Biomimetic nanoparticles targeting activated neutrophils and methods of making and using the same

Abstract

This disclosure belongs to the field of biopharmaceutical technology, and specifically relates to a biomimetic nanoparticle that targets and activates neutrophils, its preparation method, and its application. This disclosure provides a biomimetic nanoparticle that targets and activates neutrophils. The biomimetic nanoparticle consists of a nanocore and an endothelial cell membrane covering the surface of the nanocore. The endothelial cell membrane is an endothelial cell membrane expressing ICAM-1. The biomimetic nanoparticle provided by this disclosure has advantages such as specific targeting of neutrophils, good biocompatibility, long circulation time, high efficiency of inflammatory accumulation, and integrated diagnosis and treatment. It can precisely regulate excessive neutrophil activation, significantly improving the therapeutic effect and safety for diseases such as acute infections and chronic wounds.

Description

Technical Field

[0001] This disclosure belongs to the field of biopharmaceutical technology, and specifically relates to a biomimetic nanoparticle that targets and activates neutrophils, its preparation method, and its application. Background Technology

[0002] Neutrophils are the core effector cells of the body's innate immune system, playing a crucial role in the fight against infection and the repair of tissue damage. Under physiological conditions, neutrophils participate in immune surveillance in an inactive state. When stimulated by infection, trauma, or inflammation, neutrophils are activated and recruited to the site of injury, with significantly high expression of the integrin CD11b on their surface, thereby initiating activation programs such as adhesion, migration, and respiratory burst. However, in diseases such as sepsis, acute inflammation, chronic skin ulcers, and delayed wound healing, neutrophils often exhibit overactivation and abnormally sustained activation, releasing large amounts of reactive oxygen species (ROS), inflammatory cytokines, and neutrophil extracellular traps (NETs), triggering a severe amplified inflammatory response and secondary tissue damage, significantly aggravating the condition and delaying repair.

[0003] Therefore, precisely targeting and regulating overactivated neutrophils has become a key strategy for treating the aforementioned diseases. However, traditional anti-inflammatory drugs and immunomodulatory drugs have drawbacks such as poor targeting, high systemic exposure doses, and easy involvement of normal immune cells, making it difficult to achieve selective intervention on neutrophils.

[0004] In recent years, nanodelivery systems have provided a feasible approach to improve drug targeting. However, conventional synthetic nanocarriers suffer from problems such as insufficient cyclic stability, susceptibility to clearance by the immune system, and difficulty in accurately identifying activated neutrophils. Cell membrane biomimetic nanoparticles, due to their possession of surface proteins of natural biological membranes, immunocompatibility, and long-term in vivo circulation characteristics, have become a research hotspot in the field of inflammation targeting.

[0005] Currently reported biomimetic nanoparticles are mostly coated with neutrophil membranes, macrophage membranes, and erythrocyte membranes, relying primarily on inflammatory chemotaxis and adhesion properties to achieve enrichment at inflammatory sites. However, such biomimetic systems still have significant limitations: insufficient targeting selectivity, inability to effectively distinguish between inactive and activated neutrophils, lack of specific recognition ability for activated cells with high CD11b expression, unstable targeting efficiency, and limited ability to precisely regulate the inflammatory microenvironment.

[0006] Studies have confirmed that CD11b, which is highly expressed on the surface of activated neutrophils, can specifically bind to intercellular adhesion molecule-1 (ICAM-1) on the surface of endothelial cells. This interaction is the core molecular basis for neutrophil recruitment in endothelial adhesion and inflammation. Although the interaction between ICAM-1 and CD11b has been revealed, a technical approach to construct biomimetic nanoparticles using ICAM-1-expressing endothelial cell membranes to reverse-target activated neutrophils with high CD11b expression has not yet been reported.

