Preparation method and application of a neutrophil cascade targeting drug delivery system

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to a preparation method and application of a neutrophil cascade-targeted drug delivery system; the preparation method includes the following steps: S1. Coupling the neutrophil-targeted NEBP peptide (sequence: CGEAIPMSIPPEVK), the MMP9-responsive peptide (sequence: PVGLIG), and the mitochondrion-targeted polypeptide SS-31 (sequence: H2N-DArg-Dmt-Lys-Phe-NH2) to obtain the polypeptide NEMSS (sequence: DArg-Dmt-Lys-Phe-PVGLIG-GEAIPMSIPPEVK) that has MMP9 responsiveness and simultaneously targets neutrophils and mitochondria; S2. Using DSPE-PEG2K-Maleimide as a raw material, modifying the polypeptide NEMSS on the surface of DSPE-PEG2K-Maleimide to form the polymer DSPE-PEG-NEMSS; S3. Preparing the polymer, small molecule drugs, and lipid molecules into lipid nanoparticles CsA-M-Neu-NPs. The present invention provides a CsA-M-Neu-NPs lipid nanodrug with a simple structure, easy to prepare, capable of penetrating the blood-brain barrier, and capable of cascade-targeted binding to neutrophils and the mitochondria of damaged site cells, providing new ideas and new drugs for the treatment of inflammation-related diseases such as stroke.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of a neutrophil cascade-targeted drug delivery system. Background Art

[0002] Cyclosporin A (CsA) is an immunosuppressant that can bind to the cyclophilin family. By binding to the CypD protein in mitochondria, it inhibits the opening of the mitochondrial permeability transition pore (mPTP). Some studies have shown that the opening and overexpression of mPTP are important upstream signaling pathways in the mitochondrial dysfunction cascade during ischemic injury, which can cause a decrease in the production of adenosine triphosphate (ATP) and an excessive increase in ROS, ultimately leading to cell dysfunction and even death. Therefore, mPTP is considered a potential target for drug action in ischemic diseases. CsA has a definite effect of inhibiting the opening of mPTP, can reduce mitochondrial damage, change mitochondrial ATP energy metabolism, and maintain mitochondrial homeostasis, which may be an effective strategy to reduce organ ischemia-reperfusion injury.

[0003] Drug treatment for cerebral ischemia-reperfusion injury (CIRI) faces multiple consecutive obstacles (such as the blood-brain barrier (BBB), lesion site recognition, etc.), making drug delivery challenging. Studies on the brain microenvironment of CIRI have demonstrated that secondary inflammatory responses occur after CIRI, including the recruitment of leukocytes to the stroke core and penumbra regions. Neutrophils (PMNs) are the most abundant leukocytes in humans (50% to 70%), and they function in the innate immune response to infection or tissue damage through phagocytosis, degranulation, etc. Therefore, PMNs can be used to load drug molecules, and relying on their own chemotaxis and BBB-crossing ability, they can autonomously guide the targeted delivery of drugs for use in the CIRI process with inflammatory responses. In addition, during the CIRI process, the content of matrix metalloproteinase MMP-9 at the ischemic injury site increases significantly, which is a potential trigger for drug release.

[0004] Based on this, we propose a preparation method and application of a neutrophil cascade-targeted drug delivery system. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention proposes a preparation method and application of a neutrophil cascade-targeted drug delivery system.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] A preparation method of a neutrophil cascade-targeted drug delivery system, the preparation method comprising the following steps:

[0008] S1. Couple the neutrophil-targeting NEBP peptide with the sequence CGEAIPMSIPPEVK, the MMP9-responsive peptide with the sequence PVGLIG, and the mitochondrion-targeting polypeptide SS-31 with the sequence H2N-DArg-Dmt-Lys-Phe-NH2 to obtain the polypeptide NEMSS with MMP9 responsiveness and simultaneous targeting of neutrophils and mitochondria, with the sequence DArg-Dmt-Lys-Phe-PVGLIG-GEAIPMSIPPEVK;

[0009] S2. Use DSPE-PEG2K-Maleimide as a raw material to modify the NEMSS polypeptide on the surface of DSPE-PEG2K-Maleimide;

[0010] S3. Dissolve the polymer, cyclosporine A, Cy5 dye, cholesterol, and lipids in an organic solvent, spin-dry it under vacuum to form a thin film, and then add phosphate buffer PBS and sonicate for 10 minutes to obtain a neutrophil cascade-targeted drug delivery system.

