Preparation method and application of bionic nano immunomodulatory bait

Abstract

The invention discloses a preparation method and application of bionic nano immunomodulatory bait, and the preparation method comprises the following steps: firstly, obtaining neutrophils, treating the neutrophils with a cell lysis buffer solution containing a calcium ion chelating agent and a protease inhibitor, collecting supernate, and then centrifuging and purifying to obtain neutrophils membranes; then mixing an organic phase containing a polymer polylactic acid-glycolic acid copolymer and a therapeutic drug with a polyvinyl alcohol solution, and emulsifying to obtain a PLGA-therapeutic drug nano core; and finally, co-extruding the material and the neutrophile granulocyte membrane. The neutrophile granulocyte membrane is used as a camouflage shell, the nano bait capable of efficiently targeting an inflammatory site and neutralizing multiple inflammatory mediators is constructed, the nano bait is applied to brain injury after cardio-pulmonary resuscitation, efficient and active targeting on an inflammatory brain area can be achieved, the death rate of neurons can be reduced, and the nano bait has an extremely good application prospect.

Description

A method for preparing and applying a biomimetic nano-immunomodulatory decoy Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing and applying a biomimetic nano-immunomodulatory decoy. Background Technology

[0002] Cardiac arrest (CA) is one of the major public health problems worldwide. Despite improved success rates of spontaneous circulation recovery, patient outcomes remain unsatisfactory, primarily due to brain injury following cardiopulmonary resuscitation (CPR). Recent studies have shown that the imbalance between systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome is the core pathological mechanism mediating secondary brain injury. After spontaneous circulation recovery, systemic ischemia-reperfusion triggers the release of numerous injury-related molecular patterns into the bloodstream, initiating a "cytokine storm" and disrupting the blood-brain barrier (BBB). This leads to the infiltration of numerous inflammatory cells (such as neutrophils) and excessive activation of microglia, ultimately resulting in irreversible neuronal death. Therefore, precise regulation of neuroinflammation has become one of the most promising strategies for treating brain injury following CPR. However, currently, there is a lack of specific means in clinical practice to effectively intervene in this pathological process.

[0003] In related technologies, the development of nanotechnology has provided new solutions for drug delivery. For example, surface modification of nanocarriers (such as liposomes and polymer nanoparticles) (e.g., PEGylation, targeting peptides) can prolong circulation time and enhance accumulation at inflammatory sites to some extent (through the EPR effect). However, these synthetic nanomaterials still have inherent defects in dealing with the complex inflammatory environment after cardiopulmonary resuscitation, such as poor biocompatibility, insufficient "active" regulatory capacity, and limited targeting efficiency. These defects lead to the synthetic materials being easily recognized and rapidly cleared by the body's immune system (such as the mononuclear phagocytic system), requiring complex surface modifications to "disguise" them, and they cannot actively recognize and neutralize a variety of inflammatory mediators. In addition, targeting inflammation largely relies on the passive EPR effect, lacking the ability to actively "hom" to complex biological signals at inflammatory sites.

[0004] Therefore, there is an urgent need to develop a new biomimetic nanomaterial that can effectively and actively target inflamed brain regions and can efficiently and safely alleviate brain injury after cardiopulmonary resuscitation. Summary of the Invention

[0005] The first objective of this invention is to provide a method for preparing a biomimetic nano-immunomodulatory decoy.

[0006] The second objective of this invention is to provide a biomimetic nano-immunomodulatory decoy.

[0007] The third aspect of this invention aims to provide a method for preparing the above-mentioned biomimetic nano-immunomodulatory decoy or its application in the preparation of drugs for alleviating or treating brain injury.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In its first aspect, the present invention provides a method for preparing a biomimetic nano-immunomodulatory decoy, comprising the following steps: S1, obtaining neutrophils, treating them with a cell lysis buffer containing a calcium ion chelating agent and a protease inhibitor, collecting the supernatant, and then centrifuging and purifying them to obtain a neutrophil membrane; S2, mixing a polymer polylactic acid-glycolic acid copolymer and a therapeutic drug in a first solvent to obtain an organic phase, then mixing it with a polyvinyl alcohol solution, and obtaining a PLGA-therapeutic drug nanonucleus after emulsification and purification; S3, contacting the neutrophil membrane with the PLGA-therapeutic drug nanonucleus in a second solvent, and then extruding the nanonucleus to obtain the final product.

[0009] According to the preparation method of the present invention, at least the following beneficial effects are achieved: (1) The present invention first constructs a biomimetic nano-immunomodulatory decoy that has a high efficiency and active targeting effect on the inflamed brain region and can efficiently and safely alleviate brain injury after cardiopulmonary resuscitation. The core of its design is to use the natural cell membrane extracted from neutrophils as a "camouflage shell" to wrap around an inert nano core, thereby constructing a "nano-decoy" that can both efficiently target the inflamed site and broadly neutralize a variety of inflammatory mediators.

[0010] (2) Compared with traditional anti-inflammatory drugs, the biomimetic nano-immunomodulatory decoy constructed in this invention can achieve active targeting and multi-factor neutralization, avoiding systemic immunosuppression, rather than passively delivering small molecule drugs. It provides a platform for actively "trapping" inflammatory factors, with a wider and more direct effect, and avoids the systemic toxic side effects of drugs. Compared with traditional nanocarriers, this invention is not a simple synthetic material "disguise", but utilizes the natural chemotaxis of neutrophils towards inflammation. Its immune escape ability and targeting efficiency for inflammation are far superior to conventional artificially modified nanoparticles. (3) The biomimetic nano-immunomodulatory decoy of this invention does not require additional artificial special modification. Its design does not attempt to "improve" or "forcefully penetrate" the BBB, but utilizes the "channels" opened by the body itself for immune cells in the disease state, and uses biomimetic technology to make the loaded nano-drugs "disguise" as cells that can use these channels. The biomimetic nano-immunomodulatory decoy constructed using this invention avoids the complexity, uncontrollability, and potential toxicity of conventional drugs. It utilizes a natural mechanism to achieve efficient and active targeting of inflamed brain regions through the synergistic effect of multiple receptors on the neutrophil membrane. It is only activated when the permeability of the BBB increases due to inflammation and chemotactic signals are present, thereby improving the specificity of treatment and reducing off-target effects.