[0007] In summary, existing biomimetic nanodelivery systems still struggle to achieve specific recognition, precise enrichment, and controllable regulation of activated neutrophils, failing to meet the precision diagnosis and treatment needs of diseases related to neutrophil overactivation. Therefore, developing a biomimetic nanoparticle with well-defined targeting, high selectivity, good biocompatibility, and both imaging and therapeutic functions is of significant clinical value and technological necessity. Summary of the Invention

[0008] The purpose of this disclosure is to provide a biomimetic nanoparticle targeting activated neutrophils, its preparation method, and its application. Utilizing the specific receptor-ligand binding of ICAM-1 on the endothelial cell membrane to CD11b, biomimetic nanoparticles are constructed using ICAM-1-expressing endothelial cell membranes to target activated neutrophils with high CD11b expression. Specifically, a nanocore is coated with an ICAM-1-expressing cell membrane derived from endothelial cells, retaining functional membrane proteins on its surface, thus constructing biomimetic nanoparticles capable of specifically binding to activated neutrophils with high CD11b expression. Furthermore, these biomimetic nanoparticles exhibit good biocompatibility and immune escape capabilities, helping to prolong in vivo circulation time and improve accumulation efficiency at inflammatory sites.

[0009] In this disclosure, unactivated neutrophils refer to neutrophils that, after isolation, have not undergone inflammatory stimulation and exhibit basal low expression of CD11b; activated neutrophils refer to neutrophils that, after PMA-induced stimulation, show significantly upregulated expression of CD11b molecules on their surface and possess adhesion and inflammatory activation phenotypes. This study uses CD11b expression level as the criterion for distinguishing between unactivated and activated neutrophils.

[0010] The objective of this disclosure is achieved through the following technical solution: In a first aspect of this disclosure, a biomimetic nanoparticle is provided that targets and activates neutrophils. The biomimetic nanoparticle consists of a nanocore and an endothelial cell membrane covering the surface of the nanocore. The endothelial cell membrane is an endothelial cell membrane expressing ICAM-1.

[0011] In some specific embodiments of this disclosure, the endothelial cell membrane is capable of specific binding to CD11b receptors on the surface of activated neutrophils.

[0012] In some specific embodiments of this disclosure, the nanocore is a nanocarrier capable of loading diagnostic and therapeutic reagents, the diagnostic and therapeutic reagents including one or more of fluorescent probes, anti-inflammatory drugs, and antioxidants; further, the particle size of the nanocore is 50-200 nm.

[0013] In some specific embodiments of this disclosure, the mass ratio of the epithelial cell membrane to the nanocore is 1:(1~5).

[0014] In some specific embodiments of this disclosure, the particle size of the biomimetic nanoparticles is 80-250 nm; further, the particle size of the biomimetic nanoparticles is 100-230 nm.

[0015] In a second aspect, this disclosure provides a method for preparing biomimetic nanoparticles as described above, comprising the following steps: (1) Culture endothelial cells, collect cells and lyse them, and obtain endothelial cell membranes expressing ICAM-1 by centrifugation and washing. (2) Prepare nanocores by mixing the nanocores with the endothelial cell membrane obtained in step (1), and then incubating and purifying by centrifugation to obtain the biomimetic nanoparticles that target and activate neutrophils.

[0016] In some embodiments of this disclosure, the lysis involves placing endothelial cells in a hypotonic lysis buffer solution under ice bath conditions for 30 min, followed by cell disruption using a probe sonicator at 150 W for 3 min.

[0017] In some embodiments of this disclosure, the mixing is to mix the endothelial cell membrane and the nanocore in 1×PBS at a mass ratio of 1:(1~5); the incubation is to treat the aforementioned mixture using a probe ultrasonic disruptor at 150 W for 3 min.

[0018] In a third aspect of this disclosure, the disclosure provides the application of the aforementioned biomimetic nanoparticles for preparing diagnostic and therapeutic agents that target activated neutrophils, wherein the activated neutrophils exhibit high expression of CD11b on their surface and basally low expression of CD11b on the surface of unactivated neutrophils.

[0019] In some specific embodiments of this disclosure, the diagnostic reagent is used to treat diseases related to neutrophil hyperactivation, including one or more of acute infection, chronic non-healing wounds, and sepsis.

[0020] The technical solution provided in this disclosure has the following technical contributions: (1) This disclosure features precise targeting and high selectivity: relying on the specific ligand receptor binding of ICAM-1 on the endothelial cell membrane to CD11b on the surface of activated neutrophils, it can accurately identify and bind to activated neutrophils with high CD11b expression, while having a low binding rate to unactivated neutrophils, significantly reducing off-target effects and non-specific effects, thus achieving selective intervention on pathogenic activated neutrophils. Compared with nanoparticles without ligand receptor binding mechanisms, the biomimetic nanoparticles prepared in this disclosure that target activated neutrophils have a higher enrichment quantity and density on the surface of neutrophils.