[0011] Preferably, the molar ratio of the NEBP peptide, MMP9-responsive peptide, and mitochondrion-targeting polypeptide SS-31 is 1:1:1.

[0012] Preferably, the molar ratio of DSPE-PEG2K-Maleimide and NEMSS polypeptide is 1:1.

[0013] Preferably, the molar ratio of the polymer, CsA drug, and Cy5 dye is 5:1:0.2

[0014] Preferably, the organic solvent is dichloromethane.

[0015] A neutrophil cascade-targeted drug delivery system is prepared by the method for preparing a neutrophil cascade-targeted drug delivery system described above.

[0016] Preferably, the CsA-M-Neu-NPs nanodrug presents a spherical structure, and the particle size of the CsA-M-Neu-NPs nanodrug is 90 - 200 nm.

[0017] Use of the neutrophil cascade-targeted drug delivery system described above in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

[0018] Use of the above neutrophil cascade-targeted drug delivery system in the preparation of a drug carrier targeting neutrophils.

[0019] Advantages of the present invention:

[0020] 1. Through animal experiments, immunofluorescence, magnetic resonance imaging, transmission electron microscopy, and cell experimental techniques, the present invention successfully constructs a targeting polypeptide NEMSS of neutrophils and mitochondria with MMP-9 responsiveness, and loads the mPTP inhibitor CsA to self-assemble into the CsA-M-Neu-NPs nanomedicine. The ability of targeting neutrophils and mitochondria is verified through cell experiments, providing new ideas for the treatment of neutrophil-related tumors, inflammation, immunity and other diseases; meanwhile, it is verified that the CsA-M-Neu-NPs nanomedicine has a positive effect on the treatment of cerebral ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is the preparation flow chart of the neutrophil cascade targeting drug delivery system in the present invention;

[0023] Figure 2 It is the transmission electron micrograph of the neutrophil cascade targeting drug delivery system in the present invention;

[0024] Figure 3 It is the result diagram of the ability of the neutrophil cascade targeting drug delivery system in the present invention to target neutrophils and mitochondria in PC12 cells: wherein (A) the ability of the neutrophil cascade targeting drug delivery system to target neutrophils; (B) the ability of the neutrophil cascade targeting drug delivery system to target mitochondria;

[0025] Figure 4 It is the ability of the neutrophil cascade targeting drug delivery system in the present invention to penetrate the blood-brain barrier and target the damaged site;

[0026] Figure 5 It is that the neutrophil cascade targeting drug delivery system in the present invention can reduce brain tissue infarction and neurological deficits caused by MCAO: wherein (A) TTC staining of brain tissue; (B) MRI imaging of brain tissue; (C-D) quantification of infarction area; (E) EB perfusion of brain tissue; (F) body weight of MCAO / R mice; (G) Longa score; (H) number of revolutions on the rotarod; (I) survival curve;

[0027] Figure 6 It is the electron micrograph of the effect of the neutrophil cascade targeting drug delivery system in the present invention on the morphological change of brain tissue mitochondria: wherein N: represents the nucleus; Mito: represents the mitochondria; red arrow: represents the mitophagosome;

[0028] Figure 7 To study the effect of the neutrophil cascade-targeted drug delivery system of the present invention on the opening and closing of mitochondrial permeability transition pores in cells of the hypoxia-reoxygenation model;

[0029] Figure 8 The figure shows the results of the effect of the neutrophil cascade-targeted drug delivery system of the present invention on the mitochondrial membrane potential of cells in the hypoxia-reoxygenation model: where JC-1 monomers represent JC-1 as monomers, producing green fluorescence; JC-1 aggregates represent JC-1 as aggregates;

[0030] Figure 9 To study the effect of the neutrophil cascade-targeted drug delivery system of the present invention on the levels of TNF-α, IL-6, and IFN-γ in the supernatant of cells in the hypoxia-reoxygenation model. Detailed implementation methods

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0032] The sources of the raw materials and the model numbers of the instruments required for the following experiments are given.

