A neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticle, and a preparation method and application thereof

Abstract

This invention discloses a neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticle, its preparation method, and its applications. The dual-targeting magnetic biomimetic nanoparticle uses polyethyleneimine-modified Fe3O4 as a core, with a small nucleic acid drug encapsulated within the core. A phospholipid bilayer formed from natural neutrophil membrane vesicles is coated onto the surface of the nanoparticle, resulting in low-immunogenicity dual-targeting magnetic biomimetic nanoparticles. In vitro and in vivo experiments have demonstrated that the dual-targeting magnetic biomimetic nanoparticles can achieve highly efficient and precise targeting of the myocardial infarction area by leveraging the inflammatory targeting of neutrophil membrane adhesion molecules and the magnetic targeting of Fe3O4, significantly increasing drug concentration at the lesion site. By inhibiting Caspase8 expression, it reduces inflammatory responses and pyroptosis, improving cardiac damage and remodeling after myocardial infarction. This invention provides a novel technical means for the individualized and precise treatment of acute myocardial infarction, possessing significant clinical translational value and broad application prospects.

Description

Technical Field

[0001] This invention relates to a biomimetic nanoparticle formulation with dual targeting of the heart, and more particularly to a neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticle, its preparation method, and its application. This invention belongs to the field of pharmaceutical formulation technology. Background Technology

[0002] Acute myocardial infarction (AMI) is a critical cardiovascular disease caused by acute occlusion of the coronary arteries, leading to persistent myocardial ischemia and hypoxia, and ultimately myocardial necrosis. It is characterized by rapid onset, rapid progression, and high mortality, and has become a major global public health problem. According to the World Health Organization (WHO), approximately 17 million people die from cardiovascular diseases globally each year, with AMI accounting for more than 30% of these deaths. In my country, with the increasing aging population, changing lifestyles, and the high incidence of metabolic diseases (diabetes, hypertension, etc.), the incidence of AMI is rising year by year, and the age of onset is showing a trend towards younger ages, placing a heavy burden on society and families.

[0003] Current treatments for acute myocardial infarction (AMI) primarily involve thrombolysis and reperfusion therapy such as percutaneous coronary intervention (PCI). However, these treatments struggle to address the cardiomyocyte reduction caused by ischemia, leading to postoperative cardiac remodeling. In recent years, nanodelivery systems have shown promise in AMI treatment due to their advantages such as targeting and sustained release. However, existing nanocarriers often suffer from drawbacks such as high immunogenicity, short in vivo circulation time, and low lesion targeting efficiency, limiting their clinical application.

[0004] Studies have shown that neutrophils, as immune cells accounting for approximately 70% of the total white blood cells in the human body, possess a natural targeting ability for areas of acute myocardial infarction (AMI). After an AMI occurs, a large number of inflammatory factors are explosively released in the infarct border area, forming a local inflammatory microenvironment that prompts neutrophils to be recruited to the infarct site. Based on this biological characteristic, combining neutrophil membranes with nanocarriers holds promise for constructing a biomimetic delivery system that combines long-circulation characteristics with inflammatory targeting capabilities.

[0005] Therefore, this invention aims to provide a neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticle that achieves efficient loading and targeted delivery of small nucleic acid drugs through core-shell structure design, inhibits Caspase8 expression to improve cardiac damage after myocardial infarction, and provides a novel technical solution for the treatment of AMI. Summary of the Invention

[0006] The core objective of this invention is to construct a neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticle, its preparation method, and its application, thereby solving the technical problems of poor drug delivery targeting, high immunogenicity, and poor efficacy in the treatment of acute myocardial infarction.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention discloses a dual-targeting magnetic biomimetic nanoparticle coated with neutrophil membrane. The nanoparticle is prepared using Fe3O4 modified with polyethyleneimine (PEI) as the core material. The core is loaded with a small nucleic acid drug. A phospholipid bilayer formed by natural neutrophil membrane vesicles is used as a capsid to coat the surface of the nanoparticle, thereby constructing a low-immunogenic dual-targeting magnetic biomimetic nanoparticle.

[0009] Preferably, the small nucleic acid drug is siRNA, more preferably, the siRNA is Caspase8-siRNA (si-Caspase8), and preferably, the sequence of Caspase8-siRNA is: 5'-GCGCAGACCACAAGAACAA-'3'.

