A neutrophil membrane-coated metal coordination nanoparticle drug and its construction method and application

Abstract

The present invention belongs to the field of nanobiomedicine and specifically discloses a neutrophil membrane-coated metal-liganded nanoparticle drug, its construction method, and application. The neutrophil membrane is obtained from mouse bone marrow and has the ability to evade clearance by immune cells. The neutrophil membrane is coated on the surface of SRF-FePI nanoparticles obtained by reacting SRF with trivalent iron and PI. The nanomedicine described in the present invention can target tumor cells, be released in an acidic environment, have a killing effect on liver cancer cells, and have a certain ability to induce tumor immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of nano-biomedicine and relates to a neutrophil membrane-coated metal coordination nanoparticle drug and a construction method and application thereof. Background Art

[0002] Cancer is considered one of the most aggressive malignancies in humans. Common treatments for advanced liver cancer include chemotherapy and targeted therapies, depending on the status of actionable driver mutations. In addition to traditional tumor treatments, various novel nanobiomaterials have attracted attention due to their enhanced anti-tumor properties and low off-target effects. However, nanomedicines have certain limitations in clinical application, including rapid in vivo clearance, insufficient biostability, and poor targeting performance. Therefore, it is necessary to develop nano-drug delivery systems with a composition similar to that of the organism to circumvent these drawbacks.

[0003] Drug delivery systems constructed from cell membranes take advantage of the advantages of natural biological membranes and have shown great potential in the field of tumor treatment. Considering that immune cells play a role in almost every stage of tumor metastasis, people have proposed a coating method using immune cell membranes, which has unique tumor homing ability and excellent biocompatibility. Among them, neutrophils (NE) are the most abundant white blood cells in the mammalian circulation, accounting for 50-70% of the total circulating white blood cells, and are the first white blood cells recruited to the site of inflammation. Drug delivery systems based on neutrophil membranes (NEM) can improve the efficacy of cancer treatment, have advantages such as immune evasion and prolonged circulation, can enhance the accumulation of drugs at the tumor site, and improve the immunosuppression of the tumor microenvironment.

[0004] Tumor ferroptosis is a non-apoptotic form of regulated cell death characterized by the accumulation of iron-dependent lipid peroxides (LPO). Ferroptosis differs from other types of cell death in both mechanism and morphology, and is characterized by cytoplasmic swelling and cell membrane rupture, while the nucleus shows almost no morphological changes. Due to the non-apoptotic nature of the ferroptosis pathway, ferroptosis-based tumor treatment may bypass defects such as drug resistance. The Fenton reaction involved in ferroptosis requires free divalent iron to generate reactive oxygen species (ROS) represented by hydroxyl radicals. Accumulated ROS peroxidize membrane lipids, thereby causing loss of cell function and cell death. Therefore, iron is a crucial redox active component in the regulation of ferroptosis. Sorafenib (SRF), an oral multikinase inhibitor, was first approved as a standard first-line treatment for advanced liver cancer. It can inhibit vascular endothelial growth factor receptors 1 / 2 / 3 (VEGFR-1 / 2 / 3), platelet-derived growth factor receptor (PDGFR-β), Fms-like tyrosine kinase 3 (Flt-3) and stem cell growth factor (c-KIT), inhibiting tumor angiogenesis and tumor development.

[0005] In addition to directly killing tumor cells, clinical research is currently focusing on the tumor microenvironment. It is reported that the main factor for the poor effect of tumor treatment comes from the immunosuppressive microenvironment. Tumors can escape immune surveillance and easily metastasize in this environment. It has been observed that immune checkpoints are associated with the immunosuppressive environment. Immune checkpoints such as indoleamine-2,3-dioxygenase (IDO) can inhibit immune responses and promote tumor development. IDO inhibitors can reprogram the host inflammatory environment, turning "cold" tumors (non-T cell inflammatory tumors) into "hot" tumors (T cell inflammatory tumors). Tryptophan metabolism plays an important role in ferroptosis treatment. More tryptophan plays an inhibitory role in tumor development, and IDO inhibitors can inhibit the conversion of tryptophan, thereby maintaining a higher concentration of tryptophan, promoting the occurrence of ferroptosis, and exerting an enhanced anti-tumor effect of combined treatment. Summary of the Invention

[0006] In view of the problems in the above background technology, the purpose of the present invention is to provide a neutrophil membrane-coated metal coordination nanoparticle drug and its construction method and application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] A neutrophil membrane-coated metal coordination nanoparticle drug, wherein the metal coordination nanoparticles are SRF-FePI particles obtained by reacting SRF, trivalent iron, and PI (full name: 4-phenylimidazole).