[0011] In some embodiments of the present invention, in step S1, the cell membrane of the neutrophils overexpresses CXCR2.

[0012] Neutrophils, as core effector cells in the body's inflammatory response, naturally highly express a variety of chemokines and cytokine receptors on their surface, enabling them to precisely chemotactically approach inflammatory sites and participate in immune regulation. This invention discovers that neutrophil membranes overexpressing the CXCR2 membrane protein possess "inflammatory chemotaxis" properties, actively chemotactically approaching the core region of neuroinflammation, significantly increasing drug concentration at the lesion site and promoting damage repair. Simultaneously, the neutrophil membrane can broadly neutralize various excess pro-inflammatory cytokines and chemokines, rapidly interrupting the persistent damage caused by the inflammatory response. This highly matches the pathological mechanism of brain injury after cardiopulmonary resuscitation, enabling "causal treatment" of the disease mechanism. This is the first time that neutrophil membrane biomimetic technology has been applied to the immunomodulatory therapy of acute brain injury, filling a gap in the field.

[0013] In some embodiments of the present invention, the membrane proteins of the neutrophils further include either CXCR1 or CD11b.

[0014] In some embodiments of the present invention, the calcium ion chelating agent includes ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA for short).

[0015] Calcium plays a dual role in cells: it is both an essential ion and a destructive "switch" (such as an activator of proteases, phosphatases, and nucleases). During cell lysis, the presence of calcium ions can trigger a series of uncontrolled and destructive reactions, such as activating calpapsin and calmophosphatase. Once activated by calcium ions, these proteases can easily and indiscriminately degrade proteins on the cell membrane, leading to the inactivation of target proteins. This invention has found that adding a certain amount of calcium ion chelating agent helps to better retain the activity of target proteins and maintain the integrity of protein modification state and complex structure, thereby improving the "inflammatory chemotaxis" properties of neutrophil membranes.

[0016] In some embodiments of the present invention, the cell lysis buffer comprises 18-22 mM Tris-HCl, 8-12 mM NaCl and 0.8-1.2 mM MgCl2.

[0017] Preferably, the cell lysis buffer contains 20 mM Tris-HCl, 10 mM NaCl and 1 mM MgCl2.

[0018] In the cell lysis buffer of this invention, Tris-HCl is used to stabilize and maintain the pH environment, NaCl is used to create a low-osmotic environment to allow a large number of water molecules to enter the cell, preparing for physical lysis, and MgCl2 is used to stabilize the structure of the cell membrane and some membrane-associated proteins, preventing the membrane structure from excessively disintegrating in the low-osmotic environment.

[0019] In some embodiments of the present invention, in step S1, the concentration of the calcium ion chelating agent in the cell lysis buffer is 1.5~2.5mM; preferably 2mM.

[0020] In some embodiments of the present invention, in step S1, the concentration of the protease inhibitor in the cell lysis buffer is 4-6 mM; preferably 5 mM.

[0021] In some embodiments of the present invention, in step S1, the centrifugation includes first centrifuging at 300-500g for 8-15 min; collecting the supernatant; then centrifuging at 18000-22000g for 25-35 min; collecting the precipitate; and finally resuspending it in buffer solution and ultracentrifuging at 120000-180000g for 0.8-1.5 h.

[0022] Preferably, in step S1, the centrifugation includes first centrifuging at 500g for 10 min; collecting the supernatant; then centrifuging at 20000g for 30 min to collect the precipitate; and finally resuspending in buffer solution and ultracentrifuging at 150000g for 1 h.

[0023] In some embodiments of the present invention, in step S2, the first solvent is selected from dichloromethane, ethyl acetate, and acetone.

[0024] In some embodiments of the present invention, the mass ratio of lactide to glycolide in the polylactic acid-glycolic acid copolymer is 1:0.8~1.2, and the molecular weight is 10000~20000 Da.

[0025] In some embodiments of the present invention, the therapeutic agent includes a minocycline prodrug.

[0026] In some embodiments of the present invention, the minocycline prodrug is prepared by covalently linking minocycline with a thioketal compound.

[0027] Preferably, the thioketal compound comprises 3,3'-dithiodipropionic acid ketal.

[0028] In some embodiments of the present invention, the preparation method of the minocycline prodrug includes: S11, mixing the 3,3'-dithiodipropionic acid ketal, 4-dimethylaminopyridine and N,N'-diisopropylcarbodiimide in a third solvent and reacting them to obtain an activated solution; S12, suspending minocycline hydrochloride in a fourth solvent, adding a neutralizing agent, and then mixing and reacting it with the activated solution, followed by washing, drying and concentration to obtain the final product.

[0029] Preferably, the third solvent is dichloromethane; preferably, the fourth solvent is N,N-dimethylformamide; preferably, the neutralizing agent is triethylamine.

[0030] In some embodiments of the present invention, the mass concentration of the polyvinyl alcohol solution is 0.005~0.05 g / mL.

[0031] In some embodiments of the present invention, the molecular weight of the polyvinyl alcohol is 10,000 to 150,000 Da.

[0032] In some embodiments of the present invention, the volume ratio of the organic phase to the polyvinyl alcohol solution is 1:20~30; preferably 1:25.

[0033] In some embodiments of the present invention, in step S2, the emulsification is performed using ultrasonic emulsification.

[0034] Preferably, the ultrasonic emulsification power is 120~180W; the ultrasonic time is 1~5 minutes; more preferably, the ultrasonic emulsification power is 150W; the ultrasonic time is 4 minutes.

[0035] In some embodiments of the present invention, step S2, the purification includes removing the solvent from the emulsified product, followed by washing, to obtain the final product.

[0036] In some embodiments of the present invention, in step S3, the second solvent is selected from any one of phosphate buffer solution, Tris-HCl buffer solution, and HEPES buffer solution.

[0037] In some embodiments of the present invention, in step S2, the mass ratio of the neutrophil membrane to the PLGA-therapeutic drug nanonucleus is 1:2 to 20; preferably 1:3 to 15; more preferably 1:5.

[0038] In some embodiments of the invention, the extrusion is performed using an Avanti micro liposome extruder.

[0039] In some embodiments of the present invention, the extrusion temperature is 0~5°C.

[0040] In some embodiments of the present invention, step S3 further includes purification after extrusion.