[0033] Experimental materials

[0034] 1. Mice, cell lines, and liposomes

[0035] C57 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., the PC12 cell line was purchased from Wuhan Procell Life Science & Technology Co., Ltd., and DSPE-PEG2K-Maleimide was purchased from MCE.

[0036] 2. The main instrument equipment is shown in Table 1:

[0037] Table 1

[0038]

[0039]

[0040] 3. The main reagents and consumables are shown in Table 2:

[0041] Table 2

[0042]

[0043] Example 1: Preparation of MMP-9 responsive nanomedicine targeting neutrophils and mitochondria and encapsulating CsA: a neutrophil cascade-targeted drug delivery system.

[0044] S1. The NEBP peptide (sequence: CGEAIPMSIPPEVK) targeting neutrophils, the MMP9 response peptide (sequence: PVGLIG) and the mitochondria-targeted peptide SS-31 (sequence: H2N-DArg-Dmt-Lys-Phe-NH2) were coupled at a molar ratio of 1:1:1 to obtain the MMP9-responsive peptide NEMSS (sequence: DArg-Dmt-Lys-Phe-PVGLIG-GEAIPMSIPPEVK) targeting both neutrophils and mitochondria.

[0045] S2, using DSPE-PEG2K-Maleimide as a raw material, and forming a polymer with the peptide NEMSS having MMP9 response and targeting neutrophils and mitochondria prepared in step S1 on the surface of DSPE-PEG2K-Maleimide at a molar ratio of 1:1;

[0046] S3. The polymer, cyclosporin A (CsA), Cy5 dye, cholesterol and lipid are mixed in an organic solvent, vacuum dried into a thin film, and then phosphate buffered saline (PBS) is added and ultrasonicated for 10 minutes to obtain a neutrophil cascade-targeted drug delivery system.

[0047] The appearance of the neutrophil cascade-targeted drug delivery system was observed using a transmission electron microscope (TEM). An appropriate amount of CsA-M-Neu-NPs nanoparticles was dissolved in water to form a solution, then dripped onto a copper mesh, and after drying, the nanoparticle morphology was observed using a transmission electron microscope. The results were as follows: Figure 2 shown.

[0048] according to Figure 2 It can be seen that CsA-M-Neu-NPs present a spherical structure with a particle size between 95 and 105 nm.

[0049] Example 2: Neutrophil cascade-targeted drug delivery system targeting neutrophils and mitochondria

[0050] Neutrophils and PC12 cells were extracted from mouse bone marrow and seeded into 12-well plates respectively. After culturing in an incubator for 4 h, 1 μM CsA-M-Neu-NPs nano-drug was added to the 12-well plates containing neutrophils and PC12 cells respectively for co-culture for 2 h. The culture medium was discarded and the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 15 min, incubated with 0.5% Triton X-100 at room temperature for 20 min, incubated with the primary antibody overnight at 4 °C, washed with PBS, and then incubated with the Ly6G secondary antibody / mitochondrial fluorescent probe at room temperature for 1 h. The cell nuclei were counterstained with DAPI and incubated in the dark for 5 min. Finally, an anti-fluorescence quencher was added, and the samples were observed and photographed with a laser confocal microscope. The experimental results are as Figure 3 shown.

[0051] As Figure 3 can be seen: Figure 3 A indicates that the CsA-M-Neu-NPs nano-drug can target the surface of neutrophils; Figure 3 B indicates that the CsA-M-Neu-NPs nano-drug can target mitochondria.

[0052] Example 3: Evaluation of the targeting effect of a neutrophil cascade-targeted drug delivery system in the brain

[0053] In this example, a mouse model of ischemia-reperfusion (MCAO / R) was prepared using the suture method. The mice were fixed in the supine position on the operating table and continuously anesthetized with low-concentration isoflurane. The body temperature was maintained using a thermostatic blanket during the operation. A midline incision was made in the neck of the mice to expose the right external carotid artery, and an L1800 suture was inserted into the right internal carotid artery to occlude the right middle cerebral artery. After 90 min, the suture was removed for reperfusion for 6 h. The Zea Longa score was used to verify the success of the model preparation. The ability of the CsA-M-Neu-NPs nano-drug to penetrate the blood-brain barrier and target the ischemic-reperfusion injury site was recorded in this experiment. The specific operation steps are as follows:

[0054] In vivo NIR-II imaging: Mice in the sham operation group (Sham group) were injected with CsA-M-Neu-NPs via the tail vein, and mice in the ischemia-reperfusion group (MCAO / R) were also injected with CsA-M-Neu-NPs. At 1, 4, 8, and 24 h after administration, the mice were exposed to the NIR-II Kaer imaging system (KIS NIR-II, Kaer Laboratories, France) (808 nm, 50 mW cm-2) for 50 ms (n = 3 per group). Time-dependent NIR-II fluorescence images of the brain tissue were obtained using in vivo imaging. The excitation was induced with an 808 nm laser and filtered with an 808 nm long-pass filter. The fluorescence emission was collected using an 1100 nm band-pass filter, and the exposure time was 50 ms. Then the brain tissue was taken for in vivo fluorescence imaging at 24 h. The experimental results are as Figure 4 shown.

[0055] It can be seen from Figure 4 that: Compared with the sham operation group (Sham group), the NIR-II fluorescence signal intensity in the right brain of CsA-M-Neu-NPs increased significantly at 1 hour and subsequent time after CIRI, and reached the peak at 24 hours. This indicates that CsA-M-Neu-NPs cross the damaged blood-brain barrier and accumulate in the brain parenchyma, and the uptake efficiency of CsA-M-Neu-NPs in the damaged brain tissue is higher.

[0056] Example 4: Changes in body weight and neurological function evaluation of mice after administration of neutrophil cascade-targeted drug delivery system

[0057] This example records the changes in body weight and neurological function scores of mice after administration of neutrophil cascade-targeted drug delivery system. Through MRI imaging, TTC staining, EB staining, etc., it is evaluated whether the neutrophil cascade-targeted drug delivery system can better reduce brain tissue infarction and the recovery effect on the blood-brain barrier.

[0058] 4.1 TTC staining.

[0059] On the 3rd day, the mice (n = 3 per group) were sacrificed by euthanasia. The brains were cut into 6 slices, 2 mm thick, and incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC). The slices were incubated in the TTC solution at 37 °C for 20 min. The infarct area was measured using Image J analysis software. The unstained brain slices were defined as the infarct area. The infarct area was indirectly measured by subtracting the total area of the non-infarcted hemisphere of the infarcted hemisphere. The formula for calculating the percentage of the semi-brain infarct area is: infarct volume / total volume of the non-infarcted hemisphere × 100%. The experimental results are as Figure 5 A and Figure 5 C shown.

[0060] It can be seen from Figure 5 A and Figure 5 C that: Compared with the sham operation group (Sham group), the infarct area in the right brain of the ischemia-reperfusion group (MCAO / R group) and the CsA-M-Neu-NPs group increased significantly (***P < 0.001); compared with the ischemia-reperfusion group (MCAO / R group), CsA-M-Neu-NPs treatment could significantly reduce the infarct area in the brains of mice (##P < 0.01).

[0061] 4.2 Magnetic resonance (MRI) imaging

[0062] Magnetic resonance imaging: MRI was performed on a 3.0T MRI scanner (Siemens, Trio, Germany) and a 7.0T CG NOVILA system (Shanghai Chenguang Medical Technology Co., Ltd., China) to generate T2-weighted images. Under isoflurane anesthesia, mice were continuously imaged on the 3rd day after ischemia-reperfusion (5 mice per group). The infarct area of the brain was evaluated from the hyperintense region of the T2-weighted images using Image J software. The experimental results are as Figure 5 shown in Figures B and 5D.

[0063] As Figure 5 shown in Figures B and 5D: MRI was used to evaluate the effect of CsA-M-Neu-NPs on the infarct area of the brain after CIRI ( Figure 5 Figure B). We evaluated the infarct degree through the T2-weighted imaging sequence. Compared with the ischemia-reperfusion group (MCAO / R group), the infarct area in the CsA-M-Neu-NPs group was significantly reduced (##P < 0.01).

[0064] 4.3 Blood-brain barrier disruption test (EB staining)

[0065] The breakdown of the blood-brain barrier was evaluated by EB (MACKLIN, Shanghai, China) leakage technique. A 4% dye solution (2.5 mL kg-1) was injected via the tail vein on the 3rd day after CIRI. After 6 hours of circulation, heparinized saline was perfused through the heart, and then the brain was removed (n = 6 per group). A photograph of the brain was taken before dividing the brain into left and right hemispheres. Each hemisphere was weighed and homogenized in PBS. The homogenate was centrifuged at 14000 × g for 20 min at 4°C using a microcentrifuge, and then an equal volume of 50% trichloroacetic acid was added to the collected supernatant. The experimental results are as Figure 5 shown in Figure E.