[0009] The particle size range of the neutrophil membrane-coated metal coordination nanoparticle drug is about 137.2±4.1 nm.

[0010] The method for constructing the neutrophil membrane-coated metal coordination nanoparticle drug comprises the following steps:

[0011] (1) SRF solution and Fe 3+ The mixture was fully mixed with the PI solution, precipitated by adding water and centrifuged to obtain nanoparticles SRF-FePI;

[0012] (2) NE was extracted from mouse bone marrow, and NEM nanovesicles were obtained by ultrasonic centrifugation and extrusion;

[0013] (3) The nanoparticles and NEM nanovesicles are co-extruded to obtain the product.

[0014] Furthermore,

[0015] The solvent of step (1) SRF is DMSO, Fe 3+ The solvent for PI is methanol;

[0016] Fe 3+The concentration of the solution is 18-108 mg / mL; the concentration of the PI solution is 18-108 mg / mL, the concentration of the SRF solution is 11.25-108 mg / mL, and the concentration of Fe 3+ The volume ratio of solution to PI solution is 1:2~1:8, and the volume ratio of SRF to Fe 3+ The volume ratio of the solution ranges from 1:2 to 1:8.

[0017] Optimum: SRF solution is first mixed with Fe 3+ The solutions were mixed, and then the mixed solution was added dropwise to the PI solution, and the reaction was stirred at room temperature for at least 1 h.

[0018] The specific method of step (2) is as follows:

[0019] 1) Mouse bone marrow cells were harvested and resuspended in 1× PBS. Red blood cell lysis buffer was added and incubated on ice for 3 min. The cells were centrifuged at 1500 rpm for 5 min at 25°C. The supernatant was discarded and the cells were resuspended in 1× PBS.

[0020] 2) Carefully add 55%, 65%, and 78% (v / v) Percoll working solutions to a centrifuge tube in sequence (due to the different densities of the working solutions, a three-layer gradient will form, not stratify directly), and add the bone marrow cell suspension to the top layer; centrifuge at 2000 rpm for 30 minutes, collect the cells between the 65% and 78% gradients, wash, centrifuge, and resuspend in 1× PBS;

[0021] 3) Ultrasonic treatment was performed in an ice-water bath for 5 minutes, centrifuged at 5800 rpm for 5 minutes, and the precipitate was discarded; centrifuged at 15000 rpm for 30 minutes at 4°C, and the precipitate was washed with PBS;

[0022] 4) Using a liposome extruder, extrude the liposome through a 400 nm polycarbonate membrane back and forth for at least 14 times (each time it is extruded, it is returned and extruded again) to obtain NEM nanovesicles.

[0023] The specific preparation method of the Percoll working solution in step 2) is as follows: Percoll cell separation solution (purchased from Solebol) and 10×PBS are prepared in a volume ratio of 9:1 to prepare a Percoll stock solution, and then the Percoll stock solution and 1×PBS are used to prepare 55%, 65%, and 78% (v / v) working solutions.

[0024] The specific method of step (3) is as follows: 1-2 mg of SRF-FePI is weighed, 1 mL of ultrapure water is added, and ultrasonic treatment is performed for at least 5 minutes to prepare a uniformly dispersed suspension, which is then mixed with the same mass of NEM vesicles and ultrasonicated for 2 minutes (ultrasonication for 2 seconds, stop for 3 seconds, energy 30%, power 500w); then, the suspension is co-extruded at least 14 times under a 200 nm polycarbonate membrane through a liposome extruder; centrifuged at 12000 rpm for 10 minutes, and the precipitate is washed to obtain the product.

[0025] The present invention also provides the use of the neutrophil membrane-coated metal coordination nanoparticle medicine in the preparation of liver cancer therapeutic drugs.

[0026] Beneficial effects

[0027] 1. This invention has produced a drug comprising neutrophil membrane-coated metal-liganded nanoparticles for use in immunotherapy to kill liver cancer cells (HepG2) while simultaneously enhancing sensitization. Neutrophil membranes coated on the nanoparticle surface allow the drug to evade clearance by macrophages (RAW 264.7), targeting tumor cells and effectively killing liver cancer cells. Furthermore, the drug inhibits the conversion of tryptophan, thereby activating the immunosuppressive microenvironment and enhancing anti-tumor immunity.