[0010] Preferably, the average particle size of the PEI-modified Fe3O4 nanonuclei is 90-110 nm, and the final particle size after being coated with a neutrophil membrane is 110-120 nm, exhibiting good particle size uniformity.

[0011] Furthermore, this invention also proposes a method for preparing the aforementioned neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles, comprising the following steps:

[0012] ① Preparation of neutrophil membrane vesicles: Neutrophils were isolated and extracted from bone marrow of the same species of animal, and incubated in vitro with lipopolysaccharide (LPS) to induce the expression of surface adhesion molecules; phosphate-buffered saline (PBS) was added to the induced neutrophils for resuspending, homogenized on ice, and the cell membrane was extracted by differential centrifugation; the cell membrane precipitate was resuspended in triple-distilled water, frozen at -80 ℃ and lyophilized under vacuum to obtain neutrophil membranes, which were then dispersed in buffer for later use;

[0013] ② Preparation and modification of Fe3O4 nanocores: Using ferric chloride (FeCl3) as the iron source, sodium acetate (NaOAc) as the precipitant, ethylene glycol (EG) as the solvent, polyvinylpyrrolidone (PVP) as the dispersant, the mixture was magnetically stirred until a uniform orange-red solution was formed. The solution was transferred to a reaction vessel to obtain Fe3O4 nanoparticles with uniform particle size. The Fe3O4 nanoparticles were dispersed in deionized water and sonicated to ensure full dispersion. The solution was then slowly added dropwise to a PEI solution and magnetically stirred at room temperature. After centrifugation and washing to remove unreacted PEI, the nanoparticles were vacuum dried to obtain PEI-modified Fe3O4 nanoparticles. The PEI-modified Fe3O4 nanoparticles were dispersed in ultrapure water and mixed with a Caspase 8-siRNA solution. The mixture was then rotated to obtain a PEI-Fe3O4-siRNA complex.

[0014] ③ Assembly of biomimetic nanoparticles: Neutrophil membrane vesicles prepared in step ① were mixed with the PEI-Fe3O4-siRNA complex prepared in step ②. After sonication, the mixture was passed through a polycarbonate membrane using a liposome extruder to obtain neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles, named NM@PEI-Fe3O4-siRNA.

[0015] Preferably, the differential centrifugation refers to centrifugation at 1000 g for 5 min, centrifugation at 10000 g for 20 min, and centrifugation at 100000 g for 1 h.

[0016] Preferably, PEI-modified Fe3O4 nanoparticles are dispersed in ultrapure water and mixed with Caspase8-siRNA solution at a mass ratio of 5:1.

[0017] Preferably, the neutrophil membrane vesicles prepared in step ① are mixed with the PEI-Fe3O4-siRNA complex prepared in step ② at a mass ratio of 1:1, and after ultrasonic treatment at 100 Hz for 5 s, they are passed through a 100 nm polycarbonate membrane five times using a liposome extruder to obtain neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles.

[0018] Preferably, the animal is a mouse or a rat; the bone marrow is derived from the femur and tibia of a mouse or the femur and tibia of a rat.

[0019] Furthermore, the present invention also proposes the use of the neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles in the preparation of drug delivery systems loaded with small nucleic acid drugs. Preferably, the use of the neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles in the preparation of drug delivery systems loaded with Caspase8-siRNA and targeting the immune inflammatory response region of ischemic myocardial tissue in acute myocardial infarction.

[0020] Preferably, the drug delivery system is administered via intravenous injection. After administration, the drug can target the ischemic myocardial tissue in the acute myocardial infarction area through the chemotaxis of neutrophils in the immune inflammatory response area and the regulation of Fe3O4 core by an external magnetic field, thereby achieving a synergistic effect of magnetic and biological targeting.

[0021] This invention uses polyethyleneimine (PEI)-modified iron(III) oxide (Fe3O4) as a raw material to prepare nanoparticles as the core, which efficiently encapsulate small nucleic acid drugs. Natural neutrophil membranes are used as capsids to coat the nanoparticle surface, constructing a biomimetic nanocarrier system with high biocompatibility and significant dual-targeting characteristics. This nanocarrier system possesses three technological advantages: first, it achieves long-term in vivo circulation by utilizing the camouflage of the neutrophil membrane, reducing phagocytosis and clearance by mononuclear macrophages; second, it utilizes the natural recruitment characteristics of neutrophils to inflamed sites to achieve biological targeting of the myocardial infarction area; and third, through the magnetic responsiveness of the Fe3O4 core, it achieves physical targeting of the heart under the regulation of an external magnetic field. This dual targeting synergistically enhances the drug accumulation efficiency at the lesion site, thereby improving cardiac damage caused by acute myocardial infarction.