[0021] (2) The present invention has excellent biocompatibility and in vivo delivery efficiency: the endothelial cell membrane biomimetic coating structure is adopted, which enables the nanoparticles to have good biocompatibility, low immunogenicity and immune escape ability, effectively prolonging the in vivo circulation time and improving the enrichment efficiency at the inflammatory site, thus solving the problems of easy clearance and unstable targeting of traditional nanocarriers.

[0022] (3) This invention integrates diagnosis and treatment and has high application scalability: it can be equipped with fluorescent probes to realize the visual diagnosis of activated neutrophils and inflammatory lesions, while loading active ingredients to precisely regulate the overactivation of neutrophils, and has both imaging tracking and anti-inflammatory treatment functions; the system can be adapted to a variety of drugs and is suitable for a variety of diseases related to overactivation of neutrophils, such as acute infection and chronic wounds. Attached Figure Description

[0023] Figure 1 These are the results of the preparation and characterization of biomimetic nanoparticles. Figure 1 A shows scanning electron microscope and transmission electron microscope images of the nanocore alone, indicating that the nanocore was successfully obtained. Figure 1 B represents the characteristics of the extracted endothelial cell membrane under transmission electron microscopy and biomimetic nanoparticle scanning electron microscopy, which shows that the endothelial cell membrane was successfully extracted and encapsulated on the nanocore. Figure 1 C represents the relative content of ICAM-1 molecules in the endothelial cell membrane and biomimetic nanoparticles as shown by immunoblotting analysis. Compared with the simple nanoparticles without endothelial cell membrane coating, ICAM-1 is expressed in the endothelial cells themselves, the extracted endothelial cell membrane, and the biomimetic nanoparticles, indicating that the molecule is completely preserved during the preparation process. Figure 1 D, Figure 1 E represents the particle size and surface potential of the biomimetic nanoparticles. Before and after encapsulation, the diameter of the biomimetic nanoparticles increases from (184.40±4.38) nm to (203.00±3.12) nm; the surface potential increases from (7.93±0.25) mV to (17.00±1.25) mV.

[0024] Figure 2 It is an immunofluorescence detection and imaging method for the relative content of CD11b molecules after neutrophil activation. Figure 2A shows the expression of CD11b molecules (green fluorescence) in neutrophils before and after activation. The CD11b fluorescence is brighter after activation, indicating that more is expressed. Figure 2 B is neutrophil nucleus (blue fluorescence) staining. The fluorescence intensity is similar in the activated and unactivated states, which can be used as a reference to reflect that CD11b does indeed increase relatively after activation. Figure 2 C is a fluorescence fusion image of CD11b and cell nuclear staining; Figure 2 D is the average fluorescence intensity of the CD11b molecule (green fluorescence). After activation, the ratio of fluorescence intensity increases from (1.46±0.25) to (2.89±0.17).

[0025] Figure 3 It is a test for the ability to target neutrophils after endothelial cell membrane staining, used for localization analysis (for diagnosis). Figure 3 A is a potential validation of its use in imaging of diseases related to neutrophil activation. Red fluorescence is DiD staining of the endothelial cell membrane, while blue fluorescence is localization staining of the activated neutrophil nucleus. The fused image illustrates that the biomimetic nanoparticles can be adsorbed and endocytosed by neutrophils. Figure 3 B shows that the biomimetic nanoparticles expressing ICAM-1 can target and aggregate on the surface of neutrophils. The red arrows in the targeted group point to the adhered biomimetic nanoparticles, which are more numerous than those without ICAM-1 expression (non-targeted group control). Figure 3 C is the calculated surface adhesion ratio. According to electron microscopy image analysis, the adhesion ratio of the targeted group is (60.55±3.56)%, while that of the non-targeted group is (13.91±3.50)%.