[0028] 2. The nanomedicine provided by the present invention utilizes coordination bonds to form a drug delivery carrier, which is loaded with sorafenib for the treatment of liver cancer and responsively releases free drugs when it reaches the acidic environment of tumor cells.

[0029] 3. Both the free drug and the nano drug involved in the present invention have low toxic side effects on normal liver cells (L02) and have good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the infrared spectra of FeCl3·6H2O, PI, SRF and the SRF-FePI nanomedicine prepared by the present invention.

[0031] Figure 2 These are the transmission electron microscopy images (a) and elemental analysis images (b-f) of the SRF-FePI@NEM nanomedicine prepared in the present invention. The synthesized nanomedicine has a membrane-core structure.

[0032] Figure 3 This is a particle size distribution diagram of the SRF-FePI@NEM nanomedicine prepared in the present invention.

[0033] Figure 4 It is the cumulative drug release amount of the SRF-FePI and SRF-FePI@NEM nanomedicines prepared in the present invention in PBS solutions with pH 7.4 and pH 5.1.

[0034] Figure 5 This is the fluorescence intensity graph of high-content imaging detection of SRF-FePI and SRF-FePI@NEM nanomedicine uptake in HepG2 cells and RAW264.7.

[0035] Figure 5 In the experiment, the fluorescent dye Nile red (NR) was used to replace 50% of SRF as a fluorescent tracer for high-content imaging observation.

[0036] Figure 6 The present invention is to study the cytotoxicity of SRF, SRF-FePI and SRF-FePI@NEM nanomedicines to HepG2 cells and L02 cells.

[0037] Figure 7 It is a Calcein-AM / PI double staining image of HepG2 cells and L02 cells by SRF, SRF-FePI prepared by the present invention and SRF-FePI@NEM nanomedicine.

[0038] Figure 8 It is a fluorescence intensity diagram of the ROS generation ability of the SRF-FePI@NEM nanomedicine prepared by the present invention detected by high-content imaging.

[0039] Figure 9 This is a graph showing the inhibition of kynurenine by PI, SRF-FePI prepared by the present invention, and SRF-FePI@NEM nanomedicine. DETAILED DESCRIPTION

[0040] In order to better illustrate the present invention, the present invention will be further described below with reference to the embodiments.

[0041] Example 1: Preparation and characterization of nanomedicine SRF-FePI@NEM

[0042] (1) Weigh 20 mg of SRF and dissolve it in 120 μL of DMSO to obtain a drug stock solution. Weigh 33.8 mg of FeCl3·6H2O and 72.1 mg of PI and dissolve them in 1 mL of methanol. 3+ The methanol solution was mixed, and the mixed solution was added dropwise to the PI solution, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, 1 mL of pre-cooled ultrapure water was added dropwise to terminate the reaction and allow solids to precipitate. The product was washed twice by centrifugation (4°C, 10000 rpm, 20 min), and finally freeze-dried at -50°C for 5 hours to obtain a yellow solid powder product, which is SRF-FePI nanoparticles. The infrared spectroscopy test results are as follows: Figure 1 shown.

[0043] (2) ICR mice were anesthetized and killed by cervical dislocation. The femur and tibia were removed, and the bone marrow was flushed with RPMI 1640 medium and filtered through a 75μm cell strainer. The cell suspension was centrifuged at 1500rpm for 5 minutes, and the bone marrow cells at the bottom of the centrifuge tube were collected and resuspended with 1×PBS. Red blood cell lysis buffer was added, incubated on ice for 3 minutes, and centrifuged at 1500rpm for 5 minutes at 25℃. The supernatant was discarded and the cells were resuspended in 1×PBS. 55%, 65%, and 78% (v / v) Percoll working solutions were added to the centrifuge tube, and the bone marrow cell suspension was added to the top layer. Centrifuged at 2000rpm for 30 minutes, and the cells between 65% and 78% of the gradient were collected, washed, and resuspended by centrifugation. Ultrasonic treatment was performed in an ice water bath for 5 minutes. The cells were centrifuged at 3200g for 5 minutes at 4℃, and the pellet was discarded to discard unbroken cell nuclei and cells. Centrifuged at 20000g for 30 minutes at 4℃, and the pellet was washed with PBS. NEM nanovesicles were obtained by extruding the liposomes through a 400 nm polycarbonate membrane 14 times using a liposome extruder.