[0022] This invention uses Caspase8-siRNA as a model drug, and its mechanism of action is as follows: Caspase8 is a key protease activated by Caspase1 and Caspase3 / 7, and can mediate apoptosis. Excessive inflammation after acute myocardial infarction (AMI) exacerbates myocardial tissue damage. Inhibiting Caspase8 expression can significantly reduce the secretion of pro-inflammatory factors and alleviate local inflammation. Furthermore, Caspase8 can trigger pyroptosis by cleaving Gasdermin D protein, leading to cell membrane rupture and release of contents, further amplifying the inflammatory cascade. Therefore, inhibiting Caspase8 expression can also effectively inhibit pyroptosis, regulate inflammation-related signaling pathways, improve cardiac remodeling after myocardial infarction, and thus significantly reduce cardiac damage induced by acute myocardial infarction.

[0023] In this invention, Fe3O4 is selected as the nanocore, which has the following advantages: Fe3O4 itself possesses good biocompatibility and biodegradability, and exhibits excellent magnetic response characteristics, enabling targeted localization under an applied magnetic field; after PEI modification, the Fe3O4 nanoparticles have a positively charged surface, which can form a stable complex with negatively charged Caspase8-siRNA through electrostatic interaction, improving the loading efficiency and in vivo stability of small nucleic acid drugs. The diameter of the Fe3O4 nanocore is controlled at around 100 nm to ensure suitable in vivo circulation characteristics and tissue penetration ability.

[0024] The key to preparing neutrophil membrane vesicles in this invention lies in: isolating and extracting pure neutrophils from the bone marrow of the same species (mouse or rat), upregulating the expression of chemokine receptors such as CXCR2 on the membrane surface by in vitro stimulation with LPS, and extracting the cell membrane and preparing vesicles using differential centrifugation. This preparation process can effectively preserve the functional proteins on the neutrophil membrane surface, ensuring that the biomimetic nanoparticles possess inflammation-targeting capabilities similar to those of natural neutrophils.

[0025] In this invention, the expression levels of membrane proteins such as β2 integrins (e.g., LFA-1), L-selectin, chemokine receptor (CXCR2), and β1 integrins on the surface of neutrophil membranes stimulated by LPS are significantly upregulated. These adhesion molecules can specifically bind to ligands on the surface of endothelial cells in the inflammatory microenvironment of myocardial infarction area, further enhancing the targeted enrichment ability of biomimetic nanoparticles.

[0026] In this invention, the nanonucleus and neutrophil membrane vesicles are stably coated by physical compression to form core-shell structured nanoscale biomimetic particles. The PEI-modified Fe3O4 nanonucleus has an average particle size of 90-110 nm, and the final particle size after coating the cell membrane is 110-120 nm. Compared with cells at the micrometer scale, this nanoparticle size range has a larger specific surface area, which can more efficiently exert the targeting effect and drug delivery function of the membrane structure.

[0027] This invention systematically optimizes the preparation scheme of the biomimetic nanoparticles, characterizes their structure and physicochemical properties through infrared spectroscopy, zeta potential measurement, and transmission electron microscopy, and verifies their application effect through in vitro and in vivo targeting experiments and pharmacodynamic evaluation, confirming that the delivery system has excellent targeting and therapeutic effects.

[0028] The biomimetic nanoparticles of this invention are administered via intravenous injection. After administration, the Fe3O4 core can be targeted to the heart by an external magnetic field, achieving a synergistic effect of magnetic and biological targeting, significantly increasing the drug concentration in the myocardial infarction area and prolonging the drug's duration of action.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] 1. The upregulated expression of chemokine receptors on the surface of neutrophil membranes can specifically recognize the inflammatory microenvironment in the myocardial infarction area, achieving biological targeting; the Fe3O4 core can achieve physical targeting of the heart under the action of an external magnetic field. The dual targeting synergistic effect significantly improves the drug enrichment efficiency at the lesion site, and the targeting is significantly improved.

[0031] 2. The PEI-modified Fe3O4 nanocore has good positive charge properties, which can efficiently load Caspase8-siRNA through electrostatic interaction, with a drug loading rate of over 25%, and can effectively protect Caspase8-siRNA from degradation by nucleases in vivo, thus improving drug stability.