[0066] As Figure 5 shown in Figure E: To determine whether drug treatment could reverse the decrease in blood-brain barrier integrity caused by CIRI, we evaluated the permeability of the blood-brain barrier. Compared with the ischemia-reperfusion group (MCAO / R group), the EB permeability in the CsA-M-Neu-NPs group was significantly reduced on the 3rd day after CIRI, indicating that the integrity of the blood-brain barrier could be partially restored after CsA-M-Neu-NPs treatment.

[0067] Example 5: Transmission electron microscopy observation of mitochondrial morphology

[0068] After the mice were subjected to MCAO / R and drug administration according to the grouping, the brain tissues of the ischemic injury sites of the mice were taken. The brain tissues were cut into small pieces of 1 mm3 on a pre-cooled wax plate, placed in 2.5% glutaraldehyde, fixed with 1% osmium acid, dehydrated with gradient ethanol, soaked in epoxy resin 812-acetone 2:1, embedded in epoxy resin 812, sectioned, stained with saturated uranyl acetate aqueous solution, washed with water and dried, stained with lead citrate solution, and observed under an electron microscope. The results are as Figure 6 .

[0069] It can be Figure 6 seen that in the sham operation group (Sham group), the mitochondrial membrane structure of the brain tissues of each mouse was intact, the mitochondrial cristae were clear, and the morphology was normal. In the ischemia-reperfusion group (MCAO / R group) and the CsA-M-Neu-NPs group, the mitochondria were swollen, severely vacuolated, the mitochondrial cristae disappeared, and mitochondrial autophagosomes appeared. After treatment with CsA-M-Neu-NPs, the degree of mitochondrial morphological damage was reduced.

[0070] Example 6: Detection of the opening level of mPTP

[0071] After PC12 cells were subjected to oxygen-glucose deprivation / reoxygenation (OGD / R) and drug administration according to the grouping, they were washed with calcium-free Tyrode's solution, and 2 μmol·L-1 calcein-AM and 100 nmol·L-1 mitochondrial fluorescent probe (mito tracker) were added, and incubated at room temperature in the dark for 30 min. After washing, 2 mmol·L-1 cobalt chloride (CoCl2) was added and incubated for 15 min. After washing, fresh calcium-free Tyrode's solution was used, and fluorescence microscopy was used for photographing and analysis. The experimental results are as Figure 7 .

[0072] It can be Figure 7 seen that compared with the control group (Control group), the green fluorescent spots in the oxygen-glucose deprivation / reoxygenation group (OGD / R group) and the CsA-M-Neu-NPs group were significantly reduced; compared with the oxygen-glucose deprivation / reoxygenation group (OGD / R group), the green fluorescent spots were restored to varying degrees after the action of CsA-M-Neu-NPs, indicating that the opening degree of mPTP was significantly reduced.

[0073] Example 7: Detection of mitochondrial membrane potential level by JC-1

[0074] JC-1 is an ideal fluorescent probe widely used to detect the mitochondrial membrane potential (ΔΨm). It can detect the mitochondrial membrane potential of cells, tissues, or purified mitochondria. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers (J-aggregates), which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix, and at this time JC-1 is a monomer, which can produce green fluorescence. In this way, it is very convenient to detect the change of mitochondrial membrane potential through the change of fluorescence color. After PC12 cells were treated with OGD / R and drugs according to the grouping, the culture medium in each well was aspirated, the cells were washed twice with PBS, 1 mL of culture medium was added, 1 mL of JC-1 staining working solution was added, and the mixture was thoroughly mixed and incubated at 37 °C for 30 min. During the incubation, JC-1 staining buffer was prepared and placed in an ice bath. After the incubation at 37 °C was completed, the cells were washed twice with JC-1 staining buffer (ice bath) according to the operation instructions of the mitochondrial membrane potential detection kit, and observed under a fluorescence microscope. The experimental results are as Figure 8 .