[0044] (3) Weigh 2 mg of SRF-FePI using an analytical balance, add 1 mL of ultrapure water and sonicate for 5 minutes to prepare a uniformly dispersed suspension. After diluting to 900 μg / mL, mix with the same mass of NEM vesicles and sonicate for 2 minutes. Then co-extrude 14 times under a 200 nm polycarbonate membrane through a liposome extruder. Centrifuge at 12,000 rpm for 10 minutes, remove the unloaded cell membrane in the supernatant, and obtain SRF-FePI@NEM nanomedicine. The nanomedicine was dispersed in water and placed under a transmission electron microscope for observation and elemental scanning analysis. The results are as follows: Figure 2 The particle size distribution of nanomedicine was detected by dynamic light scattering. The results are shown in Figure 3 shown.

[0045] Example 2: Characterization of pH-responsive release performance of nanomedicine SRF-FePI@NEM

[0046] Prepare two release media with different pH values: (1) PBS containing 40% ethanol (pH 7.4) (2) PBS containing 40% ethanol (pH 5.1). Dilute SRF-FePI and SRF-FePI@NEM with release media to a nanoparticle concentration of 450 μg / mL. Take 1 mL and add it to the dialysis bag of each group, clamp it with dialysis clamps at 3 to 5 cm on both ends of the dialysis bag, place it in a centrifuge tube, and add 30 mL of release media with different pH values. Take out 1 mL of release medium at a specific time point and immediately add 1 mL of fresh release medium PBS with the corresponding pH value. Use high performance liquid chromatography to detect the drug content of SRF and PI. Calculate the cumulative release of the drug according to the formula, and the results are as follows: Figure 4As shown, compared with the neutral pH 7.4 environment, the release rate and cumulative release amount of the drug-loaded system in the acidic pH 5.1 environment are higher, which shows that the system constructed in the present invention has a certain pH-responsive release performance.

[0047] V n is the volume of each sample (mL); V0 is the initial volume of the release medium (mL); C n is the sample concentration of the nth sampling (μg / mL); m D is the mass of the drug initially added.

[0048]

[0049] Example 3: Cellular uptake experiment of nanomedicine SRF-FePI@NEM

[0050] (1) The cellular uptake ability of the nanodrug SRF-FePI@NEM was investigated using RAW 264.7 and HepG2 cells. Since the nanodrug itself has no fluorescence, 50% of SRF was replaced with the fluorescent dye NR during the preparation process as a fluorescent tracer marker. The centrifugal speed (8000 rpm) and centrifugation time (10 min) were reduced to remove free NR. The prepared fluorescent complex NR-FePI was then extruded to modify the neutrophil membrane on the outer layer to prepare the biomimetic nanofluorescent complex NR-FePI@NEM.

[0051] (2) RAW 264.7 or HepG2 cells were cultured at a rate of 1×10 5 / hole or 3×10 4 The cells were seeded at a density of 1 μg / mL in a 96-well plate and incubated overnight. The culture medium was discarded and the same concentration of drugs NR-FePI and NR-FePI@NEM (measured in SRF concentration) were added. After incubation for 4 hours, the culture medium was aspirated and the cells were washed twice with DPBS or PBS to remove free drugs. 100 μL of 1 μg / mL Hoechst33342 was added to stain the nuclei for 10 minutes, washed once, and finally 100 μL of DMEM was added to each well for high-content imaging. The results are shown in Figure 2. Figure 5 As shown, cell membrane-coated fluorescent composite nanoparticles NR-FePI@NEM are more readily taken up by HepG2 cells than uncoated NR-FePI. This suggests that the SRF-FePI@NEM constructed in this invention, after NEM membrane modification, possesses inflammatory targeting capabilities against liver cancer cells, facilitating targeted drug delivery. RAW 264.7 cells, on the other hand, exhibit significantly reduced uptake of cell membrane-coated nanoparticles. This is because the surface NEM, also acting as an immune cell, acts as a cell membrane camouflage, allowing it to escape phagocytosis by RAW 264.7 cells.