[0032] 3. The coating of neutrophil membranes can achieve biomimetic camouflage, effectively evading phagocytosis and clearance by mononuclear macrophages in the body, and prolonging the in vivo circulation time of nanoparticles.

[0033] 4. Neutrophil membrane vesicles do not contain genetic material or organelles and have extremely low immunogenicity; the core material Fe3O4 is an FDA-approved metal oxide that can be used in the biomedical field. After being coated with a cell membrane, its biocompatibility is further improved. No obvious toxic reactions were observed in in vivo experiments, and it has good prospects for clinical translation. Attached Figure Description

[0034] Figure 1 Infrared spectra of Fe3O4 nanoparticles and PEI-modified Fe3O4 nanoparticles;

[0035] Figure 2 Transmission electron microscopy images of NM@PEI-Fe3O4-siRNA, a dual-targeting magnetic biomimetic nanoparticle, coated on neutrophil membranes, along with statistical results on particle size and zeta potential;

[0036] In this image, A shows a transmission electron microscope image of the neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles NM@PEI-Fe3O4-siRNA; B shows the particle size distribution of the neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles NM@PEI-Fe3O4-siRNA; and C shows the Zeta potential distribution of the neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles NM@PEI-Fe3O4-siRNA.

[0037] Figure 3 Results of Coomassie brilliant blue staining and Western blot assays;

[0038] In this table, A represents the results of the Coomassie Brilliant Blue staining method; B represents the results of the Western blot method.

[0039] Figure 4 Fluorescence graph showing the uptake efficiency of Caspase8-siRNA in HeLa cells;

[0040] Figure 5 The fluorescence detection image shows the uptake of NM@PEI-Fe3O4-siRNA by primary cardiomyocytes on neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles.

[0041] Figure 6 The fluorescence intensity of various organs in mice 24 hours after intravenous injection of NM@PEI-Fe3O4-siRNA coated with dual-targeting magnetic biomimetic nanoparticles on neutrophil membranes.

[0042] Figure 7 Protein expression of Caspase 8 in the infarct border region of mice 24 hours after intravenous injection of NM@PEI-Fe3O4-siRNA-coated dual-targeting magnetic biomimetic nanoparticles;

[0043] Figure 8 Echocardiography and H&E staining results of myocardial tissue in mice 3 days after intravenous injection of NM@PEI-Fe3O4-siRNA-coated with dual-targeting magnetic biomimetic nanoparticles.

[0044] In this image, A represents echocardiography; B represents the H&E staining results. Detailed Implementation

[0045] The present invention will be further described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. All equivalent transformations made based on the content of the present invention shall fall within the scope of protection of the present invention.

[0046] Example 1: Preparation and characterization of neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles NM@PEI-Fe3O4-siRNA

[0047] method:

[0048] 1. Preparation of Fe3O4 nanoparticles: Weigh 1.35 g FeCl3·6H2O and 3.6 g NaOAc, dissolve them in 40 mL ethylene glycol, add 1.0 g PVP, and stir magnetically for 30 min until completely dissolved to form a uniform orange-red solution; transfer the solution to a 50 mL reaction vessel and place it in a 200 ℃ oven for 10 h; after the reaction is complete, cool to room temperature, open the reaction vessel, and wash the reaction product four times with alternating centrifugation of anhydrous ethanol and deionized water (centrifuge at 8000 rpm for 5 min) to remove unreacted raw materials and impurities; disperse the washed product in ultrapure water and dry it in a 60 ℃ vacuum drying oven for 12 h to obtain black Fe3O4 microsphere powder.

[0049] 2. Preparation of PEI-modified Fe3O4 nanoparticles: Weigh 0.5 g of PEI and dissolve it in 50 mL of deionized water. Stir magnetically until completely dissolved to prepare a 1% (w / v) PEI solution. Take 0.1 g of Fe3O4 microsphere powder and disperse it in 20 mL of deionized water. Sonicate the solution for 30 min to ensure thorough dispersion and a uniform suspension. Slowly add the Fe3O4 suspension dropwise to the PEI solution and stir magnetically at room temperature for 20 h to ensure that PEI and Fe3O4 are fully bonded. After the reaction is complete, centrifuge at 8000 rpm for 5 min, discard the supernatant, and wash the product four times with deionized water to remove unbound free PEI. Disperse the product in deionized water and vacuum dry to obtain PEI-modified Fe3O4 microsphere powder (PEI-Fe3O4).