[0041] Preferably, the purification process includes resuspending the extruded product, then centrifuging it with a sucrose gradient, collecting the band at the interface between 25-35% sucrose and 55-65% sucrose, and washing it to obtain the final product.

[0042] Preferably, the purification process includes resuspending the extruded product, then centrifuging it with a sucrose gradient, collecting the band at the interface between 30% sucrose and 60% sucrose, and washing it to obtain the final product.

[0043] Preferably, the centrifugal force of the sucrose gradient centrifugation is 150,000 g, and the centrifugation time is 1 hour.

[0044] Preferably, the washing process includes diluting the product collected after the sucrose gradient centrifugation with a phosphate buffer solution, centrifuging at 100,000g for 1 hour, and collecting the precipitate to obtain the final product.

[0045] In a second aspect, the present invention provides a biomimetic nano-immunomodulatory decoy, which is prepared by the preparation method described in the first aspect.

[0046] A third aspect of the present invention provides the use of the preparation method as described in the first aspect or the biomimetic nanoimmunomodulatory decoy as described in the second aspect in the preparation of a medicament for alleviating or treating brain injury.

[0047] In some embodiments of the present invention, the brain injury includes brain injury following cardiopulmonary resuscitation.

[0048] In some embodiments of the present invention, the relief or treatment of brain injury includes any of the following: A) restoring neurological function or reducing mortality; B) reducing the mortality of neurons in the CA1, CA3 and / or DG regions of the hippocampus; C) enhancing the targeting effect on brain tissue.

[0049] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0050] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 is a schematic diagram (A) of the isolation of rat peripheral blood neutrophils according to the present invention, and a magnified image of neutrophils stained under a microscope (B); Figure 2 is a diagram of the assembly process of the nano-immunomodulatory decoys of the present invention; Figure 3 is a three-dimensional image of the receptor characterization after NM-NDs assembly according to the present invention; Figure 4 is a transmission electron microscope image of the nanoparticles prepared according to the present invention, wherein the scale bar of A is 100 nm and the scale bar of B is 20 nm; Figure 5 is a Nissl staining image of each group of neurons in the CA1, CA3 and DG regions of the rat hippocampus according to the present invention. Detailed Implementation

[0051] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0052] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0053] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0054] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0055] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0056] Example 1 Preparation of Bionic Nanoscale Immunomodulatory Decoy This example provides a method for preparing a bionic nanoscale immunomodulatory decoy, specifically including the following steps: (I) Neutrophil Membrane Extraction 1. Obtaining Neutrophils: Obtain 8-week-old healthy male SD rats (SPF grade, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.), weighing approximately 250g. All rats were housed in a standard environment (temperature controlled at 21-25℃, relative humidity 50-60%), with free access to food and water. 1.5mg / kg of lipopolysaccharide was injected intraperitoneally into the rats to activate neutrophils. Peripheral blood of the rats was collected, and the original neutrophils were separated using a rat peripheral blood neutrophil separation kit (catalog number: AP9200; brand: Acmec). Then, the neutrophils were purified using density gradient centrifugation (as shown in Figure 1A), and the stained magnified image is shown in Figure 1B.

[0057] 2. Cell lysis treatment: First, wash the purified neutrophils 2-3 times with pre-cooled PBS, centrifuging at 300g for 10 minutes at 4°C each time, carefully discarding the supernatant to completely remove proteases from the serum; then resuspend the cell pellet in pre-cooled hypotonic lysis buffer (Catalog No.: M334; Brand: Amresco) containing 5mM of ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA) and protease inhibitor (product number: P1006; brand: Beyotime). Use a Dounce homogenizer to homogenize continuously 20-50 times to completely destroy the neutrophils. During this process, take a small amount of homogenate and observe it under a microscope. When more than 90% of the cells are ruptured, i.e., no intact, refractive cells are visible, stop the homogenization process to obtain the cell lysis buffer.

[0058] In this step, because neutrophils contain a large number of proteases, these enzymes will rapidly degrade key target proteins on the membrane, such as CXCR1, CXCR2 and IL-1R, thereby causing the nanodecoy to fail. Therefore, protease inhibitors need to be added to the lysis buffer and the operation should be carried out at low temperature, as low temperature can significantly reduce enzyme activity.

[0059] Then, the cell lysate was transferred to a pre-chilled centrifuge tube and centrifuged at 500g for 10 min at below 4°C. The supernatant after centrifugation contained cell membrane fragments, organelles, and cytoplasmic proteins, while the precipitate consisted of cell nuclei and incompletely lysed intact cells. The supernatant was carefully collected, and the cell precipitate was discarded. The supernatant was then centrifuged again at 20000g for 30 min at below 4°C, and the precipitate was collected. At this point, the supernatant mainly consisted of cytoplasmic proteins, and the precipitate consisted of coarse cell membrane components.

[0060] 3. Neutrophil membrane purification: The collected precipitate was resuspended in hypotonic lysis buffer containing 5 mM protease inhibitor. The precipitate was then centrifuged at 150,000 g for 1 hour at a temperature below 4°C. After centrifugation, the cell membrane fragments, due to their low density, would float at the interface between the impurities and the supernatant, forming a thin, usually milky-white band. The cell membrane at the interface was carefully aspirated with a pipette, diluted with a large amount of pre-cooled PBS or ultrapure water, and centrifuged again at 100,000 g for 1 hour at 4°C. The residual impurities were washed away, and the purified neutrophil membrane was finally obtained and stored at -80°C.

[0061] (II) Preparation of nanonuclei 1. Preparation of organic phase: Weigh 50 mg of PLGA polymer and 6.0 mg of minocycline prodrug, dissolve them together in 2 mL of dichloromethane, ethyl acetate or acetone, place on a vortex shaker or gently shake for at least 2 hours until the PLGA is completely dissolved, and obtain a clear and transparent organic phase.

[0062] In this PLGA polymer, the ratio of lactide (LA) to glycolide (GA) is 1:1, the molecular weight is controlled between 10,000 and 20,000 Da, and it is labeled with a fluorescent dye for subsequent tracking of the distribution of nanoparticles in vivo.