[0052] Example 4: Cytotoxicity experiment of nanomedicine SRF-FePI@NEM

[0053] HepG2 or L02 cells were cultured at 3×10 4 / hole or 1×10 4 / well was inoculated into a 96-well plate, and the culture medium was discarded after incubation for 24 hours. The drug was diluted with blank culture medium to 50, 25, 12.5, 6.25, 3.125, 1, and 0.1 μg / mL. 100 μL of drug-containing culture medium was added to each well of the experimental group, and a blank group (containing only blank culture medium) and a control group (cells + blank culture medium) were set up, with 5 replicate wells in each group. After incubation for 24 hours, the culture medium was discarded, and 100 μL of 10% CCK-8 solution was added and incubated for 1 hour. The absorbance at 450 nm was detected by an enzyme reader, and the results were as follows Figure 6 As shown, HepG2 cell viability decreased to varying degrees with increasing drug concentration. While free SRF exhibits some cytotoxicity against HepG2 cells, the drug-delivered system SRF-FePI provides the necessary inducer and excess iron ions for tumor ferroptosis, resulting in a more potent tumor-killing effect than free SRF. Furthermore, the cell membrane-modified drug-delivered system SRF-FePI@NEM exhibited an IC50 value of 1 to 3.125 μg / mL. At a concentration of 50 μg / mL, HepG2 cell survival was only 15.5%, demonstrating a relatively strong toxic effect against liver cancer cells. This may be due to the fact that SRF-FePI@NEM is more readily taken up by HepG2 cells, resulting in more efficient drug delivery. Furthermore, the survival rates of SRF, SRF-FePI, and SRF-FePI@NEM co-incubated with L02 cells were all above 80%, with no significant toxic side effects on normal liver cells.

[0054] Example 5: Calcein-AM / PI Experiment of SRF-FePI@NEM Nanomedicine

[0055] HepG2 or L02 cells were cultured at 3×10 4 / hole or 1×10 4 Each well was inoculated with 100 μL of 25 μg / mL or 50 μg / mL blank medium diluted with SRF, SRF-FePI, or SRF-FePI@NEM (all converted to SRF concentrations) and incubated for 24 hours. The medium was slowly aspirated after 24 hours of incubation, and 100 μL of calcein double staining reagent was added. After staining for 30 minutes, high-content imaging was performed. The results are shown in the figure. Figure 7 As shown, SRF-FePI@NEM has a better killing effect on HepG2 cells, so the red fluorescence distribution of dead cells accounts for a large proportion, and the distribution of dead cells has exceeded half, which is consistent with the cytotoxicity results.

[0056] Example 6: ROS generation experiment of nanomedicine SRF-FePI@NEM

[0057] HepG2 cells were cultured at 3×10 4 / well were inoculated in a 96-well plate, and the culture medium was discarded after incubation overnight. 100 μL of drug SRF-FePI@NEM diluted with blank culture medium (SRF final concentration was 12.5 μg / mL) was added to stimulate the cells to produce ROS, and 100 μL of blank culture medium was added to the control group. Then 100 μL of DCFH-DA probe with a final concentration of 5 μM was added, and the drug and fluorescent probe were incubated with the cells for 2 hours. After the incubation, the culture medium was aspirated and washed three times with PBS to wash away the drug that was not taken up into the cells and the excess fluorescent probe. Then 100 μL of DMEM culture medium was added for high-content imaging. The results are shown in the figure. Figure 8 As shown, the experimental group showed fluorescence, indicating that SRF-FePI@NEM produced ROS in HepG2 cells, while the blank group did not show green fluorescence.

[0058] Example 7: Kynurenine inhibition experiment of nanomedicine SRF-FePI@NEM

[0059] HepG2 cells were cultured at 1×10 4 / well was inoculated in a 96-well plate, and the culture medium was discarded after incubation for 24 hours. Drugs PI, SRF-FePI, and SRF-FePI@NEM diluted in DMEM culture medium were added, and four concentrations of 1μM, 10μM, 50μM, and 100μM were set respectively, with 3 replicates in each group. 25μL of INF-γ with a final concentration of 50ng / mL (to stimulate the expression of IDO) and 25μL of tryptophan with a final concentration of 1.2mM (to provide reaction raw materials) were added to each well. After incubation for 48 hours, 140μL of supernatant was removed to a centrifuge tube. The subsequent experimental steps remained the same as above. After color development with Ehrlich reagent, the absorbance at 490nm was measured with an enzyme reader. The results are as shown below. Figure 9 As shown, the nanodrug SRF-FePI@NEM, after modifying the cell membrane, has a good Kyn inhibition effect in HepG2 cells, and the inhibition rate gradually increases with the increase of drug concentration. The Kyn inhibition rate of SRF-FePI@NEM at a concentration of 1000μM can reach 80%. In HepG2 cells, the SRF-FePI@NEM constructed by the present invention retains a large amount of Trp reaction raw materials in the cells, indicating that the IDO pathway is inhibited. The delivered drug PI acts as an IDO inhibitor, blocking the IDO pathway by inhibiting the possibility of Trp conversion to Kyn, activating T cells, improving the ability of pathogens to clear infection, and inducing the body's immune response.

