 8:

[0050] Neutrophil-coupled EGCG / CP lipid nanoparticles include neutrophils and EGCG / CP lipid nanoparticles. Neutrophil-coupled EGCG / CP lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 220 parts of potassium chloroplatinate, 320 parts of ascorbic acid, 60 parts of sodium citrate dihydrate, 342 parts of ammonium bicarbonate, 259.2 parts of ferric chloride hexahydrate, 379.8 parts of potassium hexacyanoferrate trihydrate, 75,000 parts of ultrapure water 1, 60,000 parts of ultrapure water 2, and 120,000 parts of ultrapure water 3. The pH of phosphate buffer 1 in neutrophil-coupled EGCG / CP lipid nanoparticles is 7.4, and the mass ratios of the other components are: 26 parts of EGCG, 13 parts of CP, 246.6 parts of phospholipids, 110.6 parts of cholesterol, DSPE-PEG 2000 -Mal 138 parts, phosphate buffer 1 70000 parts, phosphate buffer 2 40000 parts.

[0051] Preparation methods: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated at 90°C for 2 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated at 190°C for 2 hours, dialyzed in the dark for 24 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated at 60°C for 1.5 hours, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 7.4 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in acetone, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a water bath at 37°C for 3 hours, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for neutrophil modification: culturing and collecting neutrophils (the number is 2.8 million), adding 1.5 mM tris(2-carboxyethyl)phosphine hydrochloride solution, incubating at 37°C for 20 minutes, centrifuging, collecting the precipitate and dispersing it in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were evenly mixed in a volume ratio of 1:14, incubated at 37°C for 30 minutes, centrifuged and the precipitate was collected to obtain neutrophil-coupled EGCG / CP lipid nanoparticles.

[0052] Embodiment 9:

[0053] M2 macrophage-coupled EGCG / CP lipid nanoparticles include M2 ​​macrophages and EGCG / CP lipid nanoparticles. The M2 macrophage-coupled EGCG / CP lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 200 parts of potassium chloroplatinate, 400 parts of ascorbic acid, 50 parts of sodium citrate dihydrate, 380 parts of ammonium bicarbonate, 216 parts of ferric chloride hexahydrate, 422 parts of potassium hexacyanoferrate trihydrate, 50,000 parts of ultrapure water 1, 50,000 parts of ultrapure 2, and 100,000 parts of ultrapure water 3. The pH of phosphate buffer 1 in the M2 macrophage-coupled EGCG / CP lipid nanoparticles is 6.8, and the mass ratios of the other components are: 20 parts of EGCG, 10 parts of CP, 274 parts of phospholipids, 85 parts of cholesterol, DSPE-PEG 2000 -Mal 120 parts, phosphate buffer 1 50000 parts, phosphate buffer 2 20000 parts.

[0054] Preparation methods: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated to 80°C for 4 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated to 180°C for 4 hours, dialyzed in the dark for 24 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated to 60°C for 1 hour, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 6.8 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in dichloromethane, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a water bath at 37°C for 2 hours, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modified M2 macrophages: culturing and collecting M2 macrophages (the number is 2 million), adding 10 ng / mL IL-4 and 1 μM lactic acid to induce for 24 hours to obtain M2 macrophages, adding 1.5 mM tris(2-carboxyethyl)phosphine hydrochloride solution, incubating at 37°C for 20 minutes, centrifuging, collecting the precipitate and dispersing it in phosphate buffer 1 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were evenly mixed at a volume ratio of 1:10, incubated at 37°C for 30 minutes, centrifuged and the precipitate was collected to obtain M2 macrophage-coupled EGCG / CP lipid nanoparticles.

[0055] Embodiment 10:

[0056] M2 macrophage-coupled EGCG / CP lipid nanoparticles include M2 ​​macrophages and EGCG / CP lipid nanoparticles. The M2 macrophage-coupled EGCG / CP lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 160 parts of potassium chloroplatinate, 440 parts of ascorbic acid, 75 parts of sodium citrate dihydrate, 608 parts of ammonium bicarbonate, 172.8 parts of ferric chloride hexahydrate, 590.8 parts of potassium hexacyanoferrate trihydrate, 45,000 parts of ultrapure water 1, 65,000 parts of ultrapure water 2, and 92,000 parts of ultrapure water 3. The pH of phosphate buffer 1 in the M2 macrophage-coupled EGCG / CP lipid nanoparticles is 6.0, and the mass ratios of the other components are: 32 parts of EGCG, 16 parts of CP, 219.2 parts of phospholipids, 149 parts of cholesterol, DSPE-PEG 2000 -Mal 240 parts, phosphate buffer 1 is 65000 parts, phosphate buffer 2 is 12000 parts.