[0063] Minocycline prodrugs are prepared by covalently linking minocycline with a sulfur-containing ketal compound, wherein the sulfur-containing ketal compound is a sulfur analog of a ketal, obtained by the condensation of a ketone with a thiol, specifically a 3,3'-dithiodipropionic acid ketal. The specific preparation steps of minocycline prodrug are as follows: S1, In a dry 100 mL round-bottom flask, 1.1 equivalents of 3,3'-dithiodipropionic acid ketal and 4-dimethylaminopyridine (as a catalyst) are dissolved in 20 mL of anhydrous dichloromethane. Under nitrogen protection, the reaction system is placed in an ice-water bath and cooled to 0-4℃. Then, N,N'-diisopropylcarbodiimide is slowly added dropwise using a constant pressure dropping funnel. After the addition is complete, the ice bath is removed, and the reaction is stirred at room temperature for 2 hours to obtain an activated solution. S2, 1.0 equivalents of minocycline hydrochloride is suspended in 10 mL of anhydrous N,N-dimethylformamide, and an equimolar amount of triethylamine is added to neutralize the hydrochloride, releasing the free base form of minocycline. Then, it is added dropwise to the activated solution, and the mixed reaction solution is stirred at room temperature, protected from light, and under nitrogen protection for 24 hours. The flask was wrapped in aluminum foil to protect it from light throughout the process; S3, the reaction progress was monitored using thin-layer chromatography. After the reaction was completed, the reaction solution was washed three times with 50 mL of saturated saline to remove N,N-dimethylformamide and neutralize the catalyst residue. The organic phase dichloromethane was separated, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at below 35°C using a rotary evaporator to obtain the minocycline prodrug.

[0064] 2. Preparation of PVA aqueous phase: Weigh 1g of polyvinyl alcohol (PVA, molecular weight 30000-50000Da), add 100mL of ultrapure water, stir in a 90℃ water bath until completely dissolved, cool to room temperature before use, pour the PVA solution into a 150ml beaker, add a magnetic stir bar, place the beaker in an ice-water bath to obtain the PVA aqueous phase.

[0065] In this step, PVA, as a surfactant, can be adsorbed at the oil-water interface, reducing interfacial tension and preventing the aggregation of nanodroplets during formation. It is key to controlling the particle size and stability of nanoparticles. An ice bath helps maintain a low temperature during ultrasonic emulsification, preventing the organic solvent from evaporating too quickly.

[0066] 3. Emulsification treatment: First, add 2 mL of the organic phase prepared above dropwise to the PVA aqueous phase that is being stirred at medium speed. Use a probe ultrasonic instrument to ultrasonically emulsify the mixture. During emulsification: immerse the probe about 1 cm below the liquid surface and move the probe position slightly during the ultrasonic process. Set the power of the probe ultrasonic instrument to 150W, the ultrasonic time to 4 minutes, and use a pulse mode of 3 seconds on and 2 seconds off to obtain a milky white primary emulsion.

[0067] The obtained colostrum was then transferred to a larger flask and placed on a magnetic stirrer. The mixture was stirred at medium speed (500 rpm) for 3-6 hours at room temperature, allowing the organic solvent to evaporate naturally. As the organic solvent continued to evaporate, PLGA gradually changed from a dissolved state to a supersaturated state and precipitated from the oil droplets to solidify, forming solid PLGA nanoparticles.

[0068] 4. Obtaining PLGA-minocycline nanonuclei: Resuspend the obtained solid PLGA nanoparticles in ultrapure water, then transfer the nanoparticle suspension to a high-speed centrifuge tube and centrifuge at 15,000 rpm for 30 minutes at 4°C. Carefully discard the supernatant, resuspend the precipitated nanoparticles in ultrapure water, and centrifuge again. Repeat this washing step 2-3 times to thoroughly remove PVA and solvent residues. The final precipitate is the purified PLGA-minocycline nanonuclei, labeled MINO-TK@NPs.

[0069] (III) Construction of Nanodecoys by Cell Membrane Encapsulation 1. Screening for the Optimal Membrane / Nucleus Ratio: In this example, a series of mass ratio gradients of neutrophil membranes and MINO-TK@NPs nanonuclei were set up. At each ratio, a fixed amount of MINO-TK@NPs nanonuclei was mixed with a corresponding amount of neutrophil membrane, and the total volume was made up to 150 μL with phosphate buffer solution. The encapsulation efficiency was evaluated by particle size change, zeta potential shift, and Western blotting membrane protein quantification to determine the optimal ratio. The experimental grouping information is as follows: Table 1:

[0070] The blank cell membrane vesicles refer to the vesicle structures formed by the neutrophil membranes prepared earlier. The specific preparation steps are as follows: Take an appropriate amount of the purified neutrophil membrane suspension prepared above, and use phosphate buffer solution to make up its volume to be the same as the subsequent nano-decoy preparation system. Using an Avanti micro-liposome extruder, following the principle of "from large to small," extrude through 400 nm and 200 nm polycarbonate membranes 11-20 times each. The extrusion process is carried out in a 4°C cold room or on ice. The extruded product is a homogeneous blank cell membrane vesicle, labeled as "blank membrane vesicle."

[0071] According to the table above, 500 μg of MINO-TK@NPs and the corresponding amount of neutrophil membrane were mixed in a microcentrifuge tube, and the volume of the mixture was brought up to 200 μL with PBS. The mixture was incubated on ice for 30 minutes, and then extruded 11 times each through a 400 nm and 200 nm polycarbonate membrane using a mini extruder. The mean hydrodynamic diameter and polydispersity index (PDI) were then evaluated using dynamic light scattering; the zeta potential was measured using laser Doppler electrophoresis; and membrane protein analysis was performed simultaneously using the following method: proteins were extracted from samples of experimental groups 1-5 with equal amounts of nanoparticles, and Western blotting was performed using an antibody targeting a characteristic protein of neutrophil membrane (specifically CXCR2, a key chemokine receptor). Experimental group 7 was used as a control.

[0072] The test results are shown in Table 2.

[0073] Table 2:

[0074] The results of mean hydrodynamic diameter and polydispersity index (PDI) analysis showed that, compared with experimental group 6, the nanoparticles coated with membrane proteins had a slightly increased particle size, with an increase of 20-50 nm. This indicates that a membrane layer was formed on the surface, with moderate particle size growth and minimal PDI, suggesting the formation of a uniform and complete core-shell structure. However, a membrane / core ratio that is too low (as in experimental groups 1 and 2) may lead to incomplete coating; while a ratio that is too high (as in experimental group 5) may lead to the presence of excess membrane vesicles, increasing the PDI.