[0050] 3. Preparation of drug-loaded nanocores: PEI-Fe3O4 microsphere powder was dispersed in ultrapure water to prepare a suspension with a concentration of 1 mg / mL; 1 mL of this suspension (containing 1 mg PEI-Fe3O4) was mixed with 0.2 mL of a 0.1 mg / mL Caspase8-siRNA solution (containing 0.02 mg Caspase8-siRNA, sequence: 5'-GCGCAGACCACAAGAACAA-'3, SEQ ID NO.1) (mass ratio 5:1), and placed in a rotary mixer to mix at 40 r / min at room temperature for 4 h to form a PEI-Fe3O4-siRNA complex (drug-loaded nanocores).

[0051] 4. Preparation and characterization of neutrophil membranes

[0052] After euthanizing 6-8 week old C57BL / 6 mice, femurs and tibias were harvested under aseptic conditions, and bone marrow cell suspensions were collected. The bone marrow cell suspensions were transferred to 50 mL centrifuge tubes, erythrocyte lysis buffer was added, and the mixture was gently pipetted and incubated at room temperature for 10 min. The mixture was then centrifuged at 300 g for 10 min. The erythrocyte lysis step was repeated once to obtain pure neutrophils.

[0053] To induce upregulation of surface-associated chemokine receptor expression in neutrophils, a solution containing 2 μg / mL was added to purified neutrophils. -1 LPS cells were incubated in RPMI-1640 medium at 37 ℃ in a 5% CO2 incubator for 12 h. Cells were collected, resuspended in 15 mL of PBS buffer, and transferred to a glass homogenizer for homogenization on ice. The homogenate was then centrifuged at 1000 g for 5 min, 10000 g for 20 min, and ultracentrifuged at 100000 g for 1 h, respectively. The supernatant was discarded, and the precipitate obtained was the neutrophil membrane. The cell membrane precipitate was resuspended in ultrapure water, frozen at -80 ℃ for 24 h, and then freeze-dried under vacuum to obtain neutrophil membrane powder, which was stored at -80 ℃ for later use.

[0054] 5. Assembly of biomimetic nanoparticles: Accurately weigh 10 mg of neutrophil membrane powder, resuspend it in 10 mL of ultrapure water, take 5 mL of cell membrane vesicle solution, mix it with 5 mL of PEI-Fe3O4-siRNA complex solution (concentration 1 mg / mL), sonicate at 100 Hz for 5 s, and then extrude it through a 100 nm polycarbonate membrane 5 times to obtain neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles, named NM@PEI-Fe3O4-siRNA, and store at 4 ℃ for later use.

[0055] result:

[0056] 1. Physicochemical characterization of dual-targeted magnetic biomimetic nanoparticles NM@PEI-Fe3O4-siRNA: The particle size, polydispersity index (PDI), and zeta potential of the nanoparticles were determined using a Malvern particle size / zeta potential analyzer. Each sample was measured in triplicate, and the average value was taken. The morphology and core-shell structure of the nanoparticles were observed using transmission electron microscopy (TEM). The samples were observed after negative staining with phosphotungstic acid. The loading rate of Caspase8-siRNA was determined using an ultra-micro UV spectrophotometer.

[0057] Figure 1 The infrared spectra of Fe3O4 nanoparticles and PEI-modified Fe3O4 nanoparticles show that PEI was successfully modified on the Fe3O4 surface. Figure 2 A is a transmission electron microscope image of the nano-drug delivery system. TEM observation results show that both nanoparticles are regular and round spheres. The cell membrane coating layer (about 20 nm thick) of the inner core of the biomimetic nanoparticle NM@PEI-Fe3O4-siRNA can be clearly observed, and the core-shell structure is well defined. Figure 2 B and 2C represent the particle size and Zeta potential statistics of the nanoparticles. The results show that the average particle size of the PEI-Fe3O4-siRNA nanocore is 105.3 nm, and the Zeta potential is +0.529 mV. After coating the neutrophil membrane, the average particle size of the biomimetic nanoparticle NM@PEI-Fe3O4-siRNA is 118.3 nm, and the Zeta potential becomes -31.07 mV, confirming that the cell membrane is successfully coated on the surface of the nanocore.