[0026] Figure 4 This describes the performance of the obtained biomimetic nanoparticles in controlling acute inflammation. Figure 4 In acute inflammatory responses, this product can scavenge reactive oxygen species, with scavenging efficiency increasing with increasing concentration, reaching over 90%. Figure 4 B and 4C are acute inflammatory factors that significantly decreased after treatment with biomimetic nanoparticles. The TNF-α content produced by activated neutrophils was (155.60±14.01) pg / mL, which decreased to (96.78±9.82) pg / mL after treatment with biomimetic nanoparticles, close to the negative control (81.60±13.56) pg / mL; while the IL-6 content after activation was (107.90±17.80) pg / mL, which decreased to (73.72±15.41) pg / mL after treatment with biomimetic nanoparticles, closer to the negative control group (52.82±11.59) pg / mL.

[0027] Figure 5 It uses biomimetic nanoparticles to alleviate the inflammatory state of chronic wounds and promote the healing of skin and soft tissue wounds. Figure 5A is the normal control group, with no obvious inflammatory cell infiltration, tissue edema, or congestion; Figure 5 B represents a chronic wound condition, where obvious inflammatory response, tissue congestion, and disordered growth can be observed. Figure 5 C represents the reduction of inflammatory infiltration, decreased congestion, and more orderly tissue after treatment with biomimetic nanoparticles. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it. It should be noted that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, all reagents used in the embodiments are commercially available analytical grade, and all experimental methods used are conventional methods.

[0029] Terminology Explanation Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting.

[0030] In this disclosure, the terms “comprising” or “including” are open-ended expressions used to refer to the phrase “including but not limited to” and are used interchangeably with it, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0031] The present disclosure is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present disclosure in any way.

[0032] 1. Preparation of nanocores One gram of Zintl-phase CaSi2 precursor was added to 100 mL of concentrated hydrochloric acid pre-cooled to -25°C, and the reaction was carried out for 7 days under magnetic stirring at 500 rpm. The resulting light green product was collected by centrifugation at 10000 g for 10 minutes and washed repeatedly with anhydrous ethanol. To obtain smaller nanocores, the above product was dispersed in 100 mL of ethanol and subjected to probe sonication under ice bath conditions for 24 hours. Subsequently, the suspension was centrifuged at 15000 g for 10 minutes to collect the smaller nanocores. Experimental results are shown below. Figure 1 A. The obtained nanocore has a uniform morphology and good dispersibility.

[0033] 2. Endothelial cell membranes expressing ICAM-1 Human umbilical vein endothelial cells (HUVECs) were used as the cell membrane-derived vascular endothelial cell line, and passaged using standard cell culture methods. The vascular endothelial cell line was incubated with DiD (80 μg / mL) for 1 hour to induce fluorescence imaging. Subsequently, the vascular endothelial cell line was placed in hypotonic lysis buffer (20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl2, pH 7.4, containing a mixture of protease inhibitors) and incubated on ice for 30 minutes. The cells were then disrupted using a probe sonicator (150 W, 3 minutes) under ice bath conditions. The homogenate was centrifuged at 8500 g for 10 minutes to remove organelles and large fragments. The supernatant was further centrifuged at 140000 g for 30 minutes to precipitate the cell membrane. The resulting membrane precipitate was washed once with washing buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4). Figure 1 Results B show the extracted endothelial cell membrane. Western blot analysis revealed ICAM-1 expression on the endothelial cell membrane (Figure 1C).

[0034] 3. Preparation of biomimetic nanoparticles At 4°C, the endothelial cell membrane and nanocore prepared above were mixed in 1×PBS at a mass ratio of 1:2. The mixture was then sonicated in a water bath sonicator for 3 minutes under the same conditions as before. Subsequently, the obtained biomimetic nanoparticles were purified by ultracentrifugation (100,000 g, 1 hour, 4°C) to obtain the final product, namely biomimetic nanoparticles with the function of targeted activation of neutrophils.

[0035] The morphology of the product was characterized using scanning electron microscopy and transmission electron microscopy, and the results are as follows: Figure 1 As shown in Figure B, biomimetic nanoparticles targeting and activating neutrophils were successfully encapsulated in the endothelial cell membrane. The relative content of ICAM-1 molecules was detected using Western blotting analysis, and the results are as follows: Figure 1 As shown in Figure C, ICAM-1 exists stably on the surface of biomimetic nanoparticles, providing a structural basis for targeted activation of neutrophils. The particle size and surface potential of the product were measured using a particle size analyzer, and the results are as follows: Figure 1 As shown in D-1E.