[0075] The Zeta potential detection results showed that: experimental group 6 exhibited a strong negative charge, experimental group 7 exhibited a weak negative charge or near neutral charge, and experimental groups 1-5 were stable and close to experimental group 7.

[0076] Membrane protein analysis results show that as the membrane / nucleus ratio increases, the membrane protein content in NM-NDs should gradually increase and tend to saturate.

[0077] In summary, the optimal membrane / nucleus ratio was chosen as the ratio that produces the minimum PDI and the optimal particle size when the Zeta potential is stable and close to the cell membrane, and the relative membrane protein content is about to plateau. This ratio, specifically in experimental group 3, resulted in the optimal membrane / nucleus ratio. At this ratio, the Zeta potential shifted significantly from -32 mV to -13 mV, approaching the cell membrane, confirming efficient membrane encapsulation. Secondly, the relative membrane protein content reached 88%, close to saturation, indicating high membrane protein loading. Finally, the low PDI (0.09) indicates high sample homogeneity, with no excessive free membrane or aggregation, and the particle size of nearly 200 nm is beneficial for long-term circulation and inflammation targeting in vivo. All subsequent in vitro and in vivo experiments will be conducted using NM-NDs prepared at this optimal ratio (1:5).

[0078] 2. Preparation of nanodecoys: Based on the optimal ratio determined above, MINO-TK@NPs nanonuclei and neutrophil membranes were added to phosphate buffer solution to obtain MINO-TK@NPs nanonuclei with a concentration of approximately 3.33 mg / mL and neutrophil membranes with a concentration of approximately 0.667 mg / mL. Gently pipette a few times to mix thoroughly, avoiding vortexing to prevent air bubbles or protein denaturation. Incubate the mixture on ice for 30 minutes to allow the membrane to make initial contact with the nanocore. Then, using the Avanti micro liposome extruder, install the polycarbonate membranes according to the principle of "from large to small". The specific steps are as follows: Add the mixture to the reservoir above the extruder with a pipette, slowly push the piston to extrude the mixture through the 400nm pore membrane 15 times. The extrudate may be uneven in the first few times, but it will become translucent and uniform in the later times. Replace with a 200nm pore membrane and extrude the sample 10-20 more times. The entire extrusion process should be carried out in a 4°C cold room or on ice to protect protein activity. The huge shear force and physical confinement through the micropores during extrusion tear the flexible cell membrane and re-encapsulate it on the surface of the rigid PLGA nanocore, forming a "core-shell" structure. Multiple extrusions ensure the integrity of the encapsulation and the uniformity of the product.

[0079] Figure 2 shows the assembly process of the nano-immunomodulatory decoy.

[0080] 3. Purification of Nanodecoys: First, dilute all the extruded samples appropriately with phosphate buffer solution. In an ultracentrifuge tube, add a high-density sucrose solution (e.g., 60% w / v), then carefully layer a low-density sucrose solution (e.g., 30% w / v) on top. Carefully add the diluted sample to the top layer of the gradient solution and ultracentrifuge at 150,000 g for 1 hour at 4°C. After centrifugation, the successfully coated nanodecoys will have a moderate density and will concentrate at the 30% / 60% sucrose interface, forming a milky white or pale yellow band. The uncoated blank membrane vesicles will have a lower density and will float on the upper layer. The uncoated PLGA nanonuclei will have a higher density and will sink to the bottom layer. Carefully aspirate the band at the interface using a pipette.

[0081] Then, the collected nano-decoys were diluted with a large amount of pre-cooled phosphate buffer solution, and centrifuged again at 100,000g for 1 hour at 4°C to precipitate the nano-decoys. The supernatant was discarded, and the precipitate (i.e., the purified neutrophil-like nano-decoys, labeled as NM-NDs) was resuspended in an appropriate amount of phosphate buffer solution and stored for a short period of time below 4°C for later use.

[0082] Test Example Performance Characterization Verification 1. Diameter, PDI and Zeta Potential Detection: The nanodecoy, pure MINO-TK@NPs core, and cell membrane vesicles prepared above were diluted with phosphate buffer solution. The hydrodynamic diameter, PDI and Zeta potential of the three were measured at 25℃ using a particle size analyzer. The particle size of the nanodecoy is larger than that of the MINO-TK@NPs core, indicating that the membrane is successfully encapsulated. PDI < 0.2 indicates that the system is homogeneous and has good monodispersity. The potential value of the nanodecoy should be between that of the PLGA core and the cell membrane vesicle.

[0083] The key physical parameters of MINO-TK@NPs, cell membrane vesicles and the final product nanodecoys (NM-NDs) are shown in Table 3. The results are expressed as mean ± standard deviation.

[0084] Table 3:

[0085] By comparing the hydrodynamic diameters, the particle size of MINO-TK@NPs was found to be approximately 198.6 nm, an increase of about 33 nm compared to the original MINO-TK@NPs. This significant and moderate particle size increase clearly indicates that a cell membrane was successfully coated onto the surface of the PLGA nanocores, forming a typical "core-shell" structure. Furthermore, the PDI results show that both the initial nanocores and the final nanodecoys exhibit very uniform particle size distribution, good monodispersity, and a stable and reliable preparation process. The cell membrane vesicles themselves have a high PDI, consistent with their natural heterogeneity, but after extrusion and coating, they form uniform particles.

[0086] The zeta potential detection results showed that MINO-TK@NPs had a strong negative charge of -31.5 mV, originating from the carboxyl groups at the ends of the PLGA polymer; the cell membrane had a weak negative charge of -10.8 mV, originating from the complex charges of membrane phospholipids and membrane proteins; NM-NDs had a zeta potential of -13.2 mV, with its value falling between that of the core and the cell membrane, and significantly shifting towards the cell membrane value. This provides the strongest electrical evidence that the surface cell membrane successfully covers the nanonucleus surface. The surface properties of the nanodecoy are no longer determined by PLGA, but are dominated by the neutrophil membrane.

[0087] The results above show that a biomimetic nanodecoy with a complete structure and uniform particle size was successfully prepared using the method of the present invention, wherein the neutrophil membrane effectively encapsulates the surface of the PLGA nanonucleus and dominates its surface properties.