[0057] Preparation methods: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated at 90°C for 4 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated at 190°C for 6 hours, dialyzed in the dark for 24 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated at 60°C for 2 hours, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 6.0 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in ethyl acetate, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a 40°C water bath for 1 hour, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modified M2 macrophages: M0 macrophages (a total of 4.8 million) were cultured and collected, 10 ng / mL of IL-4 and 1 μM of lactic acid were added to induce for 24 hours to obtain M2 macrophages, 2 mM tris(2-carboxyethyl)phosphine hydrochloride solution was added, incubated at 37°C for 30 minutes, centrifuged, the precipitate was collected and dispersed in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were mixed evenly at a volume ratio of 1:16, incubated at 37°C for 60 minutes, centrifuged and the precipitate was collected to obtain M2 macrophage-coupled EGCG / CP lipid nanoparticles.

[0058] Embodiment 11:

[0059] Neutrophil-coupled EGCG / CP lipid nanoparticles include neutrophils and EGCG / CP lipid nanoparticles. Neutrophil-coupled EGCG / CP lipid nanoparticles contain CP, and the mass ratio of each component in CP is: 320 parts of potassium chloroplatinate, 560 parts of ascorbic acid, 80 parts of sodium citrate dihydrate, 570 parts of ammonium bicarbonate, 194.4 parts of ferric chloride hexahydrate, 633 parts of potassium hexacyanoferrate trihydrate, 65,000 parts of ultrapure water 1, 80,000 parts of ultrapure water 2, and 108,000 parts of ultrapure water 3. The pH of phosphate buffer 1 in neutrophil-coupled EGCG / CP lipid nanoparticles is 6.5, and the mass ratios of the other components are: 35 parts of EGCG, 17.5 parts of CP, 397.3 parts of phospholipids, 68 parts of cholesterol, DSPE-PEG 2000 -Mal 156 parts, phosphate buffer 1 55000 parts, phosphate buffer 2 18000 parts.

[0060] Preparation method: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated at 90°C for 6 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated at 190°C for 8 hours, dialyzed in the dark for 48 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated at 80°C for 2 hours, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 6.0 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in a mixture of anhydrous ethanol and dichloromethane, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a 45°C water bath for 1 hour, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for neutrophil modification: culturing and collecting neutrophils (5 million in number), adding 2 mM tris(2-carboxyethyl)phosphine hydrochloride in phosphate buffer, incubating at 37°C for 30 minutes, centrifuging, collecting the precipitate and dispersing it in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were uniformly mixed in a volume ratio of 1:18, incubated at 37°C for 60 minutes, centrifuged and the precipitate was collected to obtain neutrophil-coupled EGCG / CP lipid nanoparticles.

[0061] Embodiment 12:

[0062] EGCG / CP lipid nanoparticles coupled to M0 macrophages include M0 macrophages and EGCG / CP lipid nanoparticles. The EGCG / CP lipid nanoparticles coupled to M0 macrophages contain CP, and the mass ratios of the components in CP are: 280 parts of potassium chloroplatinate, 360 parts of ascorbic acid, 100 parts of sodium citrate dihydrate, 456 parts of ammonium bicarbonate, 324 parts of ferric chloride hexahydrate, 464.2 parts of potassium hexacyanoferrate trihydrate, 55,000 parts of ultrapure water 1, 45,000 parts of ultrapure water 2, and 104,000 parts of ultrapure water 3. The pH of phosphate buffer 1 in the M0 macrophage-coupled EGCG / CP lipid nanoparticles is 7.0, and the mass ratios of the other components are: 40 parts of EGCG, 20 parts of CP, 315.1 parts of phospholipids, 170 parts of cholesterol, DSPE-PEG 2000 -Mal 192 parts, phosphate buffer 1 75000 parts, phosphate buffer 2 32000 parts.