[0058] 2. Characterization of membrane protein integrity using Coomassie brilliant blue staining: Neutrophils, extracted cell membranes, and subsequently prepared biomimetic nanoparticles NM@PEI-Fe3O4-siRNA were dissolved in sodium dodecyl sulfate (SDS) lysis buffer and subjected to SDS-PAGE gel electrophoresis. After electrophoresis, the cells were stained with Coomassie brilliant blue staining solution for 2 h, and destained with destaining solution until the protein bands were clear. The distribution of protein bands was observed and recorded. Figure 3 A shows the results of the Coomassie Brilliant Blue staining method, which indicates that the protein bands of the neutrophil membrane and the biomimetic nanoparticle NM@PEI-Fe3O4-siRNA are basically consistent, and the membrane protein integrity is good.

[0059] 3. Western blot was used to detect adhesion protein expression: Total protein was extracted from neutrophils, neutrophil membranes, and biomimetic nanoparticles NM@PEI-Fe3O4-siRNA before and after LPS stimulation. After measuring the protein concentration, SDS-PAGE electrophoresis was performed, and the protein was transferred to NC membranes. After blocking with 5% skim milk for 2 h, primary antibodies (CD62L, CD11b, CXCR2) were added and incubated overnight at 4 ℃. After incubation with fluorescent secondary antibody at room temperature for 1 h, the fluorescence band intensity was detected, and quantitative analysis was performed using Image Studio software. Figure 3 B represents the results of Western blot analysis, showing that proteins such as L-selectin (CD62L), CXCR2, LFA-1 (CD11b), and β1 integrin on the neutrophil membrane and the surface of the biomimetic nanoparticle NM@PEI-Fe3O4-siRNA were well preserved.

[0060] Coomassie Brilliant Blue staining results showed that the protein bands on the neutrophil membrane were basically consistent with the protein bands of NM@PEI-Fe3O4-siRNA, indicating that membrane proteins were successfully transferred to the surface of nanoparticles during cell membrane encapsulation. Furthermore, proteins such as CXCR2, CD62L, and CD11b were significantly expressed, providing a molecular basis for the inflammatory targeting ability of the biomimetic nanoparticles NM@PEI-Fe3O4-siRNA.

[0061] Example 2: Study on the uptake characteristics of neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles NM@PEI-Fe3O4-siRNA at the cellular level

[0062] 1. Qualitative analysis of cellular uptake

[0063] Using HeLa cells as a model, cells were spaced at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 24-well plates lined with cell spreaders and incubated at 37 ℃ in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was replaced with fresh medium. Caspase8-siRNA-Cy3 (free siRNA) and NM@PEI-Fe3O4-siRNA-Cy3 (bionic nanoparticle-loaded Caspase8-siRNA) solutions were added, respectively, with a nanoparticle concentration of 400 μg / mL. -1 Each group was set up with 3 replicates; after incubation for 8 hours, the culture medium was aspirated, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature for 30 min, stained with Dapi staining solution at room temperature for 10 min, washed with PBS, and observed under a fluorescence microscope to record the distribution of Cy3 fluorescence (siRNA) in the cells.

[0064] Figure 4The image shows the fluorescence spectrum of Caspase8-siRNA uptake efficiency in HeLa cells. The results indicate that only weak red fluorescence was observed in the free Caspase8-siRNA-Cy3 group, and the fluorescence was dispersed. In contrast, the red fluorescence intensity was significantly enhanced in the NM@PEI-Fe3O4-siRNA group, with fluorescence mainly distributed in the cytoplasm. This suggests that the NM@PEI-Fe3O4-siRNA effectively mediates siRNA entry into cells, significantly improving cellular uptake efficiency. This demonstrates that the intracellular delivery efficiency of siRNA mediated by the NM@PEI-Fe3O4-siRNA is significantly higher than that of free siRNA.

[0065] 2. Uptake analysis in a primary cardiomyocyte model

[0066] Primary mouse cardiomyocytes were extracted and cultured at a density of 5 × 10⁶ cells per well. 4 The nanoparticles were seeded at a density of 400 μg / mL in culture dishes and incubated for 24 hours until they adhered. -1 Observe the effect of incubation time on cell uptake.

[0067] Figure 5 shows the fluorescence detection of the uptake of biomimetic nanoparticle NM@PEI-Fe3O4-siRNA by primary cardiomyocytes. The experimental results show that primary cardiomyocytes have significant uptake of NM@PEI-Fe3O4-siRNA. The cellular uptake level reaches its maximum at 8 h and still maintains a high fluorescence level at 24 h, indicating that the siRNA has good stability after entering the cells.

[0068] Example 3: Targeting study of neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles NM@PEI-Fe3O4-siRNA in a mouse model of acute myocardial infarction.