[0088] 2. Transmission electron microscopy observation: The nano-decoy (NM-NDs) sample prepared above was dropped onto a copper grid, dried with filter paper, soaked in phosphotungstic acid or uranyl acetate solution for 1-2 minutes, dried, and observed under a transmission electron microscope.

[0089] The results are shown in Figure 4, which clearly show a typical "core-shell" structure. The dark MINO-TK@NPs core with high electron density is surrounded by a light-colored thin film (cell membrane) with low electron density, further demonstrating that the method of the present invention can effectively wrap the neutrophil membrane on the surface of the PLGA nanonucleus.

[0090] 3. SDS-PAGE electrophoresis: Neutrophil membranes, MINO-TK@NPs and the nano-decoys (NM-NDs) prepared above were taken with equal amounts of protein and subjected to SDS-PAGE electrophoresis. Coomassie brilliant blue staining was used to observe the band distribution.

[0091] The results of SDS-PAGE electrophoresis are shown in Table 4.

[0092] Table 4:

[0093] As shown in the table above, the neutrophil membrane lanes exhibited a rich variety of protein bands distributed across a wide molecular weight range, reflecting the complex protein composition of the neutrophil membrane, including various receptors, adhesion molecules, and channel proteins. The MINO-TK@NPs lanes showed no visible protein bands, demonstrating that the pure PLGA nanonuclei themselves do not contain proteins. This control group is crucial, ensuring that any protein signals from the nanodecoys in subsequent analyses originate solely from the encapsulated cell membrane, rather than from the nanonuclei themselves or contamination during the preparation process. The nanodecoy lanes displayed a protein banding pattern highly similar to that of the cell membrane lanes. All the major bands appearing in the cell membrane samples also appeared in the NM-NDs samples with similar relative intensities, indicating that the nanodecoys prepared by the extrusion method successfully carried the majority of the protein components from the source neutrophil membrane. Membrane proteins have been transferred from the pure membrane vesicle structure and integrated into the surface of the PLGA nanonuclei.

[0094] The above results indicate that the protein profile of the neutrophil membrane was successfully replicated to the nanodecoy, and the preparation process did not lead to severe loss or selective enrichment of membrane proteins, thus achieving the preservation of the membrane proteome.

[0095] 4. Detection of Characteristic Membrane Proteins: This experiment used anti-CXCR2 primary antibody (catalog number 19538-1-AP; brand Proteintech) to verify the presence of characteristic membrane proteins in the prepared nanodecoys (NM-NDs). The specific method is as follows: First, the nanodecoys were incubated with fluorescently labeled anti-CXCR2 antibody at 4°C. The fluorescence signal was detected using a nanoflow cytometer. Control groups were set up: nanodecoys without primary antibody (control group 1); nanodecoys without antibody and without staining (control group 2); and nanodecoys treated with saponin permeabilization and then stained (control group 3). The non-permeabilized sample group showed a fluorescence signal, which proved that the target protein (CXCR2) was exposed on the surface of the nanodecoy and correctly oriented, indicating functional activity; the permeabilized group showed a stronger signal, proving successful protein integration.

[0096] The receptor characterization diagram of the assembled nanodecoys (NM-NDs) prepared in this invention is shown in Figure 3. Characteristic membrane proteins were quantitatively analyzed using nanoflow cytometry, and the specific experimental data are shown in Table 5.

[0097] Table 5:

[0098] As shown in the table above, the MFI of the experimental group was significantly higher than that of the isotype control and the unstained group, indicating that the anti-CXCR2 antibody specifically binds to the CXCR2 protein on the surface of the nanodecoy. The signal intensity of the transmembrane staining group was further enhanced, indicating that the intracellular protein was also effectively labeled, which verified the integrity of the nanodecoy membrane structure and the uniformity of protein distribution.

[0099] The MFI of control group 3 was significantly higher than that of the experimental group, indicating that the saponin permeabilization treatment disrupted the membrane integrity, allowing the antibody to enter the inner side of the membrane and bind to more CXCR2 protein epitopes, thus generating a stronger fluorescence signal. This phenomenon strongly proves that the neutrophil membrane is completely wrapped around the nanonucleus, forming a closed vesicle structure, and a large amount of CXCR2 protein is successfully integrated into the membrane structure of the nanodecoy, rather than just adsorbed on the surface.

[0100] The above results indicate that the CXCR2 protein of neutrophils was successfully integrated into the membrane structure of the nanodecoy, and its key biological functions were characterized in the cellular nanodecoy.

[0101] Application Example: Application in alleviating brain injury after cardiopulmonary resuscitation. This example examines the effect of the prepared nano-decoys (NM-NDs) on alleviating brain injury after cardiopulmonary resuscitation, specifically including the following experiments.

[0102] 1. Experimental Methods and Preparation: Mature male rats were selected and placed in an induction box. Anesthesia was induced using 4% isoflurane. After anesthesia, the rats were quickly transferred to the operating table. Isoflurane was maintained at 1.5-2% via a nasal cone. Under laryngoscope guidance, endotracheal intubation was performed and connected to a small animal ventilator. Ventilator parameters were set as follows: tidal volume 8-10 mL / kg, respiratory rate 110-120 breaths / min. One femoral vein was isolated, and a siliconized endotracheal tube was inserted for intravenous and drug administration. A body temperature controller was connected, and a rectal probe was inserted into the anus to maintain the core body temperature at 37.0±0.5℃.

[0103] Experimental grouping: The rats were divided into four groups: sham operation group: rats that underwent only endotracheal intubation, arterial and venous catheterization, etc. after anesthesia; CA+vehicle group: rats that received an equal volume of physiological saline after cardiac arrest and cardiopulmonary resuscitation for 10 minutes; CA+MINO-TK@NPs group: rats that received an equal volume of MINO-TK@NPs after cardiac arrest and cardiopulmonary resuscitation; and CA+NM-NDs group: rats that received an equal volume of NM-NDs after cardiac arrest and cardiopulmonary resuscitation.