[0075] As Figure 8 can be seen: Under the fluorescence microscope, the red fluorescence of the mitochondrial membrane potential in the control group (Control group) was relatively obvious; the green fluorescence in the oxygen-glucose deprivation / reoxygenation group (OGD / R group) and the CsA-M-Neu-NPs group increased significantly; after treatment with CsA-M-Neu-NPs, the red fluorescence recovered to varying degrees, indicating that the level of mitochondrial membrane potential had recovered to some extent.

[0076] Example 9: Detection of inflammation-related factors

[0077] According to the kit instructions, the contents of TNF-α, IL-6, and IFN-γ in the cell supernatant were determined by enzyme-linked immunosorbent assay. Dilution, sample addition, washing, addition of enzyme-labeled antibody, color development, colorimetry and other operations were carried out step by step. Detection was performed using an enzyme-labeled instrument, zeroing with the blank control well, measuring the absorbance (A) of each well, and then calculating the corresponding contents of TNF-α, IL-6, and IFN-γ according to the A of the sample on the regression equation. The experimental results are as Figure 9 .

[0078] As Figure 9 can be seen: Compared with the control group (Control group), the contents of IL-6 and IFN-γ in the oxygen-glucose deprivation / reoxygenation group (OGD / R group) and the CsA-M-Neu-NPs group increased significantly; compared with the oxygen-glucose deprivation / reoxygenation group (OGD / R group), CsA-M-Neu-NPs could significantly reduce the contents of TNF-α and IFN-γ, indicating that it could reduce the contents of inflammatory factors in the oxygen-glucose deprivation / reoxygenation model.

[0079] In the description of this specification, the descriptions referring to the terms "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0080] The above has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed.

Claims

1. A preparation method of a neutrophil cascade-targeted drug delivery system, characterized in that, The preparation method comprises the following steps: S1. Couple the neutrophil-targeting NEBP peptide with the sequence of CGEAIPMSIPPEVK, the MMP9-responsive peptide with the sequence of PVGLIG, and the mitochondrion-targeting polypeptide SS-31 with the sequence of H2N-DArg-Dmt-Lys-Phe-NH2 to obtain the polypeptide NEMSS with MMP9 responsiveness and simultaneous targeting of neutrophils and mitochondria, with the sequence of DArg-Dmt-Lys-Phe-PVGLIG-GEAIPMSIPPEVK; S2. Use DSPE-PEG2K-Maleimide as the raw material to modify the NEMSS polypeptide on the surface of DSPE-PEG2K-Maleimide; S3. Mix the polymer, cyclosporin A, Cy5 dye, cholesterol, and lipid in an organic solvent, spin-dry it in vacuo to form a thin film, and then add phosphate buffer PBS and sonicate for 10 minutes to obtain a neutrophil cascade-targeted drug delivery system.

2. The preparation method of a neutrophil cascade-targeted drug delivery system according to claim 1, wherein The molar ratio of the NEBP peptide, MMP9-responsive peptide, and mitochondrion-targeting polypeptide SS-31 is 1:1:

1.

3. The preparation method of a neutrophil cascade-targeted drug delivery system according to claim 1, characterized in that, The molar ratio of DSPE-PEG2K-Maleimide and NEMSS polypeptide is 1:

1.

4. The preparation method of a neutrophil cascade-targeted drug delivery system according to claim 1, characterized in that, The molar ratio of the polymer, CsA drug, and Cy5 dye is 5:1:0.

2.

5. The preparation method of a neutrophil cascade-targeted drug delivery system according to claim 1, characterized in that, The organic solvent is dichloromethane.

6. A neutrophil cascade-targeted drug delivery system is prepared by the preparation method according to any one of claims 1 to 5.

7. A neutrophil cascade-targeted drug delivery system according to claim 6, wherein The CsA-M-Neu-NPs nano-drug presents a spherical-like structure, and the particle size of the CsA-M-Neu-NPs lipid nano-drug is 90 - 200 nm.

8. Use of a neutrophil cascade-targeted drug delivery system according to claim 6 or 7 in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

9. Use of a neutrophil cascade-targeted drug delivery system according to claim 6 or 7 in the preparation of a drug for treating inflammation-related diseases.

10. Use of a neutrophil cascade-targeted drug delivery system according to claim 6 or 7 in the preparation of a drug carrier targeting neutrophils.

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