[0063] Preparation methods: (1) Preparation method of CP: Potassium chloroplatinate and ascorbic acid are dissolved in ultrapure water 1, heated at 90°C for 6 hours, centrifuged and the product is collected, and vacuum dried to obtain solid A; sodium citrate dihydrate and ammonium bicarbonate are dissolved in ultrapure water 2, heated at 200°C for 4 hours, dialyzed in the dark for 48 hours, and vacuum dried to obtain solid B; ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B are dissolved in ultrapure water 3, heated at 80°C for 1.5 hours, centrifuged, the precipitate is collected, and vacuum dried to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: EGCG and CP are dissolved in phosphate buffer 1 with a pH of 7.0 to obtain solution C, phospholipids, cholesterol and DSPE-PEG are added. 2000-Mal was dissolved in ether, and the organic solvent was removed by rotary evaporation to form a uniform lipid film, and solution C was added, hydrated in a 45°C water bath for 1.5 hours, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modified M0 macrophages: culture and collect M0 macrophages (the number is 1.5 million), add 2mM tris(2-carboxyethyl)phosphine hydrochloride in phosphate buffer, incubate at 37°C for 40 minutes, centrifuge, collect the precipitate and disperse it in phosphate buffer 2 to obtain modified living cells; (4) The EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) were mixed evenly at a volume ratio of 1:20, incubated at 37°C for 60 minutes, centrifuged and the precipitate was collected to obtain M0 macrophage-coupled EGCG / CP lipid nanoparticles.

Claims

1. A living cell-coupled epigallocatechin gallate, platinum and carbon quantum dots co-doped Prussian blue nanozyme lipid nanoparticles, characterized in that: It includes living cells, epigallocatechin gallate, platinum and carbon quantum dots co-doped Prussian blue nanozyme lipid nanoparticles, namely EGCG / CP lipid nanoparticles; The Prussian blue nanozyme co-doped with platinum and carbon quantum dots, i.e., CP, comprises the following components, and the mass ratio of the components is: In the living cell coupled EGCG / CP lipid nanoparticles, the pH of phosphate buffer 1 is 5.5-7.4, the pH of phosphate buffer 2 is 7.4, and the mass ratios of the remaining components are: The living cells include one of M0 macrophages, M2 macrophages and neutrophils; The preparation method of the living cell coupled EGCG / CP lipid nanoparticles comprises the following steps: (1) Preparation method of CP: dissolving potassium chloroplatinate and ascorbic acid in ultrapure water 1, heating at 70-90°C for reaction for 2-8 hours, centrifuging and collecting the product, and vacuum drying to obtain solid A; dissolving sodium citrate dihydrate and ammonium bicarbonate in ultrapure water 2, heating at 180-200°C for reaction for 2-8 hours, dialyzing in the dark for 12-48 hours, and vacuum drying to obtain solid B; dissolving ferric chloride hexahydrate, potassium hexacyanoferrate trihydrate, solid A and solid B in ultrapure water 3, heating at 40-80°C for reaction for 0.5-2 hours, centrifuging, collecting the precipitate, and vacuum drying to obtain CP; (2) Preparation method of EGCG / CP lipid nanoparticles: dissolving EGCG and CP in phosphate buffer 1 with a pH of 5.5-7.4 to obtain solution C; taking phospholipids, cholesterol and DSPE-PEG 2000 -Mal, dissolved in one or more of dichloromethane, chloroform, anhydrous ethanol, methanol, acetone, ethyl acetate, and ether, and the organic solvent is removed by rotary evaporation to form a uniform lipid film, and solution C is added, hydrated in a water bath at 35-45°C for 1-3 hours, and filtered through a 0.22-0.45 μm microporous filter to obtain EGCG / CP lipid nanoparticles; (3) Preparation method for modification of living cells: culturing and collecting living cells, the number of which is 1 million to 5 million, adding 1-2 mM tris(2-carboxyethyl)phosphine hydrochloride solution, incubating at 37°C for 10-40 minutes, centrifuging, collecting the precipitate and dispersing it in phosphate buffer 2 to obtain modified living cells; (4) mixing the EGCG / CP lipid nanoparticles obtained in step (2) and the living cells obtained in step (3) at a volume ratio of 1:5 to 1:20, incubating at 37°C for 20-60 minutes, centrifuging and collecting the precipitate to obtain living cell-coupled EGCG / CP lipid nanoparticles.

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