[0104] Cardiac arrest (CA) induction: Stop isoflurane supply, administer heparin (2 IU / g) via femoral vein for anticoagulation, administer vecuronium bromide (2 mg / kg) or a similar neuromuscular blocking agent via intravenous injection to suppress spontaneous breathing, ensure complete mechanical ventilation, insert a custom electrode into the esophagus via the mouth to the back of the heart, apply alternating current stimulation until the electrocardiogram shows ventricular fibrillation or a flatline, and the mean arterial pressure (MAP) drops sharply to <10 mmHg, stop pacing, and discontinue mechanical ventilation during the 5-8 minute period of cardiac arrest.

[0105] Cardiopulmonary resuscitation (CPR): Mechanical chest compressions at a rate of 300 compressions / min, to a depth of 1 / 3 of the anteroposterior diameter of the thorax, restore mechanical ventilation, and rapidly administer epinephrine (0.5 μg / g) and normal saline (10-20 μL / g) via femoral vein catheter to increase intravascular volume. Continue compressions and administration until the electrocardiogram restores sinus rhythm and the mean arterial pressure (MAP) remains >40 mmHg. Calculate the recovery time. If CPR fails to restore normal function after 20 minutes, it is considered a failure and the animal is excluded.

[0106] Treatment: After cardiopulmonary resuscitation, mechanical ventilation was continued, and the vital signs of the rats were closely monitored for at least 1 hour. The body temperature was maintained at 37°C until the rats resumed spontaneous breathing and their breathing became regular. After extubation, the endotracheal tube was removed. 30 minutes later, a nano-decoy (NM-NDs) solution (5 mg / kg) was injected via the tail vein. The control group was injected with the same dose of naked PLGA nanoparticles (MINO-TK@NPs). The injected rats were placed alone in preheated cages and closely observed until they were fully awake. Soft food and free access to water were provided for 24 hours after the operation. Physiological saline (1 mL) and analgesics (bupreneurine, 0.1 mg / kg) were injected subcutaneously daily for at least 72 hours.

[0107] Researchers unaware of the group assignments scored the rats 24 hours after the recovery of their cardiopulmonary autonomic circulation, and on days 3 and 7 to assess their motor, sensory, reflex, and balance functions. They also recorded the survival rate and weight changes on days 1, 3, and 7 after the recovery of the major circulation, until the end of the experiment.

[0108] 2. Experimental Results (1) Effects on Neurological Function, Body Weight and Survival Rate Experimental Results: Quantitative assessment of neurological function damage and recovery after cardiopulmonary resuscitation in rats Neurological deficit scores were performed by researchers unaware of the grouping at 24 hours, 3 and 7 days after the recovery of spontaneous cardiopulmonary circulation in rats. The scores included: motor tests (0-6 points): tail lift test, assessment of forelimb flexion, walking ability and symmetry; sensory tests (0-2 points): tactile stimulation response to whiskers and trunk; balance beam test (0-6 points): ability to walk and maintain balance on balance beams of different widths; reflex tests (0-4 points): auricular reflex, corneal reflex, startle reflex, etc.

[0109] The results are shown in Table 6, and are expressed as mean ± standard deviation.

[0110] Table 6:

[0111] As shown in the table above, compared with the sham surgery group, all CA model groups showed severe neurological deficits on day 1; the CA + NM-NDs group showed slight improvement on day 1 and significant and sustained neurological recovery on days 3 and 7; the CA + MINO-TK@NPs group showed only slight improvement on day 7, indicating that the protective effect of naked nanonuclei is limited, highlighting the importance of neutrophil membrane encapsulation; the CA + Vehicle group showed slow recovery and persistent functional deficits.

[0112] Due to nerve damage and stress after cardiac arrest, animals may experience difficulty eating and drinking, leading to weight loss. Further statistical analysis was conducted on the weight ratio, which was calculated as (current weight / original weight) * 100%. The changes in rat weight are shown in Table 6 below, and the results are expressed as mean ± standard deviation.

[0113] Table 6:

[0114] The results showed that the CA + Vehicle group experienced a continuous and irreversible weight loss, indicating poor health. The CA + NM-NDs group showed the smallest weight loss, with its weight approaching baseline levels by day 7. This indicates that NM-NDs treatment effectively improved the animals' overall physiological state and recovery ability, consistent with improvements in neurological function scores and survival rates. The recovery level of the CA + MINO-TK@NPs group was between that of the Vehicle and NM-NDs groups.

[0115] CA can affect the overall condition of rats. The survival rate was calculated as (number of surviving rats / original number of rats) × 100%. The changes in rat survival rate are shown in Table 7 below. The results are expressed as mean ± standard deviation.

[0116] Table 7:

[0117] The table above shows that the CA+Vehicle group experienced a continuous and irreversible weight loss, indicating poor health. The CA+NM-NDs group showed the smallest weight loss, with its weight approaching baseline levels by day 7. This indicates that NM-NDs treatment effectively improved the animals' overall physiological state and recovery ability, consistent with improvements in neurological function scores and survival rates. The recovery level of the CA+MINO-TK@NPs group fell between that of the Vehicle and NM-NDs groups.

[0118] Therefore, it can be seen that the neutrophil-like nanodecoy prepared by the present invention can improve the neurological function of rats after cardiac arrest resuscitation, increase the survival rate of rats, and promote the overall health recovery, and the therapeutic effect is significantly better than that of the control group and the naked nanonucleus group.

[0119] (2) Effects of neuroprotective effect on experimental results: 24 hours after the recovery of spontaneous cardiopulmonary circulation in rats, cardiac perfusion was performed under deep anesthesia. The blood was first rinsed with ice-cold PBS, then fixed with 4% paraformaldehyde. The whole brain was removed and fixed for 24 hours, then transferred to 30% sucrose solution for dehydration. Coronal brain sections were prepared, and Nissl staining was performed on Nissl bodies in the neuronal cytoplasm. ImageJ software was used to quantitatively analyze the Nissl body density in the CA1, CA3, and DG regions of the hippocampus to determine the number of viable neurons. Anti-Iba1 antibody staining was used to analyze the morphological changes of neurons. A portion of fresh brain tissue (such as the cortex and hippocampus) was homogenized, centrifuged, and the supernatant was collected to detect the levels of inflammatory factors such as TNF-α and IL-1β. Neurons in the CA1, CA3, and DG regions of the hippocampus are extremely sensitive to ischemia and hypoxia and are the hallmark areas of brain injury after cardiopulmonary resuscitation.