[0036] 4. Activated neutrophils obtained through PMA stimulation Neutrophils were isolated from blood samples using density gradient centrifugation. Whole blood was centrifuged at 600g for 25 minutes at room temperature. The neutrophil layer was collected, and erythrocytes were lysed and washed twice with PBS (300g, 5 minutes). The purified neutrophils were resuspended in phenol red-free RPMI-1640 complete medium and cultured at 37°C and 5% CO2. The isolated neutrophils were activated by PMA stimulation (100 nM, 4 hours), and the relative content of CD11b molecules was detected using immunofluorescence assays. Figure 2 As shown, CD11b expression in neutrophils activated by PMA was significantly upregulated, and the fluorescence signal was significantly enhanced, indicating successful preparation of activated neutrophils. The average fluorescence intensity was compared with that of unactivated neutrophils. The experimental results showed that CD11b expression on the surface of unactivated neutrophils was basally low; while CD11b expression on the surface of activated neutrophils after induction was significantly upregulated and showed high expression, with a significant difference in expression levels between the two groups.

[0037] 5. Biomimetic nanoparticles target and activate neutrophils The isolated neutrophils were further co-cultured with the aforementioned biomimetic nanoparticles for 4 hours, and then stained with DiD dye for localization. The imaging ability of the nanoparticles targeting neutrophils was tested using a fluorescence microscope. Figure 3 As shown, DiD-labeled biomimetic nanoparticles can achieve fluorescence imaging of activated neutrophils (Figure 3A); and the biomimetic nanoparticles expressing ICAM-1 showed significantly better targeting and aggregation on the surface of activated neutrophils than those without ICAM-1 expression (using endothelial cell membranes without ICAM-1 expression coated with nanocores as non-targeting nanoparticles, which were obtained through lentiviral transfection, the specific steps of which included: 1) endothelial cell lines expressing ICAM-1 were arranged at 1×10 5Endothelial cells were seeded in 12-well plates at a density of 1 μg / mL and cultured. Cell sensitivity to puromycin was tested at concentrations of 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 10 μg / mL. After 3 days of continuous culture, all endothelial cells at the target concentration died. 2) Endothelial cell lines were seeded again in 12-well plates and cultured overnight until 50% confluence was achieved. Polybrene (10 μg / mL) was added to the culture medium, and the cells were cultured at 37°C for 1 hour. 3) With a multiplicity of infection (MOI) of 100, ICAM-1 knockout lentivirus (L27831, Beyotime) was added to the 12-well plates. After 48 hours of culture, puromycin (3 μg / mL) was used for selection. After 3 days, ICAM-1 knockout endothelial cell lines were obtained. 4) Using cells containing 3... Endothelial cells without ICAM-1 expression were amplified in a medium containing μg / mL puromycin, and the membranes of these cells were extracted to prepare non-targeting nanoparticles (Figure 3B). This confirmed that the targeting of the nanoparticles depended on the specific binding of ICAM-1 to CD11b, reflecting their diagnostic value in neutrophil-related diseases.

[0038] Subsequently, the samples were fixed at 4°C for 2 hours using 2.5% glutaraldehyde (dissolved in 0.1 M PBS, pH 7.4). After fixation, the samples underwent gradient ethanol dehydration, critical point drying, and platinum sputtering. Finally, the adhesion between activated neutrophils and biomimetic nanoparticles was observed using a scanning electron microscope. The adhesion ratio was calculated based on the percentage of surface area adhered.

[0039] 6. In vitro experiments This study investigated the role of the biomimetic nanoparticles in regulating neutrophils to control acute infection inflammation and alleviate chronic wound non-healing in vitro and in vivo. For controlling acute infection, neutrophils were stimulated with PMA (100 nM, 4 hours), and the levels of ROS, TNF-α, and IL-6 were measured. The prepared targeted biomimetic nanoparticles (100 μg / mL) were added to the culture medium and incubated overnight. Changes in acute infection markers before and after incubation were measured using a commercially available detection kit. For treating chronic wounds, an animal model of wound healing was established by removing a 1 cm section of tissue from the back of a mouse. 2 The skin is damaged, and then Staphylococcus aureus bacterial solution (10g) is evenly applied to the wound. 6 (CFU / mL, 100μL) After 1 week, a chronic wound was formed, followed by local injection of biomimetic nanoparticles for treatment. The healing of the chronic wound was assessed half a month later.