[0069] Six- to eight-week-old C57BL / 6 mice were selected, and an acute myocardial infarction model was established by ligation of the left anterior descending coronary artery. The mice were divided into three groups, and Caspase8-siRNA-Cy5.5, PEI-Fe3O4-siRNA-Cy5.5, and NM@PEI-Fe3O4-siRNA-Cy5.5 were injected into the tail vein immediately, respectively, at a dose of 1 mg / kg (calculated as Caspase8-siRNA-Cy5.5).

[0070] Figure 6The fluorescence intensity of various organs in mice 24 hours after intravenous injection of NM@PEI-Fe3O4-siRNA, a dual-targeting magnetic biomimetic nanoparticle coated with neutrophil membranes, was measured. The results showed that the fluorescence intensity in the heart region of the NM@PEI-Fe3O4-siRNA group was significantly higher than that of the group injected with free Caspase8-siRNA-Cy5.5; the strongest fluorescence intensity was observed in the NM@PEI-Fe3O4-siRNA group (2.8 × 10⁻⁶). 9 Photons / second, slightly higher than the uncoated PEI-Fe3O4-siRNA (2.0 × 10⁻⁶). 9 The concentration of photons per second (Caspase 8-siRNA-Cy5.5) was significantly higher than that of the free Caspase 8-siRNA-Cy5.5 group (1.1 × 10⁻⁶). 9 (photons / second), confirming that the synergy between magnetic and biological targeting enhances the targeting effect on the heart and can significantly improve the efficiency of drug accumulation in the cardiac region.

[0071] Example 3: Study on the effect of neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles NM@PEI-Fe3O4-siRNA on improving cardiac injury after myocardial infarction.

[0072] Six- to eight-week-old C57BL / 6 mice were selected, and an acute myocardial infarction model was established by ligation of the left anterior descending coronary artery. The sham group (Sham group) only had sutures but no ligation; the model group (MI group) was randomly divided into three groups of six mice each after successful modeling: MI group, MI+siRNA group, and MI+NM@PEI-Fe3O4-siRNA group.

[0073] Mice in the MI+siRNA group and the MI+NM@PEI-Fe3O4-siRNA group were immediately injected via tail vein with Caspase8-siRNA and NM@PEI-Fe3O4-siRNA after modeling, at a dose of 5 mg / kg (calculated as Caspase8-siRNA). Twenty-four hours after intravenous injection, Western blot was used to detect the expression levels of Caspase8 and Cleaved-Caspase8 proteins in myocardial tissue. Three days after intravenous injection, echocardiography was used to detect cardiac systolic and diastolic function, and H&E staining was used to detect cardiac pathological damage and inflammatory cell infiltration.

[0074] Figure 7Western blot was used to detect the expression level of Caspase 8 protein in the ischemic myocardium of mice 24 hours after intravenous injection of neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles NM@PEI-Fe3O4-siRNA. The results showed that, compared with the MI group, the expression levels of Caspase 8 protein in myocardial tissue were significantly downregulated in both the MI+siRNA group and the MI+NM@PEI-Fe3O4-siRNA group, with the downregulation being more significant in the MI+NM@PEI-Fe3O4-siRNA group. Figure 8 A shows the echocardiogram of mice 3 days after intravenous injection. The results showed that, compared with the MI group and the MI+siRNA group, injection of NM@PEI-Fe3O4-siRNA significantly improved cardiac function in mice with myocardial infarction. Figure 8 B shows the H&E staining results of mouse myocardial tissue 3 days after intravenous injection, indicating that inflammatory cell infiltration occurred in the myocardial tissue of the MI group. Compared with the MI group and the MI+siRNA group, injection of NM@PEI-Fe3O4-siRNA can significantly reduce inflammatory cell infiltration and myocardial structural damage after myocardial infarction.

[0075] The above results demonstrate that the neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles NM@PEI-Fe3O4-siRNA described in this invention can efficiently target the acute myocardial infarction area, reduce inflammatory response and pyroptosis by inhibiting Caspase8 expression, improve myocardial tissue pathological damage, and have a good cardioprotective effect against acute myocardial infarction.