[0120] Figure 5 shows Nissl staining in different regions of the hippocampus. Further quantitative analysis of Nissl bodies was used to directly assess the neuroprotective effect; specific experimental data are shown in Tables 8-10.

[0121] Table 8:

[0122] Table 9:

[0123] Table 10:

[0124] As shown in the table above, CA / CPR resulted in approximately 58% neuronal death in the CA1 region, 51% in the CA3 region, and 65% in the DG region of the hippocampus in the CA + Vehicle group, indicating severe neuronal death following cardiac arrest. In contrast, CA + NM-NDs treatment significantly protected neurons, increasing the survival rate of CA1 neurons from 33.6% to 76.9%, CA3 neurons from 42.9% to 69.4%, and DG neurons from 27.5% to 66.9%, demonstrating significantly better efficacy than the CA + MINO-TK@NPs group. This directly explains the structural basis for the significant improvement in neurological function scores in the NM-NDs group rats.

[0125] (3) Results of the specific targeting effect on the inflammatory area of ​​the brain: Nanoparticles and PLGA were labeled with DiR or NIR-II fluorescent dyes. Rats were anesthetized at different time points after injection (e.g., 2h, 6h, 24h) and whole-body imaging was performed. Finally, the rats were sacrificed and the main organs (heart, liver, spleen, lung, kidney, brain) were removed for in vitro organ imaging. The average fluorescence intensity of each organ was analyzed. The specific data are shown in Table 11 below.

[0126] Table 11:

[0127] As shown in the table, the brain fluorescence signal in the CA+NM-NDs group was significantly higher than that in the CA+MINO-TK@NPs group, which is the most direct evidence demonstrating the superior brain targeting ability of NM-NDs. Since PLGA nanoparticles have difficulty actively penetrating the blood-brain barrier, their weak brain signal may originate from the passive EPR effect in inflamed areas. NM-NDs, on the other hand, benefit from the natural inflammatory chemotactic properties of their surface neutrophil membranes.

[0128] In the liver and spleen, the fluorescence signal of the CA+NM-NDs group was significantly lower than that of the CA+MINO-TK@NPs group, indicating that NM-NDs have a longer in vivo circulation time. The "self-"marking" of neutrophil membranes allows them to effectively evade recognition and clearance by the immune system, primarily Kupffer cells and macrophages in the liver and spleen. The longer circulation time provides more opportunities for them to reach and accumulate at brain targets. Furthermore, there were no significant differences between the two groups in organs such as the heart, lungs, and kidneys, indicating that the nanodecoys did not produce non-specific accumulation in these sites.

[0129] The above results demonstrate that the nanodecoys of this invention exhibit specific enrichment in the inflammatory regions of the brain, with significantly higher signal intensity than the MINO-TK@NPs group (mainly distributed in the liver and spleen), directly proving their active targeting ability. This invention provides crucial pharmacokinetic and biodistribution evidence for the efficacy of NM-NDs in alleviating neuroinflammation, explaining why they exhibit excellent neuroprotective effects in subsequent experiments.

[0130] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing a biomimetic nano-immunomodulatory decoy, characterized in that, Includes the following steps: S1. Neutrophils are obtained, treated with a cell lysis buffer containing a calcium ion chelating agent and a protease inhibitor, and the supernatant is collected. After centrifugation and purification, a neutrophil membrane is obtained. S2. In a first solvent, a polymer polylactic acid-glycolic acid copolymer and a therapeutic drug are mixed to obtain an organic phase. Then, the organic phase is mixed with a polyvinyl alcohol solution, and after emulsification and purification, a PLGA-therapeutic drug nanonucleus is obtained. S3. In a second solvent, the neutrophil membrane is brought into contact with the PLGA-therapeutic drug nanonucleus, and after extrusion, the desired product is obtained.

2. The preparation method according to claim 1, characterized in that, In step S1, the cell membrane of the neutrophils overexpresses CXCR2; preferably, the membrane protein of the neutrophils also includes either CXCR1 or CD11b.

3. The preparation method according to claim 1, characterized in that, The calcium ion chelating agent includes ethylene glycol bis(2-aminoethyl ether)tetraacetic acid; and / or, the cell lysis buffer contains 18-22 mM Tris-HCl, 8-12 mM NaCl and 0.8-1.2 mM MgCl2.

4. The preparation method according to claim 3, characterized in that, In step S1, the concentration of the calcium chelating agent in the cell lysis buffer is 1.5~2.5mM; and / or the concentration of the protease inhibitor is 4~6mM.

5. The preparation method according to claim 1, characterized in that, In step S2, the first solvent is selected from dichloromethane, ethyl acetate, and acetone; and / or, the mass ratio of lactide to glycolide in the polylactic acid-glycolic acid copolymer is 1:0.8~1.2, and the molecular weight is 10000~20000 Da; and / or, the therapeutic drug includes a minocycline prodrug; and / or, the mass concentration of the polyvinyl alcohol solution is 0.005~0.05 g / mL; and / or, the volume ratio of the organic phase to the polyvinyl alcohol solution is 1:20~30.

6. The preparation method according to claim 5, characterized in that, In step S2, the emulsification is performed using ultrasonic emulsification; and / or, the purification includes removing the solvent from the emulsified product, followed by washing to obtain the final product.

7. The preparation method according to any one of claims 1 to 6, characterized in that, In step S3, the second solvent is selected from any one of phosphate buffer solution, Tris-HCl buffer, and HEPES buffer; and / or, in step S2, the mass ratio of the neutrophil membrane to the PLGA-therapeutic drug nanonucleus is 1:2~20; and / or, the extrusion is performed using an Avanti micro liposome extruder; and / or, the extrusion temperature is 0~5℃.

8. The preparation method according to claim 7, characterized in that, In step S3, the product after extrusion is further purified. Preferably, the purification process includes resuspending the extruded product, then centrifuging it with a sucrose gradient, collecting the band at the interface between 25-35% sucrose and 55-65% sucrose, and washing it to obtain the final product.

9. A biomimetic nano-immunomodulatory decoy, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 8.

10. The preparation method according to any one of claims 1 to 8 or the application of the biomimetic nano-immunomodulatory decoy according to claim 9 in the preparation of a medicament for alleviating or treating brain injury; preferably, the brain injury includes brain injury following cardiopulmonary resuscitation.

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