[0060] The results of Examples 4 to 7 above demonstrate that the present invention has an inhibitory effect on liver cancer cells.

[0061] The above embodiments are preferred implementations of the present invention and are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. It should be understood that there are many alternative solutions within the scope of the principles of the present invention, which do not affect the essential content of the present invention.

Claims

1. A neutrophil membrane-coated metal coordination nanoparticle drug, characterized in that: The metal coordination nanoparticles are SRF-FePI particles obtained by reacting sorafenib with trivalent iron and 4-phenylimidazole.

2. The method for constructing the neutrophil membrane-coated metal coordination nanoparticle drug according to claim 1, characterized in that: The following steps are involved: (1) Sorafenib solution and Fe 3+ The mixture was thoroughly mixed with 4-phenylimidazole solution, precipitated by adding water and centrifuged to obtain nanoparticles SRF-FePI; (2) NE was extracted from mouse bone marrow, and NEM nanovesicles were obtained by ultrasonic centrifugation and extrusion; (3) The nanoparticles are co-extruded with NEM nanovesicles to obtain the product.

3. The construction method according to claim 2, characterized in that Step (1) The solvent of Sorafenib is DMSO, Fe 3+ and 4-phenylimidazole, the solvent was methanol; Fe 3+ The concentration of the solution is 18~108 mg / mL; the concentration of the 4-phenylimidazole solution is 18~108 mg / mL, the concentration of sorafenib is 11.25~108 mg / mL, and the concentration of Fe 3+ The volume ratio of solution to 4-phenylimidazole solution is 1:2~1:8, sorafenib and Fe 3+ The volume ratio of the solution ranges from 1:2 to 1:

8.

4. The construction method according to claim 2 or 3, characterized in that: Sorafenib solution was first mixed with Fe 3+ The solutions were mixed, and the mixed solution was added dropwise to the 4-phenylimidazole solution, and the reaction was stirred at room temperature for at least 1 h.

5. The construction method according to claim 2, characterized in that The specific method of step (2) is as follows: 1) Obtain mouse bone marrow cells and resuspend them in 1× PBS. Add red blood cell lysis buffer and incubate on ice for 3 min. Centrifuge at 1500 rpm for 5 min at 25°C. Discard the supernatant and resuspend the cells in 1× PBS. 2) Add 55%, 65%, and 78% v / v Percoll working solutions to a centrifuge tube in sequence, and add the bone marrow cell suspension to the top layer; centrifuge at 2000 rpm for 30 minutes, collect the cells between 65% and 78% of the gradient, wash, centrifuge, and resuspend in 1× PBS; 3) Ultrasonic treatment in an ice-water bath for 5 min, centrifugation at 5800 rpm for 5 min, discard the precipitate; centrifugation at 15000 rpm for 30 min at 4°C, and wash the precipitate with PBS; 4) Use a liposome extruder to reciprocate through a 400 nm polycarbonate membrane for at least 14 times to obtain NEM nanovesicles.

6. The construction method according to claim 5, characterized in that: The specific preparation method of the Percoll working solution in step 2) is as follows: Percoll cell separation solution and 10× PBS are mixed at a volume ratio of 9:1 to prepare a Percoll stock solution. Then, the Percoll stock solution and 1× PBS are used to prepare 55%, 65%, and 78% v / v working solutions.

7. The construction method according to claim 2, characterized in that: The specific method of step (3) is as follows: weigh 1 to 2 mg of SRF-FePI, add 1 mL of ultrapure water and ultrasonicate for at least 5 min to prepare a uniformly dispersed suspension, mix with NEM vesicles of the same mass as SRF-FePI, and ultrasonicate for 2 min, ultrasonicate for 2 seconds, stop for 3 seconds, energy 30%, power 500 W; then co-extrude at least 14 times under a 200 nm polycarbonate membrane through a liposome extruder; centrifuge at 12,000 rpm for 10 min, wash the precipitate, and obtain the product.

8. Use of the neutrophil membrane-coated metal coordination nanoparticle drug prepared by the method according to claim 1 or any one of claims 2 to 7 in preparing a drug for treating liver cancer.

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