[0040] 6.1 Controlling acute inflammation The above-mentioned biomimetic nanoparticles were added to activated neutrophil culture medium and further reacted with reactive oxygen species detection reagents and inflammatory factor reagents to test their antioxidant and anti-inflammatory effects and evaluate their role in controlling acute inflammatory markers.

[0041] like Figure 4 As shown in Figure A, biomimetic nanoparticles exhibit highly efficient ROS scavenging activity, and the scavenging efficiency is positively correlated with drug concentration; after treatment, the levels of acute inflammatory factors such as TNF-α and IL-6 significantly decreased. Figure 4 (B, 4C) indicates that it can effectively inhibit acute inflammatory response.

[0042] 6.2 Alleviating chronic wound non-healing In an animal model of chronic wounds, the drug was administered via perilesional injection, and its therapeutic effect in promoting chronic wound healing was evaluated after half a month.

[0043] like Figure 5 As shown, compared with normal tissue ( Figure 5 Compared to A), chronic wounds show obvious inflammatory infiltration ( Figure 5 B); After treatment with biomimetic nanoparticles, local inflammatory infiltration of the wound significantly subsided ( Figure 5 C) Improved inflammatory microenvironment effectively promotes wound healing.

[0044] The above specific embodiments are merely illustrative of the content of this disclosure and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. A biomimetic nanoparticle for targeting and activating neutrophils, characterized in that, The biomimetic nanoparticles consist of a nanocore and an endothelial cell membrane covering the surface of the nanocore, wherein the endothelial cell membrane is an endothelial cell membrane expressing ICAM-1.

2. The biomimetic nanoparticles according to claim 1, characterized in that, The endothelial cell membrane can bind specifically to the CD11b receptor on the surface of activated neutrophils.

3. The biomimetic nanoparticles according to claim 1, characterized in that, The nanocore is a nanocarrier capable of loading diagnostic and therapeutic reagents, which include one or more of fluorescent probes, anti-inflammatory drugs, and antioxidants; furthermore, the particle size of the nanocore is 50-200 nm.

4. The biomimetic nanoparticles according to claim 1, characterized in that, The mass ratio of the endothelial cell membrane to the nanocore is 1:(1~5).

5. The biomimetic nanoparticles according to claim 1, characterized in that, The biomimetic nanoparticles have a particle size of 80-250 nm; further, the biomimetic nanoparticles have a particle size of 100-230 nm.

6. A method for preparing biomimetic nanoparticles as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Culture endothelial cells, collect cells and lyse them, and obtain endothelial cell membranes expressing ICAM-1 by centrifugation and washing. (2) Prepare nanocores by mixing the nanocores with the endothelial cell membrane obtained in step (1), and then incubating and purifying by centrifugation to obtain the biomimetic nanoparticles that target and activate neutrophils.

7. The preparation method according to claim 6, characterized in that, The lysis was performed by placing endothelial cells in a hypotonic lysis buffer solution under ice bath conditions for 30 min, and then using a probe ultrasonic disruptor at 150 W for 3 min to disrupt the cells.

8. The preparation method according to claim 6, characterized in that, The mixing involves mixing endothelial cell membranes and nanocores in 1×PBS at a mass ratio of 1:(1~5); the incubation involves treating the aforementioned mixture with a probe ultrasonic disruptor at 150 W for 3 min.

9. An application of the biomimetic nanoparticles as described in any one of claims 1-5, characterized in that, The biomimetic nanoparticles are used to prepare diagnostic and therapeutic reagents that target and activate neutrophils. The activated neutrophils show high expression of CD11b on their surface, while the unactivated neutrophils show basal low expression of CD11b on their surface.

10. The application according to claim 9, characterized in that, The diagnostic reagent is used to treat diseases related to neutrophil overactivation, including one or more of acute infections, chronic non-healing wounds, and sepsis.

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