Claims

1. A dual-targeting magnetic biomimetic nanoparticle coated with a neutrophil membrane, characterized in that, The dual-targeting magnetic biomimetic nanoparticles are core-shell structured nanoparticles. They are constructed by using nanoparticles prepared from Fe3O4 modified with polyethyleneimine as the core, which encapsulates small nucleic acid drugs. A phospholipid bilayer formed by natural neutrophil membrane vesicles is used as a capsid to coat the surface of the nanoparticles, thus constructing low immunogenicity dual-targeting magnetic biomimetic nanoparticles.

2. The neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles according to claim 1, characterized in that, The small nucleic acid drug is siRNA, preferably Caspase8-siRNA, and more preferably, the sequence of Caspase8-siRNA is: 5'-GCGCAGACCACAAGAACAA-'3'.

3. The neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles according to claim 1, characterized in that, The average particle size of the polyethyleneimine-modified Fe3O4 nanonuclei is 90-110 nm, and the final particle size after being coated with a neutrophil membrane is 110-120 nm, showing good particle size uniformity.

4. The method for preparing neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles according to any one of claims 1-3, characterized in that, Includes the following steps: ① Preparation of neutrophil membrane vesicles: Neutrophils were isolated and extracted from bone marrow of the same species of animal, and incubated in vitro with lipopolysaccharide to induce the expression of surface adhesion molecules; the induced neutrophils were resuspended in phosphate buffer, homogenized on ice, and the cell membrane was extracted by differential centrifugation; the cell membrane precipitate was resuspended in triple-distilled water, frozen at -80 ℃ and lyophilized under vacuum to obtain neutrophil membranes, which were then dispersed in buffer for later use; ② Preparation and modification of Fe3O4 nanocores: Using ferric chloride as the iron source, sodium acetate as the precipitant, ethylene glycol as the solvent, polyvinylpyrrolidone as the dispersant was added and the mixture was magnetically stirred until a uniform orange-red solution was formed. The solution was transferred to a reaction vessel to obtain Fe3O4 nanoparticles with uniform particle size. The Fe3O4 nanoparticles were dispersed in deionized water and sonicated to ensure full dispersion. The solution was then slowly added dropwise to a polyethyleneimine solution and magnetically stirred at room temperature. After centrifugation and washing to remove unreacted polyethyleneimine, the Fe3O4 nanoparticles were vacuum dried to obtain polyethyleneimine-modified Fe3O4 nanoparticles. The polyethyleneimine-modified Fe3O4 nanoparticles were dispersed in ultrapure water and mixed with a Caspase 8-siRNA solution. The mixture was then rotated to obtain a PEI-Fe3O4-siRNA complex. ③ Assembly of biomimetic nanoparticles: Neutrophil membrane vesicles prepared in step ① were mixed with the PEI-Fe3O4-siRNA complex prepared in step ②. After sonication, the mixture was passed through a polycarbonate membrane using a liposome extruder to obtain neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles, named NM@PEI-Fe3O4-siRNA.

5. The preparation method according to claim 4, characterized in that, The differential centrifugation refers to centrifugation at 1000 g for 5 min, 10000 g for 20 min, and 100000 g for 1 h.

6. The preparation method according to claim 4, characterized in that, Polyethyleneimine-modified Fe3O4 nanoparticles were dispersed in ultrapure water and mixed with Caspase8-siRNA solution at a mass ratio of 5:

1.

7. The preparation method according to claim 4, characterized in that, Neutrophil membrane vesicles prepared in step ① were mixed with the PEI-Fe3O4-siRNA complex prepared in step ② at a mass ratio of 1:

1. After ultrasonic treatment at 100 Hz for 5 s, the mixture was passed through a liposome extruder through a 100 nm polycarbonate membrane 5 times to obtain neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles.

8. The preparation method according to claim 4, characterized in that, The animal is a mouse or a rat; the bone marrow is derived from the femur and tibia of a mouse or the femur and tibia of a rat.

9. The use of the neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles according to any one of claims 1-3 in a drug delivery system for preparing small nucleic acid drugs, preferably, the use of the neutrophil membrane-coated dual-targeting magnetic biomimetic nanoparticles in a drug delivery system for preparing a drug delivery system for preparing a drug loaded with Caspase8-siRNA and targeting the immune inflammatory response region of ischemic myocardial tissue in acute myocardial infarction.

10. The use according to claim 1, characterized in that, The drug delivery system is administered via intravenous injection. After administration, it can target the ischemic myocardial tissue in the acute myocardial infarction area through the chemotaxis of neutrophils in the immune inflammatory response area and the regulation of Fe3O4 core by an external magnetic field, thereby achieving a synergistic effect of magnetic and biological targeting.

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